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in all patients with sepsis there is abundant activation of inflammatory pathways, which in demonstrable circulating levels of inflammatory cytokines and chemokines, activated inflammatory cells and other markers of increased inflammatory activity. these abnormalities range from subtle activation of coagulation, which can only be detected by sensitive markers of coagulation factor activation; to more marked activation, which may be detectable based on a small decrease in platelet count and subclinical prolongation of global clotting times; and finally to fulminant disseminated intravascular coagulation (dic), which is characterized by simultaneous widespread microvascular thrombosis and profuse bleeding from various sites. septic patients with severe forms of dic may present with thromboembolic disease or clinically less apparent microvascular failure that predominantly presents as multiple organ dysfunction. interestingly, there is a tight, bidirectional relationship between activation of inflammation and coagulation, in which inflammatory activity in activation of coagulation but activated coagulation proteases can also affect inflammatory pathways. activated protein c (apc) appears to play a central role in the pathogenesis of sepsis and associated organ dysfunction. there is ample evidence that insufficient functioning of the protein c pathway contributes to the derangement of coagulation observed in sepsis. the circulating zymogen protein c is activated by the endothelial cell bound thrombomodulin once this is activated by thrombin. apc acts in concert with its co - factor, protein s, and can proteolytically degrade the cofactors va and viiia, which are essential for coagulation; apc is therefore an effective anticoagulant. the endothelial protein c receptor (epcr) not only accelerates activation of protein c several fold but it also serves as a receptor for apc, and binding of apc to epcr may amplify its anticoagulant and anti - inflammatory effects. a recent study demonstrated that exposure of cultured endothelial cells to apc in release of microparticles that contain epcr, but the relevance of that observation to coagulation or inflammation is not yet clear. the protein c system. the solid arrows indicate the mechanisms by which the protein c system is impaired in sepsis. in patients with sepsis the apc system malfunctions at virtually all levels. first, plasma levels of the zymogen protein c are low or very low because of impaired synthesis, consumption and degradation by proteolytic enzymes such as neutrophil elastase. furthermore, significant downregulation of thrombomodulin caused by pro - inflammatory cytokines such as tumour necrosis factor- and interleukin-1 has been demonstrated, ing in diminished protein c activation. low levels of free protein s may further compromise the functioning of the protein c system. in plasma, 60% of the co - factor protein s is complexed to the complement regulatory protein c4b - binding protein (c4bbp). increased plasma levels of c4bbp, which occur as a consequence of the acute phase reaction in inflammatory disease, may in relative protein s deficiency, which further contributes to a procoagulant state during sepsis. although it has been shown that the -chain of c4bbp (which mainly governs binding to protein s) is not much affected by the acute phase response, support for this hypothesis comes from studies conducted in baboons in which infusion of c4bbp in combination with a sublethal dose of escherichia coli ed in a lethal response, with severe organ damage due to dic. finally (but importantly), in sepsis epcr has been shown to be downregulated, which may further adversely affect the function of the protein c system. apart from these effects , sepsis may induce resistance to apc by other mechanisms that are partly dependent on a sharp increase in factor viii levels (released from endothelial cells) and partly due to as yet unidentified mechanisms. administration of apc in a baboon model of intravenous e. coli administration ed in survival of all animals, whereas all control animals in the same experiment died. a similar beneficial effect was observed in rabbits with meningococcal endotoxin shock. in a rat model of septic shock, administration of apc prevented tumour necrosis factor- mediated hypotension, probably caused by modulation of the nitric oxide response. in patients with severe sepsis, administration of recombinant human activated protein c (rhapc) ed in remarkable improvement in microcirculatory perfusion. conversely, experiments conducted in baboons in which the protein c pathway was blocked with monoclonal antibodies ed in complete lethality in an otherwise sublethal model of bacteraemia. in this same model, blockade of the epcr also ed in a more severe response to sublethal e. coli bacteraemia. in addition to these observations in experimental sepsis models, apc was shown to have antithrombotic properties in experimental thrombosis models in dogs, rabbits and baboons. interestingly, apc may also affect fibrinolysis by inhibiting plasminogen activator inhibitor type 1 (a fibrinolytic inhibitor). in a rat model of dic , apc was shown to block activity of plasminogen activator inhibitor type 1, and other experiments demonstrated the ability of apc to enhance clot lysis in vivo. more definitive proof of the beneficial effect of rhapc in severe sepsis comes from clinical studies. these studies are reviewed in detail in other reviews included in this supplement and are not discussed in detail here. however, it may be of interest to review the evidence that rhapc acts as an anticoagulant agent in these studies. first, it should be noted that the selected dose of rhapc was based on the effect on d - dimer levels in a phase ii clinical trial. indeed, in the pivotal prowess (recombinant human activated protein c worldwide evaluation in severe sepsis) trial, septic patients who were treated with apc exhibited a significant decrease in d - dimer levels as compared with placebo control individuals. d - dimer levels dropped by 25% after 2 days of apc administration, which was in contrast to a 10% increase in placebo - treated patients. in a more detailed analysis of coagulation activation upon administration of rhapc, it was clearly demonstrated that markers of thrombin generation sharply dropped almost immediately after initiation of the apc infusion. second, subgroup analyses of trials including patients with severe sepsis demonstrated that patients with the most extreme coagulation abnormalities benefit the most from treatment with rhapc. the relative risk reduction in mortality among patients with sepsis and dic who received apc was 38%, as compared with a relative risk reduction of 18% observed among patients with sepsis who did not have dic. interestingly, the dynamics of coagulation abnormalities in the first days after intensive care unit admission for severe sepsis, including the response of the protein c system, is a strong predictor of outcome. it is difficult to assess whether this anticoagulant effect of rhapc translates into an antithrombotic effect. in the recently concluded xpress (xigris and prophylactic heparin in severe sepsis) study, all patients with severe sepsis received rhapc but were also randomly assigned to receive prophylactic heparin or placebo. the main of this study was that heparin was not equivalent to placebo and might have a beneficial effect on 28-day mortality. of note, this advantageous effect of heparin was completely due to a greater incidence of death and thrombotic adverse events in the patients receiving heparin but who were randomly assigned to placebo (in other words, those who stopped heparin during the trial). there was a markedly low incidence of venous thromboembolism in this study compared with previous reports (the incidence did not differ between placebo patients and patients receiving heparin), even though all patients underwent screening ultrasound for venous thrombosis at around day 6 of admission. in addition, a report of small series of patients with severe sepsis who were treated with rhapc demonstrated a lack of thrombotic obstruction of haemofiltration circuits, even in the absence of heparin or other anticoagulants. apart from the systemic response, there may be a differential localized effect of rhapc on coagulation. the localized effect of apc appears to be particularly marked in the pulmonary compartment. in experiments involving unilateral instillation of endotoxin into healthy individuals, systemic administration of rhapc ed in a marked reduction in bronchoalveolar activation of coagulation. this observation may be relevant because the vast majority of patients with severe sepsis in the various trials had a pulmonary source of infection. an interesting novel finding is the modulatory influence of alveolar epithelial cells on the protein c pathway. it is likely that the underlying mechanism involves shedding of epcr and thrombomodulin by metalloproteinases. evidence that apc acts as an important mediator in the systemic inflammatory response in sepsis comes from experiments showing that blocking the protein c pathway in septic baboons exacerbated the inflammatory response. in contrast, administration of apc ameliorated the inflammatory activation that occurred upon intravenous infusion of e. coli. similar experiments in rodents yielded identical and demonstrated a beneficial effect on inflammatory effects in various tissues. support for the notion that apc has anti - inflammatory properties comes from in vitro findings, demonstrating an apc - binding site on monocytes that may mediate downstream inflammatory processes. it also received support from experiments showing that apc can block nuclear factor-b nuclear translocation, which is a prerequisite for increased levels of pro - inflammatory cytokines and adhesion molecules. these in vitro findings are supported by in vivo studies in mice with targeted disruption of the protein c gene. in these mice with genetic deficiencies of protein c, endotoxaemia was associated with more marked increases in pro - inflammatory cytokines and other inflammatory responses than in wild - type mice. it is likely that the effects of apc on inflammation are mediated by epcr, which may mediate downstream inflammatory processes. binding of apc to epcr influences gene expression profiles of cells by inhibiting endotoxin - induced calcium fluxes in the cell and by blocking nuclear factor-b nuclear translocation. the epcr - apc complex itself can translocate from the plasma membrane into the cell nucleus, which may be another mechanism of modulation of gene expression, although the relative contributions of this nuclear translocation and cell surface signalling are unclear. some studies have also suggested that epcr binding of apc can in activation of protease activated receptor (par)-1 and thereby affect cytokine responses. in contrast, other experiments demonstrated that a significant physiological role for activation of par-1 by apc to be less probable. soluble epcr (the extracellular domain of the cell - associated epcr shed from the cell surface by the action of an inducible metalloproteinase) can bind to proteinase 3, which is an elastase - like enzyme. the ing complex binds to the adhesion integrin macrophage 1 antigen (mac-1). of considerable interest is that the crystal structure of epcr is remarkably similar to the structure of the mhc class 1/cd1 family of proteins, the majority of which are involved in inflammation. blocking the epcr with a specific monoclonal antibody aggravated both the coagulation and the inflammatory response to e. coli infusion. apart from the influence of apc on cytokine levels, remarkable effects of the agent on leucocyte chemotaxis and adhesion of circulating leucocytes to the activated endothelium have been demonstrated. this was confirmed in a hamster endotoxaemia model at concentrations of rhapc that preclude a significant anticoagulant effect. the localized effect of apc in the lung has also been shown to exhibit these anti - inflammatory properties. apc was shown to inhibit the expression of platelet - derived growth factor in the lung, which may reflect a potential mechanism underlying this localized effect. also, apc was shown to protect against disruption of the endothelial cell barrier in sepsis, probably by interfering with epcr and par-1 on endothelial cells. finally, apc can inhibit endothelial cell apoptosis, which also appears to be mediated by binding of apc to epcr and to require par-1. signalling through this pathway can affect bcl-2 homologue protein, which can inhibit apoptosis, and further suppresses p53, which is a pro - apoptotic transcription factor. inadequate functioning of the protein c system, and in particular apc, plays a central role in the pathogenesis of sepsis. attempting to restore the function of this pathway in patients with sepsis appears to be a rational approach and is supported by the beneficial effect of apc in experimental models of sepsis and in clinical studies. apart from its evident effect on the coagulation system and the ability of rhapc to correct the deranged coagulation system in severe sepsis, a series of pleiotropic modulating effects of apc on inflammatory cytokines and cells as well as protective effects on disrupted endothelium have been reported. it should be noted that many of the effects on inflammatory cells and pathways, as well as the cytoprotective effect, have mostly been demonstrated in vitro and sometimes at inordinately high concentrations of apc. the relevance of these findings to the human in vivo situation and their importance to the treatment of sepsis remain to be established. although the relative importance of the anticoagulant effect versus the inflammation - modulating effect of rhapc is not clear, it is tempting to hypothesize that a combined effect is responsible for the benefit from rhapc. indeed, strategies aimed at restoring physiological pathways with less marked effects on inflammation (such as administration of antithrombin or recombinant tissue factor pathway inhibitor) were less successful. further insight into the various mechanisms of action of rhapc on the entangled processes of inflammation and coagulation may permit more detailed dissection of the relative importance of the various pathways that contribute to the pathogenesis of sepsis, potentially culminating in improved treatment strategies. apc = activated protein c; c4bbp = c4b - binding protein; dic = disseminated intravascular coagulation; epcr = endothelial protein c receptor; par = protease activated receptor; rhapc = recombinant human activated protein c. ml and tvdp have participated in advisory boards of eli lilly and company and have participated as investigators in experimental and clinical studies with rhapc. this article is part of critical care volume 11 supplement 5: severe sepsis and drotrecogin alfa (activated).

impairment of the protein c pathway plays a central role in the pathogenesis of sepsis. administration of recombinant human activated protein c (rhapc) may correct the dysregulated anticoagulant mechanism and prevent propagation of thrombin generation and formation of microvascular thrombosis. furthermore, it may simultaneously modulate the inflammatory response. it is likely that the beneficial effect of rhapc observed in experimental and clinical studies of severe sepsis from a combination of mechanisms that modulate the entangled processes of coagulation and inflammation. this review presents an analysis of the various mechanisms of action of rhapc in sepsis.

acquired amegakaryocytic thrombocytopenia (aat) is a rare disease characterized by immunologically mediated peripheral thrombocytopenia similar to idiopathic thrombocytopenic purpura (itp). however, in contrast to itp, there is selective reduction / absence of bone marrow megakaryocytes. though multiple simultaneous / sequential peripheral nerve involvement (mononeuritis multiplex) is rarely described in itp, it is hardly ever described in aat. we are reporting such a case of mononeuritis multiplex due to a cyclical type of aat in a young man. a 34-year - old man presented in neurology service with pain in the legs, bilateral foot drop and numbness in the medial aspect of the left forearm of 2 weeks duration. examination showed pupura and on positive questioning he admitted bleeding from gums starting 2 days prior to neurological symptoms. at the age of 12 years, he had multiple blood transfusions and was told to have aplastic anemia, but a detailed medical report was not traceable. at the age of 22 years, he had an acute hepatitis b infection when the blood counts and hematocrit were documented to be normal. at the time of index admission, he had anemia and multiple purpuric skin lesions. clinical neurological examination revealed sensory motor involvement of the left radial, left ulnar, right common peroneal and left sciatic nerves suggesting a mononeuritis multiplex. an electrophysiology evaluation showed the sensory motor involvement of the left ulnar, left median, left radial, right median, right common peroneal and left sciatic nerves confirming mononeuritis multiplex. there were conduction blocks demonstrable in right peroneal and left ulnar nerves. after the neurological investigations, he was referred to a government teaching hospital where his subsequent hematological workup was done. echymotic and purpuric skin lesions in legs (arrows) motor conduction blocks (more than 50% reduction in compound muscle action potential amplitude with proximal stimulation compared to distal stimulation) in (a) right peroneal nerve and (b) left ulnar nerves his hematocrit was 33%; total leukocyte count, differential count, and erythrocyte sedimentation rates were normal. the platelet count was grossly reduced to just 5000 cells / cu mm and the bleeding time was prolonged to 9 min. corrected reticulocyte count was 2.9% and peripheral blood smear showed marked thrombocytopenia. the serum bilirubin was marginally elevated to 1.86 mg / dl with the predominant indirect component. both direct and indirect coomb's tests were negative. measurements of serum sodium, potassium, calcium, phosphorus, glucose, urea, creatinine, proteins, aspartate aminotransferase and alkaline phosphatase levels were normal. serological tests for hiv, hepatitis b, hepatitis c and syphilis were negative. peripheral blood smear (leishmans stain, 100) showing markedly reduced platelets; compare with normal smear in right panel showing platelets (arrow) a bone marrow aspiration study, after relative stabilization, showed normoblastic erythroid / myeloid series, normal lymphoid cells and marked reduction of megakaryocytes. he had profuse bleeding from the site of aspiration with a fall in hematocrit up to 17% and hence a bone marrow trephine biopsy and a nerve biopsy were not attempted. he was initiated on oral prednisolone, and since the condition was not improving, later intravenous immunoglobulin was given for 5 days, and cyclosporine a was added. gradually he stabilized, bleeding stopped and platelet count improved to 60,000 cells / cumm. at the time of hospital discharge at 6 weeks, he had only minimal bilateral foot drop and mild left grip weakness. after 1 week of discharge, he was readmitted with acute onset massive intracerebral hemorrhage, to which he succumbed within few hours. absent megakaryocytes in bone - marrow (leishmans stain, 10); compare with megakaryocytes of normal bone - marrow in right panel (arrow) we report a case of mononeuritis multiplex due to aat. our patient presented with painful bilateral foot drop along with skin lesions. the clinical and electrophysiological features were suggestive of a mononeuritis multiplex since there was asymmetric involvement of multiple peripheral nerves. skin rashes in mononeuritis multiplex are not rare, and the major differentials include collagen vascular diseases, systemic vasculitides, secondary antiphospholipid antibody syndrome, amyloidosis, sarcoidosis, lyme disease, leprosy and postviral syndromes. hepatitis b virus infection needs special mention since its long - term autoimmune manifestations like systemic lupus erythematosus, antiphospholipid syndrome, polyarteritis nodosa and rheumatoid arthritis can cause mononeutis multiplex with a skin rash. however, hepatitis b serology was negative in our patient though he had an acute infection previously. detailed evaluation revealed markedly reduced megakaryocytes in the bone marrow with relatively normal erythroid and myeloid series. this hematological picture was consistent with what is described as aat, as against itp with similar peripheral blood picture, but normal or increased megakaryocytes in bone marrow. the neuropathy in our patient was probably due to bleeding into the nerves as a of thrombocytopenia. similar neuropathy is well described in itp, where the cause was histologically proven to be intraneural hematoma beneath the epineurium, in between the fascicles. amegakaryocytic thrombocytopenia is a rare disease characterized by severe thrombocytopenia with a total absence or a selective decrease in bone marrow megakaryocytes. it may be a primary disorder or may be seen in aplastic anemia, preleukemia, systemic lupus erythematosus, congenital rubella, dengue fever, ethanol abuse, nutritional b-12 deficiency, radioiodine therapy and thrombocytopenia - absent radius syndrome. a variety of pathogenetic mechanisms were suggested, but an immune - mediated mechanism, due to an autoantibody that blocks the action of endogenous thrombopoietin on megakaryocytopoiesis, appears sensible, at least in a subset of patients. while some patients remain clinically stable, others may progress to aplastic anemia or myelodysplastic syndrome. a cyclical course has also been described, and our patient appears to be in this category though the hematological picture during the initial episode is not clear. congenital amegakaryocytic thrombocytopenia (cat) is another related rare disease presenting with isolated thrombocytopenia in infancy, developing into pancytopenia in later childhood. as against the immune - mediated mechanism in aat, defective surface expression of the thrombopoetin receptor c - mpl over hematopoietic progenitor cells due to c - mpl mutations various treatments have been tried in aat, including corticosteroids, lithium carbonate, androgens, vincristine, folic acid, platelet and erythrocyte transfusions and plasma substitution. an occasional patient may respond well to an intravenous immunoglobulin or cyclosporin a. but, the only real hope is bone - marrow transplantation using a human leukocyte antigen - matched donor.

mononeuritis multiplex involves inflammation of two or more nerves, typically in unrelated parts of the body. it has been well described in bleeding disorders like idiopathic thrombocytopenic purpura (itp) and hemophilia. acquired amegakaryocytic thrombocytopenia (aat) is a bleeding diathesis characterized by thrombocytopenia but with reduced number of megakaryocytes in the bone marrow, as against itp. though aat is a well described entity, peripheral nervous system manifestations have not been described so far. we report a young man who has presented with bleeding diathesis and mononeuritis multiplex due to aat. the mechanism of development of mononeuritis multiplex and treatment options are discussed.

obesity, hypertension, and metabolic syndrome (type 2 diabetes mellitus) are major and growing health problems and are known as high - risk factors for subsequent cardiovascular and renal complications. obesity, hypertension, diabetes, and metabolic syndrome are intimately associated , and sympathetic nervous activation is frequently observed in those conditions. thus, sympathetic nerve activation may play a major role in the onset and development of hypertension, obesity, and metabolic syndrome (diabetes mellitus) as well as cardiovascular complications in patients with hypertension, diabetes and obesity. the sympathetic nervous system plays an important role in the regulation of energy expenditure. reduced energy expenditure and resting metabolic rate are predictive of weight gain (obesity). a large part of the sympathetic nervous system - mediated energy expenditure takes place in skeletal muscle, via the coupling of catecholamines with 2-adrenoceptors. catecholamines are also powerful regulators of lipolysis and act via 1-, 2-, 3- (stimulatory), and 2- (inhibitory) adrenoceptor subtypes in adipose tissue, where their role becomes especially important during both exercise and energy restriction, when increased need for fat as a fuel exists. thus, -adrenoceptors play important roles in energy expenditure and control body weight. recently, there is evidence that human hypertension and obesity have strong genetic s. reported that about 46% of the phenotype of systolic blood pressure are determined genetically for hypertension. have reported close relationships between 2- and 3-adrenoceptor polymorphisms accompanying elevated sympathetic nervous activity, blood pressure elevation (hypertension), weight gain (obesity), and insulin resistance in a series of longitudinal study. many epidemiological studies on the relationships between -adrenoceptor polymorphisms, hypertension, obesity, and diabetes (metabolic syndrome) have still been discordant. this paper will discuss the current topics involving the contribution of the sympathetic nervous system and 2- and 3-adrenoceptor polymorphisms in the onset and the development of hypertension and metabolic syndrome (type 2 diabetes mellitus). the adrenoceptors (or adrenergic receptors) are a class of g protein - coupled receptors which specifically bind their endogenous ligands, the catecholamines (epinephrine and norepinephrine). many tissues possess these adrenoceptors, and the binding of an agonist generally elicits a typical sympathetic response (i.e., the fight - or - flight response). table 1 shows the effects of catecholamines bound to adrenoceptors (table 1) and these effects on sympathetic nervous activity are through - and -adrenergic receptors. there are several types of adrenergic receptors, but there are two main groups: -adrenoceptors (1- and 2-adrenoceptors) and -adrenoceptors (1-, 2-, and 3-adrenoceptors). table 1 also summaries the distributions and functions of the 1-, 2-, 1-, 2-, and 3-adrenoceptors. phenylephrine is a selective agonist of the -adrenoceptors (both 1- and 2-receptors), thus phenylephrine is usually used to investigate the -adrenoceptors function. downstream effects of cyclic amp include cyclic amp dependent protein kinase, which mediates the intracellular events following hormone binding. insulin resistance in hypertension has been well documented in many epidemiological and clinical studies. several investigators have reported that chronic insulin administration elevates blood pressure in rats and in humans, although insulin also has effects on vasodilation. in addition, many clinical and epidemiological studies have demonstrated the close relationships between sympathetic nerve activity, insulin resistance and hypertension. landsberg and other investigators examined the effect of feeding and starvation on sympathetic nerve activity in the cardiac tissue of animals, noting that feeding raised sympathetic nerve activity, and starvation had the opposite effect. energy intake stimulates hyperinsulinemia and sympathetic nerve activity ing in blood pressure elevations in a cycle to inhibit thermogenesis. insulin - mediated sympathetic nerve stimulation in obese subjects is a compensatory mechanism aimed at restoring the energy balance by increasing the metabolic rate. therefore, hyperinsulinemia and insulin resistance in obese subjects are all part of a response to limit further weight gain via stimulating sympathetic nerve activity and thermogenesis. on the other hand, julius et al. have hypothesized that increased sympathetic nerve activity in skeletal muscle causes neurogenic vasoconstriction, thereby reducing blood flow to muscle and consequently inducing a state of insulin resistance by lowering glucose delivery and uptake in hypertension and obesity. both blood pressure elevation and weight gain may reflect a primary increase in sympathetic nervous tone. they described that high plasma norepinephrine might predict future blood pressure elevations and weight gain accompanying deterioration in insulin resistance observed in homa - ir (homeostasis model assessments of insulin resistance). their suggest that sympathetic nerve activity might play a major role in the development of insulin resistance accompanying blood pressure elevations. reported attenuation of hemodynamic and energy expenditure responses to isoproterenol infusion in hypertensive patients, suggesting that sympathetic nerve activity - induced hypertension may subsequently lead to the development of obesity. many epidemiological studies showed close linkages of beta2- and beta3-adrenoceptor polymorphisms with obesity, hypertension, and the metabolic syndrome shown in tables 2, 3, and 4. thus, close linkages between sympathetic nerve activity and insulin resistance might depend on the -adrenoceptor polymorphisms. thus, one could speculate that the strong associations between -adrenoceptor polymorphisms and insulin resistance might provide evidence that heightened sympathetic nerve activity followed by insulin resistance might play a major role in hypertension and obesity, because -adrenoceptor polymorphisms might relate to insulin resistance through heightened sympathetic nerve activity (figure 1). the sympathetic nervous system plays an important role in the regulation of energy expenditure and blood pressure regulation. a large part of the sympathetic nervous system - mediated energy expenditure takes place in skeletal muscle, via the coupling of catecholamines with 2-adrenoceptors. catecholamines are also powerful regulators of lipolysis and act via 1-, 2-, 3- (stimulatory), and 2- (inhibitory) adrenoceptor subtypes in adipose tissue, where their role becomes especially important during both exercise and energy restriction, when increased need for fat as a fuel exists. stimulation of -adrenergic receptors by the sympathetic nervous system is a significant physiological modulator of pre- and postprandial energy expenditure and total daily energy expenditure. single nucleotide polymorphisms might have functional consequences in terms of receptor activity and regulation and hence may contribute to the pathophysiology of hypertension and obesity. on the other hand, there are few studies on the relationships between -adrenoceptor polymorphisms, hypertension, obesity, and metabolic syndrome. the 1-adrenoceptor is predominantly expressed in cardiac myocytes and adipose tissue, where its activation leads to increased heart rate and contractility and stimulation of lipolysis, respectively. the two most common 1-adrenoceptor polymorphisms are ser49gly and arg389gly, with relative allele frequencies of 0.85/0.15 and 0.70/0.30 in the caucasian population, respectively. the 1-adrenoceptor is a candidate gene for obesity because of its role in catecholamine - mediated energy homeostasis. for example, in obese individuals, the degree of weight loss during a very low calorie diet has been shown to correlate with changes in 1-adrenoceptor protein concentration in adipose tissue. a population cohort of 761 women showed that women carrying the gly49 genotype had greater increases in bmi over15 years compared to those with the ser49 genotype. conversely, the distribution of the arg389gly polymorphism is similar in lean and obese subjects and in a large cohort study including 3981 normotensive and 2518 hypertensive subjects. the factors which might explain the discrepancy of published data are shown in the later section. the 2-adrenoceptor is the dominant lipolytic receptor in white human adipose tissue and in skeletal muscle. genetic polymorphisms of the 2-adrenoceptor have been associated with hypertension, obesity, and metabolic syndrome (diabetes mellitus). the most common polymorphisms are arg16gly, with an allele frequency of 0.40/0.60, and gln27glu, with an allele frequency of 0.55/0.45, in the caucasian population. the thr164ile polymorphism is rare, occurring in only 3 to 5% of the general caucasians population. studies of agonist stimulation in cultured cells demonstrate that gly16 receptors have a greater reduction in numbers or enhanced downregulation when compared with arg16 whereas the glu27 receptor is resistant to down regulation when compared with the gln27 variant. a number of clinical studies have investigated the impact of these polymorphisms on vascular responsiveness.. found that young normotensive white men homozygous for the gly16 allele had higher blood pressure and lower peripheral vasodilation after infusion of the 2-agonist salbutamol. similar were obtained by hoit et al. using the agonist terbutaline. on the other hand, three studies investigating isoprenaline induced increase in the limb blood flow thus, volunteers homozygous for gly16 exhibited larger vasodilatory responses than did volunteers homozygous for arg16. conflicting have also been published with regard to the effects of genetic variants on the sympathetic nervous system modulation of energy expenditure. reported that the response of resting energy expenditure to nonspecific -adrenoceptor stimulation (with isoproterenol infusion) was not different between the 3 genotypes of arg16gly.. showed that individuals carrying the arg16arg variant of the 2-adrenoceptor gene have a reduced thermogenic response to selective 2-adrenoceptor activation. associations of 2-adrenoceptor polymorphisms with hypertension and metabolic syndrome have been reported in many epidemiological studies but are also discordant (summarised in tables 2 and 3). the 3-adrenoceptor, which is mainly expressed in adipose tissue, differs from the 2-adrenoceptor in two ways: it has a lower affinity for catecholamines, and it resists desensitisation (i.e., downregulation). these characteristic differences might lead to the different effects of catecholamine on 2-adrenoceptors and 3-adrenoceptors. 3-adrenoceptors stimulate the mobilization of lipids from the white fat cell and increase thermogenesis in brown fat cell. decreased function of 3-adrenoceptor in white adipose tissue slow lipolysis may contribute strongly to visceral obesity in human, and treatment of obese animal models with selective 3-adrenergic agonists reduces fat stores most effectively. many epidemiological studies have shown the strong relationships between 3-adrenoceptor polymorphisms (mainly trp54arg), hypertension, metabolic syndrome, and obesity (table 4). tables 2, 3, 4, and 5 show the discordant contributions of -adrenoceptor polymorphisms to hypertension, metabolic syndrome (type 2 diabetes), and obesity. further, haplotypes of polymorphisms have strong influence on -adrenoceptor function in each polymorphism. the role of the sympathetic nervous system 2- and 3-adrenoceptor polymorphisms in hypertension, metabolic syndrome (diabetes mellitus), and obesity is discussed through a literature review. sympathetic nervous system activity and -adrenoceptor polymorphisms (mainly 2- and 3-adrenoceptor polymorphisms) might contribute to the onset and maintenance of hypertension, metabolic syndrome, and obesity; however, the findings have been discordant. further, few studies have been performed to evaluate the relationship between 2- and 3-adrenoceptor polymorphisms and sympathetic nervous system activity in the same study. a better understanding for the relationships of genetic (polymorphisms) with sympathetic nervous system activity as the cause for hypertension (blood pressure elevation), metabolic syndrome (insulin resistance), and obesity (weight gain) might help for clinical treatment for obesity - related hypertension and metabolic syndrome. in fact, a number of studies have investigated genetic polymorphisms as determinants of cardiovascular response to antihypertensive drug therapy. but in addition, to clarify the pathogenesis and mechanisms may lead to the prevention of hypertension and metabolic syndrome in obesity.

hypertension, diabetes mellitus (especially type 2 diabetes mellitus), metabolic syndrome and obesity are rapidly growing public health problems. sympathetic nerve activation is observed in obesity, hypertension and diabetes mellitus, which have strong genetic as well as environmental determinants. reduced energy expenditure and resting metabolic rate are predictive of weight gain, and the sympathetic nervous system participates in regulating energy balance through thermogenesis. the thermogenic effects of catecholamines in obesity have been mainly mediated via the 2- and 3-adrenergic receptors in humans. further, 2-adrenoceptors importantly influence vascular reactivity and may regulate blood pressure. genetic polymorphistns of the -adrenoceptor gene have been shown to alter the function of several adrenoceptor subtypes and thus to modify the response to catecholamine. 2-adrenoceptor polymorphisms (arg16gly, gln27glu, and thr164ile) have been studied in relation to hypertension. genetic variations in the 3-adrenoceptor (i.e. try64arg variant) are also associated with both obesity and hypertension. however, the precise relationships of the polymorphisms of 2- and 3-adrenoceptor genes with sympathetic nervous system activity, hypertension, and metabolic syndrome have not been fully clarified. this paper will discuss the current topics involving the influence of the sympathetic nervous system and 2- and 3- adrenoceptor polymorphisms in hypertension and metabolic syndrome.

expansion of a tandem repeat array is responsible for disease pathology in the repeat expansion diseases, a group of genetic disorders that includes fragile x mental retardation syndrome (fxs). in fxs however, it differs from the genome - wide microsatellite instability seen in diseases like hereditary non - polyposis colorectal carcinoma (hnpcc) in showing an expansion bias (more expansions than contractions) and occurring at a single genetic locus. in addition, at least in the case of mouse models for the disorders ing from cagctg - repeat expansion, mutations in dna mismatch repair genes, like msh2, msh3 and pms2, actually decrease the frequency of repeat expansion while the opposite is true for hnpcc. different diseases in this group involve repeats with different sequences and repeat unit sizes. these repeats have the potential to form secondary structures that are thought to play a role in the expansion process. however, since not all of the repeats have the same properties, it is unclear whether all repeats expand via the same mechanism. studies in bacteria and yeast have shown that variety of mechanisms can cause repeat instability in these organisms including dna slippage during replication, errors in okazaki fragment processing as well as aberrant dna repair or recombination. however, much is currently unknown about the events responsible for expansion in humans. for example, some expansion diseases only show small changes in repeat number on intergenerational transfer, while others in alleles many times larger than the parental allele from which they are derived. the small expansions are typically seen when the repeats fall within an open reading frame, as in the case of the cagctg - repeat responsible for huntington disease (hd). large expansions, like those that cause fxs, are characteristically seen in regions outside of the open reading frame and occur almost exclusively on maternal transmission. in addition, some diseases involve significant somatic instability while others do not and it is not known if the same mechanism is responsible for both germline and somatic expansion. some studies in transgenic mice have suggested that small expansions occur premeiotically in spermatogonia, whilst others suggest that expansions occur in haploid gametes. some studies also suggest a second event occurs in the early female embryo. in some transgenic mouse models this event is an expansion and in others it is a contraction. the differences in these two models has been attributed to the different genomic context of the repeats. this would be consistent with work in bacteria, yeast and tissue culture models that have implicated orientation, proximity to origins of replication and transcription as cis - acting factors affecting expansion. in order to study the fragile x repeat in as close to normal a chromosomal context as possible , we generated a fxs premutation knock - in (ki) mouse containing 120 cggccg - repeats in the murine fmr1 gene. since the murine fmr1 gene is located in a region of the x chromosome that is syntenic with the corresponding region of the human x chromosome, differences in cis - acting signals involved in expansion may be small. as was seen in other transgenic and knockin mouse models of cggccg - repeat expansion, instability in these animals resembles what is seen in humans in that the frequency is high and shows an expansion bias. in our mouse model large expansions that generate alleles in the full mutation range (> 200 repeats) were seen, but at a much lower frequency than in humans. in fact most expansions in these mice are small, involving fewer than five repeats per generation and occurring more commonly in males than in females. in this respect these expansions resemble what is seen in human carriers of fmr1 common or intermediate sized alleles (grey - zone alleles) or those diseases such as hd that involve relatively small increases in repeat number. expansion occurring during the perigametic interval between the last premeiotic mitosis and the first post - meiotic one could explain the differences between mice and humans and the maternal cggccg - repeat expansion bias in the human fmr1 gene. this interval can last decades in human females creating a large window of opportunity for expansion to occur. in contrast, this interval lasts only months in female mice and in weeks in males of either species. depletion of the dna repair capacity for much of spermatogenesis could also exacerbate the differences in maternal and paternal expansion rates. it may also be that some of the differences between humans and mice reflect differences in the efficacy of dna damage repair or checkpoint proteins. one potential dna damage checkpoint protein that may affect expansion frequency is the ataxia - telangiectasia and rad3-related (atr) kinase. atr primarily responds to stalled replication forks and bulky dna adducts like those arising from uv irradiation or s(n)1-type alkylating agents. here we show that atr heterozygosity leads to increased maternally transmitted expansions and somatic expansions in mice of both genders that appear to involve aberrant dna repair. the atr mice were a kind gift of dr eric brown (caltech, pasadena, ca). mice were maintained in accordance with the guidelines of the niddk animal care and use committee and with the guide for the care and use of laboratory animals (nih publication no . genomic dna is prepared from mouse tail dna or homogenized mouse tissue as previously described. genotyping to detect the presence or absence of the disrupted atr gene the primer pair, frax - c and frax - f, was used to detect both wildtype (wt) fmr1 and fxs premutation alleles. the size of the cggccg - repeat tract was monitored by polymerase chain reaction (pcr) using the primers frax - m4 (5-cttgaggcccagccgccgtcggcc-3) and frax - m5 (5-cggggggcgtgcggtaacggcccaa-3). the binding sites for these primers are located immediately adjacent to the repeat tract and their 3 ends are unique to the ki allele. radiolabelled pcr products were generated by inclusion of -p - dctp in the reaction mix as previously described. the reaction products were then run on a 3130xl genetic analyzer and analysed using genemapper 3.7 (applied biosystems, foster city, ca). these bands show a gaussian distribution about the mean that is very similar for alleles in the same size range. the mean size of each allele was calculated based on the mobility of the central band in the cluster. comparison of the parental mean and the offspring's; mean determined from samples run on the same gel, allows the changes in the offspring's; allele to be reproducibly determined. statistical analysis of instability was carried out using the chi square and student's t - tests. the atr mice were a kind gift of dr eric brown (caltech, pasadena, ca). mice were maintained in accordance with the guidelines of the niddk animal care and use committee and with the guide for the care and use of laboratory animals (nih publication no . genomic dna is prepared from mouse tail dna or homogenized mouse tissue as previously described. genotyping to detect the presence or absence of the disrupted atr gene the primer pair, frax - c and frax - f, was used to detect both wildtype (wt) fmr1 and fxs premutation alleles. the size of the cggccg - repeat tract was monitored by polymerase chain reaction (pcr) using the primers frax - m4 (5-cttgaggcccagccgccgtcggcc-3) and frax - m5 (5-cggggggcgtgcggtaacggcccaa-3). the binding sites for these primers are located immediately adjacent to the repeat tract and their 3 ends are unique to the ki allele. the pcr reaction was done in one of two ways. radiolabelled pcr products were generated by inclusion of -p - dctp in the reaction mix as previously described. the reaction products were then run on a 3130xl genetic analyzer and analysed using genemapper 3.7 (applied biosystems, foster city, ca). these bands show a gaussian distribution about the mean that is very similar for alleles in the same size range. the mean size of each allele was calculated based on the mobility of the central band in the cluster. comparison of the parental mean and the offspring's; mean determined from samples run on the same gel, allows the changes in the offspring's; allele to be reproducibly determined. statistical analysis of instability was carried out using the chi square and student's t - tests. the checkpoint protein atr is responsible for activating pathways that lead to the repair of stalled dna replication forks and bulky lesions in dna. thus the effect of atr mutations on the expansions seen in fragile x premutation mice may shed light on the mechanism of repeat expansion. however, since atr heterozygous mice show a small increase in tumour incidence and a small decrease in overall survival it is apparent that some effects of atr deficiency can be seen even in the heterozygous state. we thus analysed the transmission of a fxs premutation allele with 120 cggccg - repeats in mice heterozygous for a disrupted atr gene. the obtained for the repeat length changes in the offspring of these mice are summarized in table 1. table 1.expansions of a premutation allele in wt and atr mice on paternal and maternal transmissionoffspringcross% mice with expansionsmean no. addedmale female total males females atr wt1*fmr1, atr fmr1, atr37393337 2.22fmr1, atr fmr1, atr86898396635.03*fmr1, atr fmr1, atr6161613.14fmr1, atr fmr1, atr686869675.25fmr1, atr fmr1, atr636363632.2w: wildtype fmr1 allele;: fmr1 premutation allele; -: indicates offspring from crosses that should not have either the premutation or a mutated atr allele; *: data source: entezam et al. ; numbers sharing one of these symbols were compared using the chi - squared test and shown to be significantly different with a p - value < 0.005. numbers sharing these symbols were compared using the student's t - test and shown to be significantly different with a p - value < 0.005. expansions of a premutation allele in wt and atr mice on paternal and maternal transmission w: wildtype fmr1 allele;: fmr1 premutation allele; -: indicates offspring from crosses that should not have either the premutation or a mutated atr allele; *: data source: entezam et al.; numbers sharing one of these symbols were compared using the chi - squared test and shown to be significantly different with a p - value < 0.005. numbers sharing these symbols were compared using the student's t - test and shown to be significantly different with a p - value < 0.005. atr heterozygosity had no effect on the deletion frequency and no effect of atr heterozygosity was seen on the stability of the normal mouse fmr1 repeat (data not shown). in contrast, a significant increase in the expansion frequency was seen when the premutation allele was maternally transmitted in atr mice compared to wt mice (86% versus 37% ; cross 2 versus cross 1 in table 1). this suggests that atr is normally involved in protecting the genome against intergenerational expansions in female mice carrying fxs premutation alleles. in contrast, the expansion frequency on paternal transmission by atr mice was not statistically different from mice wt with respect to atr (68% versus 61%, cross 4 versus cross 3 in table 1). there was an apparent increase in the average number of repeats added per expansion on both paternal and maternal transmission in atr heterozygotes despite the fact that no increase in expansion frequency was seen on paternal transfer. there was no significant gender bias in the expansion frequency in the offspring of atr mothers. this is similar to what is seen in with fragile x repeats in humans and differs from the male expansion bias seen in a transgenic mouse model of cagctg - expansions. atr wt males showed no increase in the transmission of an expanded cggccg - allele when crossed to atr females (cross 5 in table 1). an expansion frequency of 63% was seen in the wt progeny of females carrying the premutation who are heterozygous for atr (cross 2). this is significantly higher than the 37% seen in offspring of females wt for atr. the expansion frequency was even higher in the atr offspring of atr mothers (96%, cross 2 in table 1). to study the role of atr in somatic instability we examined the size of the repeat in different organs of young (10 weeks old) and old (18 months old) atr mice. as can be seen from figure 1a, very limited somatic instability was seen in the liver of young mice as evidenced by the very slightly skewed distribution of repeat sizes. old mice showed much more significant changes in organs like brain, testes and liver. in some instances little remained of the original allele size (see brain sample in figure 1c). we have previously shown that no such somatic instability was seen in mice of a similar age that were wt for atr. in some organs, like the male brain , the expansions ed in a shift of the average allele size without changing the basic monophasic distribution of allele sizes (figure 1c). in organs like liver and whether the biphasic distribution reflects a predisposition of certain cells within the organ to expand is currently under investigation. a biphasic distribution of somatic expansion products has been reported in the liver in a mouse model knockin mouse model for myotonic dystrophy, a cagctg - expansion disorder, where it was attributed to changes in ploidy in a subset of liver cells. figure 1.somatic instability in atr mice carrying an fxs premutation allele. genomic dna was isolated from various organs of young (10 weeks old) and old (18 months old) fxs premutation mice and the repeat tract analysed by pcr using one fam - labelled primer and an abi geneanalyzer as described in the materials and methods. the number repeats in the modal allele in the tail dna at 3 weeks of age is shown in black font and is indicated in the other organ samples by the grey dotted line. similar were obtained using pcr using p--dctp and denaturing gel electrophoresis as described previously. genomic dna was isolated from various organs of young (10 weeks old) and old (18 months old) fxs premutation mice and the repeat tract analysed by pcr using one fam - labelled primer and an abi geneanalyzer as described in the materials and methods. the number repeats in the modal allele in the tail dna at 3 weeks of age is shown in black font and is indicated in the other organ samples by the grey dotted line. similar were obtained using pcr using p--dctp and denaturing gel electrophoresis as described previously. atr heterozygosity had no effect on the deletion frequency and no effect of atr heterozygosity was seen on the stability of the normal mouse fmr1 repeat (data not shown). in contrast, a significant increase in the expansion frequency was seen when the premutation allele was maternally transmitted in atr mice compared to wt mice (86% versus 37% ; cross 2 versus cross 1 in table 1). this suggests that atr is normally involved in protecting the genome against intergenerational expansions in female mice carrying fxs premutation alleles. in contrast, the expansion frequency on paternal transmission by atr mice was not statistically different from mice wt with respect to atr (68% versus 61%, cross 4 versus cross 3 in table 1). there was an apparent increase in the average number of repeats added per expansion on both paternal and maternal transmission in atr heterozygotes despite the fact that no increase in expansion frequency was seen on paternal transfer. there was no significant gender bias in the expansion frequency in the offspring of atr mothers. this is similar to what is seen in with fragile x repeats in humans and differs from the male expansion bias seen in a transgenic mouse model of cagctg - expansions. atr wt males showed no increase in the transmission of an expanded cggccg - allele when crossed to atr females (cross 5 in table 1). an expansion frequency of 63% was seen in the wt progeny of females carrying the premutation who are heterozygous for atr (cross 2). this is significantly higher than the 37% seen in offspring of females wt for atr. the expansion frequency was even higher in the atr offspring of atr mothers (96%, cross 2 in table 1). to study the role of atr in somatic instability we examined the size of the repeat in different organs of young (10 weeks old) and old (18 months old) atr mice. as can be seen from figure 1a, very limited somatic instability was seen in the liver of young mice as evidenced by the very slightly skewed distribution of repeat sizes. old mice showed much more significant changes in organs like brain, testes and liver. in some instances little remained of the original allele size (see brain sample in figure 1c). we have previously shown that no such somatic instability was seen in mice of a similar age that were wt for atr. in some organs, like the male brain, the expansions ed in a shift of the average allele size without changing the basic monophasic distribution of allele sizes (figure 1c). in organs like liver and whether the biphasic distribution reflects a predisposition of certain cells within the organ to expand is currently under investigation. a biphasic distribution of somatic expansion products has been reported in the liver in a mouse model knockin mouse model for myotonic dystrophy, a cagctg - expansion disorder, where it was attributed to changes in ploidy in a subset of liver cells. figure 1.somatic instability in atr mice carrying an fxs premutation allele. genomic dna was isolated from various organs of young (10 weeks old) and old (18 months old) fxs premutation mice and the repeat tract analysed by pcr using one fam - labelled primer and an abi geneanalyzer as described in the materials and methods. the number repeats in the modal allele in the tail dna at 3 weeks of age is shown in black font and is indicated in the other organ samples by the grey dotted line. similar were obtained using pcr using p--dctp and denaturing gel electrophoresis as described previously. genomic dna was isolated from various organs of young (10 weeks old) and old (18 months old) fxs premutation mice and the repeat tract analysed by pcr using one fam - labelled primer and an abi geneanalyzer as described in the materials and methods. the number repeats in the modal allele in the tail dna at 3 weeks of age is shown in black font and is indicated in the other organ samples by the grey dotted line. similar were obtained using pcr using p--dctp and denaturing gel electrophoresis as described previously. we have shown that a maternal atr insufficiency leads to an increase in the frequency of intergenerational expansions. atr heterozygosity also causes the appearance of age - related expansion products in certain organs of older males and females. a number of lines of evidence support a prezygotic origin for the atr - sensitive intergenerational expansions seen in females. the elevated expansion frequency in atr wt offspring of atr heterozygous mothers (cross 2, column 5 in table 1 compared to cross 1, column 5) demonstrates that expansion can occur prior to fertilization of the oocyte. this is supported by the fact that the paternal expansion frequency in mice wt for atr is the same whether the dam is atr or wt for atr (cross 5 in table 1). despite the maternal expansion bias, this together with the fact that female mice do not show more somatic instability in adult tissues than males argues against somatic expansions that occur specifically either in the early female embryo or in the female germline prior to meiosis. we have previously shown that male mice show no bias against the transmission of large repeats. thus the female expansion bias in an atr is not likely to be due to somatic expansions that are selected against in males. furthermore, the excess of expansions in atr offspring (96% compared to 63% in atr wt offspring) is not consistent with a somatic origin in the mother either since all her somatic cells would be atr. thus the most likely origin of the additional expansions in the atr offspring is in the haploid oocyte. given that scheduled dna replication does not occur in haploid gametes, dna damage is thus likely to be responsible. the haploid gamete that gives rise to atr wt offspring would not be atr deficient. thus if expansion were confined to this stage, we would not expect the expansion frequency to be higher than that seen in offspring of a mother wt for atr. therefore, some atr - sensitive expansions in the wt offspring of atr mothers could have occurred in the oogonia or diploid oocyte. thus the window of opportunity for expansions in these cells is much larger than it is for haploid gametes although the rate of expansion may in fact be lower. it may be that the atr heterozygosity in these mice reveals the existence of two different intergenerational expansion mechanisms, the first showing a higher expansion frequency in males that is less sensitive to atr haploinsufficiency and the second, occurring predominantly in females, that is sensitive. this is not to say that atr mutations are necessary for maternal expansions in humans, but rather that an atr insufficiency in mice allows events that would have years to accumulate in humans, to be visible within the rodent lifespan. the relatively atr - insensitive mechanism may account for the paternal transmission bias seen with intermediate and grey - zone fmr1 alleles in humans. a higher mutation frequency in males is usually attributed to errors occurring during dna replication since mature sperm are the product of more rounds of cell division than ova. while it is possible that the increase in the average expansion size seen on paternal transmission reflects measurement errors, it may be that in males the atr - deficiency simply delays the resolution of the replication problem. for example, in a strand - slippage scenario, such a delay could in the incorporation of additional bases into the expanded allele without affecting the frequency with the initiation of strand - slippage occurs. atr - sensitive expansions may be more common in female mice since our data suggest that the mechanism responsible is related to the repair of dna damage and not genomic replication. expansions can thus occur at any point during gametogenesis, a process that lasts significantly longer in females. it is appealing to think of this mechanism as the basis for the strong maternal bias in the transmission of fragile x full mutation alleles in humans. since gametogenesis takes so much longer in human females, this may provide a much larger window of opportunity for such expansions to occur even in the presence of normal amounts of atr. one organ that showed evidence of significant somatic expansion is the brain. in some cases very little of the original allele was seen (see adult male brain in figure 1c). since a significant fraction of cells in the adult brain are post - mitotic, somatic expansion is also probably not limited to dividing cells. thus these expansions may also arise from an aberrant dna repair process rather than a problem with scheduled dna replication. expansion limited to some organs could be explained by differences in either the frequency with which the dna damage that initiates expansion occurs in these organs or the frequency with which such mutations are repaired or eliminated. organs such as liver and brain may be predisposed to expansion since atr is expressed at a lower level in cells with a low proliferative capacity and shows a lower affinity for chromatin in such cells. in a transgenic mouse model of cagctg - repeat expansion a deficiency of ogg1, an enzyme involved in the repair of the oxidation product of guanine, 7,8-dihydro-8-oxoguanine, reduces somatic expansion frequency. oxidative dna damage - induced expansion is likely to be high in organs like brain that in the fragile x premutation mice show elevated levels of atr - sensitive somatic mutations. however, since an ogg1 deficiency did not affect germline instability in the cag cag - mouse model, its significance for intergenerational instability in the cagctg - expansion diseases and in the etiology of fragile x syndrome is unclear. in fragile x premutation mice, an atr insufficiency may prevent error - free dna repair pathways from being activated to repair dna damage, forcing the cell to use a secondary repair pathway that in expansions. potential pathways could be non - homologous end - joining or some sort of homologous recombination. the effect of atr mutations on the cggccg - expansion frequency in female mice raises the possibility that dna damage checkpoint proteins or proteins involved in dna repair have the potential to affect expansion risk in humans. such transacting factors may explain why the risk of expansion in premutation carriers is lower in those carriers identified by general prenatal screening than in carriers from known fragile x families. it could also explain why some intergenerational instability is apparent in some families with alleles in the grey zone and not others and the transition from an allele in the normal size range to a full mutation in two generations reported in one family. our data also raise the possibility that there may be tissue variation in repeat lengths in older human premutation carriers that may be relevant for diagnosis and the severity of disease symptoms.

fragile x mental retardation syndrome is a repeat expansion disease caused by expansion of a cggccg - repeat tract in the 5 utr of the fmr1 gene. in humans, small expansions occur more frequently on paternal transmission while large expansions are exclusively maternal in origin. it has been suggested that expansion is the of aberrant dna replication, repair or recombination. to distinguish amongst these possibilities we crossed mice containing 120 cggccg - repeats in the 5 utr of the mouse fmr1 gene to mice with mutations in atr, a protein important in the cellular response to stalled replication forks and bulky dna lesions. we show here that atr heterozygosity in increased expansion rates of maternally, but not paternally, transmitted alleles. in addition, age - related somatic expansions occurred in mice of both genders that were not seen in atr wild - type animals. some atr - sensitive expansion occurs in postmitotic cells including haploid gametes suggesting that aberrant dna repair is responsible. our data suggest that two mechanisms of repeat expansion exist that may explain the small and large expansions seen in humans. in addition, our data provide an explanation for the maternal bias of large expansions in humans and the lower incidence of these expansions in mice.

struma ovarii is a rare monodermal ovarian teratoma composed predominantly of thyroid tissue.1 approximately 15% of all ovarian teratomas have a small non - significant focus of thyroid tissue. metastases of malignant struma ovarii occur in < 5% of cases,23 and frequent metastatic sites are the liver, peritoneum, lungs, and bone. we present a case of metastatic follicular carcinoma of the liver and peritoneum arising from struma ovarii. a right ovarian cyst was discovered in a 35-year - old woman during a routine follow - up examination for her second pregnancy. she underwent a right ovarian cystectomy and incidental appendectomy at 10 weeks of gestation. a histopathological examination revealed follicular carcinoma arising in a teratoma with infiltrative growth and lymphovascular invasion. after delivery in october 2010, she was transferred to our hospital for further evaluation. because there was no definite abnormal hypermetabolic lesion on positron emission tomography (pet), she was followed with regular thyroid function tests. however, her serum thyroglobulin level increased to 1,437 ng / ml (normal range : 0 - 52 ng / ml). she underwent an endoscopic total thyroidectomy in january 2012, and the histopathological examination revealed incidental nodular hyperplasia in the thyroid gland. laboratory tests, including blood cell counts and serum levels of liver enzymes, electrolytes, and creatinine were within normal limits. i-131 scans revealed increased uptake in the right upper quadrant of the abdomen (fig . 1a). abdominal computed tomography (ct) revealed an 8.56.3 cm - sized metastatic mass in the segment 6 of the liver and variable - sized seeding nodules in both the paracolic gutter and the pelvic cavity (fig . 1b), but no systemic metastatic lesions were observed on pet scans (fig . the liver had an exophytic mass and a demarcation line along segment 6, and the pelvic cavity showed multiple seeding nodules in the right salpinx and ovary . variably sized seeding nodules were discovered in the right diaphragm, small bowel mesentery, and pelvic cavity . we resected the metastatic lesions in segment 6 of the liver and the right salpinx with the infundibulopelvic ligament . the patient started a soft diet on postoperative day 3, and the percutaneous drainage tube was removed on postoperative day 5 . the histopathological examination reported that the liver mass and the nodules in the right salpinx and right diaphragm were compatible with metastatic follicular carcinoma ( fig . in particular, the pathologist mentioned that the metastatic lesion of the liver focally showed a poorly differentiated area . she has been subsequently treated with rai therapy and recent serum thyroglobulin levels were 12 ng / ml . von kalden first described struma ovarii in 1895, which is an ovarian teratoma that contains > 50% mature thyroid tissue . most patients are asymptomatic and have pelvic masses ( 45%) on screening tests, such as ultrasonography or ct. patients present with acute pelvic pain (40%), menstrual irregularities (9%), and hyperthyroidism (5 - 8%).14 a preoperative diagnosis of struma ovarii is impossible, but patients with hyperthyroidisim can be diagnosed by measuring serum thyroid - stimulating hormone, and thyroxine, and conducting thyroglobulin i-123 scintigraphy. in contrast, garg et al.3 raised the possibility of a link between pregnancy and struma ovarii, as in the present case. no consensus of opinion exists for treating a thyroid - type carcinoma arising in struma ovarii. primary surgical resection is essential for successful treatment, and various procedures have been reported in the literature (table 1). a total abdominal hysterectomy and bilateral salphingo - oopherectomy with omentectomy are suitable for postmenopausal women or premenopausal women who have completed childbearing. some investigators have demonstrated that rai therapy reduces recurrence after a total thyroidectomy in cases of metastatic struma ovarii with increased serum thyroglobulin levels. thyroidectomy is useful for excluding primary thyroid cancers with subsequent ovarian metastases and for evaluating the effectiveness of rai therapy.56 adverse effects of rai therapy treatment are transient amenorrhea and premature menopause. studies of pregnancy outcomes after rai therapy reveal no harmful effects; however, miscarriage may occur when conception occurs within 6 months of the last rai therapy.6 other adjuvant treatments after total thyroidectomy such as chemotherapy and thyroid suppression are effective for treating metastatic struma ovarii.7 serum thyroglobulin levels can be used as a marker of metastatic disease. in contrast, patients with increased serum thyroglobulin levels require a total thyroidectomy followed by rai therapy. in addition, radioiodine imaging can be used as a tool for detecting metastatic lesions.36 in this case report, the patient underwent rai therapy after total thyroidectomy, but serum thyroglobulin levels increased, indicating that rai therapy may have been ineffective for treating her disease, or that the tumour growth was too fast to respond to rai therapy. fortunately, her serum thyroglobulin level returned to the normal range after the second debulking operation. therefore, regular serum thyroglobulin measurements might be one of the surveillance tools for cases such as this. most cases of struma ovarii are benign, but malignant changes have been reported in 5 - 15% of cases.24 extraovarian spread is rare even in malignant struma ovarii and occurs in < 5% of patients.23 papillary carcinoma (21%) and follicular carcinoma (54%) are the most frequent types of carcinomas among thyroid - type carcinomas of struma ovarii.1 a typical follicular carcinoma is more likely to metastasize to the lungs, liver, and central nervous system, whereas a papillary carcinoma tends to involve the abdominal cavity and lymph nodes.8 poor prognostic factors are initial extraovarian spread, adhesion to adjacent organs, size > 5 cm, and > 50% proliferating thyroid tissue.9 our patient developed peritoneal dissemination and huge liver metastases 2 years after the ovarian cystectomy. moreover, the last pathological examination showed that the metastatic follicular carcinoma of the liver had the potential for transformation to poorly differentiated carcinoma compared to that in the initial pathological examination. therefore, this patient required close medical observation with regular thyroglobulin measurements. in , we present a rare case of metastatic follicular carcinoma to the liver and peritoneum initially arising in struma ovarii of a young woman. these metastatic lesions were successfully treated with debulking surgery and rai therapy after a total thyroidectomy. however, because she had several poor prognostic factors, close medical follow - up by serum thymoglobulin monitoring was mandatory.

a 35-year - old woman was determined to have an ovarian cyst and underwent a right ovarian cystectomy at 10 weeks of gestation. a histopathological examination revealed follicular carcinoma arising in a teratoma. no evidence of metastasis was found after delivery. she underwent a total thyroidectomy, followed by radioactive iodine (rai) therapy. however, her serum thyroglobulin level increased to 1,437 ng / ml (normal range : 0 - 52 ng / ml) after 10 months. radioiodine scintigraphy and abdominal computed tomography revealed liver metastasis and peritoneal seeding. she underwent debulking surgery of the liver, right salpinx, and peritoneal seeding nodules. a pathological examination showed metastatic follicular carcinoma with focal poorly differentiated features. adjuvant rai therapy was restarted, and her serum thyroglobulin levels returned to normal. in , metastatic lesions were successfully treated with a combination of debulking surgery and rai therapy. close medical follow - up monitoring serum thyroglobulin levels is mandatory in such patients.

the use of proteomics as a systems biology tool in cancer research continues to expand in scope and depth, as it evolves rapidly into a universally applicable method for the investigation of practically any biological process. proteomics is particularly attractive to cancer research because the complexity of tumorigenesis, cancer progression, tumor relapse, and metastasis often involves large protein networks. indeed, in a little over a decade, applications of mass spectrometry - based proteomics in cancer - related research ranging from mechanistic investigation to the discovery of novel therapeutic targets have increased exponentially. this is evidenced by the explosive growth in the number of publications on the subject matter, from 109 in 2000 to 1,349 in 2011 (http://www.ncbi.nlm.nih.gov/pubmed), with the total number of publications containing the keywords proteomics and cancer reaching over 7,500 to date. a large number of review articles have appeared in the past several years, offering excellent overviews and perspectives on novel proteomic applications in cancer. many reviews focused on different cancer types, such as breast cancer,14 pancreatic cancer,5,6 ovarian cancer,79 colorectal cancer,10,11 and glioma.1214 others have focused on sample types or subcellular components, such as tissue,1517 serum,1820 and secretome.2123 in addition, other reviews focused on the innovative proteomic methodologies or the various end goals of proteomics in cancer, such as phosphoproteomics,2427 mechanisms of drug resistance,2830 and kinome profiling.3133 finally, there appear to be more reviews on proteomic biomarker discovery for cancer diagnosis, prognosis, and therapeutic response than on any other area of focus, highlighting the intense activities in the search for new biomarkers that could be translated to clinical applications.30,3436 despite the remarkable advances in utilizing proteomics in almost every aspect of cancer research, many challenges remain. global proteomic approaches, while capable of identifying thousands of proteins (many of which are differentially expressed) are often biased towards soluble and high - abundance proteins, thus easily missing out on many low - abundance proteins that could play critical roles in signal transduction.37 most solid tumors are heterogeneous, making it difficult to identify key proteins that could be used as universal targets even in the same type of cancer.38,39 while high - throughput proteomic methods can discover unbiased cohorts of potential biomarkers and therapeutic targets that regulate disease progression, the sheer number (often in the dozens, if not the hundreds) of hits could be cumbersome, as current functional studies are limited to involving one or a few proteins at a time. thus, a large number of diagnostic and prognostic biomarkers have been identified by proteomic analysis, but very few have made it to the clinical stage, underscoring the urgent need for the convergence of functional studies and clinical sample validations. moreover, the progress in drug development has far lagged the pace of proteomic discovery of potential drug targets, as the former generally requires a prolonged developmental process from drug design to in vitro and in vivo tests for efficacy and toxicities, as well as for absorption, distribution, metabolism, and excretion (adme).40 proteomic approaches have been increasingly used in the preclinical drug discovery process to understand the effects of drug candidates on their protein targets and to shed light on the cellular mechanisms behind the observed phenotype, although many other biomaterials including deoxyribonucleic acid (dna), ribonucleic acid (rna), and carbohydrates can also be used as novel drug targets with different approaches.41 proteomic studies with the main goal of identifying new druggable targets for the treatment of cancer have been relatively few and are the primary focus of this review. for the convenience of discussion, we will attempt to group various proteomic target discovery studies into several distinct approaches for target identification. while mass spectrometry (ms)-based proteomic approaches remain the most comprehensive and versatile tool in large - scale proteomic profiling,42,43 several non - ms - based techniques, such as reverse phase protein microarrays (rpma)44 and peptide arrays45,46 have recently gained popularity. rpma is a technology platform designed for the quantitative analysis of specific phosphorylated, cleaved, or total (phosphorylated and nonphosphorylated) forms of cellular proteins from complex mixtures in multiple samples simultaneously. one microarray can accommodate a range of hundreds to thousands of samples that are printed in a series of replication. rpma is performed using either a primary or a secondary labeled antibody by chemiluminescent, fluorescent, or colorimetric assays. multiplexing is also achieved by simultaneously probing multiple arrays spotted with the same lysate with different antibodies and can be implemented as a quantitative calibrated assay.47 rpma has now been utilized for potential drug discovery / validation, as well as for advancements in understanding the disease both in the clinic and in the laboratory,48,49 and rpma is currently being integrated into human clinical cancer trials.50 the quantitative methods adopted in ms include techniques based on the stable isotope labeling of proteins / peptides, as well as label - free methods. in comparison with labeling methods, label - free methods provide higher dynamic ranges of quantification and are versatile tools that are used to estimate changes in protein abundances between different samples.5153 the utility of ms - based proteomic analyses and their applications in drug target identification have been increasingly recognized over the past decade due to their high sensitivity, specificity, and throughput. for example, gel - free isotopic labeling methods, such as silac (stable isotope labeling with amino acids in cell culture),54,55 isobaric tags for relative and absolute quantitation (itraq),5658 and isobaric tandem mass tags (tmts)59 (figure 1) can now routinely quantify several hundreds of (to a few thousand) proteins in a single analysis with high reproducibility. continued innovations and improvements in the instrumentation and bioinformatics tools will drive ms - based proteomics applications in drug discovery,60,61 complemented by streamlined and focused non - ms - based methods for targeted and repeated assays, such as protein arrays. the former will play key roles in the discovery stage, where quantitative proteomic analysis is undertaken, which compares differentially expressed proteins for potential drug targets. the array methods will be more cost effective and convenient to use for the validation of studies on drug targets and their effects. one of the greatest challenges in cancer therapy is drug resistance that occurs either intrinsically or is acquired after a certain period of treatment. resistance to targeted cancer drugs involves complex and diverse molecular adaptations by cancer cells under the selective pressure of therapeutic regimens. for example, the specific target protein in tumors may undergo mutations to become inaccessible to drugs.6264 in addition, a subset of cancer stem cells may be resistant to the cytotoxicity of drugs that target the bulk of more differentiated cancer cells.6568 moreover, chemotherapeutic agents often induce adaptive changes in the regulatory networks and signaling pathways, leading to the independence of cancer cells on the targeted protein and to the emergence of drug - resistant disease clones.69 indeed, such diverse mechanisms of drug resistance can transform cancer cells under the selective pressure of therapies, which made it extremely difficult to combat tumor recurrence; this is inevitably followed by metastatic progression of the disease and death. current cancer treatment typically consists of a monotherapy regimen targeting a specific protein or signaling pathway, (for example, selective estrogen receptor modulators for the estrogen receptor, herceptin for her2, and gefitinib for epidermal growth factor receptor). however, tumors that are initially responsive eventually develop resistance to therapy via a variety of mechanisms. switching to alternative drugs after the emergence of resistant disease clones following first - line treatment often achieves little in delaying cancer progression. resistance can develop to just one drug, or tumors may be cross - resistant to several structurally dissimilar and functionally distinct agents, a phenomenon known as multidrug resistance.70 thus, there is an urgent need to better understand the resistance mechanism and to identify novel targets for a therapeutic intervention that can be more effective in blocking the progression of cancer cells already resistant to the previous treatment. here, proteomic approaches hold the promise to identify individual proteins and interactive signaling networks that act individually or collectively in conferring drug resistance. the comparison of protein expression between drug - resistant tumor tissues or cell lines with drug - sensitive tumor tissues or cell lines is one of the most promising tools for drug target identification. targeting these proteins and signaling pathways may either resensitize tumor response to the original treatment, or it may help overcoming acquired drug resistance by blocking alternative survival signaling induced by the initial treatment. more importantly, proteomic characterization of a resistant phenotype can provide a more complete picture of signaling adaptation, as multiple drug targets for combinatorial therapeutic solutions can be identified to combat resistance and achieve longer - term improvement in disease outcomes. while advances in this application have been limited, the following examples demonstrate that proteomic approaches are inherently suitable for the discovery of targets for resistant phenotypes, and we expect an increasing number of protein targets to be validated by functional studies followed by therapeutic development. one example comes from a comparative proteomic analysis of colon cancer stem cells and differentiated tumor cells, which uncovered a prominently upregulated protein, baculoviral inhibitor of apoptosis protein (iap) repeat - containing protein 6 (birc6), one of the iaps that may play a crucial role in the chemoresistance of colon cancer stem cells. subsequent knockdown of the gene ed in resensitization of the cells to chemotherapy.71 from this proteomic study suggest that birc6 could be used as a potential therapeutic target to eradicate colon cancer stem cells contributing to colon cancer recurrence. in other cases, an itraq ms - based proteomics analysis was used to identify proteins associated with the development of docetaxel resistance by comparing docetaxel - sensitive pc3 cells and docetaxel - resistant pc3-rx cells developed by docetaxel dose escalation.56 functional validation experiments were performed using recombinant protein treatment and small interfering (si)rna knockdown experiments. the findings from this study suggested that macrophage inhibitory cytokine 1 should be further investigated as a potential biomarker and therapeutic target for acquired resistance to docetaxel treatment. however, extensive functional studies and follow - up validations are required before these proteins can be proven as viable drug targets. similarly, a quantitative proteomic investigation of hepatocellular carcinoma, a type of liver cancer known for its resistance to chemotherapy, implicated phospho - glycerate mutase 1 (pgam1) as a potential therapeutic target.72 clinicopathological analysis indicated that the overexpression of pgam1 was associated with 66.7% of hepatocellular carcinomas, and is strongly correlated with poor differentiation and decreased survival rates (p<0.01). in addition, short hairpin (sh)rna - mediated repression of pgam1 expression ed in significant inhibition in liver cancer cell growth both in vitro and in vivo. using a proteomics approach, elevated levels of prohibitin 1 (phb1) and glutathione s - transferase pi (gst) were found to be associated with paclitaxel resistance in discrete subcellular fractions of two drug - resistant sublines relative to their sensitive sublines.73 immunofluorescent staining and fractionation studies revealed an increased level of phb1 on the surface of resistant cell lines. transiently silencing either phb1 or gst gene expression using sirna in the paclitaxel - resistant cancer cell sublines partially sensitized these cells toward paclitaxel. intriguingly, silencing phb1, but not gst, ed in the activation of intrinsic apoptosis pathway in response to paclitaxel. similarly, stably silencing either phb1 or gst significantly improved paclitaxel sensitivity in a549tr cells both in vitro and in vivo. this study suggests that phb1 is a mediator of paclitaxel resistance, and this resistance may depend on the cellular localization of proteins. based on these preliminary functional studies, phb1 is proposed as a potential target for therapeutic strategies for the treatment of drug - resistant tumors. again, we have yet to see follow - up investigations of the feasibility of such novel, yet unproven, therapeutic targets. in our recent proteomic study of letrozole - resistant breast cancer, a tmt label was used for a quantitative comparison of protein expression profiles of the resistant versus sensitive mcf-7 cells overexpressing aromatase.74 this study identified fascin, among other significantly upregulated proteins, as a promising therapeutic target for the inhibition of metastasis of hormone - resistant breast cancer cells that have acquired the enhanced capacity of migration and invasion. retrospective clinical validation confirms fascin overexpression as an independent indicator of decreased survival and poor prognosis. a subsequent drug discovery effort by our group led to the discovery of a series of thiazole compounds75 as potent inhibitors of migration and invasion of metastatic cancer cells by binding to fascin. in summary, a resistant cancer population, whether inherent or acquired, is a major problem that reduces the activity of conventional and/or molecularly - targeted cancer drugs. the success of identifying drug targets in resistant cancer cells by proteomic approaches depends on reliable drug - resistant tumor tissues or cell lines. it is also critical that proteomics be combined with data from other approaches in an attempt to investigate mechanistic pathways in more detail, as well as to validate potential targets in a clinical setting. chemical proteomics is a technique that identifies proteins enriched or isolated as a of interacting with or binding to a chemical probe, usually a small molecule drug that is fixed on a solid support.7678 therefore, these affinity - based enrichment techniques, in combination with ms, have enabled the direct determination of protein - binding profiles of small molecule drugs under physiological conditions and represent one of the most direct approaches to screen for drug protein interactions.79,80 the major drawback encountered in the affinity - based chemical proteomics is the presence in the pulled - down extract of nonspecifically bound proteins. among these, a large number of proteins that bind nonspecifically to most conventional affinity matrices have been reported.81 it is, therefore, strongly recommended that green fluorescent protein be used as the tag of choice because it shows minimal nonspecific binding to mammalian cell proteins, it can be quantitatively depleted from cell extracts, and it allows for the integration of biochemical protein interaction data with in vivo measurements using fluorescence microscopy. in addition, it is also necessary to perform appropriate negative control experiments to distinguish nonspecific interactions from specific interactions. chemical proteomic approaches also include global proteomic profiling of cellular samples treated with a biologically active compound without enrichment steps. in pharmacology, chemical proteomics have been utilized to determine the specificity of drugs and their analogs, for anticipated as well as unknown targets that may also bind to the probe.41 these activity - based probes can specifically target diverse sets of enzyme families and provides direct information about the activation state of identified proteins.82 several kinds of chemical probes have been used in proteomic studies across a multitude of enzyme classes such as hydrolases, proteases, kinases, phosphatases, histone deacetylases, glycosidases, and oxidoreductases.83 thus, chemical proteomic approaches not only identify protein targets for drugs that exert known biological activities in vitro or in vivo, but they also can discover previously unknown targets for drugs of known modes of action (moa). these off - target proteins may be additional druggable targets, but they may also account for the side effects and toxicities. identification of protein targets that are responsible for toxicities may prove valuable in early drug development to minimize failure in clinical trials. in addition, chemical proteomics can identify novel drug targets by broad - action chemical probes (for example, nonselective kinase inhibitors such as staurosporine).82 in the following, we present a few examples in which chemical proteomics were used effectively for target discovery and validation. gefitinib (iressa ; astrazenca, london, uk) is the first selective inhibitor of the egfr tyrosine kinase domain.84 gefitinib used as monotherapy is an effective treatment for patients with locally advanced or metastatic nonsmall cell lung cancer (nsclc) with egfr mutations.85 gefitinib has also been tested in clinical trials in other tumors, including head and neck squamous cell carcinoma (hnscc), as monotherapy, or in combination with other chemotherapies or radiation, but it has shown limited clinical efficacy with response rates of 10%15%.8689 using cleavable isotope - coded affinity tagging (cicat)-based liquid chromatography tandem mass spectrometry (lc ms / ms) method, chen et al90 identified the tyrosine phosphorylation levels of 21 proteins between control and epidermal growth factor - treated a431 human cervical cancer cells. of these, endofin, dcbld2, and kiaa0582 were validated to be novel tyrosine - phosphorylation targets of epidermal growth factor signaling and responsive to gefitinib therapy.90 pernas et al91 observed that gefitinib sensitivity correlated with phospho (p)-akt and p - stat3 activation in hnscc cell lines and tumor specimens, thus p - akt and p - stat3 could serve as potentially useful drug targets for further development of novel therapeutic agents for hnscc. more recently, wu et al92 used quantitative chemical proteomics to identify several kinases including nek9, lyn, jak1, wee1, and epha2, which are involved in cell survival and the proliferation of hnscc cell lines. these findings may lead to new therapeutic options for hnscc patients. for inhibitors acting on multiple tyrosine kinases, chemical proteomic studies could help uncover additional unknown targets and their interacting signaling network. for example, as an inhibitor of multiple tyrosine kinases, dasatinib targets bcr - abl, src family kinases, c - kit, and platelet - derived growth factor receptor kinase, a cell - permeable kinase probe (da-2) was employed to profile potential cellular targets of dasatinib; a number of previously unknown dasatinib targets were identified, including several serine / threonine kinases (pctk3, stk25, eif-2a, pim-3, pka c-, and pkn2).93 according to another report,94 nearly 40 different kinase targets of dasatinib were discovered using quantitative phosphoproteomics. these include receptor tyrosine kinases (ephrin receptors, discoidin domain receptor 1, and egfr) and nonreceptor tyrosine kinases (frk, brk, and ack). these provided a system - level view of dasatinib action in cancer cells and suggested functional targets and rationales for therapeutic strategies. when the mechanisms of biologically active compounds against cancer cells are not fully understood, chemical proteomics could help elucidate the modes of action by mapping out protein networks perturbed by the drug treatment. in this aspect, a wealth of proteomic discoveries have seldom been translated into validated drug mechanisms, most of which stayed as preliminary findings that could lead to confirmed targets by in - depth functional studies. for example, celecoxib (celebrex ; pfizer, inc, new york, ny, usa), originally a widely prescribed nonsteroidal anti - inflammatory drug, has recently been shown to have anticancer properties95,96 with uncertain mechanisms, ranging from modulating the expression of bcl-2 family members and mitochondria - mediated apoptosis,95,97 to inhibiting nuclear factor kappa b,98,99 akt,100 and stat3101signaling pathways. proteomic analysis of human oral squamous cell carcinoma found that celecoxib treatment induced ten- to 20-fold overexpression of heterogeneous nuclear ribonuclear protein c.102 similarly, global proteomic profiles of colorectal cancer cells before and after treatment with celecoxib revealed significant alterations among multiple proteins involved in diverse cellular functions ranging from glycolysis, protein biosynthesis, dna synthesis, messenger rna processing, protein folding, phosphorylation, redox regulation, to molecular chaperon activities.103 however, none of the proteomic studies has been conclusive in the mechanistic interpretation of numerous alterations in protein expression. the wnt--catenin signaling pathway is a developmental signaling pathway that plays a critical role in regulation, differentiation, proliferation, and apoptosis; thus, aberrant wnt--catenin signaling is widely implicated in numerous cancers.104106 however, identification of targeted wnt--catenin pathway inhibitors in cancer patient treatment has been hampered by the limited number of pathway components that are amenable to small molecule inhibition. recently, huang et al107 used an itraq approach to identify a small molecule, xav939, that selectively inhibits -catenin - mediated transcription. interestingly, xav939 also inhibits the poly - adenosine diphosphate - ribosylating enzymes, tankyrase 1 and tankyrase 2, which interact with a highly conserved domain of axin and stimulate its degradation through the ubiquitin proteasome pathway.107 tankyrases, involved in fundamental cellular processes such as telomere homeostasis and wnt signaling, are potential telomere - directed anticancer targets.108,109 therefore, xav939 is not only a useful tool in understanding wnt--catenin signaling, but also a potential drug that targets wnt--catenin and telomeres. chemical proteomics aimed at characterizing the effects of drug candidates could sometimes lead to the discovery of new drug targets. for example, sulforaphane (sfn) is known to have antimicrobial and anticancer properties in experimental models.110112 sfn can modulate multiple cellular targets involved in cancer development including dna protection, inhibition of cancer cell proliferation, induction of apoptosis, inhibition of neoangiogenesis, progression of benign tumors to malignant tumors, and metastasis.112,113 mastrangelo et al114 identified serotonin receptors as the novel targets of sfn by proteomic analysis in caco-2 colon cancer cells. this finding may not only shed light on the serotonin - mediated signaling pathways in colon cancer, but it may also lead to the development of potential novel therapeutic agents targeting serotonin. in another study, phosphoglucomutase 3 was identified by proteomic analysis and may contribute to sfn - induced cell death in the lncap prostate cancer (pca) cells, which make phosphoglucomutase 3 a potential molecular therapeutic target for pca.115 while these are very early studies on the discovery of druggable protein targets in the relevant disease, they do open up new venues for the development of novel treatment regimens. although the field of chemical proteomics has proven its value in identifying novel drug targets, several challenges still remain to be overcome. first, the chemical probes must be carefully designed to covalently attach to proteins of interest and allow purification and/or identification. second, the spectrum of available activity - based probes needs to be broadened in order to target additional enzyme classes. in addition, the development of high - throughput and gel - free assays, in conjunction with activity - based probes, will be required to enhance the experimental value of chemical proteomics. the continued success of chemical proteomics depends on the design of novel probes or new probe classes that can specifically target diverse sets of enzyme families, as well as on an unbiased assessment of the full spectrum of drug target interactions and their molecular moa. proteins and their spatiotemporal distribution changes play a central role in biological processes, including cancer initiation and progression. analysis of a specific homogeneous cell type from tumor tissues could reveal molecular changes that take place in tumorigenesis since the concentration of disease - related proteins are likely to be much higher within or near the tumor areas. recently, large - scale protein identification and comparative quantitation of highly complex protein mixtures have been achieved with proteomic strategies based upon differential disease state tissues, known as tissue proteomics.35,116120 remarkable advances in tissue proteomics have been propelled by the rapid development of efficient methodologies and techniques in innovative sample preparation, sophisticated ms instrumentation, and powerful bioinformatics tools. the goals of tissue proteomic research focus on early or more accurate diagnosis, improvement of therapeutic strategies, and better evaluation of prognosis and/or prevention of a given disease, as well as on the identification of novel drug targets based upon the differential protein expression between control and case groups for the disease. a major hurdle in tissue proteomics analysis is the variability observed among the tissue samples due to the heterogeneity of tumor tissues containing cancer cells, as well as inflammatory, vascular, and connective tissue cells. detailed proteomic analyses of these clinically valuable samples require meticulous preparation procedures, which is a major current focus of tissue proteomics. so far, tissue proteomics has uncovered large numbers of proteins with altered expressions in tumor tissues by analyzing both fresh frozen biopsy samples and archived tumor tissues stabilized with formalin fixation and paraffin embedding (ffpe). however, very few, if any, of these proteins have actually become novel diagnostic biomarkers or therapeutic targets. as illustrated in the examples below, most tissue proteomic studies remain as preliminary investigations that identified differentially expressed proteins in diseased tissues. while these proteins could be potential prognostic markers or therapeutic targets, there is a general lack of in - depth functional and validated studies that follow up on these initial screening . tissue proteomics has been employed quite frequently in the study of lung cancer for molecular mechanism elucidation as well as for novel drug target discovery.121126 peng et al123 studied the protein profile changes between human pulmonary adenocarcinoma tissues and paired surrounding normal tissue with two - dimensional (2d) gel electrophoresis and esi - q - tof (electrospray ionization, quadrupole, time - of - flight) ms / ms instruments. thirty - two differentially expressed proteins (> 2-fold change ; p<0.05) were identified in pulmonary adenocarcinoma when compared to normal tissues. knockdown of pyruvate kinase isozyme 2 (pkm2), one of the overexpressed proteins, led to a significant suppression of cell growth, to the induction of apoptosis in vitro, and to tumor growth inhibition in vivo. moreover, the shrna - expressing plasmid targeting cofilin-1, another over - expressed protein, significantly inhibited tumor metastases and prolonged survival in vivo. this tissue proteomic analysis and validation indicated that pkm2 and cofilin-1 could be potential therapeutic targets for pulmonary adenocarcinoma. it should be noted that small molecule inhibitors of pkm2 were identified and shown to inhibit cancer cell glycolysis and increase cancer cell death following the loss of growth factor signaling,127 which was consistent with the proteomics - driven . cofilin-1 has long been associated with increased tumor metastasis due to its role in regulating cytoskeleton dynamics,128 and the inhibition of cofilin-1 by small molecule inhibitors has been demonstrated to enhance actin depolymerization.129 in another example, carretero et al124 performed an integrated genomic and proteomic study for the identification of genes and phosphoprotein status associated with lkb1 loss and progression to invasive and metastatic lung tumors in primary and metastatic de novo lung cancers. phosphoproteomic analysis determined that two key modulators of focal adhesion dynamics, src and focal adhesion kinase, are upregulated by lkb1 loss during nsclc progression. moreover, the combined inhibition of src, phosphoinositide 3-kinase, and mek1/2 ed in a synergistic tumor regression. these point towards a mechanism underlying the increased propensity for metastases seen in lkb1-deficient lung tumors, and they identified the src signaling pathway as a molecularly targetable pathway for the treatment of lkb1-deficient nsclc in humans. therefore, development of therapeutic src inhibitors, including dasatinib and saracatinib, will be a valid therapeutic strategy for the treatment of lkb1-deficient nsclc. proteomic analysis of pca tissues associated with multistage tumor progression also provides a valuable source of clinically relevant biomarkers and novel therapeutic targets.130,131 for example, ummanni et al132 recently reported the differential protein expression patterns from histologically characterized pca tumor tissues and surrounding benign tissues of individual pca patients based upon 2d differential gel electrophoresis coupled with ms. the study identified 118 protein spots differentially expressed in cancer (n=24) when compared to benign (n=21) prostate tissues adjacent to cancerous tissues. analysis of these gel spots by matrix - assisted laser desorption / ionization time - of - flight ms / ms revealed 79 unique proteins. moreover, system biology analysis of proteomic revealed several novel drug targets of pca development and/or progression including eif4a3, ddah1, arg2, prdx3, and prdx4, although functional validation of individual targets have yet to be performed. it is evident that proteomic analysis of multistage pca tissues could provide new insights into pca progression and potentially lead to the design of novel diagnostic and therapeutic strategies. efficient peptide / protein extraction approaches are crucial to the success of tissue proteomic analysis. two major strategies of mining proteomic information from ffpe archive tissue samples have been developed.133138 one strategy aimed to recover full - length proteins by heat treatment in suitable buffers with consequent reversion of the formaldehyde - induced cross - links. the operation conditions have been optimized by various labs since shi et al139 initially established this method. application of high temperatures and the addition of detergent sodium dodecyl sulfate were indicated as two critical conditions for enhanced protein extraction yields.140142 because the protein was deposited in large amounts as insoluble, densely packed aggregates, applications of 40,000 psi pressure were reported to recover 96% of proteins from a tissue surrogate model, compared with a 26% recovery rate at 14.7 psi.138,143 another major strategy is based upon the direct proteolytic digestion of intact ffpe archive tissue samples, followed by liquid chromatography ms / ms characterization of the complex peptide mixture.144 tissue solubilization can be achieved in various buffers including sodium dodecyl sulfate dithiothreitol,145 radioimmunoprecipitation assay,146 acetonitrile ammonium bicarbonate,147 tris - hcl148 under different temperatures, and ph conditions for the optimal yield of peptides. as a successful example , hwang et al147 characterized 428 prostate - expressed proteins from ffpe archive tissue samples for the discovery of pca biomarkers and potential drug targets using the shotgun approach. to date demonstrate that direct trypsin protein digestion is an effective sample preparation strategy for proteomic analysis of ffpe archive tissues. approximately 70% of diagnosed breast cancers express the estrogen receptor (er+) whereas er breast cancers are not well differentiated and clinically tend to be more aggressive.149152 a global proteomic characterization and quantitative comparison of er+ and er breast tumors were recently performed on fresh frozen breast tumor tissues.153 the study identified 2,995 unique proteins including a number of receptor tyrosine kinases and intracellular kinases that are abundantly expressed in both er+ and er breast cancer tissues. using a label - free quantitative approach, 236 proteins were found differentially expressed between er+ and er breast tumors. compared with er breast tumors, 141 proteins were selectively upregulated, while 95 proteins were downregulated in er+ tumors. molecular function analysis of gene ontology showed that the dehydrogenase, reductase, cytoskeletal proteins, extracellular matrix, hydrolase, and lyase categories were significantly enriched in er+ breast tumors, whereas selected calcium - binding proteins, membrane traffic proteins, and cytoskeletal proteins were enriched in er tumors. biological process and pathway analysis indicated that proteins related to amino acid metabolism, proteasome, and fatty acid metabolism were overexpressed in er+ tumors, while proteins related to the glycolysis pathway were overexpressed in er tumors. given the clinical challenges in treating er breast cancer, such in vivo findings of differentially expressed proteins in er tumors are especially significant because of their pathological relevance. these proteins may serve as potential therapeutic targets for er breast cancer if functionally validated. recently, cabezn et al154 used a systematic 2d gel - based proteomic profiling strategy, applied to the analysis of 78 fresh triple - negative breast cancer (tnbc) tissue biopsies, in combination with a three - tier orthogonal technology (2d polyacrylamide gel electrophoresis / silver staining coupled with ms, 2d western blotting, and immunohistochemistry) approach, they identified and validated one specific protein, mage - a4, which was expressed in a significant fraction of tnbc and her2-positive / er lesions. the existence of immunotherapeutic approaches specifically targeting this protein, or mage - a protein family members, provides novel management options for tnbc and her2-positive / er patients bearing mage - a4 positive tumors. although promising have been reported, there has yet to be a breakthrough that is close to becoming clinically applicable. the challenge will be to perform robustly designed, large retrospective studies including independent validation sets, followed by prospective validation studies to demonstrate the clinical benefits for patients. the development of sirna in vivo techniques will greatly speed up selection of useful proteins. characterization of discriminator proteins will provide new molecularly targeted anticancer drugs. faced with the complexity of cancer tissue proteomics and its great clinical potential, a global coordination of ongoing efforts appears to be crucial. in this review we provided an overview of three major approaches of proteomics employed in discovering potential therapeutic targets for cancer. target identification in resistant cancer populations is one of the most promising tools for drug target identification, and it can uncover multiple drug targets for combinatorial therapeutic solutions to combat resistance and achieve longer - term improvement in disease outcomes. chemical proteomics approaches not only identify protein targets for drugs that exert known biological activities, but they can also discover previously unknown targets for drugs of known moa. the tissue proteomics approach offers the advantage of direct clinical relevance and can complement mechanism - based approaches like chemical proteomics. proteomics continues to evolve rapidly both in instrumental advancement and innovative applications in biomedical research, particularly in the field of cancer. the complexity of cancer biology requires not only in - depth functional studies for mechanistic elucidation, but also unbiased system approaches for a global view of interactive signaling networks that reflect the cancer disease stage. cancer proteomics has uncovered a phenomenal number of cancer - specific proteomic alterations, shedding light on previously unknown mechanisms in tumorigenesis, cancer progression, and metastasis. perhaps more prominently, proteomics has provided an unmatched wealth of potential biomarkers that could be clinically used for the diagnosis and prediction of treatment responses. the utility of proteomics as a valuable tool for the discovery of new cancer treatment targets has been increasingly recognized in recent years. we note the remarkable progress in several directions when cancer proteomics plays an important role in drug discovery, as illustrated in figure 2. novel proteomics methodologies, both ms - based and non - ms - based, continued to be optimized and perfected using high - resolution and high - speed ms, simplified and robust array technologies for tumor tissue, cell, subcellular compartment, and other relevant biological samples. proteomics has been increasingly used to elucidate the mechanisms of drug resistance in cancer with the hope that novel protein targets could emerge for therapeutic intervention of the resistant disease. in other innovative frontiers, chemical proteomics studies examining drug - induced biological perturbations often reveal new clinically relevant biomarkers and drug targets; in dissecting mechanisms of cancer progression, proteomics also identified previously unknown or unconsidered druggable targets. however, despite the tremendous progress and potential of proteomics for target discovery, there are serious challenges. a lack of coordinated efforts to follow up with the proteomic discovery of novel therapeutic targets largely accounts for the current gap between drug development (design, synthesis, and optimization) and new drug targets emerging from numerous proteomic studies of cancer. most of the proteins identified in the proteomic analyses of drug resistance have not been validated for their roles in specific disease processes and their potential in clinical use. functional studies involving in vitro manipulations of gene expression using specific pharmacological inhibitors, antisense rna, rna interference, or gene knockout experiments should be an integral part of a proteomic study for target discovery. future investigations should place more emphasis on functional studies and on the clinical validation of novel targets for their translation in clinical trials with greater speed and higher success rates. proteomics is also expected to play a major role in the preclinical and clinical research of targeted and combinatorial therapies.

proteomic approaches are continuing to make headways in cancer research by helping to elucidate complex signaling networks that underlie tumorigenesis and disease progression. this review describes recent advances made in the proteomic discovery of drug targets for therapeutic development. a variety of technical and methodological advances are overviewed with a critical assessment of challenges and potentials. a number of potential drug targets, such as baculoviral inhibitor of apoptosis protein repeat - containing protein 6, macrophage inhibitory cytokine 1, phosphoglycerate mutase 1, prohibitin 1, fascin, and pyruvate kinase isozyme 2 were identified in the proteomic analysis of drug - resistant cancer cells, drug action, and differential disease state tissues. future directions for proteomics - based target identification and validation to be more translation efficient are also discussed.

there are growing clinical demands for controlled and sustained drug release systems to serve as implantable devices for patients with acute and chronic diseases. under these circumstances, it is not surprising that intelligent materials have emerged as a promising strategy for drug delivery. for example, many efforts have been directed toward using various stimuli - responsive biomaterials as on off controllable drug carriers in which the bioactive cargos are released via changes in ph, temperature, or input of electrical or uv energy. at the present time, electrical stimulation appears to be one of the more suitable approaches for clinical translation in that the electrical signal can be triggered using portable equipment, not requiring significant cost or sophisticated technologies, and the generated signal can be tuned using a variety of exposure times and current intensities. in this regard, conductive polymers have emerged as one of the more useful drug - delivery platforms. in particular, polypyrrole (ppy) has become a candidate material due to its lack of toxicity, favorable biocompatibility, and reversible electrochemical properties. for instance, polypyrrole demonstrated excellent in vivo biocompatibility, with similar to teflon when implanted as a neural prosthetic. both glial and neuronal cells were found to be in intimate contact with the ppy material. other studies noted that ppy extracts exhibited no hemolytic, allergenic, or mutagenic properties whereas sciatic nerve implants elicited only a minor inflammatory response 6 months postimplantation. functionally, the electrostatic interaction of ppy in response to electric current provides a controllable switch for the release of tethered cargo, providing in situ delivery of nerve growth factors, anti - inflammatory drugs, or adenosine triphosphate. prior investigations demonstrate that time- and site - specific release profiles can be obtained by modifying electrical or magnetic pulse patterns and durations. electromagnetic fields (emf) have been further discussed as another potential form of stimulus for drug delivery and was first realized in carbon nanotubes. we have previously outlined the fabrication and physiochemical details that advance the potential of ppy in medical practice. however, two obstacles that prevent the practical use of the ppy polymer systems are the following: the amount of a drug s cargo is limited when using typical flat thin - film fabrication and delivery of the cargo within the human body requires percutaneous electrodes to deliver the required level of electric current (i.e., a physical electrical contact with the ppy substrate). this latter obstacle must be understood in the context of chronic applications where drug release may be desirable over many days until the supply within the film is exhausted. during this time , percutaneous wires carry the possibility of infection by retrograde tracking along the insertion path where normal movement vitiates a perfect seal between tissues and the insulated electrodes. in this work, we detail a new ppy paradigm that overcomes the limits of payload and invasive delivery. we demonstrate that a three - dimensionally nanostructured ppy platform impregnated with a model test drug (dexamethasone, dex) exhibits outstanding drug loading efficiency. moreover, noninvasive and on - demand drug release has been demonstrated by exposing the ppy nanowires to high - frequency pulsed electromagnetic fields (emf). subsequent studies using a lipopolysaccharide - challenged bv-2 glial cell line showed that the dex released by emf stimulation remained bioactive and ameliorated both oxidative damage and the inflammatory response. the putative inductive coupling between the dex - doped polypyrrole nanowires (dex / ppynws) and emfs bypasses the requirement for direct electrical contact with ppy and opens the door to ppy embodiments that can be placed in vivo in which the cargo can be delivered controllably and noninvasively for many weeks. ppy flat films and nanowires were fabricated using common electropolymerization techniques. the first step in this manufacturing process was the preparation of the templates. for flat ppy films, indium tin oxide (ito) glass slides with 515 resistivity (delta technologies) were washed in acetone for 30 min, followed by ethanol and milli - q water in an ultrasonic bath. for ppynws experiments, an anodic aluminum oxide template (aao, figure 1) with a 0.2 m pore size and 60 m thickness (whatman) was obtained. the aao templates were subsequently coated with a 100-nm - thick gold layer on one side using a varian e - beam evaporator. briefly, an aqueous mixture consisting of 0.2 m pyrrole (py, sigma), 0.025 m dexamethasone 21-phosphate disodium salt (dex, sigma), and 0.05 m 10 nm nanoxact spherical gold nanoparticles (aunps, nanocomposix, ca) was mixed. for comparison, 0.2 m pyrrole, 0.025 m dex, and 0.1 m poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (pss, sigma) were also mixed for synthesis. note that for the in vitro experiment the concentration of dex within the solutions was decreased to 5 mm. prepared aao templates were subsequently incubated into their respective mixtures for 30 min. cleaned ito slides and aao templates were separately connected to the working electrode from a ch instruments model 604 electrochemical analyzer / workstation, with a platinum counter electrode and ag / agcl reference electrode placed in the synthesis solution. the one - step electropolymerization of dex / ppy was accomplished by applying a constant potential of 1 v using a potentiostat. following polymerization, the final films were rinsed thoroughly with milli - q water for 5 min. the aao templates were removed by placing the films in 3 m sodium hydroxide and then rinsed with milli - q water. each cutaway diagram is an illustration of the aao template. at the far left the third step illustrates the filling and diffusion of the solution consisting of pyrrole monomer, aunps or pss, and dex in the template pores. the fourth step is the dex - conjugated polypyrrole electropolymerization process. finally (far right), the ppy nws are shown attached to the gold base after the dissolution of the aao template with sodium hydroxide. ppy samples were prepared by first sputter coating with platinum for 60 s. sem images were taken with an fei nova nano sem via field emission scanning using et and tld at high vacuum at an acceleration voltage of 5 kv. tem images were taken using a fei / philips cm-10 transmission electron microscope operated at 100 kv, a 200 m condenser aperture, and a 70 m objective aperture. multiple factors must be considered when designing the emf stimulation system and pulse conditions. these parameters include the waveform shape, pulse duration, pulse magnitude, duty cycle, and so forth. we originally chose a narrow pulse width (500 ns) stimulating regime as it allowed us to experiment with various coil geometries with minimal power consumption. the square wave was chosen in order to maximize the induced electric fields within the ppy since that is the hypothesized stimulus for drug release. our original pilot measurements were made using a three - turn coil which had a very low inductance allowing testing that set the limits for overheating. this data (not shown) then permitted the construction of a 15-turn coil, which was used in all experiments reported here. briefly, awg 16 copper wire was wound 15 times to create a 15-turn coil. the geometry of the coil was 2.3 2.8 cm inside dimensions and 3.0 3.8 cm outside dimensions. the coil was then stimulated with a pulsing regime using a custom - made circuit. each input pulse was on only long enough to saturate the coil and then was turned off for the duration required to unload the coil completely (10 s). additionally, we tested several stimulation patterns such as oscillating the polarity, grouping the same polarity pulses in different temporal patterns, and so forth while maintaining a 5% duty cycle. as we did not find significant differences in drug release in the initial trials (data not shown), we utilized the waveform described in figure s2 in order to minimize heat generation. (a) the top illustration shows the integrated system used to create the electromagnetic field (emf) to induce ppynws activity. the stimulator is connected to a wire - wound coil in which a standard cuvette is placed in the center. following emf stimulation, aliquots of the solution were assayed for drug concentration via uv vis spectrophotometry. the left inset describes the dimensions of the stimulating coil, where h = 1.2 cm, l = 3.8 cm, and w = 3.0 cm. the recordings in (b), left and right, are probe measurements of the magnetic fields (mf) and electric fields (ef) at the center of the coil (36 g and 4000 v / m, respectively). the insets describe the idealized mf and ef waveforms based on the input square - pulse stimulation regime. using the waveform patterns in figure s1 as the input signal, the real time magnetic and electric field outputs from the stimulation coil were measured using high - frequency emc 100b (magnetic) and 100d (electric) probes (beehive electronics, ca). probes were connected to a tektronics tds 2012b oscilloscope and terminated with a 50 resistor. for data collection, each probe was positioned in the stimulation coil center (origin). the probes were directional and oriented in the direction of the maximum expected reading. this value was taken to be the output probe value and was converted to dbm. the recorded data points from the oscilloscope were imported into ms excel in which the fast fourier transform (fft) of the waveforms was calculated. from the ffts, the fundamental frequency and with the output dbm power value were used with the antenna gain equation (figure s1) provided for each probe to estimate ac field magnitudes. both potentiostat electrical stimulation and the setup was the same as for the electropolymerization process with three electrodes, but the dex - conjugated ppy film was connected to a working electrode and subsequently placed in pbs solution containing 0.80% w / v nacl. a constant voltage of 0.1 v was applied for 5 h using a ch instruments model 604 electrochemical analyzer / workstation. for comparison, the dex / ppy platforms were also stimulated by an induced emf generated by our custom circuit and coil stimulator system. the supernatants were collected after 1, 3, and 5 h of stimulation for both the potentiostat and the pulsed magnetic field generator. figure 2 depicts the experimental setup and the essential components of the emf stimulation system used to remotely trigger ppy drug release. the ppy samples were placed in a 12.5 mm 12.5 mm 45 mm standard cuvette filled with phosphate buffer solution. the cuvette was positioned in the center void of the stimulation coil but was not in physical contact with the coil. the coil was subsequently energized using square wave pulse trains as described in figure s1. experiments comparing the dex - conjugated polypyrrole compositions and the drug release effects of stimulation were continued for up to 16 days using emf stimulation. to test if the stimulated release of dex might be influenced by the heating of stimulated samples, we placed the dex - doped flat polypyrrole templates within a controlled incubator at 37 c. this temperature was slightly higher than ambient temperature in the vicinity of the active coil, which was measured to be about 30 c. for all 16 days of dex release, the sample solutions were collected after 1, 3, 5, 7, 10, 13, and 16 days of continuous emf stimulation (n = 5). a calibration curve of dex (y(od) = 23.8x 0.04 ) was also prepared to detect the drug release. to determine the characteristics of pulsatile emf stimulation, on and off experiments were performed on polypyrrole nanowires synthesized with gold nanoparticle (ppynws aunps) samples. the dex release profile recordings were begun after 10 h of stimulation by the pulsed emf. then, pulsed emf stimulation was turned on (referred to as the on time) for 2 h and discontinued (off time) for 2 h. this regimen was continued for four cycles for each sample. ppy samples of about 0.51.0 cm were prepared and submitted for xps analysis. the surface chemical composition of the samples was analyzed by x - ray photoelectron spectroscopy (xps) using a kratos axix ultra dld spectrometer. the survey and high - resolution spectra were obtained in constant pass energy mode with pass energies of 160 and 20 ev, respectively (survey, 1 ev / step ; high - resolution spectra, 0.05 ev / step). the bv-2 cells were cultured in 75 cm flasks in dulbecco s modified eagle s medium (sigma - aldrich) with 10% fetal bovine serum, 100 iu / ml penicillin, and 100 g / ml streptomycin. cells were maintained at 37 c in a humidified incubator with 5% co2, harvested by trypsinizing (0.25% trysin / edta in pbs), and reseeded (at approximately 1 10 density) in 12-well dishes for 24 h to detect ros. cells were incubated in the medium containing 1g / ml lipopolysaccharides (lps) from escherichia coli 026:b6 (sigma - aldrich) for 6 h. then, bv-2 cells were treated with 1 g / ml dex after the application of lps for 1 h. to detect dex release by pulsed emf, ppynws - aunps films (approximately 30 - 40 mm in unit area) were stimulated for 30 min and 1 h after lps treatment in 1 h increments. in these experiments, the coil was placed flat directly below the petri dish, aligned with the well and touching the bottom surface of the dish. after 6 h of lps treatment, cells were trypsinized, centrifuged, and resuspended in a 10 m oxidative stress indicator (cm - h2dcfda, molecular probes) in pbs. suspended cells were then placed in the incubator for 1 h, and the measurement of ros was performed with a plate reader at 480 nm/530 nm. measurement was also performed with a fluorescence microscope (olympus ix 81 inverted). the cells were illuminated with an x - cite series 120pcq fluorescence illumination source. additionally, another strong oxidizing reagent, hydrogen peroxide (h2o2), was used to confirm the lps (oxiselect intracellular ros assay kit ; cell biolabs, inc .). briefly, bv-2 cells were seeded in black 96-well plates for 12 h and then incubated in 1 mm dcfh - da solution for 1 h. hydrogen peroxide (20 m) was incubated in these plates for 30 min, and then dex treatments were applied for 15 min. for emf - stimulated dex release , the ppynws - aunps films were divided into smaller pieces (approximately 30 mm), placed in each well, and stimulated for 15 min. in these instances , the coil was placed below the 96-well plate touching the bottom surface, with wells in a 3 3 matrix being stimulated. bv-2 cells were cultured on poly - d - lysine (sigma) precoated round cover glasses (no . 1.5 thickness, 12 mm) in 12-well plates for 24 h. after experimental treatment with lps, dex, and released dex, bv-2 cells were fixed with 4% paraformaldehyde for 30 min at room temperature. a blocking solution, 1% albumin from bovine serum (sigma - aldrich), was applied to the samples for 1 h. antinitric oxide synthase ii antibody (emd millipore) was diluted in a ratio of 1:400 with antibody dilution buffer (sigma). cells were then incubated in a diluted primary antibody solution overnight at 4 c. the next day, these cells were incubated in 1:100 diluted cy3 conjugated goat antirabbit igg antibody solution (emd millipore) for 2 h. dried cover glasses were mounted on glass slides using a vectashield hardset mounting medium with dapi (vector lab). these samples were all imaged with a confocal microscope (leica sp5/sted / mp system). all of the data are shown with the standard error of the mean sem. comparative tests used were the conventional student s t test and one - way anova, with significance determined by a p value of 0.05. nanowire architecture was revealed by sem and tem (figures 3 and 4). polypyrrole wires (10 m long) were completed in about 13001400 s of deposition using the potentiostat (figure 2a), while shorter lengths were realized for shorter deposition times (figure s3b). the desired structure of ppynws was achieved for 200300 s of deposition (figure 3b). as an aside, smaller ppynws ppynws (200 nm long) were fabricated using different aao templates with 0.02 m pores (figure s3a), but these were not used in this study. the free - standing, vertically aligned ppynws were generally arranged into mats in unit areas of up to 1 to 2 cm. (a) scanning electron micrograph showing a top (dorsal) view of polypyrrole wires (200 nm diameter and 10 m length) produced with 13001400 s of deposition. (b) a similar micrograph where 200300 s of deposition was used. scale bars: (a) 10 m and (b) 2 m. figure 4a, b shows the distribution of gold nanoparticles within the bulk of 500-nm- to 2-m - long ppynws. note that several gold bases are shown due to the folding of the ppynws. (b) higher - magnification tem of individual nanowires showing the details of gold nanoparticle deposition. these wires range from approximately 500 to 2000 nm in length and 150 nm in diameter. the real - time magnetic and electric field output waveforms at the coil center were plotted in figure 2. the measurements revealed that the magnetic field output was similar to the input field, with some oscillation noise present in the square waveform. fft decomposition of the measured signal showed that the fundamental frequency of the magnetic field was 3.2 mhz. this converts to an average peak amplitude of 36 g when using the antenna gain equation supplied by the probe vendor (figure s1). moreover, the amplitude of the magnetic field did not vary by more than 20% within the coil. the highest field amplitudes were located near the corners in the x y plane containing the coil center. along the z axis , there was a small decrease in intensity as the probe was moved away from the coil center. overall, the magnetic field was focused and concentrated within the internal space of the coil. however, outside of the coil, the magnetic field became divergent and weak, with the field roughly following a cubic decay. therefore, ppy drug - release experiments were tested with the films situated within the coil. on the basis of the obtained mf probe readings, the ppy films were exposed to peak magnetic fields within the range of 2540 g. similarly, the raw electric field data obtained for each coil is also depicted in figure 2b. note that the measured electric field corresponded to the derivative of the measured magnetic field, as expected by faraday s law. the electric field waveform exhibited oscillatory behavior with sharp peaks primarily concentrated in the ramp - up and switch off phases of the emf. the 15-turn coil produced a peak e field of 4700 v / m and a fundamental frequency of 65 mhz. thus, the estimated electric field magnitude in which the ppy fabrications resided is in the 30005000 v / m range for the 15-turn coils. we emphasize that due to the high - frequency pulsed nature of the stimulation pattern these are the estimated peak emf values during the 500 ns pulse duration. the time - averaged emf values are much lower when considering the duty cycle and would be less than 5% of these values. the distance dependence of the electric field from the coil center followed that of the magnetic field. note that for bv-2 cell culture experiments, the coil was placed on the bottom of the petri dish since it was not possible to place the culture chamber within the coil. in such situations, the measured peak fields to which the ppy substrates were exposed were between 320 g and 3002500 v / m for the mf and ef, respectively. drug release characteristics of the ppynws to conventional flat ppy films are shown in figure 5. (a) the release profiles of dex from both a conventional flat ppy film and ppy nws within 5 h, with one group stimulated electrically using a potentiostat and the others by emf. again, there was only marginal release of dex (< 0.5 mg / cm) from flat ppy films. (c) active (emf stimulation) vs passive (no stimulation) of both flat ppy films and ppynws. note that an extraordinary release of dex occurs after emf stimulation of the ppynw - aunps. the small amount of dex detected at the off times was likely due to passive diffusion from the source. * p 0.05, * * p 0.01, and * * * p 0.001. figure 5a reveals the of drug release monitored over a 5 h period. potentiostat stimulation of flat ppy - aunps was not significantly different from that of emf stimulation. upon exposure to emf stimulation, there was a statistically significant increase in the release levels of ppynws compared to that of the flat ppy film (* p 0.05). also note that the emf stimulation of ppynws - aunps ed in more release of dex than did potentiostat stimulation. the inclusion of gold nanoparticles greatly improved the loading capacity and the rate of release. this trend was preserved in both flat ppy thin films and in the ppynws, when compared to the pss - doped ppy films. at 1, 7, and 16 days, these differences were almost double for both ppy platforms (* p 0.05 between ppynws and * * p 0.01 between flat ppy). also, ppynws showed a very significant enhancement of dex release triggered by emf stimulation when compared to flat ppy (on days 1 and 16, * * p 0.01, and on day 7, * * * p 0.001 between aunps ; at days 7 and 16, * * p 0.01 between psss studied, figure 5b). release studies (no stimulation) showed that the outward diffusion of dex was below 0.11 mg / cm for ppynws and 0.3 mg / cm for flat ppy films on the last day of recording. this reveals a significant drug release of emf stimulation compared to that without emf stimulation. (on day 1, * * p 0.01, and on days 7 and 16, * * * p 0.001 between ppynws ; on day 1, * p 0.05, and on days 7 and 16, * * p 0.01 between flat ppy films, figure 5c). figure s4 depicts the dex release profiles of flat ppy as a function of temperature. some passive release was found, but it did not reach the levels of active emf stimulation. further switching experiments where the emf was turned on for 2 h and then turned off for 2 h are shown in figure 5d. in such cases, dex release fell off precipitously after the removal of emf stimulation and was regained when emf coupling was resumed. such consistency over multiple unloading cycles demonstrated excellent reproducibility and reversibility that is a hallmark of controlled release. x - ray photoelectron spectroscopy (xps) was used to verify the existence of dex and to verify the escape of entrapped drug (figure 6). xps surface analysis was conducted on the ppynws before and after (16 days) pulsed electromagnetic stimulation. as expected, high - resolution spectra of f 1s and p 2p revealed changes in the peak magnitudes, denoting an obvious release of dex upon exposure to the emf. specifically, these spectrographs detail signatures of fluorine and phosphorus elements in the dex molecule, which essentially vanish after emf stimulation. for ppynws - aunps, the prestimulation f 1s and p 2p atomic percentages were 1.53 and 1.54%, respectively, while the poststimulations were 0.00 and 0.17%. for ppynws - pss, the prestimulation f 1s and p 2p atomic percentages were 0.60 and 1.31%, respectively, while the poststimulations were 0.00 and 0.15% xps spectra for the surface analysis of the dex presence on ppy templates before and after pulsed emf stimulation. (a) xps recording of dex - doped ppynws - aunps before (green) and after (red) dex release by emf. (b) xps data of dex - doped ppynws - pss before (green) and after (red) 16 days of emf stimulation. these show details of signature fluorine and phosphorus elements in the dex molecule before stimulation, respectively. note that these elemental signatures vanish after emf stimulation, indicative of dex release from the surface. the bottom graph shows the molecular structure of dex, with fluorine (f) and phosphorus (p) highlighted in red. two groups of toxin - challenged murine neonatal microglial cells (bv-2) were used to evaluate ros production and to determine whether such damaged cells could be rescued via emf - associated drug release. in one group, the challenge was bacterially derived lps (figure 7a g), and in the other, the cells were directly insulted with hydrogen peroxide (figure 7h). (b) bv-2 cells insulted by lps, with intense green labeling of ros by 2,7-dichlorodihydrofluorescein (dcf). (c) ros was eliminated by the direct introduction of 1 g / ml dex suspended in the medium. (d) 30 min of emf stimulation from dex - doped ppynws - aunps, demonstrating reduced ros production. (e) with 1 h of emf stimulation, ros production was nearly undetectable. (f) in contrast, ppynws not subjected to emf stimulation again showed significant ros production in the tested cell population. (g) corresponding graph describing the quantitation of ros production in panels a f. (h) additional ros data using h2o2 as a positive control to induce ros. the graph confirms that both applied and stimulated dex suppressed ros production and that dex retained its bioactivity after emf stimulation. * p 0.05 and * * p 0.01. bv-2 cells treated with lps released pro - inflammatory cytokines and ros via mapk signaling pathways. cm - h2dcfda is one such indicator compound for ros and was used as a metric for ros production. lps (1 g / ml)-treated cells exhibited bright green fluorescence in the cytoplasm, marking the production of significant oxidative stress (figure 7b). the addition of dex (1 g / ml) to these lps - induced microglia effectively suppressed ros production during the inflammatory cascade (figure 7c). the dex - doped ppynws - aunps platform also suppressed inflammation byproducts during emf stimulation (figure 7e). a weaker inhibition of ros activity occurred when shorter stimulation times were applied (figure 7d). in contrast, nonstimulated dex / ppynws - aunps films did not show signs of ros scavenging (figure 7f, g). similarly, exposure to 20 g / ml h2o2 ed in more ros byproducts in bv-2 cells (figure 7h). this enhanced production of ros was reduced either by direct application of 10 g / ml dex or by 15 min of emf stimulation to the dex - coupled ppynws - aunps. microglial cells were stained intensely with cy3 (red fluorescent signal) when exposed to lps in a dose - dependent manner (figure 8). inos expression in bv-2 cells after lps challenge (without stimulation) also showed positive cy3 staining, which is in agreement with the ros (figure 7). in contrast, emf stimulation of dex - conjugated ppynws - aunps ed in a strong suppression of inos, indicating a reduced level of nitric oxide. inos expression in bv-2 cells is shown with red fluorescence (cy3 column). the row labeled as control does not show any inos expression due to the absence of lps treatment. in contrast, the row lps 10g / ml shows significant upregulation of inos, and the merged confocal micrograph details that the staining is primarily localized in the cytoplasm. / ml shows less upregulation at a reduced lps concentration while ppynws - aunps not stimulated with emf (dex w / o sti) showed a similar response. / ml / dex 1g / ml ), were comparable to the untreated control and highlighted the suppression of inos to lps - challenged bv-2 cells. finally, ppynws - aunps stimulated with emf (1 h) produced inos comparable to direct dex application. the electroactive properties and biocompatibility of ppy are desirable features in designing programmable drug - delivery systems. previously, our group used topographically modified ppy films that increased the available surface area for drug inclusion and release. while the drug capacity was improved, these films were difficult to fabricate and highly porous, making them susceptible to damage. we have thus introduced another form of conductive ppy by shaping the polymer into solid nanowire arrays for further functionalization. moreover, we propose the use of electromagnetic fields as the stimulus for ppy drug release. this is a significant departure from all previous forms of drug - doped ppy, in which direct electrical connection (i.e., wire electrodes) to the ppy was required to induce current flow and subsequent drug release. experimental showed that the ppynws - aunps possessed excellent drug - carrying capabilities as well as a controllable switching response to electromagnetic fields. xps analysis confirmed that the model drug, dexamethasone, was successfully incorporated onto the surface of the ppy nanowires. when exposed to pulsatile emfs, the dex cargo was released from the ppy surface, a process that was verified by changes in the surface chemistry and the free drug concentration. the amount of drug that could be eluted from emf stimulation was superior to direct stimulation via a potentiostat. while the mechanism of emf - coupled drug release is still unclear, we hypothesize that the phenomenon is similar to ppy stimulation via direct current. for instance , it is well known that ppy undergoes an electrical and conformational change via an oxidation / reduction reaction in response to applied voltages. furthermore, the electroactive ppy swells during oxidation and shrinks (mechanical actuation) during reduction to satisfy charge balance. this reversible volume change along with the electrostatics of moving charges within the ppy governs how charged molecules move in and out of the ppy (i.e., drug deposition and elution). a multitude of cytokines / drugs such as nerve growth factors, analgesics, and adenosine triphosphate have been released in situ based on this functionality. in the proposed polymer platform, charge balance is obtained when the anionic dex is electrostatically entrapped within the cationic pyrrole chain during the electropolymerization process. when a sample is exposed to a time - varying em field, an oscillating electric current is induced in the ppy and is posited to drive the redox reaction. during the reduction state, charge neutralization of the pyrrole backbone eliminates the electrostatic bonding to the dex, causing the dex to migrate out of the ppy matrix. indeed, prior evidence has shown that dex desorbs from ppy in the reduction state. therefore, the cumulative reduction states that occur during the inductive cycling of ppy facilitate dex movement. the concomitant mechanical actuation or pumping action from the redox reaction may further play a role in forcing the dex out of the ppy. not surprisingly, this polymer electroactive effect should be more pronounced if the conductivity of the ppy is enhanced, which was indeed observed when the ppy nanowires were impregnated with gold. as a , it appears that dex elution occurs regardless of whether current flow is direct or electromagnetically induced. it has been noted that ppy is susceptible to overoxidation whereby the electroactivity can diminish due to high applied voltages or continued stimulation. this fatigue behavior was not detected as evidenced by the prolonged dex release over a period of 16 days in vitro. the extended electrochemical stability of ppynws - aunps may be partially due to the low duty cycle of the applied em fields and its rapid oscillatory nature or by the enhanced conductivity conferred by the gold nanoparticles. for instance, the addition of multiwalled carbon nanotubes improves the conductivity and electrostability of ppy neural prosthetic coatings. however, induction may cause joule heating and subsequent drug release, an observation that was described for carbon nanotubes. to address passive thermal effects, we performed additional studies at elevated temperature (37 c). show that temperature played only a small role in the release kinetics. follow - up cyclical stimulation also revealed that dex was released only during on stimulation states. it is further unlikely that ph changes were responsible for drug elution since these experiments were conducted in physiologically buffered solutions. to verify the bioactivity of the released dex cargo , we performed several cell culture assays using the bv-2 glial line exposed to lipopolysaccharide (lps) toxin. lipopolysaccharide - challenged microglia have been shown to induce neurotoxicity through the production of pro - inflammatory mediators such as tumor necrosis factor-, interleukin-1(il-1), ros, and inos, which catalyze the production of nitric oxide (no). since a focus area within our laboratory is neurodegenerative diseases of the central nervous system (cns) (reviewed in ref ), we chose this in vitro neuroinflammatory model to assess the therapeutic activity of emf - released dex. the showed that the addition of dex (1 g / ml) to lps - exposed microglia suppressed ros production. similarly, the dex - doped ppynws - aunps platform also suppressed inflammation byproducts during emf stimulation. in contrast, the nonstimulated dex / ppynws - aunps films did not confer signs of ros scavenging. similarly, exposure to the h2o2 positive control ed in ros byproducts in bv-2 cells. this production of ros was also reduced either by direct application of 10 g / ml dex or with 15 min of emf stimulation to the dex - coupled ppynws - aunps. these findings were consistent with inos measurements, with a marked reduction in inos expression in the presence of soluble dex. while we can not eliminate the possibility that emf alone may reduce ros - associated damage in bv-2 cells, this is highly unlikely since the nanosecond pulse widths and stimulation times used were much shorter than the emf waveforms commonly employed in bioelectromagnetics. therefore, we conclude that the mitigation of the inflammatory cascade was due to the availability of free dex. even as the proposed geometric scheme of ppy improves with drug - loading capacity, the amount of drug delivered may still be considered to be miniscule in comparison to systemic delivery. rather, the intent of nanoscaled reservoirs is to deliver extraordinarily high concentrations of potent drugs to a localized microenvironment in order to produce therapeutic responses in adjacent, contiguous cells. in essence, nanoconstruction aims to release drugs into only specified target areas while escaping systemic circulation and side effects, a critical advancement in therapies involving potent or toxic drug cargos. examples of such indications include early detected tumors, a restricted lesion in the nervous system (such as a localized region of acute cns embolism or trauma), and the local region of small - scale crush injury to cns or pns axons that completely inhibit action potential propagation across the injury zone. these minor spatial defects produce profound and catastrophic behavioral, functional, and cognitive consequences. here we show that the release of drugs from ppynws can occur for more than 2 weeks and may serve as an ideal candidate for such applications. we finally note that the reported emf stimulation protocol is not fully optimized, and we are refining methods in which this release system can be employed for broad biomedical and pharmaceutical applications. as the putative mode of drug release is emf - ppy induction coupling, we aim to develop oscillating waveforms that maximize the induction current without significant joule heating. the phenomenon of joule heating requires special attention in vivo since the thermal energy must be dissipated. additional design parameters will also account for the attenuation of the em signal through skin and connective tissues. various theoretical and empirical models that consider tissue permeability, conductivity, and imposed emf frequency have been developed and will be used as a reference for design. however, the pulsing frequencies used in the present system are effective in vitro within a couple of centimeters of the coil center and will serve as a basis for further in vivo study. future improvements may also include synthesizing degradable forms of ppy which may be resorbed within the body. nonetheless, we reveal for the first time that a ppy system can be used as a type of programmable drug - delivery reservoir that responds to electromagnetic fields. this action at a distance potentially provides a new direction for noninvasive controlled drug release. conductive polymer polypyrrole was fashioned into a nanowire architecture and doped with the drug dexamethasone. this geometry enabled high - capacity drug entrapment and subsequent sustained release for over 2 weeks. more importantly, the release of dexamethasone could be triggered on demand via an externally applied pulsed electromagnetic field. dexamethsone remained bioactive, as demonstrated by its ability to ameliorate damage to toxin - challenged glial cells. this is the first demonstration of a noninvasive mode of drug delivery using polypyrrole.

in this work, we introduce a free - standing, vertically aligned conductive polypyrrole (ppy) architecture that can serve as a high - capacity drug reservoir. this novel geometric organization of ppy provides a new platform for improving the drug - loading efficiency. most importantly, we present the first formal evidence that an impregnated drug (dexamethasone, dex) can be released on demand by a focal, pulsatile electromagnetic field (emf). this remotely controlled, on off switchable polymer system provides a framework for implantable constructs that can be placed in critical areas of the body without any physical contact (such as percutaneous electrodes) with the ppy, contributing to a low foreign body footprint. we demonstrate this possibility by using a bv-2 microglia culture model in which reactive oxygen species (ros) and inducible nitric oxide synthase (inos) expression was attenuated in response to dex released from emf - stimulated ppy.

it is well known that hearing levels progressively deteriorate with age which is the most common factor associated with acquired hearing loss. age - related hearing loss is prevalent in the elderly regardless of age, gender, or race. the prevalence of hearing loss is more than 50% in people older than 50 years in the u.s.. in europe, more than 55% of males have a hearing loss of more than 30 db hl by the age of 80 years. age - related hearing loss is characterized that high - frequency sounds are more affected than low - frequency sounds. the deterioration of hearing thresholds is mainly due to aging of the peripheral and central auditory system and exposure to environmental noise. the degree of hearing loss varies among individuals, although the degree of hearing loss is larger in males than in females. the age - related variation in hearing thresholds was defined by international organization for standardization (iso) 7029, providing the expected hearing threshold distribution in population aged from 18 to 70 years. however, iso 7029 was based upon the hearing thresholds for european and north american populations between the 1950s and the 1970s. some authors have indicated the problems with iso 7029 in terms of specific population data, subject selection criteria, outdated calibration and test procedures, and restricted test frequencies. authors have proposed to revise the norms of hearing thresholds as a function of age, and as a , iso 7029 is currently under revision by the iso technical committee 43. in that study, 2492 adults (1250 males and 1242 females) with an age range of 20 - 59 years who were otologically normal were included. the primary goal of this study is to provide the distribution of hearing thresholds as a function of age in korean individuals from 60 to 84 years of age who have been screened by the procedure described in the iso 8253 - 1. the hearing threshold levels in this study were compared with those provided by the iso 7029. also, the hearing thresholds for each signal frequency according to gender and age groups were compared. we anticipate that these data will be used for preparing new standards of hearing thresholds as a function of age in korea and for updating the iso 7029 under revision. data were collected from a total of 526 ears (from 112 males and 151 females aged 60 - 84 years). the age criteria were stratified into 60 - 64, 65 - 69, 70 - 74, 75 - 79, and 80 - 84 groups in 5-year intervals as shown in table 1. all subjects were recruited from various areas in korea (seoul, chuncheon, gwangju, daegu, and mokpo) in order to ensure sampling from the entire country. all of the participants received an a - type tympanogram and did not report a history of hearing loss, ear surgery, otologic disease, or occupational noise exposure (iso 8253 - 1, 2000). a gsi 61 audiometer (grason - stadler, eden prairie, mn, usa) was used for measuring pure - tone thresholds in a soundproof room. according to the criteria of iso 8253 - 1, the maximum permissible sound pressure level for ambient noise in the room was sufficiently low. the test tones were calibrated first by the method of iso 389 - 5 and 389 - 8 and then presented to participants through tdh-50 headphones (telephonics, farmingdale, ny, usa). a sound level meter (type 2250-l ; b&k, denmark) was used for the calibrations with a microphone (type 4192 ; b&k) and a preamplifier (type 2690-a-0s1 ; b&k). informed consent was obtained from the participants and a questionnaire was additionally completed before audiometric tests. both ears were tested, and the first ear (right or left) was chosen randomly in all subjects. if hearing thresholds in right and left ears differed by more than 20 db hl at a given frequency, only better hearing thresholds in the ear were obtained. pure - tone air conduction audiometry was conducted over the range of 250 - 8000 hz in the order of 1000, 2000, 3000, 4000, 6000, 8000, 500, and 250 hz with a combined ascending and descending approach following a standardized protocol (iso 8253 - 1). an audiologist presented stimuli, and the participants were asked to press a button when they heard any beep sound. , armonk, ny, usa ). the hearing thresholds for each signal frequency for each age band were compared by gender using an independent t - test. the dependent variable was the hearing thresholds for the frequencies, and the independent variable was gender. to compare average hearing thresholds in each age band for males and females, one - way analysis of variance (anova) the between factor was each age band, and the independent variable was average hearing threshold. iso 7029 presents the equation hmd, y= (y-18 years) for computing the expected age - related hearing loss. h refers to the deviation from the median hearing threshold level at an age of y years based on a reference age of 18 years (0 db hl). data were collected from a total of 526 ears (from 112 males and 151 females aged 60 - 84 years). the age criteria were stratified into 60 - 64, 65 - 69, 70 - 74, 75 - 79, and 80 - 84 groups in 5-year intervals as shown in table 1. all subjects were recruited from various areas in korea (seoul, chuncheon, gwangju, daegu, and mokpo) in order to ensure sampling from the entire country. all of the participants received an a - type tympanogram and did not report a history of hearing loss, ear surgery, otologic disease, or occupational noise exposure (iso 8253 - 1, 2000). audiometric tests were conducted by trained audiologists. a gsi 61 audiometer (grason - stadler, eden prairie, mn , usa) was used for measuring pure - tone thresholds in a soundproof room. according to the criteria of iso 8253 - 1, the maximum permissible sound pressure level for ambient noise in the room was sufficiently low. the test tones were calibrated first by the method of iso 389 - 5 and 389 - 8 and then presented to participants through tdh-50 headphones (telephonics, farmingdale, ny, usa). a sound level meter (type 2250-l ; b&k, denmark) was used for the calibrations with a microphone (type 4192 ; b&k) and a preamplifier (type 2690-a-0s1 ; b&k). informed consent was obtained from the participants and a questionnaire was additionally completed before audiometric tests. both ears were tested, and the first ear (right or left) was chosen randomly in all subjects. if hearing thresholds in right and left ears differed by more than 20 db hl at a given frequency, only better hearing thresholds in the ear were obtained. pure - tone air conduction audiometry was conducted over the range of 250 - 8000 hz in the order of 1000, 2000, 3000, 4000, 6000, 8000, 500, and 250 hz with a combined ascending and descending approach following a standardized protocol (iso 8253 - 1). an audiologist presented stimuli, and the participants were asked to press a button when they heard any beep sound. the hearing thresholds for each signal frequency for each age band were compared by gender using an independent t - test. the dependent variable was the hearing thresholds for the frequencies, and the independent variable was gender. to compare average hearing thresholds in each age band for males and females, one - way analysis of variance (anova) the between factor was each age band, and the independent variable was average hearing threshold. iso 7029 presents the equation hmd, y= (y-18 years) for computing the expected age - related hearing loss. h refers to the deviation from the median hearing threshold level at an age of y years based on a reference age of 18 years (0 db hl). table 2 lists the numerical data related to the hearing threshold distributions (5, 10, 25, median, 75, 90, and 95 percentiles) for males and females in each age band. the of the independent t - test showed that there were no significant differences between genders in low frequencies (250 and 500 hz), but there were significant differences in high signal frequencies. the average hearing thresholds were computed by averaging the hearing sensitivity at 500, 1000, and 2000 hz (table 4), and 4000, 6000, and 8000 hz (table 5). for comparing the mean hearing thresholds of each age band, the independent variable was the average hearing thresholds and the between factor was the age band. of mean thresholds of 500, 1000, and 2000 hz revealed that there were significant differences among the age bands for males and for females. of mean thresholds of 4000, 6000, and 8000 hz also showed that there were significant differences among the age bands for males and for females. table 6 and table 7 provide summary of post hoc tests for mean thresholds of 500, 1000, 2000 hz and those of 4000, 6000, 8000 hz, respectively. table 8 presents the mean alpha coefficient values for the present study and those for iso 7029 at each signal frequency for males and females. figs. 1 and 2 show the mean hearing thresholds of each age band for the males and females. since iso 7029 does not provide hearing thresholds according to age bands, the median hearing thresholds at each age were computed by the equation and means of the hearing thresholds for each age band were calculated for comparison. revealed that the elderly korean population had worse hearing thresholds than the estimated hearing thresholds based on the iso 7029 equation applied to the same age band. in particular, korean males had similar hearing thresholds at high frequencies and worse hearing thresholds at low frequencies than those based on the iso 7029. korean females had worse hearing thresholds at all frequencies than those based on the iso 7029. table 2 lists the numerical data related to the hearing threshold distributions (5, 10, 25, median, 75, 90, and 95 percentiles) for males and females in each age band. the of the independent t - test showed that there were no significant differences between genders in low frequencies (250 and 500 hz), but there were significant differences in high signal frequencies. the average hearing thresholds were computed by averaging the hearing sensitivity at 500, 1000, and 2000 hz (table 4), and 4000, 6000, and 8000 hz (table 5). for comparing the mean hearing thresholds of each age band, the independent variable was the average hearing thresholds and the between factor was the age band. of mean thresholds of 500, 1000, and 2000 hz revealed that there were significant differences among the age bands for males and for females. of mean thresholds of 4000, 6000, and 8000 hz also showed that there were significant differences among the age bands for males and for females. table 6 and table 7 provide summary of post hoc tests for mean thresholds of 500, 1000, 2000 hz and those of 4000, 6000, 8000 hz, respectively. table 8 presents the mean alpha coefficient values for the present study and those for iso 7029 at each signal frequency for males and females. figs. 1 and 2 show the mean hearing thresholds of each age band for the males and females. since iso 7029 does not provide hearing thresholds according to age bands, the median hearing thresholds at each age were computed by the equation and means of the hearing thresholds for each age band were calculated for comparison. revealed that the elderly korean population had worse hearing thresholds than the estimated hearing thresholds based on the iso 7029 equation applied to the same age band. in particular, korean males had similar hearing thresholds at high frequencies and worse hearing thresholds at low frequencies than those based on the iso 7029. korean females had worse hearing thresholds at all frequencies than those based on the iso 7029. table 2 lists the numerical data of hearing threshold distributions for the male and female participants. additionally, figs. these patterns were consistent with iso 7029 as well as with those in previous studies. a previous study showed that significant differences between genders occurred between 3000 and 8000 hz. in the present study, hearing thresholds across age bands in males were significantly higher than those in females from 1000 to 8000 hz. we also found that pure - tone threshold averages (ptas) of low (500, 1000, and 2000 hz) and high frequencies (4000, 6000, and 8000 hz) were gradually elevated in males as compared to those in females with increasing age. again, greater pta differences in high frequencies were observed in males than in females. this excessive noise exposure is one of the main reasons of elevated hearing - frequency hearing loss. most korean males of 60 years and above were required to serve in the military for at least 21 months. recent studies have reported that noise - induced hearing loss and tinnitus were increased due to noise exposure during military service. therefore, it is possible that exposure to excessive noise during military service affected the elevated hearing sensitivity of korean males.. taken together, these suggest a greater amount of age - related hearing loss in males than in females and more hearing loss at higher frequencies than at lower frequencies. compared with the alpha coefficients of the iso 7029, the alpha coefficients of the present study were higher for both genders except for high frequencies in males. in particular, the alpha coefficients of the present study for both males and females at low frequencies were higher than those of the iso 7029. these are consistent with previous studies. in the study by wiley, et al. , the hearing sensitivity for individuals aged 48 - 65 years was compared with the iso 7029 median thresholds for males and females. the revealed that the expected hearing thresholds based on the iso 7029 were better than those in the previous data. the differences between the data and the iso values were larger for males than for females. in the study by stenklev and laukli, alpha coefficients were significantly higher than the iso values, especially at 125 - 4000 hz, for females. our also indicated that the differences between the data and the iso values were larger for females across all frequencies. table 2 lists the numerical data of hearing threshold distributions for the male and female participants. additionally, figs. these patterns were consistent with iso 7029 as well as with those in previous studies. a previous study showed that significant differences between genders occurred between 3000 and 8000 hz. in the present study, hearing thresholds across age bands in males were significantly higher than those in females from 1000 to 8000 hz. we also found that pure - tone threshold averages (ptas) of low (500, 1000, and 2000 hz) and high frequencies (4000, 6000, and 8000 hz) were gradually elevated in males as compared to those in females with increasing age. again, greater pta differences in high frequencies were observed in males than in females. this excessive noise exposure is one of the main reasons of elevated hearing - frequency hearing loss. most korean males of 60 years and above were required to serve in the military for at least 21 months. recent studies have reported that noise - induced hearing loss and tinnitus were increased due to noise exposure during military service. therefore, it is possible that exposure to excessive noise during military service affected the elevated hearing sensitivity of korean males. furthermore, the alpha coefficients were larger at high frequencies than at low frequencies. taken together, these suggest a greater amount of age - related hearing loss in males than in females and more hearing loss at higher frequencies than at lower frequencies. compared with the alpha coefficients of the iso 7029, the alpha coefficients of the present study were higher for both genders except for high frequencies in males. in particular, the alpha coefficients of the present study for both males and females at low frequencies were higher than those of the iso 7029. these are consistent with previous studies. in the study by wiley, et al. , the hearing sensitivity for individuals aged 48 - 65 years was compared with the iso 7029 median thresholds for males and females. the revealed that the expected hearing thresholds based on the iso 7029 were better than those in the previous data. the differences between the data and the iso values were larger for males than for females. in the study by stenklev and laukli, alpha coefficients were significantly higher than the iso values, especially at 125 - 4000 hz, for females. our also indicated that the differences between the data and the iso values were larger for females across all frequencies. in , we presented the data of hearing thresholds for a korean screened population aged from 60 - 84. it is difficult to compare our data directly with those of previous studies due to differences in sample size, age range, and etc. we conclude that 1 ) hearing threshold sensitivity increases with age, 2 ) greater hearing losses at high frequencies appear across all age groups, and 3 ) greater hearing losses are seen in males than in females. as discussed above, these data will be used for establishing a new korean standard with data from 2003 and for updating the iso 7029 which is presently under revision.

and objectivesthe purpose of the present study was to provide the hearing threshold levels in the elderly korean population, and to compare korean data with that in the international organization for standardization (iso) 7029.subjects and methodsdata were collected from a total of 526 ears from 112 males and 151 females aged 60 - 84 years. all participants were screened otologically by the procedure given in iso 8253 - 1. showed that the pure - tone average was gradually elevated with increasing age. the amount of hearing loss was greater in males than in females, and the high frequency hearing thresholds were worse than the low frequency hearing thresholds in males and females. the hearing threshold levels were higher at low frequencies in males and at all frequencies in females than the norms of iso 7029. from this study will be partly used for standardization of hearing thresholds as a function of age in korea and for updating the iso 7029.

neurofibromatosis type 1 (nf1) is an autosomal dominant genetic disorder that affects approximately 1 in 3000 births. individuals with nf1 are predisposed to developing abnormalities in a number of body systems; individually and cumulatively they can have a significant impact on quality of life. for example, skeletal deformities such as scoliosis and pseudarthroses, are uncommon but can be associated with considerable morbidity. benign, malignant, and disfiguring tumors (termed neurofibromas) are characteristic of the condition and can be challenging to manage. neuropsychological impairments in nf1, such as executive dysfunction, inattention, specific learning disorder and reduced social competency, can in reduced social participation and social isolation. the clinical diagnosis of nf1 relies on fulfilling at least two of the seven diagnostic criteria; caf au lait macules, skinfold freckling, neurofibromas, lisch nodules, optic pathway tumors, bone dysplasia or a family history. notably, some of these features are congenital in origin, while some manifest over time at characteristic developmental stages. skeletal muscle and motor deficits, such as reduced muscle size, muscle weakness, and poor co - ordination however, recent preclinical and clinical studies have indicated a primary role for the nf1 gene product, neurofibromin, in muscle growth and metabolism. neurofibromin has been characterized as a gtpase activating protein that is a negative regulator of the ras gtpase. loss of functional neurofibromin leads to a dose - dependent increase in ras signaling, which can profoundly affect cell proliferation, differentiation, and function and impairment on the beery test of visuomotor integration, which requires subjects to draw increasingly difficult shapes and figures. individuals with nf1 also present with deficits in a range of functional tasks that likely have significant impact on quality of life. for example, significant impairment in balance, muscle strength, and upper limb co - ordination in nf1 has been observed using the bot-2 test of motor proficiency. mild to moderate deficits in manual dexterity, balance, and ball handling skills, deficient motor timing and reaction time, reduced fine motor speed, and impaired handwriting in nf1 children has also been reported. gait assessment was performed in 46 children and adolescents with nf1 (ages 7 - 17yrs) using the gaitrite electronic walkway, and were compared with a battery of cognitive tests, including the wechsler intelligence scale for children and sub - tests from the cambridge automated neuropsychological test battery. the largest correlations were found between deficits in gait width and spatial working memory (r=0.594, p<0.01), and deficits in running speed and agility with impaired strategy generation (r=0.549, p<0.01), supporting speculation of a link between nf1 motor deficits and abnormal central nervous system (cns) function. while the mechanisms of interaction between the cns and peripheral nervous system (pns) and motor dysfunction are unclear, structural and functional brain abnormalities are a feature of the condition that may contribute to motor impairments. neurofibromin plays a significant role in the developing brain, with loss of expression ing in increased cell proliferation and differentiation, ultimately impacting morphology. a recurrent finding has been an increase in total brain volume, involving both grey and white matter. diffusion weighted magnetic resonance imaging (mri) in individuals with nf1 indicates reduced white matter integrity in a number of regions closely linked to motor function. these include the: corpus callosum, caudate nucleus; a sub - region of the basal ganglia involved in goal - direct behavior and voluntary movement, and thalamus; a sub - cortical nuclear complex that receives and relays cortical and sub - cortical inputs that sub - serve sensory and motor mechanisms, as well as cognitive abilities. volumetric studies report these structures as abnormally large in nf1 cohorts, suggesting a reduced signal - to - noise ratio. moore et al have further shown significant associations between increased corpus callosum size and reduced motor performance in children with nf1. there is also mounting evidence for functional abnormalities within the thalamus and caudate in individuals with nf1. both adult and pediatric positron emission tomography studies report thalamic hypometabolism, suggesting reduced thalamic signal processing, and a recent event - related functional mri study identified abnormal right caudate activation during a spatial working memory task; a cognitive ability significantly associated with impaired gait in nf1. individuals with nf1 also commonly show focal areas of high t2 signal intensity on mri, which have been associated with reduced fine motor skill. mechanistically, one clinical study has examined sensory and motor neuropathy in nf1 as possibly contributory. electrophysiological measures in 39 individuals with nf1 aged 10 - 56 revealed motor polyneuropathy was a rare manifestation, and while abnormalities in multimodal evoked potentials (visual, auditory and sensory) were seen commonly, many were associated with tumors or lesions. further clinical studies correlating neurological assessments with strength and other motor outcomes are needed to define the influence of the cns and pns in the nf1 motor phenotype. while preclinical mouse models have been used to investigate mechanisms underlying some neurocognitive deficits, a relationship between cognition and motor performance has not yet been defined in these systems. abnormalities such as the learning and attention deficits have been extensively examined in the nf1 mouse. for example, increased gaba release in the hippocampus has been shown to underlie learning deficits, and can be rescued by inhibition of erk signaling, while lovastatin treatment has been shown to rescue deficits in learning and attention. however, interactions between these deficits and motor function remain unknown. the most significant motor deficit seen in this mouse line was impaired grip strength using a hanging wire test, and there was no demonstration of a neurocognitive basis for this . furthermore, this line failed to show any deficiencies in motor performance tests linked to cerebellar function. decreased dopamine levels in the striatum have also been identified in a further nf1 mouse model with bi - allelic nf1 inactivation in glia. these mice exhibited reduced exploratory behaviors as well as selective and non - selective attention abnormalities, which were rescued by pharmacologic intervention to restore dopamine levels. while speculative , abnormal dopamine levels may underlie some of the motor impairments observed in individuals with nf1. indeed some gait characteristics identified in a pediatric nf1 cohort, including a shorter step length and longer step time, resemble those seen in early parkinson disease; a disorder associated with reduced dorsal striatal dopamine levels. further insight into the interactions between neurocognitive development and motor deficits may come from more advanced mouse models of conditional double inactivation of nf1 in neurons. in 2005, stevenson et al. published data from an analysis of 40 individuals with nf1 using peripheral quantitative computed tomography (pqct) scanning. individuals with nf1 presented with a significant reduction in muscle cross - sectional area compared to age matched controls. comparative findings were seen in a pediatric nf1 cohort where lean tissue was measured using dual - energy x - ray absorptiometry (dexa). children with nf1 had a significantly reduced lean tissue mass. while reduced muscle size may imply a reduction in strength, muscle functional outcomes were not assessed in these primarily radiographic studies. however, in a seminal 2009 study, souza et al performed hand grip dynamometry testing of 21 subjects (age 7 - 60 yrs) with nf1 compared to gender, aged, and physical activity matched controls, and demonstrated a significant reduction in grip strength in the nf1 cohort. findings of muscle functional impairment in nf1 have been subsequently reported in clinical studies (table 2). reduced strength of the hip extensor muscles has been described using hand held dynamometry. likewise, a 2013 trial investigating lower body muscle function in nf1 children, found that jumping force (n / kg) and jumping power (w / kg) were both significantly reduced. a cohort of 17 individuals with nf1, along with gender, age, and bodyweight matched control subjects, underwent maximal oxygen consumption (vo2 max) testing, a measure of maximal aerobic exercise capacity. individuals with nf1 had a reduced vo2 max as well as a reduced maximal systolic blood pressure. while the authors acknowledged that there was some difficulty in recruiting activity matched controls, it remains to be thoroughly investigated whether reduced physical activity accounts for reduced exercise capacity in nf1. given the mounting evidence for motor and muscular deficits in nf1, continued exercise studies of this kind are warranted, particularly those assessing quality of life outcome measures. muscle size and muscle function in nf1. insight gained from research into nf1 muscle function may be applicable to other related genetic diseases. nf1 belongs to the rasopathy family of diseases, which includes costello syndrome, cardiofaciocutaneous syndrome, and noonan syndrome. in a 2012 clinical study, reduced grip strength and muscle weakness was identified as a common feature of them all. it is unclear whether these conditions share a common mechanism for muscle weakness downstream of altered ras signaling. however, if this is the case, any successful pathway - targeted interventions that improve nf1 muscle performance, may have broad clinical applicability to other rasopathies. early evidence suggesting a critical role for nf1 in skeletal muscle development came from in vitro experiments assessing gene expression during myoblast differentiation. levels of nf1 mrna, and neurofibromin were elevated during differentiation, and a concomitant decrease in activated p21-ras was observed. double inactivation of nf1 was found to be embryonically lethal, and nf1 null embryos showed underdeveloped cardiac and skeletal muscle. however, it remained unclear whether these effects on muscle were secondary to some other failure in the developmental program, and it was nt until recently that a key role for nf1 in muscle was demonstrated in an animal model. developed a limb - specific nf1 knockout mouse (nf1prx1) using a prx1-cre transgene to drive deletion of nf1 in cells of the mesenchymal lineage. furthermore, consistent with in vitro data, analysis of mutant embryos revealed hyperactive ras / mapk signaling, and impaired myoblast differentiation in the developing limbs. while the nf1prx1-/- mouse model demonstrated the requirement of nf1 for normal limb muscle development, prx1-cre driven recombination is not restricted to the muscle lineage. thus, the inactivation of nf1 in other mesenchymal tissues including adipocytes, connective tissue, and bone, were potential confounders to interpretation. to overcome this, a skeletal muscle specific nf1 knockout mouse (nf1myod) was subsequently generated, by crossing the myod - cre transgenic mouse with the nf1-flox line. homozygous nf1 inactivation in muscle was lethal in the first week of life, and while pups were born at normal bodyweight, they had stunted growth and a high rate of maternal infanticide was observed. electron microscopy (em) imaging of nf1myod muscle specimens showed no evidence of cytoarchitectural abnormalities, including protein aggregates, myofibrillar disruption, or z - line streaming. while nf1prx1-/- muscle has been described as dystrophic, this may be a misnomer, as neither mouse model nor patient muscle biopsies have been characterized as having progressive loss of cytoskeletal or membrane protein integrity. em of 3-day old nf1myod muscle samples unexpectedly revealed excessive accumulations of intramyocellular lipid, which was subsequently confirmed by oil red o staining. this led to speculation that nf1 may have a key role in the regulation of muscle lipid metabolism. analysis of adult nf1prx1-/- muscle samples revealed similarly elevated triglyceride levels, 10-fold that of controls. increased fatty acid synthesis may underlie these accumulations in the nf1prx1-/- mice, as a substantive increase in the expression of fatty acid synthase was observed. metabolic dysregulation was also seen for a range of mitochondrial enzymes, including succinate dehydrogenase (sdh), -hydroxyacyl - coa dehydrogenase (bhad), and medium - chain acyl - coa dehydrogenase (mcad). the expression of mitochondrial fatty acid transport protein carnitine palmitoyl transferase-1 (cpt-1), and membrane transport proteins, cd36, and fatty acid transport protein 4 (fatp4) were also reduced. for example, it is difficult to separate any primary deficits responsible, from any downstream or compensatory metabolic changes in the mature nf1prx1-/- muscle. interestingly, analysis of neonatal nf1myod muscle showed only the intramyocellular lipid phenotype, suggesting that lipid accumulation may be the initiating factor, and that subsequent molecular and metabolic dysregulation is temporal in nature. although the precise mechanisms remain unknown, these mouse data demonstrate a novel metabolic regulatory role for neurofibromin in muscle. one possibility is that nf1 muscle has commonalities with the lipid storage myopathies (lsms), which also present with progressive muscle weakness and muscle lipid accumulation. if parallels are found to exist with the lsms, this may provide insight into potential interventions for nf1. for example, primary carnitine deficiency (pcd) leads to an impairment of lipid transport into the mitochondria, a ant accumulation of lipid droplets, and muscle weakness. pcd patients have been successfully treated with high dose l - carnitine supplementation. while such speculation is attractive to entertain, evidence for lipid accumulation in human nf1 samples has not been firmly established. identifying lipid accumulation in human nf1 muscle biopsies will be an important goal for researchers in order to demonstrate the relevance of these murine models. a number of historical and recent studies raise important questions regarding the role of the nf1 gene in muscle development and function. in the 1990s, gutmann et al identified cardiac and skeletal muscle isoforms of nf1. to date, the functional importance of these isoforms remains unclear. it is possible that they have a unique role in the regulation of muscle development and/or metabolism. isoform - specific knockout models may be able to provide insight into the role of alternatively spliced variants of neurofibromin in muscle. furthermore, the genetics of nf1 in muscle are yet to be elucidated. some manifestations of nf1 are associated with heterozygosity (haploinsufficiency) and others with double inactivation. for example, local double inactivation has been observed in nf1 tumors as well as in tibial pseudarthrosis tissue. to date, no studies have addressed the potential double inactivation of nf1 in muscle. myofibers are multinucleated cells where sporadic double inactivation in individual nuclei could have unpredictable effects. analyzing double inactivation in myofibers may be challenging, but fluorescent in situ hybridization for nf1 on human muscle biopsies could be a feasible approach. one confounding factor in interpretation of clinical data is that the cognitive, motor control and psycho - social effects of nf1 may indirectly influence physical activity. recent data indicates that children with nf1 have reduced participation in formal and informal physical activities. finding ways to accommodate this into both studies of the underlying biological weakness and strategies for exercise - based intervention may have its own challenges. one intriguing possibility is that nf1 mutations and associated muscle weakness may increase the risk or severity of other related or unrelated conditions. for example, spinal complications such as scoliosis can be a major source of morbidity in nf1. late onset scoliosis has been described in adolescents with nf1 with no underling bone abnormalities, and it is possible that weakness or hypotonia may be contributory. in addition, two recent case reports suggest the potential for interactions between nf1 and other muscle - related genetic conditions. one report describes an individual with digeny for mutations in nf1 and ryanodine receptor 1, ing in myopathy. the other shows a previously unreported mutation in an nf1 locus leading to mitochondrial complex i deficiency, hypotonia, and developmental delay. continued identification of clinical presentations of this kind may provide useful insight. from a therapeutic standpoint , it will be critical to ascertain the capacity of exercise regimes to modify the muscle and motor phenotypes in nf1. the current literature is limited to a single case study that reports improved jumping and throwing performance in children with nf1, following a plyometric training program. however, this study was small (n=3), the children were of variable ages and genders, and the study lacked a control cohort. larger randomized and controlled exercise intervention studies are greatly needed to answer questions regarding the effects of exercise training on motor control, muscle size and strength, fatigue, and quality of life outcomes in individuals with nf1. while physical therapies are often favored if they can produce significant benefits, pathway - specific pharmacological interventions remain a potential treatment for those found unresponsive to exercise. the ras - mek - erk pathway is the canonical pathway in nf1, but this signaling cascade is also recognized for its role in muscle. constitutively active mek has been shown to directly bind and repress myogenic transcription factors, inhibiting myogenic differentiation in vitro. furthermore, in a cancer setting, mek / erk inhibition has been shown to be anabolic for skeletal muscle in humans and mice. this pathway is likely to be of particular relevance for interventions aiming to improve muscle function in nf1. souza et al. can be credited for initiating an explosion of activity in the field of nf1 muscle research in 2009. since then, a range of clinical studies have confirmed reduced motor performance and/or muscle impairment in individuals with nf1. while there are associations between neurological abnormalities and the nf1 muscle / motor phenotype, the mechanisms underlying these interactions are yet to be elucidated, and remain an area for further research. mechanistically, recent studies using genetically modified mouse models have provided strong evidence for a metabolic regulatory role for neurofibromin in muscle, likely contributing to the phenotype. this review has also reflected on a number of historically overlooked or potentially undervalued studies. the key roles for nf1 in muscle development are preceded by studies showing increases in nf1 gene and protein expression during myogenic differentiation. the findings of deficits in muscle strength are similarly preceded by radiographic studies showing decreases in muscle mass. however, it is the studies describing muscle - specific nf1 isoforms that are perhaps the most relevant to revisit, as these isoforms may have as yet undefined roles in the nf1-muscle phenotype. in summary, there have been significant advances in our understanding due to a co - ordination of basic and clinical research studies.

neurofibromatosis type 1 (nf1) is a genetic neurocutaneous disorder with multisystem manifestations, including a predisposition to tumor formation and bone dysplasias. studies over the last decade have shown that nf1 can also be associated with significant motor deficits, such as poor coordination, low muscle tone, and easy fatigability. these have traditionally been ascribed to developmental central nervous system and cognitive deficits. however, recent preclinical studies have also illustrated a primary role for the nf1 gene product in muscle growth and metabolism; these findings are consistent with clinical studies demonstrating reduced muscle size and muscle weakness in individuals with nf1. currently there is no evidence - based intervention for nf1 muscle and motor deficiencies; this review identifies key research areas where improved mechanistic understanding could unlock new therapeutic options.

mosquitoes can transmit more diseases than any other group of arthropods and affect million of people throughout the world. who has declared the mosquitoes as mosquito borne diseases are prevalent in more than 100 countries across the world, infecting over 700,000,000 people every year globally and 40,000,000 of the indian population. they act as a vector for most of the life threatening diseases like malaria, yellow fever, dengue fever, chikungunya ferver, filariasis, encephalitis, west nile virus infection, etc. , in almost all tropical and subtropical countries and many other parts of the world. to prevent proliferation of mosquito borne diseases and to improve quality of environment and public health the major tool in mosquito control operation is the application of synthetic insecticides such as organochlorine and organophosphate compounds. but this has not been very successful due to human, technical, operational, ecological, and economic factors. in recent years , use of many of the former synthetic insecticides in mosquito control programme has been limited. it is due to lack of novel insecticides, high cost of synthetic insecticides, concern for environmental sustainability, harmful effect on human health, and other non - target populations, their non biodegradable nature, higher rate of biological magnification through ecosystem, and increasing insecticide resistance on a global scale23. thus, the environmental protection act in 1969 has framed a number of rules and regulations to check the application of chemical control agents in nature4. it has prompted researchers to look for alternative approaches ranging from provision of or promoting the adoption of effective and transparent mosquito management strategies that focus on public education, monitoring and surveillance, source reduction and environment friendly least - toxic larval control. these factors have ed in an urge to look for environment friendly, cost - effective, biodegradable and target specific insecticides against mosquito species. considering these , the application of eco - friedly alternatives such as biological control of vectors has become the central focus of the control programmme in lieu of the chemical insecticides. one of the most effective alternative approaches under the biological control programme is to explore the floral biodiversity and enter the field of using safer insecticides of botanical origin as a simple and sustainable method of mosquito control. further, unlike conventional insecticides which are based on a single active ingredient, plant derived insecticides comprise botanical blends of chemical compounds which act concertedly on both behavourial and physiological processes. identifying bio - insecticides that are efficient, as well as being suitable and adaptive to ecological conditions, is imperative for continued effective vector control management. botanicals have widespread insecticidal properties and will obviously work as a new weapon in the arsenal of synthetic insecticides and in future may act as suitable alternative product to fight against mosquito borne diseases. roark5 described approximately 1,200 plant species having potential insecticidal value, while sukumar et al6 listed and discussed 344 plant species that only exhibited mosquitocidal activity. shallan et al in 20057 reviewed the current state of knowledge on larvicidal plant species, extraction processes, growth and reproduction inhibiting phytochemicals, botanical ovicides, synergistic, additive and antagonistic joint action effects of mixtures, residual capacity, effects on non - target organisms, resistance and screening methodologies, and discussed some promising advances made in phytochemical research. table i summarized the mosquitocidal activities of various herbal products from edible crops, ornamental plants, trees, shrubs, herbs, grasses and marine plants according to the exaction procedure developed in eleven different solvent systems and the nature of mosquitocidal activities against different life stages of different vector species as a ready reference for further studies.. applications of phytochemicals in mosquito control were in use since the 1920s8, but the discovery of synthetic insecticides such as ddt in 1939 side tracked the application of phytochemicals in mosquito control programme. after facing several problems due to injudicious and over application of synthetic insecticides in nature, re - focus on phytochemicals that are easily biodegradable and have no ill - effects on non - target organisms was appreciated. since then, the search for new bioactive compounds from the plant kingdom and an effort to determine its structure and commercial production has been initiated. at present phytochemicals botanicals are basically secondary metabolites that serve as a means of defence mechanism of the plants to withstand the continuous selection pressure from herbivore predators and other environmental factors. several groups of phytochemicals such as alkaloids, steroids, terpenoids, essential oils and phenolics from different plants have been reported previously for their insecticidal activities7. insecticidal effects of plant extracts vary not only according to plant species, mosquito species, geographical varities and parts used, but also due to extraction methodology adopted and the polarity of the solvents used during extraction. a wide selection of plants from herbs, shrubs and large trees was used for extraction of mosquito toxins. phytochemicals were extracted either from the whole body of little herbs or from various parts like fruits, leaves, stems, barks, roots, etc., of larger plants or trees. in all cases where the most toxic substances were concentrated upon, found and extracted for mosquito control. more than 2000 plant species have been known to produce chemical factors and metabolites of value in pest control programmes. members of the plant families- solanaceae, asteraceae, cladophoraceae, labiatae, miliaceae, oocystaceae and rutaceae have various types of larval, adulticidal or repellent activities against different species of mosquitoes7. human beings have used plant parts, products and secondary metabolites of plant origin in pest control since early historical times. vector control has been practiced since the early 20th century. during the pre - ddt era, reduction of vector mosquitoes mainly depended on environmental management of breeding habitats, i.e., source reduction. during that period, some botanical insecticides used in different countries were chrysanthemum, pyrethrum, derris, quassia, nicotine, hellebore, anabasine, azadirachtin, d - limonene camphor, turpentine, etc7. from the early 1950s, ddt and other synthetic organochloride and organophosphate insecticides were extensively used to interrupt transmission of vector borne diseases by reducing densities, human - vector contact and, in particular, the longevity of vector mosquitoes. in the mid-1970s, the resurgence of vector borne diseases, along with development of insecticide resistance in vector population, poor human acceptance of indoor house spraying and environmental concerns against the use of insecticides led to a rethinking in vector control strategies10. as a , emphasis was given on the application of alternative methods in mosquito control as part of the integrated mosquito management (imm)11. integrated mosquito management (imm) is a decision - making process for the management of mosquito populations, involving a combination of methods and strategies for long - term maintenance of low levels of vectors. the purpose of imm is to protect public health from diseases transmitted by mosquitoes, maintain healthy environment through proper use and disposal of pesticides and improve the overall quality of life through practical and effective pest control strategies. the main approaches of imm include: (i) source reduction and habitat management by proper sanitation, water management in temporary and permanent water bodies, and channel irrigation. vegetation management is also necessary to eliminate protection and food for mosquito larvae; (ii) larviciding by application of dipteran specific bacteria, insect growth regulators, surface films and oils, expanded polystyrene beads, phytochemicals, organophosphates and organochlorides, (iii) adulticiding by application of synthetic pyrethroids, organophosphates and synthetic or plant derived repellents, insecticide impregnated bed nets, genetic manipulations of vector species, etc., (iv) use of mosquito density assessment in adult and larval condition and disease surveillance; and (v) application of biological control methods by using entomophagous bacteria, fungi, microsporidians, predators and parasites. of the above avenues of imm, larviciding approach is the more proactive, proenvironment, target specific and safer approach than controlling adult mosquitoes. application of larvicide from botanical origin was extensively studied as an essential part of imm, and various mosquito control agents such as ocimenone, rotenone, capllin, quassin, thymol, eugenol, neolignans, arborine and goniothalamin were developed7. the efficacy of phytochemicals against mosquito larvae can vary significantly depending on plant species, plant parts used, age of plant parts (young, mature or senescent), solvent used during extraction as well as upon the available vector species. sukumar et al6 have described the existence of variations in the level of effectiveness of phytochemical compounds on target mosquito species vis - - vis plant parts from which these were extracted, responses in species and their developmental stages against the specified extract, solvent of extraction, geographical origin of the plant, photosensitivity of some of the compounds in the extract, effect on growth and reproduction. changes in the larvicidal efficacy of the plant extracts occurred due to geographical origin of the plant (in citrus sp18396465, jatropha sp132021, ocimum sanctum22356582, momordica charantia222449, piper sp54638995 and azadirechta indica65); response in the different mosquito species (in curcuma domestica26, withania somnifera13, jatropha curcas1320, piper retrofractum63, cestrum diurnum58, citrullus vulgaris5071, and tridax procumbens3031); due to variation in the species of plant examined (in euphorbia sp22283751, phyllanthus sp20, curcuma sp36, solanum sp16295760757996, ocimum sp23356582, eucalyptus sp22283751, plumbago sp20, vitex sp5093, piper sp54638995, annona sp485469, and cleome sp3178) and between plant parts used to study the larvicidal efficacy (in euphorbia tirucalli2851, solanum xanthocarpum16, azadirechta indica65, solanum villosum57607996, annona squamosa485469, withania somnifera13, melia azedarach45, moringa oleifera46 and ocimum sanctum3582). however, the principal objective of the present documentation is to report the changes in larvicidal potentiality of the plant extracts due to change of the particular solvent used during extraction. variation of the larvicidal potential of the same plant changed with the solvents used as evidenced in case of solanum xanthocarpum16, euphorbia tirucalli2851, momordica charantia222449, eucalyptus globules14152883, citrullus colocynthis13, azadirechta indica65, annona squamosa485469 and solanum nigrum2975. it has been shown that the extraction of active biochemical from plants depends upon the polarity of the solvents used. polar solvent will extract polar molecules and non - polar solvents extract non - polar molecules. this was achieved by using mainly eleven solvent systems ranging from hexane/ petroleum ether, the most non polar (polarity index of 0.1 that mainly extracts essential oil) to that of water, the most polar (polarity index of 10.2) that extracts biochemical with higher molecular weights such as proteins, glycans, etc. chloroform or ethyl acetate are moderately polar (polarity index of 4.1) that mainly extracts steroids, alkaloids, etc. it has been found that in most of the studies solvent with minimum polarity have been used such as hexane or petroleum ether or that with maximum polarity such as aqueous/ steam distillation. however, those biochemical that were extracted using moderately polar solvents were also seen to give good as reported by a few bioassay. thus, different solvent types can significantly affect the potency of extracted plant compounds and there is difference in the chemo - profile of the plant species. in table i, the lowest lc50 value was reported in solenostemma argel against cx. several other plants such as nyctanthes arbotristis38, atlantia monophylla57, centella asiatica40, cryptotaenia paniculata76 were also reported with promising lc50 values. these extracts may be fractioned in order to locate the particular bioactive toxic agent responsible for larval toxicity. table i also reported that most of the studies were carried out on culex mosquitoes and aedes was the least frequently chosen mosquitoes for all the experiments. in several studies, instead of a particular solvent, combination of solvents or serial extraction by different solvents according to their polarity has also been tried and good larvicidal potentiality found as a 96. the plant world comprises a rich untapped pool of phytochemicals that may be widely used in place of synthetic insecticides in mosquito control programme. kishore et al97 reviewed the efficacy of phytochemicals against mosquito larvae according to their chemical nature and described the mosquito larvicidal potentiality of several plant derived secondary materials, such as, alkanes, alkenes, alkynes and simple aromatics, lactones, essential oils and fatty acids, terpenes, alkaloids, steroids, isoflavonoids, pterocarpans and lignans. they also documented the isolation of several bioactive toxic principles from various plants and reported their toxicity against different mosquito species (table ii). identification of various bioactive toxic principles from plant extract and their relative mosquitocidal efficacy generally the active toxic ingredients of plant extracts are secondary metabolites that are evolved to protect them from herbivores. the insects feed on these secondary metabolites potentially encountering toxic substances with relatively non - specific effects on a wide range of molecular targets. these targets range from proteins (enzymes, receptors, signaling molecules, ion - channels and structural proteins), nucleic acids, biomembranes, and other cellular components98. this in turn, affects insect physiology in many different ways and at various receptor sites, the principal of which is abnormality in the nervous system (such as, in neurotransmitter synthesis, storage, release, binding, and re - uptake, receptor activation and function, enzymes involved in signal transduction pathway)98. rattan98 reviewed the mechanism of action of plant secondary metabolites on insect body and documented several physiological disruptions, such as inhibition of acetylecholinestrase (by essential oils), gaba - gated chloride channel (by thymol), sodium and potassium ion exchange disruption (by pyrethrin) and inhibition of cellular respiration (by rotenone). such disruption also includes the blockage of calcium channels (by ryanodine), of nerve cell membrane action (by sabadilla), of octopamine receptors (thymol), hormonal balance disruption, mitotic poisioning (by azadirachtin), disruption of the molecular events of morphogenesis and alteration in the behaviour and memory of cholinergic system (by essential oil), etc. of these, the most important activity is the inhibition of acetylcholinerase activity (ache) as it is a key enzyme responsible for terminating the nerve impulse transmission through synaptic pathway; ache has been observed to be organophosphorus and carbamate resistant, and it is well - known that the alteration in ache is one of the main resistance mechanisms in insect pests99. several studies have documented the efficacy of plant extracts as the reservoier pool of bioactive toxic agents against mosquito larvae. but only a few have been commercially produced and extensively used in vector control programmes. the main reasons behind the failure in laboratory to land movements of bioactive toxic phytochemicals are poor characterization and inefficiency in determining the structure of active toxic ingredients responsible for larvicidal activity. for the production of a green biopesticide, the following steps can be recommended during any research design with phytochemicals: (i) screening of floral biodiversity in search of crude plant extracts having mosquito larvicidal potentiality; (ii) preparation of plant solvent extracts starting from non - polar to polar chemicals and determination of the most effective solvent extract; (iii) evaporation of the liquid solvent to obtain solid residue and determination of the lethal concentration (lc50/lc100 values); (iv) phytochemical analysis of the solid residue and application of column chromatography and thin layer chromatography to purify and isolate toxic phytochemical with larvicidal potentiality; (v) determination of the structure of active principle by infra red (ir) spectroscopic, nuclear magnetic resonance (nmr) and gas chromatography and mass spectroscopy (gcms) analysis; (vi) study of the effect of active ingredient on non target organisms; and (vii) field evaluation of the active principle before its recommendation in vector control programme and commercial production. human beings have used plant parts, products and secondary metabolites of plant origin in pest control since early historical times. vector control has been practiced since the early 20th century. during the pre - ddt era, reduction of vector mosquitoes mainly depended on environmental management of breeding habitats, i.e., source reduction. during that period, some botanical insecticides used in different countries were chrysanthemum, pyrethrum, derris, quassia, nicotine, hellebore, anabasine, azadirachtin, d - limonene camphor, turpentine, etc7. from the early 1950s, ddt and other synthetic organochloride and organophosphate insecticides were extensively used to interrupt transmission of vector borne diseases by reducing densities, human - vector contact and, in particular, the longevity of vector mosquitoes. in the mid-1970s, the resurgence of vector borne diseases, along with development of insecticide resistance in vector population, poor human acceptance of indoor house spraying and environmental concerns against the use of insecticides led to a rethinking in vector control strategies10. as a , emphasis was given on the application of alternative methods in mosquito control as part of the integrated mosquito management (imm)11. integrated mosquito management (imm) is a decision - making process for the management of mosquito populations, involving a combination of methods and strategies for long - term maintenance of low levels of vectors. the purpose of imm is to protect public health from diseases transmitted by mosquitoes, maintain healthy environment through proper use and disposal of pesticides and improve the overall quality of life through practical and effective pest control strategies. the main approaches of imm include: (i) source reduction and habitat management by proper sanitation, water management in temporary and permanent water bodies, and channel irrigation. vegetation management is also necessary to eliminate protection and food for mosquito larvae; (ii) larviciding by application of dipteran specific bacteria, insect growth regulators, surface films and oils, expanded polystyrene beads, phytochemicals, organophosphates and organochlorides, (iii) adulticiding by application of synthetic pyrethroids, organophosphates and synthetic or plant derived repellents, insecticide impregnated bed nets, genetic manipulations of vector species, etc., (iv) use of mosquito density assessment in adult and larval condition and disease surveillance; and (v) application of biological control methods by using entomophagous bacteria, fungi, microsporidians, predators and parasites. of the above avenues of imm, larviciding approach is the more proactive, proenvironment, target specific and safer approach than controlling adult mosquitoes. application of larvicide from botanical origin was extensively studied as an essential part of imm, and various mosquito control agents such as ocimenone, rotenone, capllin, quassin, thymol, eugenol, neolignans, arborine and goniothalamin were developed7. the efficacy of phytochemicals against mosquito larvae can vary significantly depending on plant species, plant parts used, age of plant parts (young, mature or senescent), solvent used during extraction as well as upon the available vector species. sukumar et al6 have described the existence of variations in the level of effectiveness of phytochemical compounds on target mosquito species vis - - vis plant parts from which these were extracted, responses in species and their developmental stages against the specified extract, solvent of extraction, geographical origin of the plant, photosensitivity of some of the compounds in the extract, effect on growth and reproduction. changes in the larvicidal efficacy of the plant extracts occurred due to geographical origin of the plant (in citrus sp18396465, jatropha sp132021, ocimum sanctum22356582, momordica charantia222449, piper sp54638995 and azadirechta indica65); response in the different mosquito species (in curcuma domestica26, withania somnifera13, jatropha curcas1320, piper retrofractum63, cestrum diurnum58, citrullus vulgaris5071, and tridax procumbens3031); due to variation in the species of plant examined (in euphorbia sp22283751, phyllanthus sp20, curcuma sp36, solanum sp16295760757996, ocimum sp23356582, eucalyptus sp22283751, plumbago sp20, vitex sp5093, piper sp54638995, annona sp485469, and cleome sp3178) and between plant parts used to study the larvicidal efficacy (in euphorbia tirucalli2851, solanum xanthocarpum16, azadirechta indica65, solanum villosum57607996, annona squamosa485469, withania somnifera13, melia azedarach45, moringa oleifera46 and ocimum sanctum3582). however, the principal objective of the present documentation is to report the changes in larvicidal potentiality of the plant extracts due to change of the particular solvent used during extraction. variation of the larvicidal potential of the same plant changed with the solvents used as evidenced in case of solanum xanthocarpum16, euphorbia tirucalli2851, momordica charantia222449, eucalyptus globules14152883, citrullus colocynthis13, azadirechta indica65, annona squamosa485469 and solanum nigrum2975. it has been shown that the extraction of active biochemical from plants depends upon the polarity of the solvents used. polar solvent will extract polar molecules and non - polar solvents extract non - polar molecules. this was achieved by using mainly eleven solvent systems ranging from hexane/ petroleum ether, the most non polar (polarity index of 0.1 that mainly extracts essential oil) to that of water, the most polar (polarity index of 10.2) that extracts biochemical with higher molecular weights such as proteins, glycans, etc. chloroform or ethyl acetate are moderately polar (polarity index of 4.1) that mainly extracts steroids, alkaloids, etc. it has been found that in most of the studies solvent with minimum polarity have been used such as hexane or petroleum ether or that with maximum polarity such as aqueous/ steam distillation. however, those biochemical that were extracted using moderately polar solvents were also seen to give good as reported by a few bioassay. thus, different solvent types can significantly affect the potency of extracted plant compounds and there is difference in the chemo - profile of the plant species. in table i, the lowest lc50 value was reported in solenostemma argel against cx. several other plants such as nyctanthes arbotristis38, atlantia monophylla57, centella asiatica40, cryptotaenia paniculata76 were also reported with promising lc50 values. these extracts may be fractioned in order to locate the particular bioactive toxic agent responsible for larval toxicity. table i also reported that most of the studies were carried out on culex mosquitoes and aedes was the least frequently chosen mosquitoes for all the experiments. in several studies, instead of a particular solvent, combination of solvents or serial extraction by different solvents according to their polarity has also been tried and good larvicidal potentiality found as a 96. the plant world comprises a rich untapped pool of phytochemicals that may be widely used in place of synthetic insecticides in mosquito control programme. kishore et al97 reviewed the efficacy of phytochemicals against mosquito larvae according to their chemical nature and described the mosquito larvicidal potentiality of several plant derived secondary materials, such as, alkanes, alkenes, alkynes and simple aromatics, lactones, essential oils and fatty acids, terpenes, alkaloids, steroids, isoflavonoids, pterocarpans and lignans. they also documented the isolation of several bioactive toxic principles from various plants and reported their toxicity against different mosquito species (table ii). generally the active toxic ingredients of plant extracts are secondary metabolites that are evolved to protect them from herbivores. the insects feed on these secondary metabolites potentially encountering toxic substances with relatively non - specific effects on a wide range of molecular targets. these targets range from proteins (enzymes, receptors, signaling molecules, ion - channels and structural proteins), nucleic acids, biomembranes, and other cellular components98. this in turn, affects insect physiology in many different ways and at various receptor sites, the principal of which is abnormality in the nervous system (such as, in neurotransmitter synthesis, storage, release, binding, and re - uptake, receptor activation and function, enzymes involved in signal transduction pathway)98. rattan98 reviewed the mechanism of action of plant secondary metabolites on insect body and documented several physiological disruptions, such as inhibition of acetylecholinestrase (by essential oils), gaba - gated chloride channel (by thymol), sodium and potassium ion exchange disruption (by pyrethrin) and inhibition of cellular respiration (by rotenone). such disruption also includes the blockage of calcium channels (by ryanodine), of nerve cell membrane action (by sabadilla), of octopamine receptors (thymol), hormonal balance disruption, mitotic poisioning (by azadirachtin), disruption of the molecular events of morphogenesis and alteration in the behaviour and memory of cholinergic system (by essential oil), etc. of these, the most important activity is the inhibition of acetylcholinerase activity (ache) as it is a key enzyme responsible for terminating the nerve impulse transmission through synaptic pathway; ache has been observed to be organophosphorus and carbamate resistant, and it is well - known that the alteration in ache is one of the main resistance mechanisms in insect pests99. several studies have documented the efficacy of plant extracts as the reservoier pool of bioactive toxic agents against mosquito larvae. but only a few have been commercially produced and extensively used in vector control programmes. the main reasons behind the failure in laboratory to land movements of bioactive toxic phytochemicals are poor characterization and inefficiency in determining the structure of active toxic ingredients responsible for larvicidal activity. for the production of a green biopesticide, the following steps can be recommended during any research design with phytochemicals: (i) screening of floral biodiversity in search of crude plant extracts having mosquito larvicidal potentiality; (ii) preparation of plant solvent extracts starting from non - polar to polar chemicals and determination of the most effective solvent extract; (iii) evaporation of the liquid solvent to obtain solid residue and determination of the lethal concentration (lc50/lc100 values); (iv) phytochemical analysis of the solid residue and application of column chromatography and thin layer chromatography to purify and isolate toxic phytochemical with larvicidal potentiality; (v) determination of the structure of active principle by infra red (ir) spectroscopic, nuclear magnetic resonance (nmr) and gas chromatography and mass spectroscopy (gcms) analysis; (vi) study of the effect of active ingredient on non target organisms; and (vii) field evaluation of the active principle before its recommendation in vector control programme and commercial production. today, environmental safety is considered to be of paramount importance. an insecticide does not need to cause high mortality on target organisms in order to be acceptable but should be eco - friedly in nature. phytochemicals may serve as these are relatively safe, inexpensive and readily available in many parts of the world. several plants are used in traditional medicines for the mosquito larvicidal activities in many parts of the world. according to bowers et al100, the screening of locally available medicinal plants for mosquito control would generate local employment, reduce dependence on expensive and imported products, and stimulate local efforts to enhance the public health system. the ethno - pharmacological approaches used in the search of new bioactive toxins from plants appear to be predictive compared to the random screening approach. the recently developed new isolation techniques and chemical characterization through different types of spectroscopy and chromatography together with new pharmacological testing have led to an interest in plants as the source of new larvicidal compounds. synergestic approaches such as application of mosquito predators with botanical blends and microbial pesticides will provide a better effect in reducing the vector population and the magnitude of epidemiology.

mosquitoes act as a vector for most of the life threatening diseases like malaria, yellow fever, dengue fever, chikungunya ferver, filariasis, encephalitis, west nile virus infection, etc. under the integrated mosquito management (imm), emphasis was given on the application of alternative strategies in mosquito control. the continuous application of synthetic insecticides causes development of resistance in vector species, biological magnification of toxic substances through the food chain and adverse effects on environmental quality and non target organisms including human health. application of active toxic agents from plant extracts as an alternative mosquito control strategy was available from ancient times. these are non - toxic, easily available at affordable prices, biodegradable and show broad - spectrum target - specific activities against different species of vector mosquitoes. in this article, the current state of knowledge on phytochemical sources and mosquitocidal activity, their mechanism of action on target population, variation of their larvicidal activity according to mosquito species, instar specificity, polarity of solvents used during extraction, nature of active ingredient and promising advances made in biological control of mosquitoes by plant derived secondary metabolites have been reviewed.

in total, we received six blackbirds, five owls, and one swallow for investigation. at necropsy (estimated postmortem times between 24 h and 48 h), paraffin - embedded tissue samples were immunostained with a polyclonal mouse antibody to wnv (b. murgue, institut pasteur, paris) and a polyclonal rabbit antibody to tick - borne encephalitis virus (strain neudoerfl, h. holzmann, klinisches institut fr virologie, vienna) using the avidin - biotin complex technique. rna was extracted from 140-l organ homogenates or cell culture suspensions by using the qiaamp viral rna mini kit (qiagen gmbh, hilden, germany). after aligning available nucleotide (nt) sequences of various mosquito - borne flaviviruses and determining highly conserved genomic regions, we designed three pairs of oligonucleotide primers (to amplify a wide range of mosquito - borne flaviviruses) and used them in the reverse transcription - polymerase chain reaction (rt - pcr) assays: 5-tacaacatgatgggvaaragagaga-3 (nt position 90319055 of wnv genbank accession no . nc 001563) and 5-agcatgtcttcygtbgtcatccayt-3 (nt position 1011510091), ing in a 1,084-bp amplification product; 5-gartggatgacvacrgaagacatgct-3 (nt position 1009010115) and 5-ggggtctcctctaacctctagtcctt-3 (nt position 1083210807), amplifying a 743-bp pcr product; and 5-gccaccggaagttgagtaga-3 (nt position 1046010479 of wnv no . nc 001563) and 5-gctggttgtgcagagcagaa-3 (nt position 1090810889), ing in a 449-bp amplicon. reverse transcription and amplifications were performed in a continuous rt - pcr method by using the qiagen onestep rt - pcr kit (qiagen gmbh). each 25-l reaction mixture contained 5 l 5x buffer (final mgcl2 concentration 2.5 mm), 0.4 mm deoxynucleoside triphosphate (dntp), 10 u recombinant rnasin ribonuclease inhibitor (promega, madison, wi), 40 pmol forward and reverse primers, 1 l enzyme mix, and 2.5 l template rna. reverse transcription was performed for 30 min at 50c. following an initial denaturation for 15 min at 95c, the reaction mixture was subjected to 45 cycles of heat denaturation at 94c for 30 s, primer annealing at 60c for 30 s, and dna extension at 72c for 1 min, completed by a final extension of 10 min at 72c. following rt - pcr, we performed electrophoresis on 20 l of the amplicons in a 1.2% tris acetate - edta - agarose gel. the gel was stained with ethidium bromide, and the bands were observed under uv light. the pcr products were sequenced in both directions by using the abi prism 310 genetic analyzer automated sequencing system (perkin elmer instruments, wellesley, ma). the nucleotide sequences were compiled and aligned with the corresponding sequences deposited in the genbank database. finally, we constructed a phylogenetic tree based on a 1,035-nt fragment in the ns5 genomic region. the following sequences have been included in the phylogenetic analysis: af013384, koutango virus; af013413, yaounde virus; d00246, kunjin virus; af202541, wnv (new york 1999); m12294, wnv; af013367, cacipacore virus; u15763, jev; af013360, alfuy virus; af013389, mvev; af013412, usuv (south africa); af452643, usuv (austria); af013416, slev; m93130, denv (type 3); and af013417, yfv. the phylogenetic analysis was carried out by using phylogeny interference program package (phylip), version 3.57c (available from : url : http://evolution.genetics.washington.edu/phylip.html). distance matrices were generated with the dnadist / neighbor - joining program; a translation / transversion ratio of 2.0. paraffin - embedded tissue samples were processed as described. for detection of wnv nucleic acid, an antisense digoxigenin - labeled riboprobe complementary to nt 49665439 of wnv strain ny1999 was generated from plasmid pwnny-88b-14 (w.i . the final concentration of the probe was approximately 0.5 ng/l . for detection of usuv nucleic acid , we used a digoxigenin - labeled oligonucleotide probe with the sequence : 5-tcgcataactttcaccaccttgtgtttgtaggtcagctc-3 , which is complementary to nt 367 - 328 of the accessible partial sequence of the ns5 gene of usuv ( genbank accession no . necropsy showed grossly swollen livers and spleens, as well as seromucous enteritis in all blackbirds and owls ; histology showed various degrees of multifocal acute necrosis in liver and spleen . although the blackbirds did not show obvious histologic brain lesions, the owls had encephalitis, predominantly shown as multifocal areas of neuronophagia and microgliosis ( figure 1, a and d). pathologic and immunohistochemical investigation of the swallow did not yield useful because of severe autolysis. histology and detection of viral signals in paraffin - embedded tissue sections of birds infected with usutu virus (usuv). f, great gray owl; a, no histologic lesions present, hematoxylin and eosin staining; b immunohistochemistry, using a polyclonal antibody to west nile virus, shows numerous positive neurons; c, in situ hybridization with usuv - specific oligonucleotide probe shows a staining pattern comparable with that in b; d, microglial nodule within the cerebral cortex, hematoxylin and eosin staining; e, immunohistochemistry shows single positive neurons within a glial nodule; f, in situ hybridization shows several positive neurons next to a glial nodule. original magnification, x 130 (a c), x 200 (d f). immunohistochemistry (ihc) with polyclonal antibodies to wnv was positive in 10 of 11 brains, showing reaction products in neurons and their processes and in the cytoplasm of microglial cells in glial nodules (figure 1, b and e). positive reactions were also present in kidney, spleen, liver, lung, and autonomous ganglia of the gastrointestinal tract. brain and kidney samples from wnv - infected birds from the united states and israel, respectively, were positive controls; blackbirds that died from trauma were negative controls. ihc with polyclonal antibodies to tbev, another flavivirus found in central europe, showed negative . rt - pcr with wnv - specific primers and ish with a wnv - specific probe were negative. infection with a flavivirus related to wnv would account for the cross - reactivity of a polyclonal antibody used in ihc and the negative outcome of the wnv - specific assays. after 2448 hours, a cytopathic effect of cell rounding could be observed; 1 to 2 days later, the affected cells detached and floated in the medium. rt - pcrs with universal flavivirus primers ed in clear pcr amplification products of the expected lengths. the primers were designed to amplify overlapping pcr products in the ns5 genomic region of mosquito - borne flaviviruses. rt - pcrs were performed both on the original organ homogenates and on cell culture suspensions, with identical . despite a poor state of preservation, organ homogenates of the swallow the pcr products (1,084 bp, 743 bp, and 449 bp) were directly sequenced in both directions; the compiled nucleotide sequences (a stretch of 1,877 bp, representing approximately 17% of flavivirus genome) were aligned and compared with other sequences by using the basic local alignment search tool (blast) search (national center for biotechnology information, national institutes of health, bethesda, md). the sequence obtained from the austrian dead birds was 97% identical with a 1,035-nt fragment of usuv (genbank accession no . af013412 ;), an african mosquito - borne flavivirus in the jev group. to investigate the genetic relationship of the austrian usuv with other flaviviruses in the jev antigenic complex , we constructed a phylogenetic tree by using the programs in the phylip package; we also included in the phylogenetic analysis three other important mosquito - borne flaviviruses (yfv, slev, and denv3). the phylogenetic tree (figure 2) demonstrates the close genetic relationship between usuvs isolated in south africa and in austria; therefore, we classified the austrian usuv as part of the jev group of flaviviruses. at the amino - acid level, the austrian and the south african usuv isolates proved (in the investigated 1,035-nt region) to be 100% identical. phylogenetic analysis of several members of the japanese encephalitis virus (jev) group and selected other mosquito - borne flaviviruses demonstrates the close genetic relationship of the austrian usutu virus (usuv) isolate with the south african usuv (red underlay); well - known members of the jev group are highlighted in red; distinct branches are formed by saint louis encephalitis virus, dengue virus (type 3), and yellow fever virus. the partial nucleotide sequence of the austrian usuv isolate used in the phylogenetic tree has been deposited in the genbank database under accession no. af452643. with an oligonucleotide probe specific for usuv, ish showed presence of viral nucleic acid in the cytoplasm of neurons in a distribution pattern closely matching ihc (figure 1, c and f). regarding other organs, however, kidneys of only two birds were positive, probably reflecting rna degradation due to postmortem times > 24 h. we demonstrated the presence of a mosquito - borne flavivirus, never before observed outside tropical and subtropical africa, in the continental climate of central europe, where winter temperatures are as low as 20c. since we also detected usuv nucleic acid in a barn swallow, the virus was probably introduced to the austrian bird population by swallows or other migrating birds. bird die - offs in various bird species in different areas of austria suggest that the virus has already adapted to local mosquito species, which are probably transmitting the virus. isolating the virus from local mosquitoes has not been attempted thus far but is planned. during a retrospective survey of paraffin - embedded blackbird tissues by ihc and rt - pcr , we also detected usuv in a blackbird that died a year earlier. a partial nucleotide sequence of this usuv proved to be 100% identical to the sequences of the 2001 usuv isolates. although no severe bird die - offs were observed in 2000, we think that usuv may already be established and be overwintering in austria (rather than being newly introduced in 2 consecutive years). comparable with the introduction of wnv to north america in 1999, where the virus propagated in local mosquito species and then rapidly spread from new york to > 20 states in the united states and canada, we foresee a similar scenario for usuv in europe. this study shows for the first time that usuv is highly pathogenic for several different species of birds. because full sequence data of usuv isolates are not available (only a short fragment of one usuv isolate has been deposited in the genbank database) we can not provide information on amino acid changes that might contribute to altered pathogenic properties. for the closely related wnv, the pathogenicity for birds seems to depend on the virus strain and whether the virus affects a previously exposed or unexposed population. the wnv strain that appeared in north american birds in 1999 is closely related to a strain isolated in israel; this strain is associated with avian deaths in both countries. certain other wnv strains, such as those responsible for recent outbreaks in romania and russia in humans and in italy and france in equines, were not associated with avian deaths. also, the fact that certain avian species, such as eurasian blackbirds, great gray owls, and barn swallows in austria, are especially vulnerable to usuv infection, is reminiscent of the observation that wnv in north america has primarily affected american crows and blue jays. the emergence of wnv in the united states in 1999 and usuv in central europe in 20002001 is an indication of future virus activity. the next mosquito - borne flavivirus, which might be introduced to regions far from its original habitats, may be highly pathogenic for humans, farm animals, or pets, as many strains of jev group are. we could speculate whether global warming or other environmental factors may have contributed to the introduction and maintenance of usuv, formerly restricted to tropical and subtropical areas, in a much colder climate. as a consequence of the introduction of usuv to central europe, surveillance programs for mosquito - borne flaviviruses in general (based on virus detection in mosquitoes and dead birds, as well as epidemiologic investigations) should be established in europe, like those initiated in the united states after the first occurrence of wnv. moreover, we will have to fully sequence usuv, establish serologic test systems, and evaluate the spread and pathogenic potential to control this new virus infection in central europe.

during late summer 2001 in austria, a series of deaths in several species of birds occurred, similar to the beginning of the west nile virus (wnv) epidemic in the united states. we necropsied the dead birds and examined them by various methods; pathologic and immunohistologic investigations suggested a wnv infection. subsequently, the virus was isolated, identified, partially sequenced, and subjected to phylogenetic analysis. the isolates exhibited 97% identity to usutu virus (usuv), a mosquito - borne flavivirus of the japanese encephalitis virus group; usuv has never previously been observed outside africa nor associated with fatal disease in animals or humans. if established in central europe, this virus may have considerable effects on avian populations; whether usuv has the potential to cause severe human disease is unknown.

the recommended treatments for acne include topical medications and systemic use of antibiotics, hormones, and retinoids, according to severity. tretinoin or isotretinoin is very effective for the treatment of intractable acne but its use has been limited due to the potential for teratogenicity. topical photodynamic therapy (pdt) with topical application of sensitizers in the treatment of different kinds of skin diseases is of increasing research interest. pdt is based on the activation of light - sensitive molecules (photosensitizers) to cause cell death by producing cytotoxic oxygen radicals. 5-aminolevulinic acid (ala) is frequently used in topical pdt because it is converted into protoporphyrin (ppix), an extremely active photosensitizer, upon irradiation with red light. recent studies have suggested that ala - pdt is also effective in treatment of acne vulgaris. with the increasing use of ala - pdt for patients with acne vulgaris, adverse effects have increased, including pain, burning sensation, itching, prickling, erythema, edema, pustules, acneiform eruption, and excoriations , with causes related to the concentration of photosensitizer, the energy of light, and skin conditions of patients. therefore, we studied an optical fiber intra - tissue irradiation 5-aminolevulinic acid photodynamic therapy (ofi - ala - pdt) in the treatment of moderate to severe acne in patients treated in our department from january 2014 to june 2015. patient inclusion criteria were according to the diagnosis and classification in the cunkuffe classification method. these classifications areas are: light (mainly whiteheads and blackheads), medium (mainly inflammatory papules and pustules), and severe (inflammatory papules, nodules, and inflammatory cysts). exclusion criteria were: internal or external use of antibiotics within the last 4 weeks; systemic use of a retinoid in the last 6 months; photosensitive or keloid history; pregnant or liver function unusual; can not complete the course; staff directly participating in the study; and participants currently in other clinical studies or who participated in another study within the last 3 months. the study was upon the approval of the hospital ethics committee and all the patients signed informed consent. these 60 patients with moderate to severe acne were treated from january 2014 to june 2015 in our dermatology department. rash was limited to the face, and the main symptoms were inflammatory papules, pustules, nodules, and cysts, with different degrees of pigmentation and scarring. the treatment group (ofi - ala - pdt) included 12 males and 18 females, aged 1244 years, with an average of 26.85.2 years; the course of disease ranged from 3 to 120 months with an average of 30 months, with no drop - outs. in the control group , there were 16 males and 14 females, aged 1542 years with an average of 27.34.8 years; the course of disease ranged from 4 to 108 months, with an average of 33 months, with no drop - outs. no significant differences were observed between the 2 groups in sex, age, or course of disease (p>0.05). in the treatment group (ofi - ala - pdt) (figure 1), 3.6% aminolevulinic acid was evenly applied to the rashes and the surrounding 0.5 to 1.0 cm of normal skin. after 1.5 h of incubation shielded from light, we wiped off the remaining photosensitizer, disinfected, inserted disposable optical fiber needles into the skin lesions with inflammatory papules and nodules, and imported the red light irradiation to the tissues located 3 mm below the follicular orifice (including the sebaceous glands) for 5 min. a dose of 4.5 j / cm (dose at skin, detected by a vlp-200 laser power meter, changchun feimiao tech ., ltd) was given for the first time (this dose was based on our previous data in an animal model, data not shown), and was adjusted to 324 j / cm in the following irradiations according to adverse reactions. irradiations were carried out every 710 days, and each patient received 6 irradiations in total. in the control group, the traditional skin surface irradiation was used. for irradiation, a dose of 54 j / cm at skin was used with a fixed - power density of 45 mw / cm for 20 min, and the distance between the light panel and patient s apex nasi was set at 10 cm. effective criteria were the number of skin lesions and curative effect as recorded and judged by the same dermatologist. cure was 90% or more of the skin lesions disappeared; remarkably effective was 60% to 89% of the skin lesions disappeared; effective was 20% to 59% of skin lesions disappeared; and invalid was less than 20% of skin lesions disappeared. effective rate was the percentage of cured cases plus remarkable cases divided by the total cases. treatment effects and adverse reactions were recorded during each treatment, before the next treatment and in the subsequent follow - up period. these adverse reactions include itching, pain, pustules, blisters, edematous erythema, pigmentation, reactive acne, and desquamation. we recorded the appearing and fading away time, severity, and actions used to combat these adverse reactions. follow - up was performed at 4, 8, and 16 weeks after the last irradiation. the chi - square test was used to compare different groups and the rank - sum test was used to compare adverse reactions between groups, age, and disease courses. patient inclusion criteria were according to the diagnosis and classification in the cunkuffe classification method. these classifications areas are: light (mainly whiteheads and blackheads), medium (mainly inflammatory papules and pustules), and severe (inflammatory papules, nodules, and inflammatory cysts). exclusion criteria were: internal or external use of antibiotics within the last 4 weeks; systemic use of a retinoid in the last 6 months; photosensitive or keloid history; pregnant or liver function unusual; can not complete the course; staff directly participating in the study; and participants currently in other clinical studies or who participated in another study within the last 3 months. the study was upon the approval of the hospital ethics committee and all the patients signed informed consent. these 60 patients with moderate to severe acne were treated from january 2014 to june 2015 in our dermatology department. rash was limited to the face, and the main symptoms were inflammatory papules, pustules, nodules, and cysts, with different degrees of pigmentation and scarring. the treatment group (ofi - ala - pdt) included 12 males and 18 females, aged 1244 years, with an average of 26.85.2 years; the course of disease ranged from 3 to 120 months with an average of 30 months, with no drop - outs. in the control group , there were 16 males and 14 females, aged 1542 years with an average of 27.34.8 years; the course of disease ranged from 4 to 108 months, with an average of 33 months, with no drop - outs. no significant differences were observed between the 2 groups in sex, age, or course of disease (p>0.05). in the treatment group (ofi - ala - pdt) (figure 1), 3.6% aminolevulinic acid was evenly applied to the rashes and the surrounding 0.5 to 1.0 cm of normal skin. after 1.5 h of incubation shielded from light, we wiped off the remaining photosensitizer, disinfected, inserted disposable optical fiber needles into the skin lesions with inflammatory papules and nodules, and imported the red light irradiation to the tissues located 3 mm below the follicular orifice (including the sebaceous glands) for 5 min. a dose of 4.5 j / cm (dose at skin, detected by a vlp-200 laser power meter, changchun feimiao tech ., ltd) was given for the first time (this dose was based on our previous data in an animal model, data not shown), and was adjusted to 324 j / cm in the following irradiations according to adverse reactions. irradiations were carried out every 710 days, and each patient received 6 irradiations in total. in the control group, the traditional skin surface irradiation was used. for irradiation, a dose of 54 j / cm at skin was used with a fixed - power density of 45 mw / cm for 20 min, and the distance between the light panel and patient s apex nasi was set at 10 cm. effective criteria were the number of skin lesions and curative effect as recorded and judged by the same dermatologist. cure was 90% or more of the skin lesions disappeared; remarkably effective was 60% to 89% of the skin lesions disappeared; effective was 20% to 59% of skin lesions disappeared; and invalid was less than 20% of skin lesions disappeared. effective rate was the percentage of cured cases plus remarkable cases divided by the total cases. treatment effects and adverse reactions were recorded during each treatment, before the next treatment and in the subsequent follow - up period. these adverse reactions include itching, pain, pustules, blisters, edematous erythema, pigmentation, reactive acne, and desquamation. we recorded the appearing and fading away time, severity, and actions used to combat these adverse reactions. follow - up was performed at 4, 8, and 16 weeks after the last irradiation. the chi - square test was used to compare different groups and the rank - sum test was used to compare adverse reactions between groups, age, and disease courses. the treatment are shown in figure 2. by the end of the 4 irradiation, the effective rate of the ofi - ala - pdt group was 90%, which is significantly higher than the control group s 66.7% (=4.812, p<0.05). however, there was no significant difference between these groups when all the 6 irradiations were finished (93.3% in the treatment versus 90% in the controls) (table 1). furthermore, follow - up at 4, 8, and 16 weeks after all the treatment showed no improvement in the ofi - ala - pdt group (table 1). there were 84 adverse reactions recorded in the ofi - ala - pdt group during treatment, which is significantly less than the 242 recorded in the control group (table 2). after the treatment, there are 98 adverse reactions in the treatment group and 255 in the control group (table 2). overall, 182 adverse reactions in the ofi - ala - pdt group and 497 in the control group were recorded, indicating that ofi - ala - pdt treatment performed better in decreasing the number of adverse reactions (p<0.01). the treatment are shown in figure 2. by the end of the 4 irradiation, the effective rate of the ofi - ala - pdt group was 90%, which is significantly higher than the control group s 66.7% (=4.812, p<0.05). however, there was no significant difference between these groups when all the 6 irradiations were finished (93.3% in the treatment versus 90% in the controls) (table 1). furthermore, follow - up at 4, 8, and 16 weeks after all the treatment showed no improvement in the ofi - ala - pdt group (table 1). there were 84 adverse reactions recorded in the ofi - ala - pdt group during treatment, which is significantly less than the 242 recorded in the control group (table 2). after the treatment, there are 98 adverse reactions in the treatment group and 255 in the control group (table 2). overall, 182 adverse reactions in the ofi - ala - pdt group and 497 in the control group were recorded, indicating that ofi - ala - pdt treatment performed better in decreasing the number of adverse reactions (p<0.01). the clinical treatment for moderate to severe acne has mainly focussed on the systematic usage of antibiotics and retinoids. however, this kind of treatment is limited due its obvious adverse effects. in recent years, a large number of studies have confirmed that ala - pdt by red light is a highly effective treatment for acne. unfortunately, with the extensive use of ala - pdt, more and more adverse effects had been reported. almost all photodynamic therapy patients experience various ranges of adverse reactions, including moderate to severe pain, erythema, pustules, and peeling. the presently known factors affecting photodynamic therapy efficiency and safety include the concentration of photosensitizer, photosensitizer packet time, photosensitizer type, light type, and light parameters. the conventional method used to reduce adverse reactions is to avoid light and sunshine, and cold spray or cold repair facial mask after photodynamic therapy, but the effect is limited. one study used different concentrations of ala for the treatment and found that the higher concentration was related to improved curative effect but caused severe adverse reactions. some studies reduced the ala packet time from a few hours to 1 hour and observed low incidence of adverse reactions, especially in the case of pigmentation, but its curative effect is reduced to a certain degree. at the same time, the use of new photosensitizers is also a popular research topic. jang introduced new photosensitizers (indocyanine green and indole-3-acetic acid) in photodynamic therapy for acne. the study achieved a good effect, and only produced slight erythema (6/34) and pruritus (8/34) after each irradiation, which resolved within a few hours. the photodynamic approach currently used in the ala - pdt uses the external exposure mode. in this mode, to get to the hair follicle sebaceous glands, the red light has to penetrate the skin epidermis layer. however, this is bound to increase the light attenuation, and the epidermis layer may absorb more light, leading to local adverse effects. on the other hand, the hair follicle sebaceous glands would absorb less energy and the effect would be limited. therefore, to address this problem, we used disposable optical fiber needles to penetrate the skin 3 mm to import the red light into subcutaneous layers of the target site. this method was expected to achieve accurate, minimally invasive, targeted therapy in the tissues and reduce damage to the normal skin surrounding the lesions, so it can alleviate the related adverse effects. to determine whether this irradiation method could improve the therapeutic effects, as expected, by the end of the 4 irradiation, the effective rate of the ofi - ala - pdt group reached 90%, which is significantly higher than the 66.7% achieved in the control group (=4.812, p<0.05). these indicate the ofi - ala - pdt contributed to the effective rate in the early stage of irradiation but did not help the long - time therapy. moreover, 182 adverse reactions in the ofi - ala - pdt group and 497 in the control group were recorded, indicating that ofi - ala - pdt treatment performed better in decreasing the adverse reactions (p<0.01). overall, the ofi - ala - pdt treatment developed in this study showed improved treatment effective rate in the early stage of irradiation and fewer adverse reactions.

to treat moderate to severe acne vulgaris, we developed an optical fiber imported intra - tissue photodynamic therapy: the optical fiber irradiation 5-aminolevulinic acid photodynamic therapy (ofi - ala - pdt). the aim of this study was to compare the treatment effect and tolerability of ofi - ala - pdt versus traditional ala - pdt in the treatment of moderate to severe acne vulgaris.material/methods60 patients with facial acne enrolled into this study were randomly divided into an ofi - ala - pdt group and a traditional ala - pdt group, with 30 patients in each group. the difference between these 2 groups was the red light irradiation methods used. in the ofi - ala - pdt group we used intra - tissue irradiation (import the red light directly into the target lesion with optical fiber) for 5 min, while the traditional ala - pdt group received whole - face irradiation for 20 min. all patients received 1 irradiation every 7 to 10 days for a total of 6 irradiations. treatment effects and adverse reactions were recorded after the 4th and 6th irradiation, and at 4, 8, 16 weeks after the entire treatment.after the 4th irradiation, significantly different effective rates were observed in these groups (90.0% for the ofi - ala - pdt group and 66.7% for the control group). however, no significant difference in effective rate was recorded in the later observations. there were 182 adverse reactions in the ofi - ala - pdt group and 497 in the control group, which showed a significant difference (p<0.05).ofi - ala - pdt showed improved treatment effective rate in the early stage of irradiation, and it had fewer adverse reactions.

tyrosine hydroxylase (th ; tyrosine 3-mono - oxygenase ; ec 1.14.16.2 ; chromosome 11p15.5), which catalyzes the formation of dopa from dopamine, is generally considered to be the rate - limiting enzyme in the catecholamine biosynthetic pathway (flatmark and stevens 1999). although a single - copy gene, th produces four different types of mrna through alternative splicing of a single primary transcript (grima et al . substantial loss of th enzymatic activity has profound consequences both in humans ( flatmark and stevens 1999) and in mice with targeted ablation of the th locus (zhou et al . the human th locus bears several examples of common natural allelic variation, such as the tetranucleotide repeat in its first intron, which associate with essential hypertension ( sharma et al ., 1998). the th tetranucleotide repeat (tcat)n occurs in 511 head - to - tail copies. found that (tcat)6 and (tcat)10i alleles seemed to be cardioprotective by association with attenuation of the hemodynamic response to stress with increasing age, while (tcat)7 seemed to be deleterious by their association with higher resting systolic blood pressure and greater hemodynamic response to stress with increasing body mass index. sharma et al. found that the (tcat)10 (e) allele was over - represented in hypertensive subjects, whereas the (tcat)9 (d) allele frequency was increased in control (normotensive) subjects. wei et al. found (tcat)9 to be associated with higher norepinephrine levels. in order to probe the role of th polymorphism in stress - induced disease pathways 2002 ). exploring the effects of particular (tcat)n alleles on intermediate phenotypes, we stratified each individual on the basis of number of copies of the allele. we found that the two most common alleles, (tcat)6 and (tcat)10i, influenced a number of autonomic traits, both biochemical and physiological: (tcat)6 copy number affected basal pulse interval and heart rate, and post - stress heart rate. (tcat)10i copy number affected basal pulse interval, plasma epinephrine, and renal norepinephrine excretion (zhang et al . we documented the heritability of blood pressure, and found that ( tcat)6 allele frequencies differed among individuals stratified by genetic risk (family history) of hypertension: in particular, family history - positive individuals were less likely to bear the (tcat)6 allele. since increasing (tcat)6 allele copy number was associated with lower basal and stress - induced heart rates, the obtained suggested a mechanism whereby (tcat)6 alleles may be protective against the future development of hypertension. the (tcat)n motif can bind such transcription factors as ap1 or znf191 (albanese et al . 2001) and may function as a transcriptional enhancer when tested in transfected / expressed promoter / reporter plasmids. however, in transfected th promoter / intron / reporter studies (albanese et al . 2001), the (tcat)n repeat silences transcription in a copy number - dependent way; by contrast, in vivo we observed directionally opposite associations of common (tcat)n alleles with autonomic function: (tcat)10i with activation and (tcat)6 with diminution of sympathetic outflow (zhang et al . ( tcat)n could not mechanistically explain common variation in human autonomic function. therefore, we carried out systematic polymorphism discovery at the locus. in order to probe the possible underlying impact of th variation on stress - induced disease pathways, we re - sequenced ~1.2 kbp of 5 promoter as well as all 13 exons and adjacent intronic regions for rare and common variants in 80 ethnically diverse subjects as well as 422 twins. plasma and urine catecholamines were measured by radioenzymatic assay (kennedy and ziegler 1990). in the coding region, we found two common bi - allelic variants, only one of which was non - synonymous: val81met (at 37.4%) which did not associate with the autonomic traits we studied. we also found 12 unusual coding region variants, but the non - synonymous variants had minor allele frequencies of only 0.30.6%, not sufficient to account for the common population associations. thus, we turned to potential regulatory (non - coding) variants and found 10 snps in the proximal promoter region, four of which (c-824 t, g-801c, a-581 g, and g-494a) were common (minor allele frequencies > 10%). statistically, c-824 t (rs10770141) and a-581 g (rs10770140) seemed to influence both catecholamine secretion and the blood pressure response to environmental stress. a multivariable analysis further indicated that c-824 t became the most significant predictor of change in dbp during cold stress. the -824 t allele is associated with increased catecholamine production, increased blood pressure increments in response to stress, and extreme blood pressure values in the population. it was tempting to speculate that the functional variation that we observed in -824 t carriers could be the outcome of environmental selective pressures on alleles augmenting catecholaminergic function (rao et al . more recently, we performed additional studies to test whether such th promoter variants were by themselves functional ( zhang et al . first, common haplotypes ( at least 15% frequency) of the th promoter were generated by site - directed mutagenesis, then verified by sequencing, and inserted into luciferase reporter vectors. after transfection into pc12 cells, such human th promoter haplotypes showed substantial differences in luciferase reporter activity. by two - way anova on luciferase activity, two of the four common variants (c-824 t and a-581 g) altered transcriptional activity. comparison of these in cells with the effects of the same 4 variants in vivo indicates that the same variants (c-824 t and a-581 g) that exert the greatest effect on th transcription in cells also have the most pronounced effect on human catecholamine secretion in vivo. in addition, the -824 t allele displayed an augmented response to typical chromaffin cell secretory stimuli, such as nicotine or pacap. under basal circumstances, greater activity of the t allele was observed, and when stimulated by nicotine, c-824 t responded differentially, with an increased effect of the drug on the t allele; when stimulated by pacap, the increased response of the t allele was even more apparent. the -581 g allele also responded differentially to pacap. in the basal state, greater activity of the g allele was apparent; during nicotine stimulation, greater activity of the g allele was noted, as was the case during response to pacap. we further probed the functional significance of c-824 t and a-581 g by co - transfection of sequence - predicted trans - activating factors in cells: mef2, sry, and foxd1 differentially activated c-824 t, whereas the g / c - rich binding factors sp1, ap2, and egr1 differentially activated a-581 g. at c-824 t, the co - transfected human mef2 increased th promoter expression to 131.3% on the t allele, but had little effect on the c allele. the mef2 dominant - negative mutant mmef2 decreased th promoter expression down to 71.3% on the t allele, but had little effect on the c allele. thus, at c-824 t, factor mef2 acted in a directionally coordinate fashion (at t > c) to explain the in vivo trait associations, whereas at a-581 g, factors sp1, ap2, and egr1 displayed similar differential actions (at g > a). chromatin immunoprecipitation (chip) confirmed the interaction of the endogenous transcription factors with these motifs in the nucleus ( zhang et al. we conclude that inter - individual variability in catecholamine secretion is controlled in substantial part by genetic variation in the adrenergic pathway encoding catecholamine synthesis, especially at the classical rate - limiting step, th. c-824 t and a-581 g are not only statistical predictors of catecholamine secretory and stress bp response traits in vivo, but also causally responsible for alterations in transcriptional efficiency of the th gene. our thus document novel pathophysiological links between a key adrenergic locus, catecholamine metabolism, and blood pressure, and suggest new strategies to approach the mechanism, diagnosis, and treatment of autonomic dysfunction as well as systemic hypertension.

the catecholamine biosynthetic pathway consists of several enzymatic steps in series, beginning with the amino acids phenylalanine and tyrosine, and eventuating in the catecholamines norepinephrine (noradrenaline) and epinephrine (adrenaline). since the enzyme tyrosine hydroxylase (th ; tyrosine 3-mono - oxygenase ; ec 1.14.16.2 ; chromosome 11p15.5) is generally considered to be rate - limiting in this pathway, probed as to whether common genetic variation at the th gene occurred, and whether such variants contributed to inter - individual alterations in autonomic function, either biochemical or physiological. we began with sequencing a tetranucleotide (tcat) repeat in the first intron, and found that the two most common versions, (tcat)6 and (tcat)10i, predicted heritable autonomic traits in twin pairs. we then conducted systematic polymorphism discovery across the ~8 kbp locus, and discovered numerous variants, principally non - coding. the proximal promoter block contained four common variants, and its haplotypes and snps (especially c-824 t, rs10770141) predicted catecholamine secretion, environmental stress - induced bp increments, and hypertension. finally, we found that two of the common promoter variants, c-824 t (rs10770141) and a-581 g (rs10770140), were functional in that they differentially affected transcriptional activity of the isolated promoter, disrupted recognition motifs for specific transcription factor binding, altered the promoter responses to the co - transfected (exogenous) factors, and bound the endogenous factors in the chromatin fraction of the nucleus. we concluded that common variation in the proximal th promoter is functional, giving rise to changes in autonomic function and consequently cardiovascular risk.

the aluminum concentration and ryznar index increased and the ph decreased in a small appalachian water supply reservoir following acid precipitation runoff episodes. concomitant increases in tapwater aluminum and decreases in tapwater ph were also observed at two homes in the water distribution system. lead concentrations in the tapwater of one home frequently exceeded recommended levels, although spatial and temporal variation in tapwater copper and lead concentrations was considerable. since source water and reservoir water copper and lead concentrations were much lower, the increased copper and lead concentrations in tapwater were attributed to corrosion of household plumbing. tapwater copper concentration correlated well with tapwater ph and tapwater temperature. asbestos fibers were not detected in tapwater. the asbestos - cement pipe in the water distribution system was protected by a spontaneous metallic coating that inhibited fiber release from the pipe. several simultaneous reactions were hypothesized to be taking place in the distribution system that involved corrosion of metallic components and coating of asbestos - cement pipe components in part with corrosion products and in part by cations of watershed origin. greater water quality changes might be expected in areas of higher atmospheric deposition.imagesfigure 5.figure 6.

whilst rubella is usually a mild disease in adults and children, maternal infection with rubella, especially early in pregnancy, can cause severe defects in the developing foetus, ing in congenital rubella syndrome (crs). the constellation of anomalies of crs includes ophthalmic, auditory, cardiac, and craniofacial defects. crs is common in developing countries, affecting about 110,000 infants annually in these countries. in 2009 in addition, 165 cases of crs were reported to who by 123 member countries in the same year. in the western pacific region, the number of rubella cases increased 12-fold from 5475 in 2000 to 73077 in 2009. with rubella a growing problem in the western pacific region, there are concerns that crs may also be on the rise in this region. the relationship between the incidence of rubella and the incidence of crs has not been clearly shown, although some studies from resource poor settings, such as romania, have shown clusters of children with crs after rubella outbreaks. in fiji, the incidence of rubella has ranged between 1 and 30 cases per 100,000 population with outbreaks noted in 1995, 2002, 2006, and 2011. the most recent rubella outbreak, in july 2011, has highlighted the need to carry out surveillance of crs in fiji. vaccination against rubella was introduced in 1975 for females (at 12 years of age), and in 2004 this was extended to include both males and females at primary school entry. however , a lack of information on the incidence of crs in fiji limits our ability to assess the effectiveness of these vaccination campaigns. to address this information gap, this study seeks to (a) report on the incidence of crs in fiji and the relationship between the incidence of crs and the incidence of notified cases of rubella since 1995 and (b) document how crs cases are classified (suspected ( possible), clinically confirmed (probable), or laboratory confirmed (definite) ) over the period 19952010. this was a descriptive study involving a retrospective review of all recorded congenital abnormalities associated with live births in fiji over a 16-year period. fiji is an island nation located in the south - west pacific with a population of approximately 837,271. it consists of approximately 332 islands covering a total land area of about 18,333 sqkm. the ministry of health (moh) in fiji provides decentralized health services through a three tier structure of primary, secondary, and tertiary care. fiji's health system comprises three divisional hospitals, 17 subdivisional hospitals, 78 health centres, and 103 nursing stations. there is a hierarchical referral mechanism from nursing stations to health centres to subdivisional and divisional hospitals. infants born with congenital defects requiring intervention are likely to be referred to the paediatric departments in the three divisional hospitals colonial war memorial (cwm) hospital, lautoka hospital, and labasa hospital. infants with newly diagnosed congenital anomalies are routinely admitted to intensive care wards at these hospitals. the study population included all live births in fiji with a congenital anomaly registered between january 1, 1995, and december 31, 2010. between november 2011 and april 2012 all newborn infants with congenital anomalies were identified from the congenital anomalies registers when these registers were available, and when not available, from a review of the neonatal intensive care unit registers. data on the number of live births and the annual population numbers were obtained from moh consolidated monthly reports (cmr) for the period 19952008 and from the moh public health information system (phis) for the period 2009 - 2010. data on the annual number of reported cases of rubella were obtained from the national notifiable disease surveillance system reports for the period 19952010. the cmr and phis records were used to allow calculation of the incidence rates of crs and rubella. for each recorded case of crs the following data were collected: hospital, date registered, date of birth, sex, ethnicity, and description of congenital defects. the presence or absence of specific congenital defects was recorded, as specified in the who diagnostic classification for crs and cases were classified as suspected, clinically confirmed, or laboratory confirmed where these notes were available, they were reviewed to verify the diagnosis that had been recorded in the congenital anomalies book or the neonatal intensive care unit register. the incidence of crs was expressed per 1000 live births, and the incidence of rubella was expressed as cases per 100,000 population for each year. the association between the incidence of rubella and the incidence of crs was estimated by linear regression using epi - info, version 3.5.1. the congenital anomalies book was not available for lautoka hospital and for labasa hospital before 2010. data was also missing for cwm hospital for 1998 and june december for 2000. based on the available data, a total of 977 babies with congenital anomalies were recorded between 1995 and 2010 in fiji and initially 294 of these cases were found to meet the criteria for crs. folders were only available for 38 (13%) of these 294 cases. in 33 cases the diagnosis was not changed but the classification was changed from suspected to clinically confirmed crs. in two cases crs cases comprised more males than females and i - taukei was the most common ethnic group followed by fijians of indian ethnic descent (table 1). between 1995 and 2010, there was a significant linear increase in the incidence of crs (odds ratio 1.045 per year, 95% ci 1.019 to 1.071, p < 0.001), with this incidence ranging from 0.4 cases per 1000 live births in 1995 to about 1.7 cases per 1000 live births in 2010 (figure 1). whilst the peak in incidence of rubella and the incidence of crs coincided in 2002 , there was no significant association between incidence of crs and rubella (p = 0.3) (figure 1). the majority of crs cases (n = 278, 95%) were classified as suspected thereafter, the annual proportion of clinically confirmed cases was consistently less than 20% of total crs cases. the most common presentations in babies with crs were congenital heart disease (80%) followed by jaundice (10%). this study demonstrates an increasing trend in the incidence of suspected crs over the past 16 years. however, very few cases were clinically confirmed and there were no laboratory confirmed cases of crs. there was no significant relationship between the incidence of crs and the incidence of rubella. this study is based on data for crs recorded in all four divisions of fiji. we have ensured that the data are comparable among the hospitals and with international reports by using a standard criteria, as recommended by who, to classify the cases. there was a lack of information on clinical and laboratory aspects of the diagnosis in the registers. hence, we were not able to adequately assess the validity of the registers as a source of information on the diagnosis of crs. furthermore, not all hospitals had a congenital anomalies book and, where this did not exist, we could only obtain the diagnostic information by searching the neonatal intensive care admission book. the information contained in this book was also limited as it did not include laboratory or detailed clinical findings. the range in incidence of possible crs recorded in fiji is similar to that reported in developing countries during epidemics at about 0.62.2 per 1000 live births. this range of incidence is also similar to that of industrialized countries prior to vaccination. whilst there was no significant relationship found between the incidence of rubella and incidence of crs in fiji, rubella outbreaks leading to increased incidences of crs have been documented in other countries such as panama, oman, and sri lanka. the majority of the crs cases are suspected cases in which the clinical finding was congenital heart disease. improvements in diagnosis of congenital heart disease over time could be contributing to increasing numbers of cases of suspected crs reported during this period. the low numbers of clinically confirmed cases reported may be due to difficulties in assessment at birth particularly for features that become more apparent at a later stage of life, for example, deafness, blindness, and mental retardation. the lack of a significant relationship between the incidence of crs and the incidence of rubella may be caused by underreporting of rubella due to shortcomings with data recording and management at the hospitals. the increasing incidence of suspected crs in fiji demonstrated through this study has implications for both clinical management and surveillance purposes. the absence of any laboratory confirmed crs cases suggests the need to develop standard operating procedures and guidelines for confirming diagnosis through serological tests so that we are able to accurately estimate the burden of crs in fiji. considering the challenges in documenting the extent of crs in the population due to great variation in the manifestations of crs in the first year of life and diagnostic issues related to detecting some of these features, a system would need to be established for close followup of these infants in maternal and child health clinics in order to identify features of crs that may not be possible to assess at birth. this study also suggests the need to monitor adequacy of rubella prevention, through the current rubella vaccination programme carried out at primary school entry. who recommends coverage rates to be maintained above 80% to ensure herd immunity. low coverage rates may indicate the need for serological surveys among women of child bearing age. furthermore, assessment of the sensitivity, specificity, and predictive value of the different clinical definition of crs may need to be carried out. the very small proportion of patient folders obtained for validation through this study suggests the need to improve information management and record keeping at health facilities. ultimately an improvement in surveillance of crs would allow evaluation of our disease control efforts towards elimination of rubella and crs. this highlights the need to strengthen surveillance for crs through improvements in clinical and laboratory diagnosis to confirm or exclude suspected cases.

setting. a nationwide study in fiji. objective. to describe the incidence of congenital rubella syndrome (crs) and its relationship to the incidence of notified cases of rubella in fiji from 1995 to 2010. design. descriptive, retrospective review of all recorded congenital abnormalities associated with live births in fiji over 16 years. . there were 294 infants who met the criteria for crs. of these, 95% were classified as suspected cases, 5% were clinically confirmed, and none were laboratory confirmed cases. there was a significant linear increase over the study period in the incidence of crs (odds ratio 1.045 per year, 95% ci 1.019 to 1.071, p 0.001). there was no significant association between the incidence of crs and the reported incidence of rubella (p = 0.3). . there is a rising trend in reports of suspected crs cases in fiji. this highlights the need to strengthen surveillance for crs through improvements in clinical and laboratory diagnosis to confirm or exclude suspected cases. it is also important to ensure high coverage of rubella vaccination in fiji.

primary hypothyroidism is a common medical condition, and may be a significant cause of morbidity if left untreated. the symptoms of hypothyroidism, including weight gain, constipation, fatigue, cold intolerance, hair loss, and poor concentration, can be debilitating. typically, the symptoms associated with this disorder are effectively treated with an oral replacement of thyroxine at an average daily dose of 1.6 mcg / kg ideal body weight.1 the correct dosing is usually confirmed with the resolution of symptoms and normalization of thyroid - stimulating hormone (tsh) levels within 68 weeks. one would expect that the debilitating nature of this disorder and the improvement with treatment would be reason enough for patients to comply with therapy. however, there have been a number of cases described where patients intentionally fail to take their prescribed thyroid medication.27 these patients are believed to suffer from an underlying psychiatric disorder; more specifically, they are believed to suffer from factitious disorder, or as it has been termed by the authors in these cases, pseudomalabsorption of thyroxine. we describe a case report of a patient with a persistently elevated tsh level despite high doses of oral thyroxine who subsequently developed an enlarged pituitary and hyperprolactinemia. this article will highlight the factors that may interfere with thyroxine treatment, the consequences of untreated hypothyroidism and management of patients with pseudomalabsorption of levothyroxine. a 32-year - old woman presented with a 2-year history of elevated tsh levels (> 100 mu / l, normal range 0.325.00 mu / l) and undetectable free thyroxine / triiodothyronine (ft4/ft3) levels despite increasing doses of oral thyroxine. at the time of presentation, she had been prescribed a thyroxine dose of 0.5 mg / day and liothyronine 0.125 mg daily. she had been on these doses for more than 6 months. on presentation, the patient complained of vague symptoms of weakness, but clinically there was no evidence of profound hypothyroidism. the patient was subsequently found to have an elevated prolactin level and enlarged pituitary on magnetic resonance imaging. table 1 provides details of the investigations and that were carried out to rule out various factors that may have interfered with the absorption and clearance of thyroxine in this patient. with no evidence of malabsorption and absence of another medical condition to explain the lack of response to the high doses of levothyroxine , arrangements were made for supervised treatment with 0.5 mg of thyroxine treatment initially and then daily loading with 0.1 mg of levothyroxine and liothyronine 25 mcg for 5 days (table 2). a 32-year - old woman presented with a 2-year history of elevated tsh levels (> 100 mu / l, normal range 0.325.00 mu / l) and undetectable free thyroxine / triiodothyronine (ft4/ft3) levels despite increasing doses of oral thyroxine. at the time of presentation, she had been prescribed a thyroxine dose of 0.5 mg / day and liothyronine 0.125 mg daily. she had been on these doses for more than 6 months. on presentation, the patient complained of vague symptoms of weakness, but clinically there was no evidence of profound hypothyroidism. the patient was subsequently found to have an elevated prolactin level and enlarged pituitary on magnetic resonance imaging. table 1 provides details of the investigations and that were carried out to rule out various factors that may have interfered with the absorption and clearance of thyroxine in this patient. with no evidence of malabsorption and absence of another medical condition to explain the lack of response to the high doses of levothyroxine, arrangements were made for supervised treatment with 0.5 mg of thyroxine treatment initially and then daily loading with 0.1 mg of levothyroxine and liothyronine 25 mcg for 5 days (table 2). within 5 days of treatment, the patient s thyroid hormone levels normalized and tsh levels decreased to 7 mu / l. with tsh levels maintained at < 15 mu / l for 2 weeks , there was a reduction in the prolactin level and normalization of the pituitary on magnetic resonance imaging (table 2, figure 1). although poor compliance with medication is the most common cause for treatment failure in patients with hypothyroidism,8 there are a number of pharmacological agents and disease processes that are known to affect the absorption or metabolism of levothyroxine (table 3). a number of studies have shown that both iron and calcium supplements can potentially decrease the absorption of thyroxine through the formation of a nonabsorbable complex in the gut.9,10 given the increased incidence of hypothyroidism in the elderly population and the association of both osteoporosis and anemia with this condition, it is important to consider the interaction of these supplements with thyroxine as a potential cause for treatment failure. to eliminate this interaction and to maximize the benefit of each treatment, other studies have shown that gastrointestinal disorders and anatomical gastrointestinal abnormalities, such as untreated celiac disease and short - gut syndrome, may affect the absorption of thyroxine.11,12 celiac disease is an important consideration in this group of patients, considering the autoimmune association between these two disorders and the frequency in which they occur in patients simultaneously. in terms of patients with celiac disease, normalization of thyroid hormone levels and tsh have been observed once a gluten - free diet has been implemented.11 normal absorption of thyroxine is also known to be affected by changes in the acidic environment of the stomach. specifically, it has been shown that patients with impaired gastric acid secretion, whether secondary to helicobacter pylori infection or atrophic gastritis, required higher doses of thyroxine - replacement therapy to become euthyroid.13 similar but reversible findings were also seen in patients treated with omeprazole, a proton - pump inhibitor used to suppress acid secretion.13 other common medications to consider in this group of patients are estrogen - replacement therapy and the selective estrogen receptor modulator raloxifene. arafah showed that a group of 18 postmenopausal women treated with thyroxine - replacement therapy demonstrated increases in serum thyroxine and thyroxine - binding globulin (tbg) concentrations while receiving estrogen treatment.14 however, patients with a diagnosis of hypothyroidism had decreased ft4 levels (22 5 pmol / l to 18 4 pmol / l) and increased serum thyrotropin levels (0.9 1.1 to 3.2 3.1). the coadministration of raloxifene and levothyroxine has also ed in increased dosing requirements for thyroxine.15 it is not clear whether the increased dosing of thyroid - replacement treatment is related to an absorption problem or related to a change in the tbg level. garwood et al report a case where separation of these medications by 12 hours enabled their patient to reduce her thyroxine dosage, suggesting that it may be related to absorption, although the mechanism is unclear.15 in younger female patients, pregnancy should also be considered as a potential cause for increasing tsh levels and the need for an increase in thyroxine dosing, particularly in previously stable patients. similar to patients on estrogen - replacement therapy, the increased thyroid replacement dosing is attributed to an increase in tbg, ing in decreased levels of ft4 and ft3 and subsequent rises in tsh levels.16 the increased thyroxine dosage requirements have been estimated at approximately 45% higher than their regular dose of thyroxine.16 once each of the various factors discussed above has been thoroughly investigated and eliminated, pseudomalabsorption of thyroxine should be considered in patients failing to achieve normal tsh despite high levels of thyroxine. this diagnosis and normal intestinal absorption can be confirmed through a supervised loading doses of up to 1 mg of oral thyroxine.5 it is often recommended that this be completed when patients are fasting, as uptake of thyroxine has been found to be greater in a fasting state.2 patients in these situations should respond with a rapid lowering of their tsh levels and an increase in ft4 levels. it has been reported that most levothyroxine absorption takes place within the first few hours of administration and that serum ft4 levels peak within 2 hours of administration.26 patients such as the one described in this case report are often emphatic about their compliance with the prescribed treatment. the obvious consequence of untreated hypothyroidism is the persistence of the common symptoms of fatigue, weight gain, constipation, and poor concentration, and the morbidity associated with each of these. however, there are more serious and potentially life - threatening consequences of hypothyroidism if left untreated. specifically, myxedema coma is a rare but serious medical emergency and definitely the most severe complication of decompensated hypothyroidism. it is often the of coexisting medical conditions or drugs, and has been found to have a mortality rate of approximately 80% in untreated patients.1 another rare and potentially severe complication of untreated hypothyroidism is the development of a pituitary hyperplasia and hyperprolactinemia, as was seen in our patient. although there have been no documented cases where specifically the intentional noncompliance with thyroxine treatment has led to the development of pituitary hyperplasia, it is well established that one of the potential causes for hyperprolactinemia and pituitary hyperplasia is hypothyroidism.26,27 in these situations, the increase in pituitary size and elevated prolactin secretion are felt to be secondary to increased synthesis of thyroid releasing hormone (trh) in response to chronically low thyroid hormone levels. the development of pituitary hyperplasia in itself has the potential for quite severe complications, including headaches, neurological deficits, and visual loss. prolonged hyperprolactinemia may lead to infertility, hypopituitarism, and low bone density.28 although hypothyroidism is clearly not the only cause of a pituitary hyperplasia and elevated prolactin levels, resolution of these findings with appropriate treatment of the hypothyroidism suggests that this is the most likely explanation in the case presented here. success has been reported with weekly observational administration of thyroxine.3 specifically, grebe et al demonstrated through their study that once - weekly dosing was an effective means of maintaining tsh levels and thyroid hormone levels within relatively normal ranges, and did not appear to place the patient at risk of toxic complications associated with high doses of thyroxine.28 kubota et al explain that with this type of arrangement, the patient maintains their status as a patient, the hypothyroid condition is treated, and the risks of serious complications are avoided.3 a number of studies have shown that both iron and calcium supplements can potentially decrease the absorption of thyroxine through the formation of a nonabsorbable complex in the gut.9,10 given the increased incidence of hypothyroidism in the elderly population and the association of both osteoporosis and anemia with this condition, it is important to consider the interaction of these supplements with thyroxine as a potential cause for treatment failure. to eliminate this interaction and to maximize the benefit of each treatment, other studies have shown that gastrointestinal disorders and anatomical gastrointestinal abnormalities, such as untreated celiac disease and short - gut syndrome, may affect the absorption of thyroxine.11,12 celiac disease is an important consideration in this group of patients, considering the autoimmune association between these two disorders and the frequency in which they occur in patients simultaneously. in terms of patients with celiac disease, normalization of thyroid hormone levels and tsh have been observed once a gluten - free diet has been implemented.11 normal absorption of thyroxine is also known to be affected by changes in the acidic environment of the stomach. specifically, it has been shown that patients with impaired gastric acid secretion, whether secondary to helicobacter pylori infection or atrophic gastritis, required higher doses of thyroxine - replacement therapy to become euthyroid.13 similar but reversible findings were also seen in patients treated with omeprazole, a proton - pump inhibitor used to suppress acid secretion.13 other common medications to consider in this group of patients are estrogen - replacement therapy and the selective estrogen receptor modulator raloxifene. arafah showed that a group of 18 postmenopausal women treated with thyroxine - replacement therapy demonstrated increases in serum thyroxine and thyroxine - binding globulin (tbg) concentrations while receiving estrogen treatment.14 however, patients with a diagnosis of hypothyroidism had decreased ft4 levels (22 5 pmol / l to 18 4 pmol / l) and increased serum thyrotropin levels (0.9 1.1 to 3.2 3.1). the coadministration of raloxifene and levothyroxine has also ed in increased dosing requirements for thyroxine.15 it is not clear whether the increased dosing of thyroid - replacement treatment is related to an absorption problem or related to a change in the tbg level. garwood et al report a case where separation of these medications by 12 hours enabled their patient to reduce her thyroxine dosage, suggesting that it may be related to absorption, although the mechanism is unclear.15 in younger female patients, pregnancy should also be considered as a potential cause for increasing tsh levels and the need for an increase in thyroxine dosing, particularly in previously stable patients. similar to patients on estrogen - replacement therapy, the increased thyroid replacement dosing is attributed to an increase in tbg, ing in decreased levels of ft4 and ft3 and subsequent rises in tsh levels.16 the increased thyroxine dosage requirements have been estimated at approximately 45% higher than their regular dose of thyroxine.16 once each of the various factors discussed above has been thoroughly investigated and eliminated, pseudomalabsorption of thyroxine should be considered in patients failing to achieve normal tsh despite high levels of thyroxine. this diagnosis and normal intestinal absorption can be confirmed through a supervised loading doses of up to 1 mg of oral thyroxine.5 it is often recommended that this be completed when patients are fasting, as uptake of thyroxine has been found to be greater in a fasting state.2 patients in these situations should respond with a rapid lowering of their tsh levels and an increase in ft4 levels. it has been reported that most levothyroxine absorption takes place within the first few hours of administration and that serum ft4 levels peak within 2 hours of administration.26 patients such as the one described in this case report are often emphatic about their compliance with the prescribed treatment. the obvious consequence of untreated hypothyroidism is the persistence of the common symptoms of fatigue, weight gain, constipation, and poor concentration, and the morbidity associated with each of these. however, there are more serious and potentially life - threatening consequences of hypothyroidism if left untreated. specifically, myxedema coma is a rare but serious medical emergency and definitely the most severe complication of decompensated hypothyroidism. it is often the of coexisting medical conditions or drugs, and has been found to have a mortality rate of approximately 80% in untreated patients.1 another rare and potentially severe complication of untreated hypothyroidism is the development of a pituitary hyperplasia and hyperprolactinemia, as was seen in our patient. although there have been no documented cases where specifically the intentional noncompliance with thyroxine treatment has led to the development of pituitary hyperplasia, it is well established that one of the potential causes for hyperprolactinemia and pituitary hyperplasia is hypothyroidism.26,27 in these situations, the increase in pituitary size and elevated prolactin secretion are felt to be secondary to increased synthesis of thyroid releasing hormone (trh) in response to chronically low thyroid hormone levels. the development of pituitary hyperplasia in itself has the potential for quite severe complications, including headaches, neurological deficits, and visual loss. prolonged hyperprolactinemia may lead to infertility, hypopituitarism, and low bone density.28 although hypothyroidism is clearly not the only cause of a pituitary hyperplasia and elevated prolactin levels, resolution of these findings with appropriate treatment of the hypothyroidism suggests that this is the most likely explanation in the case presented here. success has been reported with weekly observational administration of thyroxine.3 specifically, grebe et al demonstrated through their study that once - weekly dosing was an effective means of maintaining tsh levels and thyroid hormone levels within relatively normal ranges, and did not appear to place the patient at risk of toxic complications associated with high doses of thyroxine.28 kubota et al explain that with this type of arrangement, the patient maintains their status as a patient, the hypothyroid condition is treated, and the risks of serious complications are avoided.3 this case highlights the serious complication of pituitary hyperplasia that may occur with prolonged and untreated hypothyroidism in the context of pseudomalabsorption of levothyroxine. recognizing pituitary hyperplasia and hyperprolactinemia as a potential complication of this disorder may also prevent the potential misdiagnosis of a prolactinoma leading to unnecessary investigations and inappropriate treatment. supervised loading of levothyroxine can help to confirm this disorder and avoid extensive and unnecessary investigations. also unique to this case report patient awareness of this serious complication and the rapid, demonstrable resolution with adequate thyroid hormone replacement may provide the motivation to comply with levothyroxine treatment.

objectivethe pseudomalabsorption of thyroxine has been used to describe patients with hypothyroidism who fail to comply with their treatment. we describe a unique case of a 32-year - old with hypothyroidism who developed pituitary hyperplasia and hyperprolactinemia secondary to the pseudomalabsorption of thyroxine.investigations and treatmentafter baseline thyroid - function tests were performed, the patient was administered levothyroxine 0.5 mg under the supervision of a registered nurse. thyroid function testing was repeated at 30, 60, 120, and 180 minutes. arrangements were made for further daily supervised loading of levothyroxine 0.1 mg.with the administration of 0.5 mg levothyroxine, free thyroxine levels increased by 120 minutes, and with daily supervised dosing of 0.1 mg there was normalization of the thyroid hormone levels and a reduction of thyroid - stimulating hormone levels. maintenance of thyroid - stimulating hormone < 15 mu / l for 2 weeks led to a reduction in prolactin levels and regression in the size of the pituitary on magnetic resonance imaging.if left untreated, these patients face significant morbidity and are at risk of developing pituitary hyperplasia, complications from an increase in pituitary size, hyperprolactinemia, and potentially myxedema coma. recognizing pituitary hyperplasia and hyperprolactinemia as a complication from the pseudomalabsorption of levothyroxine may prevent the potential of a misdiagnosis of a prolactinoma leading to unnecessary investigations and inappropriate treatment. patient awareness of this serious complication and the rapid, demonstrable resolution with adequate thyroid hormone replacement may provide motivation to comply with supervised dosing of levothyroxine. it has also been suggested that supervised treatment enables the individual to maintain their patient status, which may be in part the motivation behind this disorder.

for instance, exhaustive search becomes unreasonable as the number of variables increases; employing a multiple regression search produces over one billion possible models for data with 30 explanatory variables. in ecological studies, one of the commonly used methods for selection is stepwise regression, with forward or backward variable selection algorithms. these methods have been criticized for lacking the ability to truly pick the best model for several reasons (boyce et al ., 1974 ; wilkinson, 1989). one problem is that the choice by which the variables enter the selection algorithm is not justified theoretically. in addition, the probabilities for the selection procedure are chosen arbitrarily, which may lead to a poorly selected model. since these methods employ local search, it is unlikely that the global maximum set of variables will be found (mantel, 1970 ; hocking, 1976, 1983 ; moses, 1986). we propose the use of genetic algorithms (gas) to determine the subset of variables with the highest goodness of fit for a multiple regression model. due to their global search capabilities, the ga based model building is not prone to the problems associated with local search method, hence is a wise choice for this procedure. we now explain the basics of gas briefly; a thorough one can be found in goldberg. genetic algorithms are a set of optimization techniques inspired by biological evolution, operating under natural selection. first developed by holland, they have grown in popularity because of the ability of the algorithm to perform well on many different types of problems. in a ga, possible solutions are coded using binary strings, which are called chromosomes. each chromosome has a fitness value associated with it based on how well the string model parameters predicts the dependent variables. during each generation, which is the time step of the algorithm, a population of chromosomes compete to have their genes the selection step is used to pick the chromosomes for the next generation based on their fitness. those selected enter the mating pool, where two chromosomes mate using crossover. during this phase, parts of each parent string are swapped to form two new chromosomes that have certain aspects of their parents. mutation occurs with a small probability and is defined by a change from 0 to 1 or 1 to 0 in the binary string. mutation allows the introduction of new genes that were either lost from the population or were not there to start with. through successive generations, increasingly better chromosomes come to dominate the population, and the optimal solution (or something very close) is realized. a key component of a ga is the method to evaluate the fitness of a chromosome. thus, in order to use a ga for model selection in multiple regression, a way to evaluate the chromosomes is needed. more specifically the fittest chromosome is the set of parameters that maximizes the explanatory power of the model with minimum number of parameters. bozdogan considered complexity as a measure of fitness, which can be described as follows: the complexity of a system (of any type) is a measure of the degree of interdependency between the whole system and a simple enumerative composition of its subsystems or parts. the concept of information complexity was first introduced by akaike as a measure of the complexity of a model: it is a relative measure of the information lost when a given model is used, and can be described as a function of the precision and accuracy of the model. the expression for aic is given as where l(k) denotes the maximum likelihood function, ^k is the maximum likelihood estimate of parameter vector k, and m(k) is the number of parameters in the model. the first term of aic gives the lack of fit of the model, and the second term is a penalty for the number of parameters in the model. the model with the lowest aic value is considered the best, because the model successfully determines the underlying stochastic process with the least number of parameters. although aic does take into account the problem of over - fitting, where other measures such as r - square do not, aic is not sensitive to parameter dependency, which is an important component for model selection. if a model with both low variance and low covariance can be produced, then the parameters can be better estimated, as they will not be correlated. as an alternative to aic, we consider icomp as a complexity measure which considers variance and covariance, and accounts for the problem of over - fitting the model. it is calculated by where l(k) again denotes the maximum likelihood function, ^k is the maximum likelihood estimate of parameter vector k under the model mk, c is a real - valued complexity measure, and ^model is the estimated covariance matrix of the parameters of the model. the main difference between the two measures of complexity is that aic only considers the number of parameters as a penalty, whereas icomp considers the covariance between parameters. in predictive model building, this value for icomp is based on the inverse - fisher information matrix (ifim). for multiple regression , the value of icomp can be directly calculated after regression is implemented, and is given by n is the number of parameters in the model, q is the number of observations, ^2=sse / n, tr(^2(xx)1 ) is the trace of the observation matrix multiplied by its inverse and then scaled by ^2, and |^2(xx)1| is the determinant of the previous matrix. since the model with the lowest icomp value is considered the best, the ga chooses strings biased toward those with the lowest value. a commonly used method to form the mating pool is proportional selection, which depends on selecting strings for the mating pool with a probability proportional to their fitnesses. in proportional selection, the first step of the calculation of the fitness values is subtracting the icomp value of each string in that generation from the maximum value of icomp in the population. that is, for each i = 1,2, ,n, where n is the size of the population. then the average icomp difference (the average fitness) for the total population is calculated as finally, each string is given a fitness value that is the ratio of its icomp difference and the average fitness of the population: here we consider the implementation of ga's for predictive model selection and discuss possible improvements. the first step to implementing a ga for any optimization problem is to encode the input variable into binary strings. in the case of multiple linear regression we wish to fit the data to where y is an n 1 response vector, x is an n q matrix of the data points, is a q 1 coefficient matrix, and is an n 1 error vector with entries from independent normal distributions. the encoding is done by creating a binary string which has n + 1 bits, where each bit represents a different parameter of the model and an intercept. the last n bits correspond to the n explanatory variables contained in the dataset, whereas the first bit is the intercept for the linear model. a parameter is included in the model if the value of the bit for that parameter is a 1 and is excluded if it is a 0. for example, suppose we have a dataset where we are interested in predicting the reproductive fitness of a species of trees. the possible explanatory variables may include: density of trees in the surrounding area, average temperature of environment, average rainfall of environment, circumference of trunk, longitude of environment, latitude of environment, prevalence of disease in environment. in this case for example, the string 10010111101 would represent a model which includes the intercept, soil ph, average temperature of environment, average rainfall of environment, circumference of trunk, longitude of environment, and prevalence of disease in environment. similarly, the string 00001000110 is a model that has no intercept, and includes density of trees in the surrounding area, longitude of environment, and latitude of environment (see table 1). the probability that a string will be chosen for the mating pool is proportional to its fitness value. note that the string with the worst icomp value will never be picked for the mating pool, as its fitness will be 0. now that we have a method of encoding information and a method to evaluate the fitness values, we have to determine the remaining parameters of the ga. the first one we consider is the method of creating the initial population and determining its size. unless previous knowledge about the problem is given, it is commonplace in gas to randomly generate binary strings (goldberg, 1989). however, in the case of model selection, a user may want to force a parameter(s) to be included, even if it is not part of the model with the lowest complexity. in this case , the initial population can be generated in such a way that certain parameters are always in the model. in addition to determining the method to generate the population, the user must choose the size of the initial population. generally the size should not be too large, as this will slow the algorithm, and should not be so small that genetic drift takes over the course of evolution of the population. in typical gas, the size of the population stays the same; however, this may not be an effective use of computation. we will see in the next section that starting with a larger size then reducing it may be more effective. finally, we discuss the genetic operators which allow the algorithm to find the optimal model. first, the probability of crossover, pc, is chosen. in the mating pool, a pair of strings are chosen along with a random number from. if that number is less than the probability of crossover, crossover occurs. thus, if pc = 1, then every pair will cross, and if pc = 0, then no strings will be altered by crossover. after the choice of pc, the number of crossover points must be chosen. then the bits from the parent strings are swapped to create two new offspring strings (see figure 1). the purpose of crossover is to bring together models which have components that reduce complexity. recall the previous example about trees, where we specified two strings, which we will call parent 1 and parent 2. applying crossover to the two parents creates two offspring (see figure 1), where offspring 1 represents a model with an intercept, soil ph, average temperature of environment, longitude of environment, latitude of environment, and prevalence of disease in environment, and offspring 2 represents a model that includes density of trees in the surrounding area, average rainfall of environment, circumference of trunk, and longitude of environment. through successive generations and application of crossover of low complexity models, the algorithm is able to find the least complex model (or something close to it) to explain the data. but, what happens if the actual least complex model includes a parameter that is not present in the population, that is, the position in the string that represents the parameter is fixed at 0? this value gives the probability that at each location in the string the bit will be flipped. flipping is defined as a change of a 0 to 1 or a 1 to a 0. however, strings used for other applications of ga's are usually longer than the ones used for determining least complex models. although there are ongoing studies on determining optimal crossover and mutation rates (such as nested gas, self - adjusting parameterless gas), these rates can be determined by trial and error or by pilot runs before the actual data set has been used to build a model. we conclude this section with a pseudo code for a ga used to find the least complex model that sufficiently describes the data. generate initial population while (t < max generations or the maximum number of computations have not been executed) (a)calculate icomp for the model each string encodes (b)select strings for the mating pool (c)create a new population using crossover (d)mutate new population while the use of a typical ga for model selection already proves to be more efficient than stepwise regression, with a few modifications, the process can show a 10-fold increase in accuracy given the same amount of computation. first, we discuss the modifications, and then we explain the study done to determine the effectiveness of these modifications. the first modification is changing how the initial population was created. according to fisher's fundamental theorem of natural selection (fisher, 1930), the increase in mean fitness is equal to the variance in fitness. for model selection using gas, the easiest way to increase variance in fitness would be to allow every model to be represented in the population. of course, this is impossible for a model with a large number of possible explanatory variables, and would amount to doing an exhaustive search. we believe that the next best procedure is to force the population to start with the highest variance in each position of the chromosome. since each position is either a 0 or 1, this would imply that at each position there are the same amount of 0 s and 1 s across the entire population. to implement this procedure, the other half is generated by taking each of the chromosomes in the first half and changing each bit from 1 to 0 or 0 to 1. in addition to increasing variance at each position, this procedure guarantees that within one generation, recombination alone could generate the best model. this does not imply that mutation is not necessary, as selection acts on the entire string, not individual positions. since selection will reduce variance at each position, mutation is still required to maintain some variance. the second modification is starting with a larger initial population and then reducing it in size. we have used a reduction method that adapts to the changes in the algorithm in this study. adaptively reducing the population is done by calculating the change in the best fitness between two consecutive generations and then reducing the population based on this change. more specifically, the population is reduced by the percentage increase in best fitness up to some limit. clearly, there must be a limit to the percentage of reduction, since the population should not be reduced too much, and also because the percent change can be more than 100. here the amount of population reduction depends on the complexity of the problem, that is the type of the fitness function (such as mse, aic, icom, mallow 's cp and so on) used. this limit on the reduction may be determined by pilot studies. the percent change in fitness at generation t the change is calculated by the formula ftbest=|ft1bestft2best|/|ft2best|. the population size n(t) at each generation is given by the recursive relation when using the adaptive method, elitism was also implemented. elitism is a procedure commonly used in gas in order to pass the best chromosome, or a group of the best chromosomes, to the next generation without any modifications. using elitism guarantees that the change in best fitness is always non - negative. as a , since we wished to minimize icomp, we set the fitness of each chromosome to be the negative of the icomp value. the choices of these parameters should be done by considering characteristics of the problem such as the expected increase in fitness over time. this is typically a difficult characteristic to determine. generally, as the number of variables increase, the value of fmaxbest should decrease. as the number of variables increases, so does the number of possible values of icomp, and the likelihood that the population will evolve slower. the value of min_popsize should be chosen so that it is quite small, regardless of the number of variables. as a side note, the ga with no population reduction is a special case of the adaptive method where fmaxbest=0. the final modification to a ga for multiple regressions is the use of binary tournament instead of proportional selection. in this selection scheme, two chromosomes are chosen at random, and the one with the lower icomp value is selected for the mating pool. then both chromosomes are put back into the pool of contestants of the tournament. one advantage of this technique is that icomp values need only be calculated for the chromosomes that participate in the tournament. for models with few explanatory variables, this gain in computation may be negligible. on the other hand, for those models with many variables, the reduction in computation means that more generations can be used, or the initial population can be larger. when the population is being reduced, genetic drift may be amplified, since the sampling space for the next generation decreases. proportional selection may increase this effect because a few chromosomes with extremely high fitness are expected to be picked often for the mating pool. however, selection to participate in the tournament is random, avoiding the over - selection of chromosomes with extremely large fitness values. to test the benefits of these modifications , we used the data set in bozdogan where the predictive model is constructed for body fat and 13 explanatory variables. in order to determine how well the ga was performing, all subsets of the variables (2 1 = 16,383 subsets) were used to generate a model, and then the icomp value was determined. testing was done to ensure the same icomp values were being generated for the matlab and java code. these cases differed in the value of fmaxbest, and as a in the initial population size. all trials were allowed 600 computations, where a computation is the total number of chromosomes summed over every generation. each different ga scheme ran through 200 trials and the number of times the correct model was selected was recorded. parameters that were the same for all genetic algorithm schemes. the frequency of the correct model being selected over 200 trials. the first step to implementing a ga for any optimization problem is to encode the input variable into binary strings. in the case of multiple linear regression we wish to fit the data to where y is an n 1 response vector, x is an n q matrix of the data points, is a q 1 coefficient matrix, and is an n 1 error vector with entries from independent normal distributions. the encoding is done by creating a binary string which has n + 1 bits, where each bit represents a different parameter of the model and an intercept. the last n bits correspond to the n explanatory variables contained in the dataset, whereas the first bit is the intercept for the linear model. a parameter is included in the model if the value of the bit for that parameter is a 1 and is excluded if it is a 0. for example, suppose we have a dataset where we are interested in predicting the reproductive fitness of a species of trees. the possible explanatory variables may include: density of trees in the surrounding area, average temperature of environment, average rainfall of environment, circumference of trunk, longitude of environment, latitude of environment, prevalence of disease in environment. in this case for example, the string 10010111101 would represent a model which includes the intercept, soil ph, average temperature of environment, average rainfall of environment, circumference of trunk, longitude of environment, and prevalence of disease in environment. similarly, the string 00001000110 is a model that has no intercept, and includes density of trees in the surrounding area, longitude of environment, and latitude of environment (see table 1). the probability that a string will be chosen for the mating pool is proportional to its fitness value. note that the string with the worst icomp value will never be picked for the mating pool, as its fitness will be 0. now that we have a method of encoding information and a method to evaluate the fitness values, we have to determine the remaining parameters of the ga. the first one we consider is the method of creating the initial population and determining its size. unless previous knowledge about the problem is given, it is commonplace in gas to randomly generate binary strings (goldberg, 1989). however, in the case of model selection, a user may want to force a parameter(s) to be included, even if it is not part of the model with the lowest complexity. in this case , the initial population can be generated in such a way that certain parameters are always in the model. in addition to determining the method to generate the population, the user must choose the size of the initial population. generally the size should not be too large, as this will slow the algorithm, and should not be so small that genetic drift takes over the course of evolution of the population. in typical gas , the size of the population stays the same; however, this may not be an effective use of computation. we will see in the next section that starting with a larger size then reducing it may be more effective. finally, we discuss the genetic operators which allow the algorithm to find the optimal model. first, the probability of crossover, pc, is chosen. in the mating pool, a pair of strings are chosen along with a random number from. thus, if pc = 1, then every pair will cross, and if pc = 0, then no strings will be altered by crossover. after the choice of pc, the number of crossover points must be chosen. then the bits from the parent strings are swapped to create two new offspring strings (see figure 1). the purpose of crossover is to bring together models which have components that reduce complexity. recall the previous example about trees, where we specified two strings, which we will call parent 1 and parent 2. applying crossover to the two parents creates two offspring (see figure 1), where offspring 1 represents a model with an intercept, soil ph, average temperature of environment, longitude of environment, latitude of environment, and prevalence of disease in environment, and offspring 2 represents a model that includes density of trees in the surrounding area, average rainfall of environment, circumference of trunk, and longitude of environment. through successive generations and application of crossover of low complexity models, the algorithm is able to find the least complex model (or something close to it) to explain the data. but, what happens if the actual least complex model includes a parameter that is not present in the population, that is, the position in the string that represents the parameter is fixed at 0? this value gives the probability that at each location in the string the bit will be flipped. flipping is defined as a change of a 0 to 1 or a 1 to a 0. however, strings used for other applications of ga's are usually longer than the ones used for determining least complex models. although there are ongoing studies on determining optimal crossover and mutation rates (such as nested gas, self - adjusting parameterless gas), these rates can be determined by trial and error or by pilot runs before the actual data set has been used to build a model. we conclude this section with a pseudo code for a ga used to find the least complex model that sufficiently describes the data. generate initial population while (t < max generations or the maximum number of computations have not been executed) (a)calculate icomp for the model each string encodes (b)select strings for the mating pool (c)create a new population using crossover (d)mutate new population while the use of a typical ga for model selection already proves to be more efficient than stepwise regression, with a few modifications, the process can show a 10-fold increase in accuracy given the same amount of computation. first, we discuss the modifications, and then we explain the study done to determine the effectiveness of these modifications. the first modification is changing how the initial population was created. according to fisher's fundamental theorem of natural selection (fisher, 1930), the increase in mean fitness is equal to the variance in fitness. for model selection using gas, the easiest way to increase variance in fitness would be to allow every model to be represented in the population. of course, this is impossible for a model with a large number of possible explanatory variables, and would amount to doing an exhaustive search. we believe that the next best procedure is to force the population to start with the highest variance in each position of the chromosome. since each position is either a 0 or 1, this would imply that at each position there are the same amount of 0 s and 1 s across the entire population. to implement this procedure, the other half is generated by taking each of the chromosomes in the first half and changing each bit from 1 to 0 or 0 to 1. in addition to increasing variance at each position, this procedure guarantees that within one generation, recombination alone could generate the best model. this does not imply that mutation is not necessary, as selection acts on the entire string, not individual positions. since selection will reduce variance at each position, mutation is still required to maintain some variance. the second modification is starting with a larger initial population and then reducing it in size. we have used a reduction method that adapts to the changes in the algorithm in this study. adaptively reducing the population is done by calculating the change in the best fitness between two consecutive generations and then reducing the population based on this change. more specifically, the population is reduced by the percentage increase in best fitness up to some limit. clearly, there must be a limit to the percentage of reduction, since the population should not be reduced too much, and also because the percent change can be more than 100. here the amount of population reduction depends on the complexity of the problem, that is the type of the fitness function (such as mse, aic, icom, mallow 's cp and so on) used. this limit on the reduction may be determined by pilot studies. the percent change in fitness at generation t is denoted by ftbest and the limit is denoted by fmaxbest. the population size n(t) at each generation is given by the recursive relation when using the adaptive method, elitism was also implemented. elitism is a procedure commonly used in gas in order to pass the best chromosome, or a group of the best chromosomes, to the next generation without any modifications. using elitism guarantees that the change in best fitness is always non - negative. as a , the population never increases in size. since we wished to minimize icomp, we set the fitness of each chromosome to be the negative of the icomp value the choices of these parameters should be done by considering characteristics of the problem such as the expected increase in fitness over time. this is typically a difficult characteristic to determine. generally, as the number of variables increase, the value of fmaxbest should decrease. as the number of variables increases, so does the number of possible values of icomp, and the likelihood that the population will evolve slower. the value of min_popsize should be chosen so that it is quite small, regardless of the number of variables. as a side note, the ga with no population reduction is a special case of the adaptive method where fmaxbest=0. the final modification to a ga for multiple regressions is the use of binary tournament instead of proportional selection. in this selection scheme, two chromosomes are chosen at random, and the one with the lower icomp value is selected for the mating pool. then both chromosomes are put back into the pool of contestants of the tournament. one advantage of this technique is that icomp values need only be calculated for the chromosomes that participate in the tournament. for models with few explanatory variables, this gain in computation may be negligible. on the other hand, for those models with many variables, the reduction in computation means that more generations can be used, or the initial population can be larger. when the population is being reduced, genetic drift may be amplified, since the sampling space for the next generation decreases. proportional selection may increase this effect because a few chromosomes with extremely high fitness are expected to be picked often for the mating pool. however, selection to participate in the tournament is random, avoiding the over - selection of chromosomes with extremely large fitness values. to test the benefits of these modifications , we used the data set in bozdogan where the predictive model is constructed for body fat and 13 explanatory variables. in order to determine how well the ga was performing, all subsets of the variables (2 1 = 16,383 subsets) were used to generate a model, and then the icomp value was determined. testing was done to ensure the same icomp values were being generated for the matlab and java code. these cases differed in the value of fmaxbest, and as a in the initial population size. all trials were allowed 600 computations, where a computation is the total number of chromosomes summed over every generation. each different ga scheme ran through 200 trials and the number of times the correct model was selected was recorded. parameters that were the same for all genetic algorithm schemes. the frequency of the correct model being selected over 200 trials. while model selection remains to be a difficult procedure in case of a large number of parameters, using a ga to find the least complex model can be quite helpful. we have shown that our modifications to the original ga for model selection can yield strong . additionally, the ga approach (because of the use of icomp) is better at handling data in which collinearity exist than the traditional selection methods such as forward, backward, and stepwise selection. in particular it is clear that the modifications had a large effect on the accuracy of the ga. this seems to indicate that we may reduce computation and still get statistically the same accuracy if we employ diversification. in all trials, along with the facts presented above and the fact that diversification is easy (and not costly) to implement, it is our recommendation that it be used for model selection using gas. the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

we implement genetic algorithm based predictive model building as an alternative to the traditional stepwise regression. we then employ the information complexity measure (icomp) as a measure of model fitness instead of the commonly used measure of r - square. furthermore, we propose some modifications to the genetic algorithm to increase the overall efficiency.

monilethrix is an autosomal dominant disorder of hair shaft with regular thinning of hair shaft with fracture of the hair shaft at constricted points, which is commonly caused due to human hair keratin hhb6 gene located on long arm of chromosome 12. patients have short and fragile hair not requiring haircut since childhood commonly over the occipital and temporal scalp, but may also involve eyebrows, eyelashes, axillary, pubic or body hair. holt - oram syndrome is a syndrome characterized by limb defects associated with cardiovascular anomalies. it is an autosomal dominant disorder caused due to mutations of tbx5 gene located on a long arm of chromosome 12. the cardiovascular defects are usually in the form of septal defects commonly atrial septal defect. limb defects range from a hypoplastic thumb, metacarpals, carpals, radius to an absent limb. a 2-month - old female child, first born of second degree consanguineous marriage was brought with complaints of absence of hair over the scalp, eyebrows and body since birth. examination revealed absence of hair from the scalp, eyebrows and body with presence of hair over eyelashes. other examination findings included a hypoplastic thumb on the right side with normal nails. clinical photograph showing absence of hair over scalp and eyebrows clinical photograph showing hypoplastic thumb dermoscopic examination revealed presence of numerous empty follicles, small broken hairs, black dots and hair with uniform nodal dilatations with intermittent constrictions at which there was shaft breakage. dermoscopy of scalp showing hairs with monilethrix microscopic hair shaft examination could not be carried out as hair were too short for plucking. echocardiography showing atrial septal defect thus, a diagnosis of holt - oram syndrome with monilethrix was arrived at. it can be transmitted as an isolated condition, which has an autosomal dominant inheritance. it can also be associated with papular atrichia, keratosis pilaris, vitamin d resistant rickets, ectodermal dysplasia, moynahan's syndrome, hypotrichosis simplex, marie - unna hereditary hypotrichosis, pseudo - thalidomide syndrome, amino acid metabolism and hair shaft disorders. the common hair shaft disorders, which may present as hypotrichosis include monilethrix, pili torti and pili annulati. monilethrix (omim # 158000) is an uncommon hair shaft defect, which can manifest as congenital hypotrichosis. monilethtix is associated with trichorrhexis nodosa, nail and teeth defects, retarded growth and juvenile cataracts. the conditions where a hypoplastic thumb can be associated with atrial septal defect include holt - oram syndrome and vacterl association. holt - oram syndrome (omim # 142900) is associated with limb defects such as hypoplastic thumb, metacarpals, carpals or radius defects and cardiovascular anomalies especially septal defects. vacterl association includes vertebral anomalies, anal atresia, cardiovascular anomalies, especially septal defects, trachea - esophageal fistula, renal defects and limb defects like hypoplastic thumb, syndactyly, polydactyly and forearm bony defects like radial aplasia. the presentation of monilethrix as congenital hypotrichosis is not very common. in this patient, the association of holt - oram syndrome with monilethrix, in spite of the causative genetic mutations being on the same chromosome, has not been reported to the best of our knowledge.

congenital hypotrichosis may be due to a number of causes and may have multiple systemic associations. a child born of second - degree consanguineous marriage was found to have monilethrix as the cause of congenital hypotrichosis. a detailed systemic evaluation in the child revealed atrial septal defect and a hypoplastic right thumb leading to a diagnosis of coexisting holt - oram syndrome.

tobacco smoking is the most attributable and preventable risk factor for adult mortality and morbidity in japan.1, 2 tobacco smoking has been confirmed as an independent risk factor for many disorders, such as cancer and cardiovascular diseases, with dose - response verifications.3, 4 heavy smokers are more likely to suffer from tobacco - related harm than light smokers. smoking cessation and tackling regional and socioeconomic inequalities in smoking are key public health targets throughout the world. the world health organization (who)'s commission on social determinants of health recommended, in its final report, monitoring and evaluating socioeconomic inequalities in health and health behavior, including smoking. japan's new health promotion strategy, health japan 21 (second term), follows the who recommendations and includes monitoring socioeconomic inequalities in tobacco smoking and other public health targets. when monitoring socioeconomic inequality in smoking, using educational attainment as a socioeconomic indicator is important. educational attainment is a representative socioeconomic factor that barely changes in adulthood after around 25 years of age, whereas other socioeconomic variables, such as income and occupation, could change considerably during a life - course. moreover, educational attainment could reflect key determinants of the initiation and habituation of smoking and other health behaviors, such as health literacy. although some japanese studies have investigated smoking inequalities according to socio - economic factors, such as income and occupation,9, 10 no study has examined smoking according to education in japan. recent european studies have consistently shown higher prevalence of tobacco smoking among poorly educated populations, whereas patterns of smoking prevalence in terms of sex and age groups vary across regions. although there is relatively rich evidence from western countries, data from asian regions are scarce. moreover, data on educational inequality in smoking in older persons, especially those aged 75 years or more, are scarce even worldwide.3, 11 to start continuous monitoring of educational inequality in health, in 2010, the comprehensive survey of living conditions of people on health and welfare (cslc), a large nationally representative population - based cross - sectional survey in japan, collected information on education, in addition to health behavior indicators, including smoking status. thus, our objective in this study was to investigate the magnitude of educational inequality in smoking and the prevalence of current and heavy smoking according to sex, age, and education among japanese adults. utilizing the large nationally representative dataset, we sought to provide detailed evaluations of age- and sex - specific variations in smoking inequality in japan, covering the whole range of adult age groups from 25 to 94 years old. we used data from a nationally representative cross - sectional survey: the 2010 cslc, conducted by the japanese ministry of health, labour and welfare (mhlw). out of 940,000 inhabited census tracts (the sampling unit for national census in 2005), 5510 were randomly sampled across japan in 2010 for the collection of data from all household members within each census tract. data were used with permission from mhlw. the study was reviewed and approved by the research ethics committee of the osaka medical center for cancer and cardiovascular diseases. levels of completed education were categorized as six groups: junior high school (9 years of mandatory education) was defined as persons who graduated junior high school without graduating high school; high school (12 years of education) was defined as persons who graduated high school without graduating further educational steps; technical school (1019 years of education) 2-year college (14 years of education) was defined as persons who graduated 2-year college without going to 4-year college; 4-year university (16 years of education) was defined as persons who graduated 4-year university without going to graduate school; graduate school (1722 years of education) was defined as persons who graduated graduate school having previously graduated 4-year university. current smokers were those who smoked cigarettes regularly at the time of survey, including daily and sometimes smokers. among daily smokers (93.8% of the current smokers in the data), heavy smokers were those who smoked more than 20 cigarettes per day. we analyzed japanese adults aged 2594 years because education status was less likely to change after 25 years of age. we compared current smoking prevalence and heavy smoking proportions among daily smokers according to sex, age, and education group. the percentages are shown with 95% confidence intervals (cis) calculated by the wald method. to show the summarized relationship between education and smoking, the age - adjusted smoking prevalence for young and middle - aged adults (2564 years) was also calculated via the direct standardization method using population figures from the 2010 japanese census. following recent recommendations, to evaluate educational inequality in current and heavy smokers, we calculated multiple health disparity indicators, including absolute indicators (rate difference and between - group variance) and relative indicators (rate ratio, index of disparity, and mean log deviation),15, 16, 17 using hd*calc software, version 1.2.4 (the national cancer institute, rockville, md, usa). detailed explanations of these indicators are given in the supplementary data (eappendix 1) and elsewhere.19, 20 population weight was used to calculate measures of inequality, because the population size differed according to the education categories, reflecting educational distributions. the proportion of highly educated people in the general population has been increasing over time. such a demographic shift has an impact on population health and needs to be considered for the assessment of inequalities. inequality measures of index of disparity, mean log deviation, and between - group variance accounted for the population size of the groups in the calculation. subject numbers according to sex, age, and education group are shown in etable 1 (for current smoking prevalence) and etable 2 (for heavy smoking prevalence among daily smokers). to maintain precision of estimates, we did not calculate the smoking prevalence (proportion) of the groups that included fewer than 100 subjects. although there are no convincing criteria for the sample size cut - off point, we chose a sample size of 100 based on statistical considerations of the relationship between sample size and precision. according to machin et al , the width of a 95% ci depends on the size of the point estimate. however, a sample size of 100 can maintain at least 20% point width of the 95% ci regardless of the size of the point estimates. this choice of sample size cut - off ed in some groups for which we did not estimate smoking prevalence (proportion). we evaluated the educational inequality in smoking where smoking prevalence (proportion) data were available for at least three education groups. all statistical analyses, except for the inequality index calculation, were performed using sas version 9.3 (sas institute inc ., cary, nc, usa). we used data from a nationally representative cross - sectional survey: the 2010 cslc, conducted by the japanese ministry of health, labour and welfare (mhlw). out of 940,000 inhabited census tracts (the sampling unit for national census in 2005), 5510 were randomly sampled across japan in 2010 for the collection of data from all household members within each census tract. data were used with permission from mhlw. the study was reviewed and approved by the research ethics committee of the osaka medical center for cancer and cardiovascular diseases. levels of completed education were categorized as six groups: junior high school (9 years of mandatory education) was defined as persons who graduated junior high school without graduating high school; high school (12 years of education) was defined as persons who graduated high school without graduating further educational steps; technical school (1019 years of education) was defined as persons who graduated technical professional school without going to college; 2-year college (14 years of education) was defined as persons who graduated 2-year college without going to 4-year college; 4-year university (16 years of education) was defined as persons who graduated 4-year university without going to graduate school; graduate school (1722 years of education) current smokers were those who smoked cigarettes regularly at the time of survey, including daily and sometimes smokers. among daily smokers (93.8% of the current smokers in the data), we analyzed japanese adults aged 2594 years because education status was less likely to change after 25 years of age. we compared current smoking prevalence and heavy smoking proportions among daily smokers according to sex, age, and education group. the percentages are shown with 95% confidence intervals (cis) calculated by the wald method. to show the summarized relationship between education and smoking, the age - adjusted smoking prevalence for young and middle - aged adults (2564 years) was also calculated via the direct standardization method using population figures from the 2010 japanese census. following recent recommendations, to evaluate educational inequality in current and heavy smokers, we calculated multiple health disparity indicators, including absolute indicators (rate difference and between - group variance) and relative indicators (rate ratio, index of disparity, and mean log deviation),15, 16, 17 using hd*calc software, version 1.2.4 (the national cancer institute, rockville, md, usa). detailed explanations of these indicators are given in the supplementary data (eappendix 1) and elsewhere.19, 20 population weight was used to calculate measures of inequality, because the population size differed according to the education categories, reflecting educational distributions. the proportion of highly educated people in the general population has been increasing over time. such a demographic shift has an impact on population health and needs to be considered for the assessment of inequalities. inequality measures of index of disparity, mean log deviation, and between - group variance accounted for the population size of the groups in the calculation. subject numbers according to sex, age, and education group are shown in etable 1 (for current smoking prevalence) and etable 2 (for heavy smoking prevalence among daily smokers). to maintain precision of estimates , we did not calculate the smoking prevalence (proportion) of the groups that included fewer than 100 subjects. although there are no convincing criteria for the sample size cut - off point, we chose a sample size of 100 based on statistical considerations of the relationship between sample size and precision. according to machin et al , the width of a 95% ci depends on the size of the point estimate. however, a sample size of 100 can maintain at least 20% point width of the 95% ci regardless of the size of the point estimates. this choice of sample size cut - off ed in some groups for which we did not estimate smoking prevalence (proportion). we evaluated the educational inequality in smoking where smoking prevalence (proportion) data were available for at least three education groups. all statistical analyses, except for the inequality index calculation, were performed using sas version 9.3 (sas institute inc ., cary, nc, usa). table 1 shows the current smoking prevalence according to sex, age, and education group in japan. among men aged 2534 years , junior high school graduates had highest current smoking prevalence at 68.4% (95% ci, 66.0%70.6%), and graduate school graduates had the lowest at 19.4% (95% ci, 17.2%21.9%). these figures were lower in the higher age groups: among men aged 6574 years, the corresponding figures were 27.6% (95% ci, 26.7%28.6%) and 12.2% (95% ci, 7.9%17.7%) for junior high school graduates and graduate school graduates, respectively. among men aged 7594 years, the differences in current smoking across educational categories were small, though we did not calculate the smoking prevalence of graduate school graduates in that age group because of the small sample size. a similar but steeper educational gradient in current smoking was observed among women. among women aged 2534 years, junior high school graduates had the highest current smoking prevalence at 49.3% (95% ci, 46.3%52.3%), and graduate school graduates had the lowest at 4.8% (95% ci, 2.9%7.4%). age - adjusted rates of young and middle - aged adults (2564 years) also showed a similar gradient among both sexes. the inequality indicators for educational inequality in current smoking according to sex and age groups are shown in fig. 1 (corresponding values are shown in etable 3). compared with older age groups, such as 6594 years, younger age groups, such as 2554 years, had higher estimates of inequality indicators for educational inequality in smoking among both sexes, except for one outlier of the index of disparity for women aged 7584 years. table 2 shows the prevalence of heavy smoking (%) among daily smokers according to sex, age, and education groups. among both men and women aged 2544 years, prevalence of heavy smoking was highest among junior high school graduates; for example, among men aged 2534 years, 27.7% (95% ci, 25.0%30.5%) of daily smokers who were junior high school graduates were heavy smokers, while the prevalence was 13.6% (95% ci, 9.0%19.4%) among graduate school graduates. heavy smokers were most prevalent among daily smokers aged 5564 years in both men (33.4%) and women (12.1%). 2 shows the inequality indicators for educational inequalities in heavy smoking among daily smokers (corresponding values are shown in etable 4). compared with younger age groups, older age groups, especially those aged 5584 years for men and 4564 years for women, had lower educational inequality in heavy smoking. we found that educational inequalities in current and heavy smoking were apparent and large in the young population compared with older generations; these disparities were consistently shown using multiple indicators of inequality. the inequality in smoking among young women is strikingly large, calling for urgent political measures to address this disparity. among those aged 2544 years , junior high school graduates had considerably higher current and heavy smoking prevalence than other education groups: e.g., nearly half of young women who were junior high school graduates currently smoked (49.3%47.5%), a rate which is dramatically higher than other education groups. heavy smoking prevalences were the highest among those aged 5564 years in both men and women, but the educational inequalities in heavy smoking in that group were smaller than in other age groups. first, education may capture the social class of younger generations more sharply than older generations. as in most other developed countries, junior high school graduates are a minority in the younger generations in japan. they have a definite disadvantage in earning power, the job market, and having partners. the less educated among the young generations are likely to lose self - efficacy, self - stigmatize, suffer chronic psychosocial stress, and have a strong sense of relative deprivation, which may cause smoking.22, 23 among older generations, on the other hand, graduating only elementary school or junior high school was common. for example, being a junior high school graduate might not mean being disadvantaged for older age groups, especially those over 75 years old. second, the large educational gradient in smoking among the young may reflect inequality in the initiation of smoking. among men, when currently old people were young, smoking was a more common practice, and its health risks were less acknowledged. until the 1970s, nearly 80% of japanese men smoked.24, 25 although data on educational inequality in smoking in those periods are not available, it is plausible that highly educated people continued to smoke more in those periods than current years. third, the opportunities to quit smoking are fewer among younger and less educated people. although the present study indicated a snapshot of smoking in 2010, an individual's smoking trajectory is related to the accumulation of social disadvantage over the entire life - course.3, 26 less educated smokers may be more likely to fail at quitting and to become more addicted; thus, educational inequality in smoking among the young generation may be large. further, those who had few opportunities to quit smoking, such as partner's and/or social cessation supports, and smoking - related diseases onset (as an opportunity to quit) through their life course, might smoke currently when they became old. moreover, since mortality is higher among the lower socioeconomic group, older people in the low - education group may be in the selected population (survivors) who tend to be non - smokers / ex - smokers. multiple factors other than education throughout a life - course may be associated with smoking, especially in a geriatric population; therefore, educational inequality among the old population is small. the strength of this study is the provision of data with fine resolution in terms of age groups and educational attainments; previous studies on educational inequality in smoking prevalence have used only four or fewer categories for both educational attainments and age groups.3, 11 in the present study, the large sample size enabled us to analyze sex- and age - specific data on educational inequality in smoking using seven age groups encompassing participants 2594 years old and six educational levels from junior high school graduates to graduate school graduates. data on the geriatric population is especially valuable because such data are scarce worldwide.3, 11 our findings are consistent with studies from the western region of the world showing an inverse association between educational attainments and smoking prevalence. however, the age- and sex - specific trends have variations across countries or with existing data. the american surgeon general report in 2014 reported educational gradients in smoking using four education levels among adults aged 18 years or older: 31.5% of adults (36.2% of men and 26.5% of women) who had education of less than high school smoked currently, compared with 10.4% of college graduates (11.1% for men and 9.7% for women). in europe, an inverse association of education with smoking was also seen, especially among men in northern countries. for men, studies in ireland and the united kingdom showed inequalities in all generations; studies in finland, denmark, germany, belgium, and spain showed inequalities among middle - aged and younger men; and studies in other countries only showed inequalities among young adults. lag time in educational inequalities in smoking between northern and southern countries, and also a lag between women and men, were observed in the late 1990s to early 2000s in europe and interpreted as smoking epidemic patterns (within country): first, male smoking prevalence increases, and then female smoking prevalence increases. however, female smoking in most asian and african countries is uncommon. in the present study the study may contribute to a better understanding of socioeconomic patterns in the smoking epidemic in an asian region, because the female smoking pattern in asia is considered to be different from europe. first, the smoking variables were self - reported without biomarker validation; however, the quality of self - reported smoking has been noted to be high.27, 28 nevertheless, we could not exclude the possibility that reporting error occurred and that it may be differential according to education. second, this is a cross - sectional study, so we could not refer to causality between education and smoking, although both directions of causality were assumed. we compared calculated values of inequality indices between different age groups to evaluate the magnitude of educational inequality in smoking. because evaluable levels of education were different among heavy smokers according to age group, the evaluation of the inequality would in an underestimation among old population with fewer evaluable education levels (due to sample size). furthermore, graduate school graduates showed the lowest prevalence in most sex and age categories, and their sample size was relatively small, especially among the old and among women. because some inequality indicators were calculated using the lowest smoking prevalence with a wide ci, these indicators may be unstable. fourth, we need to explain the nature of inequality indicators; inequality indicators were calculated using the smoking prevalence sequence of highest to lowest across education categories. however, the educational rank sequence was not considered in the calculation of inequality indicators. the current study provided basic data on the educational inequalities in smoking among japanese adults, which contributes to the existing literature examining smoking inequalities according to other socio - economic positions, such as income and occupation.9, 10 educational inequalities in smoking, especially among young adults, have been observed in japan as well as the rest of the world.3, 11 in japan, health japan 21 (second term) asks for a reduction of health inequality, including smoking inequality. to achieve this goal, given the findings of this study several tobacco control measures, such as tobacco taxation and media campaigns, may possibly reduce smoking inequality due to socioeconomic factors and age,26, 29, 30, 31, 32 because the tobacco price is considered to be very low in japan according to the affordability index and anti - tobacco television media campaigns have not been conducted by the japanese government. we need to continue to monitor smoking prevalence and inequality and implement effective tobacco control measures to reduce smoking inequalities. first, the smoking variables were self - reported without biomarker validation; however, the quality of self - reported smoking has been noted to be high.27, 28 nevertheless, we could not exclude the possibility that reporting error occurred and that it may be differential according to education. second, this is a cross - sectional study, so we could not refer to causality between education and smoking, although both directions of causality were assumed. we compared calculated values of inequality indices between different age groups to evaluate the magnitude of educational inequality in smoking. because evaluable levels of education were different among heavy smokers according to age group, the evaluation of the inequality would in an underestimation among old population with fewer evaluable education levels (due to sample size). furthermore, graduate school graduates showed the lowest prevalence in most sex and age categories, and their sample size was relatively small, especially among the old and among women. because some inequality indicators were calculated using the lowest smoking prevalence with a wide ci fourth, we need to explain the nature of inequality indicators; inequality indicators were calculated using the smoking prevalence sequence of highest to lowest across education categories. however, the educational rank sequence was not considered in the calculation of inequality indicators. the current study provided basic data on the educational inequalities in smoking among japanese adults, which contributes to the existing literature examining smoking inequalities according to other socio - economic positions, such as income and occupation.9, 10 educational inequalities in smoking, especially among young adults, have been observed in japan as well as the rest of the world.3, 11 in japan, health japan 21 (second term) asks for a reduction of health inequality, including smoking inequality. to achieve this goal, given the findings of this study, tobacco control measures should focus more on younger generations with low educational attainment. several tobacco control measures, such as tobacco taxation and media campaigns, may possibly reduce smoking inequality due to socioeconomic factors and age,26, 29, 30, 31, 32 because the tobacco price is considered to be very low in japan according to the affordability index and anti - tobacco television media campaigns have not been conducted by the japanese government. we need to continue to monitor smoking prevalence and inequality and implement effective tobacco control measures to reduce smoking inequalities.

few studies have investigated differences in age- and gender - specific educational gradients in tobacco smoking among the whole range of adult age groups. we examined educational inequality in smoking among japanese adults aged 2594 years.methodsusing a large nationally representative sample (167,925 men and 186,588 women) in 2010, prevalence of current smoking and heavy smoking among daily smokers and their inequalities attributable to educational attainment were analyzed according to sex and age groups.among men aged 2534 years, junior high school graduates had the highest current smoking prevalence at 68.4% (95% confidence interval , 66.0%70.6%), and graduate school graduates had the lowest at 19.4% (95% ci, 17.2%21.9%). high school graduates had the second highest current smoking prevalence (e.g., 55.9% ; 95% ci, 54.9%56.8% in men aged 2534 years). among men aged 7594 years , the difference in current smoking across educational categories was small. a similar but steeper educational gradient in current smoking was observed among women. among women aged 2534 years , junior high school graduates had the highest current smoking prevalence at 49.3% (95% ci, 46.3%52.3%), and graduate school graduates had the lowest at 4.8% (95% ci, 2.9%7.4%). compared with older age groups, such as 6594 years , younger age groups, such as 2554 years, had higher estimates of inequality indicators for educational inequality in both current and heavy smoking in both sexes.educational inequalities in current and heavy smoking were apparent and large in the young population compared with older generations. the current study provides basic data on educational inequalities in smoking among japanese adults.

autogenous bone is considered the best choice and the gold standard due to the presence of viable cells and its osteogenic potential. use of the host's osseous tissue requires an additional surgical procedure and has some limitations; in addition, it can not supply adequate graft material in multiple and extensive defects. on the other hand, unwanted consequences (810%) and root resorption may occur during or after harvesting of autogenous bone, and many patients do not allow manipulation of other parts of their body to harvest grafts. allografts are human - derived materials, which have the properties of autografts but are acellular. they resemble human tissues more than other animal - derived graft materials or synthetic materials. allografts are abundantly available in tissue banks and are inexpensive; they are sterilized and stored and can be used in pure form or in combination with autogenous bone or other materials. allografts contain growth factors and osteogenic proteins such as bone morphogenetic proteins (bmps) with possible osteoinductive properties. the major problem associated with the use of allografts is the potential of virus transmission. the use of allografts in orthopedics was first reported in 1908 and then many studies reported the use of many types of allografts. the aim of the present study was to evaluate and compare the regenerative potential of these allografts. after obtaining ethical approval from the animal studies ethics committee of the implant research center, tehran university of medical sciences, 12 mature male white new zealand rabbits with a mean weight of 2.5 g were used in this study by observing all the ethical principles. the animals were kept for two weeks in the animal center for acclimatization under standard and similar dietary and environmental conditions. the surgical area was disinfected using 10% povidone - iodine solution and the hair in the animals head was shaved. a 78-cm straight incision was made in the midline in an antero - posterior direction; then two lateral mucocutaneous flaps were elevated to expose the area and gain access to the parietal bone. a trephine bur, which measured 8 mm in internal diameter in a rotary handpiece under irrigation and cooling with saline solution was used to create four round symmetrical bicortical defects with 8 mm diameter and almost 1 mm depth at the two sides of the calvarium midline. because of low thickness of the cortical plate and the possibility of rupture of the brain membrane, care was exercised to prevent traumatizing the meningeal membrane. one of the defects was filled with itb allograft manufactured by the iranian tissue bank (iranian tissue bank research and preparation center, tehran, iran), the second defect was filled with cenobone allograft manufactured by hamanand saz baft kish company (trc corporation, kish, iran); the third defect was filled with grafton manufactured by the american tissue bank (osteotech inc ., one defect was left empty without any graft material as a control ( figs . 1 and 2). four defects measuring 8 mm in diameter were created in the calvarium of each rabbit. one of the defects was not manipulated and designated as the control and the remaining three defects were filled with three different allografts. the properties of the allografts (demineralized freeze dried bone allograft, dfdba) used were as follows: itb / dfdba/ demineralized bone matrix (dbm) gel / cortico - cancellous/75125 mccenobone / dfdba /particle /cortico - cancellous /150500 mgrafton /dfdba /dbm putty / < 300 m itb / dfdba/ demineralized bone matrix (dbm) gel / cortico - cancellous/75125 m ccenobone / dfdba /particle /cortico - cancellous /150500 m grafton /dfdba /dbm putty / < 300 m the type of the material placed in each defect was recorded. to avoid bias in relation to the location of defects, randomization was used to place allografts in the first animal and then clockwise rotation was used to change the sites in which the allografts were placed in the other rabbits. finally, the periosteal flap was returned to its original location and the periosteum and the skin were sutured separately with resorbable 04 vicryl sutures (supa, tehran, iran) in two layers internally and externally. after the surgical operation, a pharmaceutical regimen consisting of subcutaneous injections of antibiotic (0.6 ml enrofloxacin) and analgesic (0.1 ml ketoprofen daily for three days) was administered. in addition, the animals underwent routine daily examinations to evaluate their postoperative status. during the healing period after the surgical operation, one of the animals in group one was lost; therefore, the sample size in this group decreased to five samples. in order to sacrifice the animals the defect sites were sectioned by a saw and a notch was produced in the occipital area to assist in identifying the direction of the defects. the samples were placed in 10% formalin solution (neutral buffered formalin) in closed containers and submitted to the laboratory in a blind manner. the blocks were placed in 10% formalin (sigma aldrich chemie gmbh, taufkirchen, germany) for five days, followed by placement in 5% formic acid (bayer ag, leverkusen, germany) for decalcification. then, the samples were dehydrated in ethanol and immersed in paraffin. during the decalcification process, which usually takes 10 days to complete, fresh acid solution was used every day and the extent of decalcification was evaluated until it reached the desired level. at least ten 5-mm thickness cross - sections were made from each defect and all the cross - sections were carefully evaluated by two oral pathologists twice. all the cross - sections were photographed under a binocular light microscope (bx51, olympus, tokyo, japan) connected to a camera (dp25, olympus, tokyo, japan). all the parameters were measured using computer software (dp2-bsw, olympus, tokyo, japan). in case of disagreement, the cross - section was reevaluated by the two pathologists and the was recorded after a consensus was reached. spss version 20 was used for statistical analysis and processing of data (spss inc . friedman 's test was used to compare qualitative variables and also bone regeneration due to deviation from normal distribution . in cases in which friedman 's test was significant, adjusted p values statistical significance was set at p<0.05 and p values between 0.06 and 0.10 were considered as marginally significant . spss version 20 was used for statistical analysis and processing of data ( spss inc . friedman 's test was used to compare qualitative variables and also bone regeneration due to deviation from normal distribution . in cases in which friedman 's test was significant, adjusted p values statistical significance was set at p<0.05 and p values between 0.06 and 0.10 were considered as marginally significant . the inflammation severity was recorded in a descending order in the control, grafton, itb and cenobone samples, respectively . also, there were statistically significant differences in the inflammation severity between the first and second months ( p=0.02 and p=0.03, respectively). maximum differences were observed between cenobone and control group in the first month and cenobone and grafton in the second month (tables 1 and 2). analysis of the data and parameters in the first month i= itb allograft manufactured by the iranian tissue bank k= cenobone allograft manufactured by hamanand saz baft kish company (trc corporation) g= grafton manufactured by the american tissue bank (osteotech inc . , eatontown, nj, usa) e= empty sites without any graft material as control analysis of the data and parameters in the second month i= itb allograft manufactured by the iranian tissue bank k= cenobone allograft manufactured by hamanand saz baft kish company (trc corporation) g= grafton manufactured by the american tissue bank (osteotech inc ., eatontown, nj, usa) e= empty sites without any graft material as control no foreign body reaction was detected except one in the first month in the control group and one in the second mouth in the grafton group. there were no statistically significant differences in foreign body reaction between the two intervals and among the study groups (both ps=0.39, tables 1 and 2). lamellar (mature) bone was not observed in any defect in the first interval, and the difference in the regenerated bone type was marginally significant (p= 0.06). in the second interval, only one of the defects filled with itb exclusively showed lamellar bone and in all other defects, the regenerated bone was mixed (a mixture of mature and immature bone). in the second interval, the four defect types did not exhibit any significant differences in the quality of regenerated bone (p=0.60, tables 1 and 2). the mean amount of regenerated bone was 29.46%, 3.55% and 0.93% in defects filled with grafton, itb and cenobone, respectively in the first interval, and 35.23%, 25.29% and 25.36%, respectively, in the second interval. in all defects filled with grafton, bone regeneration was observed in both intervals (table 3). comparison of the mean bone regeneration by time and materials i= itb allograft manufactured by the iranian tissue bank k= cenobone allograft manufactured by hamanand saz baft kish company (trc corporation) g= grafton manufactured by the american tissue bank (osteotech inc . , eatontown, nj, usa) e= empty sites without any graft material as control the difference in the amount of regenerated bone during the first month between cenobone and itb groups was trivial, but the difference in the amount of regenerated bone between the grafton and cenobone groups was partially significant (p=0.06). at the two - month interval, no significant differences were observed among the groups in relation to bone regeneration (p=0.62). the mean amount of residual material was measured and reported in square - millimeters (mm). on the whole, the maximum and minimum means of the residual graft material were observed in defects filled with grafton and cenobone, respectively, with no statistically significant difference (p=0.19 and p=0.20, respectively for the first and second intervals). osseous regeneration is one of the most important therapeutic aims and has widespread application in periodontal and implant treatments. the main goal of the present study was to evaluate the regenerative value of two types of allografts manufactured in iran in comparison with a more commonly used product in critical - size defects in rabbit calvarium. chang et al, in 2011 created four 8-mm defects in the calvaria of rabbits to evaluate the biological effects of calcium phosphate combined with cyanoacrylate. produced four 8-mm defects in the calvaria of rabbits in order to evaluate the efficacy of three different types of allografts. rabbits have been widely used by medical researchers due to their easy use, high metabolic rate and maturation in six months. the rate of bone remodeling in rabbits is three times the rate in humans; therefore, a 4-week time interval appears to be adequate for evaluation of initial healing responses in bone. the rabbit skull is a favorable bone model to carry out experiments with the use of bone graft materials. in addition , rabbit calvarium is considered an experimental site comparable to the alveolar bone because of its intramembranous osteogenesis process. histological evaluations carried out in the current study showed that in the first interval, the most common inflammation grade was mild; however, in the second interval, the predominant situation was absence of inflammation. this finding, along with the absence of foreign body reaction in the majority of defects, demonstrates a favorable healing process in the area. none of the defects filled with cenobone exhibited acute inflammation, indicating a high rate of adaptation and minimal provocation of the immune system by this material. in studies in which synthetic materials or xenografts are used, acute inflammation and presence of a dense infiltrate of polymorphonuclears are common occurrences. materials that are resorbed slowly (such as hydroxyapatite) lead to a chronic inflammatory reaction and they might even be surrounded by a fibrotic encapsulation. acute inflammation can interfere with the regeneration process and can even in dehiscence of the margins of the wound, which did not occur in the current study. previous researches showed that placing a demineralized allograft (dbm) in non - skeletal areas ed in the formation of ossicles, a phenomenon that was termed osteoinduction. there are differences between the products of different tissue banks in relation to their capacity to induce bone regeneration. osteoinductivity depends on different factors, such as the age and gender of the donor, form, size and origin of the particles and the technique used in the tissue bank to prepare the allograft. it has been reported that dfdba should be harvested from donors under 50 years of age and the best age is under 30 years old. pinholt and solheim placed dbm in the muscular and subcutaneous tissues of mice and reported that osteoinduction potential increased from birth up to puberty but decreased afterwards in addition, donor - related genetic factors and prolongation of the interval between death and harvest of bone affect the regenerative potential of allografts. the best are achieved with the use of allografts with a particle size of 250750 m. allografts are classified based on their mineral content into mineralized (freeze - dried bone allografts, fdba) and decalcified (dbm or dfdba) groups. decalcification in the release of factors such as bmps, which induce the differentiation of undifferentiated mesenchymal cells of the recipient site into osteoprogenitor cells. complete decalcification compromises the physical properties of the allograft, although it increases the amount of bmps available. compared dfdba and fdba for the repair of intraosseous periodontal defects and reported no significant differences between them. simion et al. reported favorable with the use of dfdba based on the guided bone regeneration (gbr) philosophy and use of membranes. analyses carried out in the current study showed that the amount of regenerated bone in the first interval decreased from defects containing grafton to defects containing itb and cenobone, respectively and there were great differences between the minimum and maximum amounts of regenerated bone (table 3). similarly, in the second interval, the maximum amount of bone regeneration was observed in defects containing grafton; however, the amount of bone regenerated in areas containing itb and cenobone increased by almost similar amounts. although the amount of regenerated bone in all the defects did not exhibit significant differences at the end of the study, osteogenesis during the first and second months did not show a uniform pattern. in other words, there was a delay in bone healing processes in areas containing cenobone and itb. in the grafton group, the maximum bone regeneration occurred during the first month but in the itb and cenobone groups, maximum bone regeneration occurred after the first month. in other words, with an increase in duration of healing and during the two - month period, bone regeneration in areas containing cenobone and itb increased to the level of grafton (graphs 1 and 2). major differences in osteogenesis among the different groups during the first month minor differences in osteogenesis among the different groups during the second month in a study by lee et al, the osteogenic effects of cortical allografts were compared with those of mixed cortico - cancellous and dbm allografts using the micro - computed tomography technique. the of the current study were consistent with those of lee et al, with regard to the chronology of regeneration processes. ziran et al. used grafton with lyophilized cancellous bone allograft chips for orthopedic repair of bone traumatic defects and nonunion areas and concluded that such a composite is a good alternative for autogenous bone but should be used with caution in smokers. used cenobone and dembone allografts for the treatment of dehiscence defects around implants and concluded that application of these two dfdba allografts under the collagen membrane and use of gbr technique did not increase the bone - implant contact. sarkarat et al. carried out a human study and compared two types of dfdba allografts, including cenobone and osseo+ (imtec corporation) in extraction tooth sockets and concluded that the mounts of viable, non - viable, trabecular and amorphous bone were the same in both groups after three months and even the number of osteoblasts was significantly higher in cenobone biopsies compared to osseo+ biopsies. therefore, both materials were considered suitable to preserve the alveolar ridge after tooth extraction. al, in 2009 evaluated the cell behavior in response to osteoinductive effect of dbm using immunohistochemistry techniques. also, a type of allograft, referred to as dynagraft (dbm) has been reported to induce osteogenesis in defects. kaya et al, in 2009 showed that dbm in the form of particles or putty had a similar effect on hard and soft tissue parameters. the of a human study by abolfazli et al. showed that the reaction of soft tissue to cenobone allograft was favorable and comparable to that of autogenous grafts. in addition, there were no significant differences in bone regeneration and soft and hard tissue parameters with the use of cenobone and autografts in the periodontal defects. in general, since dbm can cause ectopic osteogenesis in muscle and subcutaneous tissues, it is considered osteoinductive. also, dbms contain bmps and low concentrations of growth factors. growth factors play an important role in induction and regulation of wound healing processes and influence cellular functions such as osteogenicity, mitogenicity, chemotaxis, differentiation and metabolism. evaluation of residual materials in the defects in different groups in our study showed that a significant portion of allografts was resorbed during the first month. the resorption rate of allografts depends on the cortical bone content, processing technique, the form and size of the particles, porosity of particles, inflammation and various other factors. decalcification process decreases the antigenicity of bone; therefore, dbm is less immunogenic than mineralized allografts, which might be a reason for a low inflammatory response to itb and cenobone allografts. higher resistance of grafton to resorption and more inflammation around it might be attributed to its higher degree of mineralization compared to cenobone and itb. based on the of the current study, the highest and lowest percentage of residual materials belonged to grafton and cenobone, respectively, indicating a higher rate of osseous regeneration in defects containing grafton versus the lowest numerical value of regeneration in defects containing cenobone. which exhibited the highest resorption rate. in other words, longer presence of allograft in the defect means longer presence of an osteoconductive scaffold and osteoinductive growth factors and thus, in higher osteogenesis. itb allograft ranked between the two other allografts in relation to the amount of resorption and bone regeneration. however, the of the current study showed that there was an inverse relationship between allograft resorption rate and the severity of inflammation. in other words, the minimum inflammation and highest resorption rate, and the maximum inflammation and lowest resorption rate were observed with cenobone and grafton allografts, respectively. it appears that easy and fast resorption of cenobone without severe provocation of the immune system decreased the rate of osteogenesis in early stages of healing; however, in later stages, regeneration was accelerated and osteogenesis improved by the release of growth factors. however, despite differences in the amount of allograft resorption, which were evaluated in the current study, there were no significant differences among the study groups in the amount of residual allograft materials. histological evaluation showed that osteogenesis normally spread from the peripheral areas toward the center of the defects. in some situations, osseous regeneration began at both the central and peripheral sites of the defects and in some defects a bridge or a lateral connection was observed between the central and peripheral areas. this was consistent with the of studies by khoshzaban et al, and lee et al. lee et al. demonstrated that in the control defects, which had no graft material, bone regeneration occurred only at the periphery and never extended to the central areas ( fig. , in most areas, the regenerated bone was of the immature or woven type and only in limited areas of the defects lamellar bone was seen ( fig. no mature bone was found in the one - month samples; however, mature bone was observed in all the samples in the second interval. comparison of the type of regenerated bone among the groups showed that bone maturation rate in samples of itb was higher than that in other groups. it appears that longer evaluation intervals ed in more bone maturation, and higher percentages of lamellar bone formation in all defects. except for the control defects, in some cases , disturbances in bone regeneration were observed in defects containing biomaterials, which might be attributed to the collapse of the periosteum or the dermal flap toward the defect duo to an increase in intracranial pressure and advancement of meningeal membrane into the defects. the main disadvantages of allografts include transmission of human immunodeficiency virus and hepatitis b and c viruses and creutzfeldt - jacob disease. the prevalence of transmission of viruses by allografts was reported to be 1 in 1.5 millions in 1990. however, it decreased to 1 in 8 millions by drying and freezing the allografts. advances in molecular technology for the control of diseases have significantly decreased the risk of transmission of viral diseases. compared the effects of different techniques for sterilization of allografts on the inductive properties of dbm and concluded that there were no significant differences between radiation with gamma rays and use of ethanol oxide and ethanol; however, autoclaving decreased the inductive potential of allografts. under the limitations of the present study, the showed that the allografts evaluated are favorable materials for osseous regeneration and have the potential to regenerate osseous defects in vivo. although there were differences in the amount of bone regeneration in the samples, the differences were not significant. in other words, the did not indicate significant differences in the amount of bone regeneration among the study groups. therefore, the allografts manufactured in iran, which were evaluated in the current study, can be considered as safe and efficient alternatives for bone grafts, with properties comparable to those of grafton. further animal and human studies are necessary with larger sample sizes and longer follow - ups so that other aspects of the effects of these allografts on the regeneration of bone can be elucidated.

objectives: the aim of this study was to histologically compare the regenerative properties of two allografts manufactured by two iranian companies.materials and methods: in this study, four 8-mm defects were produced in the calvaria of 12 rabbits. in three defects, three types of allografts namely itb, cenobone and grafton were placed and one defect served as control. samples were prepared and histomorphometric evaluations were carried out after healing periods of four weeks (interval 1) and eight weeks (interval 2). qualitative and quantities variables were compared and analyzed with spss software.:mild inflammation was observed in 45% and 12.5% of the samples in the first and second intervals, respectively. foreign body reaction was observed in only 5% of the samples. the quality of regenerated bone was immature, mixed and lamellar in 54.5%, 15.9% and 4.5% of the samples, respectively. the rate of allograft resorption was the highest and lowest in the cenobone and grafton samples, respectively. the mean amount of regenerated bone was higher in areas containing grafton; however, the differences were not statistically significant.:despite the differences in the numerical values of bone regeneration, there were no statistically significant differences in bone generation among the material groups, and allografts manufactured in iran can be suitable alternatives to grafton with the same good properties. further studies are necessary to clarify the efficacy of these allografts.

deep sternal wound infection (dswi) is a rare but serious and potentially fatal complication after median sternotomy for cardiac surgery, with an incidence rate 0.5% to 4% and an associated mortality rate between 14% and 47%. presently, management of this problem is still controversial. some reports emphasize that more efforts should be taken to prevent it rather than to treat it, especially for those patients who have high risk factors such as obesity, comorbidities of diabetes mellitus, or chronic obstructive pulmonary disease. however, the introduction of various flaps has led to a significant decrease in morbidity and mortality over the past 30 years. according to the severity and dimensions of the dswi, the therapeutic methods range from conservative treatment to radical surgical debridement followed by negative pressure wound therapy or 1-stage flap coverage. current sternal wound management involves debridement of any devitalized infected soft tissues and bones, administration of culture - specific antibiotics, and the use of a vascularized flap to fill the wound cavity. at present, the pectoralis major muscle or myocutaneous flap remains the most common method for closing the majority of the infected median sternotomy wound. however, this flap does not have the capacity to resurface the entire length of the sternal defect when the thoracic defects are larger than half of the sternum. the greater omentum has the ability to cover the entire wound and control the sternal infection, but perhaps, leads to the spread of the infection to the abdomen. the rectus abdominis myocutaneous flap (ramf) has the major advantages of easy dissection, proximity to the infected sternal wound, and provision of ample skin island to repair the extensive thoracic skin deficit. however, several authors reported that this flap may not be considered as a viable option for sternal defects reconstruction when the ipsilateral or bilateral internal mammary artery (i m a) had been harvested for coronary artery bypass grafting (cabg). when the ramf is rotated by 180 into the thoracic defects, the inferior epigastric artery (iea) can be anastomosed to the intercostal artery or the i m a perforator. thus, this flap possesses a double blood supply. in this study, we aimed to describe our experience with the ramf for large sternal wound management, especially in the absence of 1 or 2 internal mammary arteries. to our knowledge, this is the first introduction of the particular ramf with a double blood supply. between october 2010 and january 2016, 9 consecutive patients who had received sternal reconstruction with the ramf after radical surgical debridement were referred. the general data (table 1) was collected by reviewing the medical records of the patients. according to the american center of disease control norm , dswi was defined as clinical infection requiring surgical debridement up to and including the sternum, with or without positive microbiology. the 9 patients in this study included 7 males and 2 females, with a mean age of 59 years (range : 4865 years). patients weights ranged from 43 to 95 kg, with a mean weight of 78 kg. all selected patients were received at least 2 debridements (range : 24 times) before sternal defect closure, and did not undergo any other flap procedures to repair the defects before the ramf procedure which was performed by the same surgeon (zhao zheng). the second intercostal artery or the second i m a perforator was identified with the handheld doppler probe preoperatively and marked (to decide which artery was anastomosed to the iea). next, the superior epigastric artery (sea) and the iea were marked, then the sea iea axis after microbiological specimens were collected, all infected necrotic tissues and bones were removed until healthy solid bone with bleeding margins was found. we carefully protected the internal mammary vessels during resection of the infected bones and cartilaginous structures. the dimensions of the thoracic defects were templated made and marked on the abdominal skin and off the midline. , the inferior epigastric vessels were viewed as a pedicle left at the inferior axis of the flap and the pedicle length was about 8 to 10 cm. then, the flap was rotated by 180 and filled into the sternal defects over suction drains. the abdominal donor defects were repaired by suturing the margin of the remaining anterior rectus sheath to its lateral component and then the skin was closed by relaxation suture. the iea was anastomosed to the second intercostal artery or the second i m a perforator. at the same time, the inferior epigastric vein was anastomosed to the concomitant vein. therefore, the ramf had a double blood supply (the anastomotic artery and the sea). the iea anastomosis was selected according to the following indication: the ipsilateral or bilateral i m a had been harvested previously or the contralateral i m a was ligated during previous cabg and the wound almost encompassed the full length of the sternum. after the operation, between october 2010 and january 2016, 9 consecutive patients who had received sternal reconstruction with the ramf after radical surgical debridement were referred. the general data (table 1) was collected by reviewing the medical records of the patients. according to the american center of disease control norm , dswi was defined as clinical infection requiring surgical debridement up to and including the sternum, with or without positive microbiology. the 9 patients in this study included 7 males and 2 females, with a mean age of 59 years (range : 4865 years). patients weights ranged from 43 to 95 kg, with a mean weight of 78 kg. all selected patients were received at least 2 debridements (range : 24 times) before sternal defect closure, and did not undergo any other flap procedures to repair the defects before the ramf procedure which was performed by the same surgeon (zhao zheng). all procedures were performed under general anesthesia. the second intercostal artery or the second i m a perforator was identified with the handheld doppler probe preoperatively and marked (to decide which artery was anastomosed to the iea). next, the superior epigastric artery (sea) and the iea were marked, then the sea after microbiological specimens were collected, all infected necrotic tissues and bones were removed until healthy solid bone with bleeding margins was found. we carefully protected the internal mammary vessels during resection of the infected bones and cartilaginous structures. the dimensions of the thoracic defects were templated made and marked on the abdominal skin and off the midline. , the inferior epigastric vessels were viewed as a pedicle left at the inferior axis of the flap and the pedicle length was about 8 to 10 cm. then, the flap was rotated by 180 and filled into the sternal defects over suction drains. the abdominal donor defects were repaired by suturing the margin of the remaining anterior rectus sheath to its lateral component and then the skin was closed by relaxation suture. the iea was anastomosed to the second intercostal artery or the second i m a perforator. at the same time, the inferior epigastric vein was anastomosed to the concomitant vein. therefore , the ramf had a double blood supply (the anastomotic artery and the sea). the iea anastomosis was selected according to the following indication: the ipsilateral or bilateral i m a had been harvested previously or the contralateral i m a was ligated during previous cabg and the wound almost encompassed the full length of the sternum. after the operation, nine patients who had the extensive thoracic defects were treated by the plastic surgery team during the study period. overall preoperative status six patients were in asa grade 3 and 3 patients in asa grade 4 (table 1). of the 8 patients who received the cabg, 7 patients had been harvested of 1 or 2 internal mammary arteries and 1 patient had aorto - coronary arterial saphenous vein bypass grafting (table 1). the mean operation time was 176 minutes (range : 155228 minutes) including the vascular anastomosis and the debridement. there was no recurrent infection after the thoracic defects were covered with the ramf combined with antibiotic therapy. one patient who did not have the double blood supply flap suffered from a distal part necrosis of the flap 7 days later and was taken back to the operation room. then, the distal 10% of the flap was debrided and reconstructed with a split - skin graft. the other patient had a seroma at the abdomen donor site and was managed conservatively. the mean overall hospitalization time was 32 days (range : 2356 days) after referral to the plastic surgery department. the mean follow - up time was 6 months (range : 312 months). the schematic diagram of the double blood supply ramf for coverage of the entire sternum is shown in fig. a 60-year - old man received coronary artery bypass grafting and his bilateral internal mammary artery had been harvested. after this operation, deep sternal wound infection occurred and 2 debridements were performed by cardiac surgeons. because of the large defect area (25 cm10 cm), the patient was recommended into our department. defect reconstruction was achieved by rectus abdominis myocutaneous flap combined with the inferior epigastric artery anastomosed to the second intercostal artery perforator. (a) large sternal defect after 2 debridements by the cardiac surgeon. (b) mediastinal organs were exposed after radical surgical debridement (c) defect reconstruction with a double blood supply rectus abdominis myocutaneous flap (the inferior epigastric artery was anastomosed to the second intercostal artery perforator). (d) at a 6-month follow - up, no wound infection was found and the patient was satisfied with his own state. the schematic diagram shows the double blood supply rectus abdominis myocutaneous flap for coverage of the entire sternum. (a) the red arrow indicates the inferior epigastric vessels, which were left at the inferior axis of the rectus abdominis myocutaneous flap. (b) when the rectus abdominis myocutaneous flap was rotated by 180 into the sternal defects, the inferior epigastric artery (the red arrow) was anastomosed to the second intercostal artery. at the same time, the inferior epigastric vein was anastomosed to the concomitant vein. treatment of the dswi has improved significantly over the past half century. during the 1960s, the infected sternal wound was managed by local debridement with closed catheter antibiotic irrigation, which led to mortality rate up to 20%. in 1976, the greater omentum was first reported to cover the extensive thoracic defects after extensive bone and cartilage debridement. later, various muscle or myocutaneous flaps became the primary treatment for reconstruction of the sternal defects. in the 1990s, vacuum - assisted closure dressings occurred and was readily viewed as an adjunct to facilitate sternal wound healing, even as the sole method for definitive treatment in specific cases. today, repairing the wound with well - vascularized muscle or myocutaneous flap after the early radical surgical debridement becomes an effective method. in this study, we used the ramf to repair the extensive thoracic defects containing almost the entire sternum, especially in those patients whose ipsilateral or bilateral i m a had been divided for reasons such as cabg. the rectus abdominis received dual - dominant blood supply from the superior and inferior epigastric arteries, which consisted of a complex perfusion pattern. the sea is a continuation of the i m a and the iea comes from the external iliac artery. with the superior epigastric vessels as the sole blood supply , the rectus abdominis with or without the overlying skin can be raised as a local flap to repair the sternal wound from the manubrium sterni to the xiphoid. the most common complication is the distal part necrosis of the flap because the distal flap can not receive enough blood supply from the pedicle. however, this complication did not occur when the iea was anastomosed to the second intercostal artery or the second i m a perforator in our case series. several studies reported that prior harvesting of ipsilateral or bilateral i m a may preclude the use of the ramf. indeed, we can speculate that the collateral circulation will be strengthened from the anterior intercostal arteries when the i m a has been ligated previously. furthermore, the i m a divides distally behind the sixth or seventh interspace into the sea and the musculophrenic artery. if the i m a is ligated on the distal bifurcation point, the musculophrenic artery will provide a rich supply of collateral circulation to the sea. based on this , some previous studies reported that the rectus abdominis muscle or the myocutaneous flap was used successfully for median sternotomy wounds after ipsilateral i m a ligation. however, this flap is limited to repair the lower half of small sternal wound, which requires a pectoralis major muscle flap for the superior part of the wound. in our study, the entire sternal wound was repaired successfully when we took advantage of the iea to provide additional blood supply to the ramf. there are also other alternative methods to reconstruct the extensive thoracic defects which comprise almost the entire sternum and the significant skin defect after radical surgical debridement. for example, pectoralis major muscle advancement for upper sternum with rectus abdominis muscle turn - over for the coverage of lower sternum is another choice for the entire sternal wound reconstruction. however, we rarely consider the combined use of 2 or more local flaps in our study when a single local flap is sufficient to repair the defect. the greater omentum flap is also a useful reconstructive option because it contains rich vascular supply and large amount of available soft tissues which can reach the entire sternal defects and fill irregular defects sufficiently. the emergence of laparoscopic omental harvest has gained great attention on omental flap use in extensive thoracic defects after dswi. in addition, several previous literatures suggested that the use of omentum may be associated with lower mortality and fewer complications when compared with the pectoralis muscle flap.. moreover, omentum may not be available in those patients with intra - abdominal adhesion after previous surgery. lastly, secondary split - thickness skin graft is necessary when compared with the ramf. more recently, the free myocutaneous flap has been described for sternal wound closure and viewed as a new choice to repair the extensive thoracic defects after dswi. in our case the complication rate of 22% (2/9) is comparable to that found in previous studies of 25% to 30%. the tip necrosis is the major complication associated with the ramf, which has been reported in a lot of literatures. but this complication did not occur in our patients whose iea had been anastomosed to the second intercostal artery or the i m a perforator. furthermore, even when the ipsilateral or bilateral i m a has been harvested, the particular ramf also has enough blood supply and heals well. despite the satisfactory that all patients were healed and did not have recurrent infection first, because of the small number of patients included in the study, we can not have enough data for further analysis and make a stronger . in fact, the small number of eligible patients is largely due to the rare prevalence of the extensive thoracic defects after dswi. in addition, when the bilateral i m a and 1-sided iea are harvested, it may decrease the abdominal wall perfusion and increase the risk of abdominal wall herniation. in , our study suggests that the ramf may be suitable to repair the large sternal wound up to the entire sternum. when one or both internal mammary arteries have been ligated for cabg or other reasons, the iea can be anastomosed to the second intercostal artery or the i m a perforator to provide the flap with a double blood supply.

abstractdeep sternal wound infection is a severe complication after open heart surgery. according to the different severity and dimensions of the deep sternal wound infection , the treatment method is different. in this study , we aimed to describe our experience with the rectus abdominis myocutaneous flap for large sternal wound management, especially when 1 or 2 internal mammary arteries were absent.between october 2010 and january 2016, a retrospective review of 9 patients who suffered from the extensive thoracic defects after deep sternal wound infection was conducted. all of these sternal defects encompassed almost the full length of the sternum after debridement. defect reconstruction was achieved by covering with a rectus abdominis myocutaneous flap. when the ipsilateral or bilateral internal mammary artery had been harvested previously, we took advantage of the inferior epigastric artery to provide additional blood supply to the rectus abdominis myocutaneous flap. thus, this flap had a double blood supply.there was no recurrent infection in all 9 patients. three patients received the rectus abdominis myocutaneous flap with a double blood supply. flap complications occurred in 2 patients (22%). one patient who did not have the double blood supply flap suffered from necrosis on the distal part of the flap, which was then debrided and reconstructed with a split - skin graft. the other patient had a seroma at the abdomen donor site and was managed conservatively. none of the patients died during the hospital stay.this study suggests that the rectus abdominis myocutaneous flap may be a good choice to repair the entire length of sternal wound. when 1 or 2 internal mammary arteries have been harvested, the inferior epigastric artery can be anastomosed to the second intercostal artery or the internal mammary artery perforator to provide the rectus abdominis myocutaneous flap with a double blood supply.

because of the exponential growth of available sequence data, the development of accurate computational strategies for functional site identification has become one of the most important post - genomic challenges. although attractive owing to their relative simplicity, conservation - based approaches frequently in too many false positives to be satisfactory. in addition, sequence regions with significant variability can also be critical to function, especially when their composition may define subfamily specificity. frequently, these regions correspond to residues that are critical in molecular recognition and binding specificity. in this report pms are sequence alignment regions that conserve the overall phylogeny of the complete family. through comparison with structural and biochemical data , we have shown that pms represent good functional site predictions in a wide variety of protein systems. our indicate that, despite little overall proximity in sequence, pms are structurally clustered around key functionality across a wide variety of structural examples. pms correspond to a variety of structural features, including solvent exposed loops, active site clefts and buried regions surrounding prosthetic groups (figure 1). our also indicate that pms are generally conserved in sequence, indicating that pms tend to be motifs in the traditional sense. consequently , pm bridge evolutionary and traditional motif approaches. in spite of the small alignment window size if sequences are unaligned, miner will align them for the user by using clustalw. a sliding sequence window algorithm is used to quantitatively evaluate the phylogenetic similarity between each sequence region and the whole sequence. phylogenetic similarity is based on tree topology, which is calculated using the partition metric algorithm. overlapping sequence windows scoring past some preset phylogenetic similarity z - score (psz) threshold are identified as pms. empirically, we have determined that a window width between 5 and 10, and a psz threshold between 1.5 and 2.2 (lower scores indicate greater similarity) represent ideal default parameters for functional site prediction.. however, the user retains the option to handle gaps as described previously. subsequently, partition around medoids clustering of the similarity scores assesses those sequence fragments whose annotation remains in doubt. the accuracy of the approach has been confirmed through comparisons with our manual . a preprint more thoroughly describing the automated algorithm is available at the miner website. with the automated algorithm in hand , we have precomputed all pms for the most recent version of the cog database. miner is available as standalone (command - line based) software and through the web via a user - friendly interface. after the web - based calculation is complete, miner sends an email with a hyperlink directing the user to their . miner is part of the larger protein motif analysis portal at california state polytechnic university, pomona. however, we recommend using 25 or more sequences to ensure sufficient evolutionary diversity. with the exception of gaps, all non - alphabetic characters found in the input will be purged. optionally, a protein data bank (pdb) structure may be submitted to better highlight pm regions. miner will automatically add the pdb sequence to a dataset of unaligned sequences if it does not exist. however, user - provided alignments must already include the pdb sequence as part of the alignment. there are several default miner options that can be customized before submission (figure 2). although masking is optional, we find that eliminating these positions significantly increases the quality of functional site predictions, especially in more divergent families. miner also provides three methods for identifying motifs. by default, miner identifies functional sites as described above. alternatively, miner also provides the option to identify traditional motifs using the false positive expectation (fpe) of a regular expression or profile. when used in conjunction with the pm , these alternative approaches often provide synergistic information. in addition, the width of the sliding window can also be modified. by default, the width is set to five alignment positions, which we find it to be ideal for identifying functional sites. however, large windows are more appropriate when exploiting motif - ness (e.g. using pms to de - orfan uncharacterized sequences). the z - score threshold is automatically determined by default, but can be manually set any value 1. finally, either jmol (default) or chime viewers can be used for interactive structure visualization. the miner output is a framed html file (figure 3) that provides (i) phylogenetic similarity versus window number plots, (ii) an annotated structure and (iii) an annotated msa. pm regions in the pdb structure are annotated by writing the psz to the temperature factor column. furthermore, interactive structural visualization of the identified pms is achieved with the option of using either jmol or chime. each pm within the alignment is hyperlinked such that clicking it will highlight the corresponding structural region. pm sequence logos, generated by weblogo, are also hyperlinked from the msa. in all cases, the raw data are available for easy export to auxiliary programs. with the masking feature enabled, regions of the msa colored light gray represent alignment positions that have been purged before pm identification. at the miner website, the tutorial guides one through the output of triosephosphate isomerase , which is the center of discussion in our previous reports. miner utilizes a sliding sequence window algorithm to systematically evaluate all regions of an msa input. phylogenetic similarity is determined by comparing tree topology, which is calculated using the partition metric algorithm consequently ing in a psz value. the sensitivity in the pm identification is constrained using a psz threshold, which is automatically determined by default. the ing miner output uses jmol or chime pdb viewers allowing protein structure and corresponding pm regions to be interactively visualized. the four best - scoring (psz threshold = 1.5) pms identified from the myoglobin protein family are mapped onto structure (pdbid : 1mba). the -carbons of the pms are shown as black spheres; the heme is shown in gray. the sequence window (which can toggle between aligned and ungapped) is hyperlinked to the structure viewer and weblogos. the upper - left window can toggle between both pm (red) and fpe (green) . in all cases,

miner is web - based software for phylogenetic motif (pm) identification. pms are sequence regions (fragments) that conserve the overall familial phylogeny. pms have been shown to correspond to a wide variety of catalytic regions, substrate - binding sites and protein interfaces, making them ideal functional site predictions. the miner output provides an intuitive interface for interactive pm sequence analysis and structural visualization. the web implementation of miner is freely available at. source code is available to the academic community on request.

abetalipoproteinemia (abl ; omim 200100) is a very rare hereditary autosomal recessive metabolic disorder of fat and fat - soluble vitamins absorption, which is caused by microsomal triglyceride transfer protein (mttp ; omim 157147) deficiency and characterized by absence of plasma apolipoprotein b and (apo b)-containing lipoproteins. mttp is a chaperone protein found in enterocytes and hepatocytes endoplasmic reticulum (er) and includes three structural domains (n - terminal -barrel, -helix and c - terminal) and three functional domains (transfer activity, membrane interaction and lipid binding). the molecular basis of this disorder is the inheritance of two mutations in mttp gene which is located on chromosome 4q23 and encodes the large subunit of mttp (a 97-kda protein containing 894 amino acids) that forms a heterodimer with the endoplasmic reticulum enzyme protein disulfide isomerase (pdi) and accelerates the transfer of lipids onto apolipoprotein b, ing in assembly and secretion of apo b in the formation of vldl and chylomicrons in the liver and intestine respectively. the syndrome was first described by bassen and kornzweig in 1950. according to the signs and absence of lipoproteins, salt and colleagues named the disorder as abetalipoproteinemia afterwards. children are usually asymptomatic at birth but develop digestive symptoms including diarrhea, steatorrhea and failure to thrive in infancy. subsequently retinopathy, ataxic neuropathy, acanthocytosis and fatty liver may appear in childhood due to fat - soluble vitamin deficiency, especially vitamin e and beta - carotene that are at extremely low levels in patients with abl. herein, we describe the history and clinical features of a patient with abl who was referred to our hospital and followed until the age of five. a 12-months - old male infant was referred to our hospital because of failure to thrive. he was born from consanguineous parents and his birth weight was 3.4 kg and he failed to gain weight appropriately since two months of age. at the first visit his weight was 6 kg and his height and head circumference were 61 and 44 cm, respectively. he also had developmental delay as he held his neck at 6 months and sat at 9 months of age. he had frequent defecations (8 - 9 times a day) that sometimes were steatotic. he had no family history of similar problems. on physical examination only widening of fontanels was observed. laboratory tests showed: cholesterol=43 mg / dl, triglycerides=6 mg / dl, alanine transaminase (alt)=17 u / l, aspartate transaminase (ast) = 60 u / l, total protein=5 g / dl and albumin=3.8 g / dl. stool examination showed fat droplets (+) < 40 drop / hpf. thyroid function test showed tsh=6.5 miu / l and t4=135 nmol / l that established diagnosis of subclinical hypothyroidism. abdominal sonography demonstrated normal size of liver and spleen but there were multiple small stones in both kidneys. macroscopic findings in endoscopy included normal mucosa of esophagus and stomach but snow like appearance and pathologic findings were found in bulb of duodenum. crypts were unremarkable and there was mild infiltration of lymphoplasmacells and some eosinophils (3 - 16/hpf) in lamina properia alongside edema (figure 1). crypts were unremarkable and there was mild infiltration of lymphoplasmacells and some eosinophils (3 - 16/hpf) in lamina properia alongside edema. it would have been beneficial to perform molecular diagnosis but unfortunately the parents did not agree to do genetic study. in the presence of symptoms, the most probable differential diagnosis included cystic fibrosis (cf) and pediatric celiac disease, but severe hypocholesterolemia and hypotriglyceridemia suggested other conditions including abl, homozygous familial hypobetalipoproteinemia (fhbl) with dominant transmission (mutations in apob gene) and chylomicron retention disease (cmrd) with recessive transmission (mutation in sar1b gene). normal levels of tissue transglutaminase antibodies and sweet chloride test along with microscopic studies ruled out celiac disease and cf. family lipid profile was useful in delineating between other conditions. while obligate heterozygote parents of homozygous fhbl patients have decreased levels of plasma ldl - cholesterol and apo b, asymptomatic parents of our patient with normal plasma cholesterol levels suggested a recessive disorder and ruled out fhbl. undetectable amounts of plasma apo b and low density lipoproteins ruled out cmrd and confirmed the diagnosis of abl. our patient was treated with caprilon formula, medium - chain triglycerides (mct) oil 1cc / kg / day, vitamin a 10000 iu / day, vitamin e 2000 iu / day, vitamin d 400 iu / day, vitamin k 5 mg daily and levothyroxine 50 - 100 mcg / day. kidney stones resolved with medical treatment and intraventricular brain cyst remained at the same size. liver aminotransferases were elevated (ast 160 and alt 250 u / l) but reinforcing of accurate fat - limited diet ed in normal ranges of these enzymes levels during follow - ups. in last visit he was 5 years old and his weight was 18 kg and his height was 105 cm. abl is a very rare recessive metabolic disorder characterized by the absence of apob - containing lipoproteins in plasma. malabsorption of fat and fat - soluble vitamins leads to a variable clinical phenotype that presents in early childhood with steatorrhea and failure to thrive and may include progressive multi - system abnormalities as the patient ages. most patients are diagnosed in the 2nd to 4th decades and few others in 1st and 6th decades. earlier presentation of symptoms may be due to a more severe phenotype and may be more resistant to medical treatment ing in poor outcomes. on the other hand, later presentation and longer period of untreated disease may be associated with poor outcomes due to consequences of fat - soluble vitamins deficiency. most reported patients with abl had lipid malabsorption, remarkably low serum lipid levels and gastrointestinal manifestation including diarrhea, steatorrhea and oral fat intolerance. retinitis pigmentosa, myopathy and spinocerebellar ataxia have also been reported in most patients due to fat - soluble vitamins deficiency. our patient had signs and symptoms of early onset abl including fat intolerance, diarrhea, steatorrhea, growth retardation, developmental delay, low tg and cholesterol levels. gastrointestinal manifestations including fat intolerance, diarrhea and steatorrhea are consistent features in most reported patients. brain ct scan showed intraventricular cyst which has not been previously reported despite numerous reported neurological involvement. in abl, both central and peripheral nervous systems can be affected and may be the most serious clinical manifestation. the primary pathology is progressive demeylination of the nervous system which is related to abnormal lipid peroxidation of the highly unsaturated phospholipid of myelin, probably due to prolonged vitamin e deficiency. several studies showed that early high dose vitamin e (100 iu / kg) therapy prior to the age of 2 years can prevent progressive neurological dysfunction and diminish neurological sequelae. pigmentory retinal degeneration is progressive and often causes slowly enlarging annular scotomas with macular sparing. patients may have reduced night vision or color vision, early in the course of disease. previous studies showed that high dose vitamin e therapy or combined vitamin e and vitamin a treatment before 2 years of age can prevent the development and progression of retinopathy. the absence of retinitis pigmentosa in our patient must be attributed to the fact that retinopathy may appear at any time during the first 2 decades of life. red cell acanthocytosis is the hematologic manifestation of abl which is reported in several cases but was not seen in our case. nephrolithiasis was present in our patient which was also been reported previously. in patients with abl fat malabsorption causes combination of unabsorbed fatty acids with calcium ions in the intestinal lumen leading to excessive absorption of dietary oxalate and kidney stone formation. management included dietary fat restrictions, fat soluble vitamins supplements, specific formula and polycitrate that resolved the problem in the patient. our patient had subclinical hypothyroidism, the association between abl and subclinical hypothyroidism was previously reported in another case. the hepatic manifestation of our patient was elevated levels of serum transaminases, probably due to hepatosteatosis which resolved with accurate treatment. in early diagnosis and accurate treatment are necessary to avoid the complications following fat - soluble vitamin deficiencies.

abetalipoproteinemia (abl) is a very rare autosomal recessive disorder caused by mutations in the microsomal triglyceride transfer protein gene (mttp). abl is characterized by lack of lipids and apolipoprotein b (apob) in plasma, fat malabsorption and various clinical manifestations. we describe a 12-month - old infant boy, born from consanguineous parents and presented with diarrhea, steatorrhea, growth retardation, hypothyroidism, intraventricular brain cyst and kidney stones. the patient was diagnosed to have abl and treated with dietary modification and oral fat - soluble vitamin replacement and followed until he reached 5 years of age.

pain management during transurethral procedures is a major concern. apart from the standard general anaesthesia, nevertheless, the administered type of anaesthesia is ultimately based on the anaesthesiologist's decision. recently, local anesthesia with infiltration of the bladder wall or periprostatic nerve blockage was reported. additionally, sedoanalgesia which is the combination of local anaesthesia with sedation or even the use of virtual reality for pain management has been examined. the selection of anaesthesia in a transurethral prostatectomy (tur - p) or a transurethral bladder tumor resection (tur - b) has been investigated meticulously in previous reports. in general, regional or local anaesthesia demonstrates distinct advantages and disadvantages in terms of postoperative morbidity. the aim of the present study was to compare general and spinal anaesthesia in terms of postoperative pain mitigation by recording the patient's pain perception during the critical first 24 postoperative hours and to investigate a potential correlation of clinical and pathological data with the pain induced by the transurethral procedures. distribution of the patients is depicted in the consort flow diagram (figure 1). patient age and distribution of the population according to stage and grade of the transitional cell carcinomas (tcc) are summarized in table 1. administration of 1 - 2 g / kg of propofol, followed by 1 - 2 mg / kg of fentanyl along with 1 - 2 mg / kg of suxamethonium. administration of 0.5 mg / kg of atracurium (n2o at 50% o2) and inhaled desflurane at mac 1 (minimum alveolar concentration). spinal anaesthesia was administered as a single shot of 2 ml bupicaine and 1 ml lidocaine without adrenaline. patients with bladder or prostatic capsule perforation, which was identified intraoperatively, were excluded from the study. a 22-f, 3-way dufour catheter was placed in all patients, and bladder irrigation was standard. bladder irrigation was stopped after the completion of the 24-hour observation period after verifying the absence of intravesical clotting by manual irrigation and suction. postoperative pain severity was assessed and recorded using an 11-point visual analogue scale (vas). the vas was given to each patient at the time of his / her arrival in the recovery room, which was considered as hour 0. each patient recorded his / her pain tolerance at postoperative hours (h) 0, 2, 4, 8, 12, and 24. no pain scored 0 points, while worst possible pain for the patient was scored with 10 points. score > 3 during the observation period, 500 mg of paracetamol combined with 20 mg of hyoskine n - butylbromide (buscopan) were administered. stratification of pain scoring was made using several parameters, such as age, gender, stage, grade, and location of the tcc's. statistical analysis was performed using the spss 14.0 for windows statistical package (spss, inc ., clinical and demographical characteristics were compared using the mann - whitney u test for continues variables and the chi - square test for categorical variables . kruskal - wallis test was used to estimate equality of population medians among groups and the mann - whitney u test for comparison between the groups . the spearman correlation coefficient ( when appropriate vas scores, with 95% confidence interval ( ci) in relation to postoperative time are shown in figure 2. no statistical difference was detected in the tur - p patients at any postoperative time between the two anaesthetic methods. in the tur - b patients, mean (95% ci) analogue vas score was greatest at 0 h for general versus spinal anaesthesia (p = 0.027). at 8 h and 12 h, general anaesthesia's analgesic efficacy was increased significantly (p = 0.017 and p = 0.007, resp .) after adjusting for gender in the tur - b group, male patients under general anaesthesia experienced more pain at 0 h and 2 h. however, in the female patients, vas score was distributed differently, since spinal versus general anaesthesia's analgesic efficacy was lost at 4 h , 8 h and 12 h (p = 0.005, p = 0.004, p < 0.001, resp .), suggesting a better analgesic efficacy for general anaesthesia. a separate analysis was performed in male patients comparing tur - p versus tur - b. no difference in vas score was recorded between the two surgical approaches when general anaesthesia was chosen as analgesic method. in the spinal group, however, tur - b patients presented lower mean vas score than tur - p patients at 0 h. this pattern changed at 8 h and at 12 h,. interestingly, after adjusting for the covariates stage and grade in the tur - b patients, a higher vas score was observed for pt2 patients compared to pta, pt1 (tnm), and patients with negative pathology report (figure 3 and table 2). stage and tumor grade were highly correlated (p < 0.001), but grade did not present any statistical significance with vas score at any postoperative hour. resected tumor volume was used as a categorical variable, and patients were divided in those with < 10 cm and > 10 cm mean 28.1 (n = 15) ]. the cut - off point of 10 cm was set by the statistical analysis. those with resected tumor volume > 10 cm presented vas score > 2 at 8h and 24 h, which was statistically significant (p = 0.050, p = 0.036, resp .) (figure 4). adjusting for the method of anaesthesia, subjects with bladder tumors larger than 10 cm that received general anaesthesia presented vas score <3 at 4 h, (or = 6 95% ci 1.0135.04, p = 0.035). however at 24 h, vas > 3 was more common in patients with tumor volume < 10 cm who underwent general anaesthesia (or = 3.5 95% ci 1.0911.02, p = 0.008). apart from the 24 h, tumor located in the lateral bladder walls induced more pain than those situated in the trigone. moreover, tumor multifocality had the highest vas scores in all observation periods (table 3). several papers compare the effect of these methods in terms of peri- and postoperative morbidity (blood loss, side effects, and possible complications). to our knowledge, this is one of the few attempts of comparing the 2 methods by recording the patient's pain perception and tolerance. firstly, in patients undergoing tur - p, we found that none of the 2 methods of anaesthesia prevailed during the 24-hour observation period. another report by fredman and colleagues advocated general anaesthesia as the method of choice in transurethral procedures. again, patient satisfaction was recorded, but the main limitation was that no adjustment for the type of the transurethral procedure was made. suggested that regional anaesthesia reduces the incidence of catheter - related pain, although being of similar efficacy to oral diazepam and thus is more advantageous than general anaesthesia. on the other hand, differences were recorded in tur - b patients. to be more specific, spinal was more efficient than general anaesthesia in the first 2 h after surgery, while general proved to be better in the later time points. this fact can be explained by the major implications induced in bladder function by regional anesthesia. indeed, catheter - related pain and detrusor muscle spasm elicited by bladder irrigation can be managed efficiently with regional anaesthesia in the first 2 postoperative hours. however, it simultaneously causes a clinically significant disturbance of bladder function due to interruption of the micturition reflex. additionally, it has a greater effect on bladder compliance and lowers the intraabdominal pressure. as a , painful bladder overdistention or even acute retention might occur after the removal of the catheter, due to the long - lasting recovery of the normal bladder function. based on the clinical experience, the incidence of such an event is rather rare, thus we might implicate the catheter and bladder spasm as the most distressing causes of postoperative pain. however, an interesting analgesic approach could have been the combination of spinal anaesthesia with antimuscarinic pretreatment, such as oxybutnin or tolterodine, or the administration of gabapentin during general anaesthesia, which is shown to significantly reduce catheter - related pain. the above urodynamic effects might be interpreted better in the female patients than in men, since in women there are no prostatic symptoms that could bias the bladder pain, and we should also acknowledge the fact that females perceive pain better than men, as recent reports suggest. in the female population of our study, spinal was less effective than general anaesthesia after the first 4 hours, verifying the possible implications of regional anaesthesia in the female bladder. a very surprising observation is exhibited in the statistical analysis, concerning the bladder tumor stage. patients with higher stage experienced a higher level of pain than the ones with localized disease (pta - pt1). one can speculate that infiltrative disease stimulates more nociceptors (highly specialized free endings of sensory nerve fibers). even though this is a random , we believe that it is worth mentioning, since it exhibited statistical significance, and it might motivate a better anaesthetic approach of patients that present with pt2 disease cystoscopically. it is obvious that verification is required for the analysis of a large cohort of patients, which should be also stratified by the type of the applied anaesthesia. we also found that the resected bladder tumor volume becomes a significant parameter of pain induction after a tur - b, when more than 10 cm are resected. on the other hand, when we adjusted our analysis to the type of anaesthesia, general anaesthesia seemed to be more efficient at 4 h and 24 h. tumor volume was the only parameter favoring the efficiency of general anaesthesia independently of the postoperative time of observation. the use of vas scale for the documentation of pain measurement introduces a study bias per se, since this is a subjective method. to date, no objective recording of pain perception has been described, even though several pain scales assessing the patient's agitation level by recording the facial expression, leg movement, and muscle tension, or even the brain electrophysiologic activity through specialized electroencephalograms (eeg) are presented as promising. yet, the use of vital signs, such as heart rate and arterial pressure or even pupil reactions could reflect partially the pain status, but they could be affected also by the postsurgical stress, the patient's comorbidities, or the prescribed medication. nevertheless, clinically significant differences between vas scores might be considered realistic anecdotally, when they exceed 4 - 5 points. thus, in our study, clinical significant differences were observed in different bladder tumor stages and multiple versus single tumors. the dosage of epidural regimens has an effect of approximately one and a half hours. therefore, it could not have altered the vas scoring, since the 0 h point was set at the arrival in the recovery room. the selection of paracetamol and hyoskine n - butylbromide as rescue analgesics in patients, reporting vas > 3 postoperatively, was made due to their immediate action and their short half - lives , which could not have affected the perception of pain during the 2-hour and 4-hour intervals of vas recording. the patients were not randomized due to the fact that the selection of analgesia was made by the anaesthesiologists individually for each patient, according to their performance status. another drawback could be the lack of analysis of the time of the operation and the prostatic volume resected. even though the resected adenoma can not be rationally associated with pain induction, since the major causes of tur - p pain are bladder spasm, catheter - related pain, and capsule offense, a future study could incorporate this parameter. as for the time of the operation , we believe that it could not have affected the , since the scoring started after the completion of the operation, during which pain is managed sufficiently. the analysis of the advantages and disadvantages of regional compared to general anaesthesia is immensely complicated. as can easily be imagined, the secondary effects of both types of anaesthesia are varied, and realistically, they must be considered for each and every patient, due to the unique clinical profiles presented. this study attempted to establish the potency of general and spinal anaesthesia during transurethral procedures by recording the patient's pain perception. it seems that spinal anaesthesia is more effective during the first 2 postoperative hours, while general prevails at later stages and at larger traumatic surfaces. finally, we incidentally found that tumor stage plays a significant role in postoperative pain, a point that requires further verification. we advocate a closer cooperation of the urologist with the anaesthesiologist in terms of providing more specific information about the patient's disease and tailoring the type of anaesthesia to the patient's needs.

we compared the analgesic efficacy of spinal and general anaesthesia following transurethral procedures. 97 and 47 patients underwent transurethral bladder tumour resection (tur - b) and transurethral prostatectomy (tur - p), respectively. postoperative pain was recorded using an 11-point visual analogue scale (vas). vas score was greatest at discharge from recovery room for general anaesthesia (p = 0.027). the pattern changed significantly at 8 h and 12 h for general anaesthesia's efficacy (p = 0.017 and p = 0.007, resp .). a higher vas score was observed in pt2 patients. patients with resected tumour volume > 10 cm3 exhibited a vas score > 3 at 8 h and 24 h (p = 0.050, p = 0.036, resp .). multifocality of bladder tumours induced more pain overall. it seems that spinal anaesthesia is more effective during the first 2 postoperative hours, while general prevails at later stages and at larger traumatic surfaces. finally, we incidentally found that tumour stage plays a significant role in postoperative pain, a point that requires further verification.

an 18-year old male presented with recurrent palpitations associated with occasional presyncope for the preceding 3 months. nonsustained episodes of narrow qrs tachycardia with sinus pauses at termination were noted on the electrocardiogram (ecg) (fig . holter monitoring showed sustained and nonsustained episodes of tachycardia, with a cumulative duration of 14 h in a 24-h recording, and intervening sinus pauses ( fig . 1c) lasting up to 4.5 s. his left ventricular ejection fraction was 35% at presentation. during electrophysiological study, sinus cycle length was 734 ms with normal atrio - hisian (ah) and his - ventricular (hv) intervals. the termination and re - initiation of the tachycardia are shown in fig. 2. the ecg in fig. 1a and b show a narrow qrs long rp tachycardia with negative p waves in inferior leads. the episodes of this nonsustained tachycardia are initiated with sinus beats, and the second qrs complex of the tachyarrhythmia shows right bundle branch block morphology. the morphology and axis of the subsequent p waves (p) are different from the sinus beats. occasionally, the isolated sinus beat is followed by a single p wave, without continuing as tachycardia. the differential diagnosis of this long rp tachycardia includes atrial tachycardia (at) originating from inferior atrium, av nodal reentrant tachycardia (avnrt) of fast - slow sub - form and persistent form of junctional reciprocating tachycardia (pjrt). during at , the p waves of the initial and subsequent beats tend to be similar, unlike what is observed in this case. the fast - slow type of avnrt is usually initiated following ventricular premature beat and the incessant nature is unusual. orthodromic reentrant tachycardia using an accessory pathway (ap) with decremental retrograde conduction properties, i.e., pjrt can be initiated by sinus beat, and this tachyarrhythmia tends to be incessant. termination on either antegrade or retrograde limb can occur in both reentrant mechanisms, but to terminate with a p wave is unusual for at. 2 helps to confirm pjrt as the mechanism in this case. the initial 2 cycles of tachycardia in fig. 1 bracketing at proximal coronary sinus (cs) bipole (cs 7 - 8). the tachycardia terminates following a paced beat from the right ventricular apex, timed 30 ms later than the his signal. the morphology of this paced complex suggests qrs fusion between the paced and tachycardia beat. termination following a paced ventricular beat not conducted to atrium and ing in qrs fusion suggests orthodromic av reentry as the tachycardia mechanism. the prolonged local ventriculoatrial (va) interval of 210 ms even at the site of earliest retrograde atrial activation during the tachycardia suggests slow conduction through the ap. immediately following its termination, the next sinus notably, the first local v v interval at cs 7 - 8 after the re - initiation is 370 ms, as compared to 360 ms during its stable phase. the increment of 10 ms is contributed by a delay in the ah interval by 60 ms (40100 ms) despite the shortening of the conduction time across the ap by 40 ms (210170 ms). despite a delay in conduction down the his bundle , the anterograde impulse finds the right bundle in refractoriness, and conducts down the left bundle with an hv interval similar to that in other tachycardia beats. this functional block and its mitigation in the subsequent beats follows that the recovery period in the bundle branch is directly related to the previous rr interval. the conduction times through the pathway, as measured by the local va interval in cs 7 - 8 bipole, showed progressive prolongation (170, 190 and 210 ms), in response to a shortening of the preceding local v the progressive reduction in the time interval of the retrograde impulses at the ventricular end of the ap during the re - initiation, related to the conduction delay in the av node in the second beat, unmasked the decremental properties of the ap in this case. similarly, the decremental nature of the conduction in the av node accounted for the minimal changes in av interval in the initial cycles during the re - initiation. frequently, the sinus beats were followed by single atrial echo beats, nonconducted down the av node ( fig. however, if this echo beat could find av node during its relative refractory period and conduct with a prolonged ah interval and bundle branch aberrancy, the reentry could be initiated. during the following cycles, the minimal but incremental prolongation in the conduction time through the ap due to its decremental nature helped the retrograde impulse the tachycardia was incessant during the study and could be terminated only with ventricular pacing. the ap was successfully ablated at cs, within 1 cm of its ostium. holter monitoring done 3 months after the study showed no pauses or tachycardia and the ventricular function recovered completely to suggest tachycardiomyopathy at presentation. symptomatic sinus pauses at the termination of the tachycardia episodes, mimicking a tachy - brady syndrome is unusual in pjrt. suppression of the sinus node related to chronic atrial arrhythmias and its reverse remodeling over weeks to months following successful ablation of the culprit arrhythmia has been described,. however, the complete recovery of sinus node function immediately after the ablation makes this mechanism unlikely. a compensatory vagotonia in response to the initial hypotension and ant sympathetic activity has been proposed as the dominant mechanism in spontaneous and early termination of supraventricular tachyarrhythmias. however, no significant sinus pauses occurred at the termination of tachycardia episodes on pacing protocols or successful ablation. this also would suggest that a common denominator like vagotonia accounted for the spontaneous termination of tachycardia and the sinus pauses followed. like the av node, the ap mediating pjrt is also known to be vagal sensitive. interestingly, the termination with a block on the antegrade limb was more commonly noted in this case presumably related to a higher sensitivity of the av node to vagotonia compared to the ap.

a young male presented with incessant narrow qrs tachycardia and left ventricular dysfunction. 24-holter monitoring revealed multiple episodes of sustained and nonsustained episodes of tachycardia with prolonged sinus pauses at termination. the analysis of the electrocardiogram, followed by an invasive electrophysiological study, suggested an unusual mechanism for this tachy - brady syndrome.

while trying to integrate multiple data sets collected by different researchers, we noticed that the sample names were frequently entered inconsistently. most of the variations appeared to involve punctuation, white space, or their absence, at the juncture between alphabetic and numeric portions of the cell line name. reasoning that the variant names could be described in terms of mutations or deletions of character strings, we implemented a simple version of the needleman - wunsch global sequence alignment algorithm and applied it to the cell line names. incorrect matches only occured when a cell line was present in one data set but not in the other. a simple application of the needleman - wunsch global sequence alignment algorithm provides a useful first pass at matching sample names from different data sets.

: while trying to integrate multiple data sets collected by different researchers, we noticed that the sample names were frequently entered inconsistently. most of the variations appeared to involve punctuation, white space, or their absence, at the juncture between alphabetic and numeric portions of the cell line name.:reasoning that the variant names could be described in terms of mutations or deletions of character strings, we implemented a simple version of the needleman - wunsch global sequence alignment algorithm and applied it to the cell line names. all correct matches were found by this procedure. incorrect matches only occured when a cell line was present in one data set but not in the other. the raw match scores tended to be substantially worse for the incorrect matches.:a simple application of the needleman - wunsch global sequence alignment algorithm provides a useful first pass at matching sample names from different data sets.

angle class iii malocclusion has raised controversies among researchers concerning diagnosis, prognosis, and treatment. it affects 5% of the brazilian population, with a greater incidence in people of asian origin. in terms of etiology , this problem can have either a genetic origin, with a more unfavorable prognosis, or an environmental origin caused by more anterior and inferior tongue positioning, habits, and oral breathing. class iii malocclusion can be classified as dentoalveolar, skeletal, or functional, which will determine the diagnosis and prognosis. ideally, diagnosis of this malocclusion should be made as early as possible, still during deciduous dentition. early recognition of this discrepancy depends on a careful observation of a sequence of facial, occlusal, and cephalometric characteristics. it is known that class iii malocclusion exacerbates during growth, mainly starting at adolescence. therefore, in children, this malocclusion is not totally defined, and the not yet established facial and occlusal features can complicate the diagnosis. the earlier the interceptive phase is initiated, the greater the orthopedic effects will be to the detriment of the unavoidable orthodontic effects. moreover, an early benefit in terms of esthetics for the child implies improved self - esteem, considering the psychological factor. among the approaches for treating class iii malocclusion is the use of orthopedic appliances, such as chincups, facial masks, functional orthopedic appliances of the jaws, preventive orthodontic appliances (e.g. : eschler arch and porter appliance or " w " arch), multibracket fixed appliances, and a combined orthodontic and orthognatic surgery protocol. the correct indication of orthodontic therapy with a chincup depends on a precise diagnosis of class iii malocclusion. it is advisable to initiate the treatment at an early age, using an upward and backward force from 350 g to 500 g. a cephalometric study performed by graber showed that the use of a chincup promoted a backward movement of point b, due to a clockwise rotation of the mandible. the length of the mandible also decreased about 1 mm due to the pressure transmitted by the chincup to the condyle, which generated, on the other hand, a delay in vertical growth. in another study, evaluated the skeletal changes produced before, during and after chincup therapy. the authors concluded that chincup therapy would be a very useful and efficient method for correcting class iii with mandibular prognathism. verified that an early treatment with a chincup produced positive orthopedic effects on the mandible; however, they did not assure an improvement in the skeletal profile. another possibility of early interception in class iii treatment consists of an orthopedic / orthodontic appliance, the so - called eschler arch. this appliance has a modified labial bow, which will gently touch the lower incisor labial surface, and an acrylic occlusal bite - raising appliance, which affords normal growth of the maxilla, and helps the correction of the negative overjet. should it be necessary to correct the upper incisor inclination, finger springs can be used for their protrusion. considering the aforementioned therapeutic possibilities, the aim of this study was to describe and discuss the treatment of a patient with angle class iii malocclusion, treated according to a two - stage approach (interceptive and corrective), and a long - term follow - up period. a 9-year - old female patient in the mixed dentition stage (second transitional period) was referred for treatment with a chief complaint of an anterior crossbite. during the clinical interview, facial evaluation showed lack of development of the middle third, which is an apparently normal feature for asians. intraoral examination revealed a forward shift of the mandible, with a marked mesial molar relationship, and a crossbite of the four permanent incisors, thus characterizing a functional class iii malocclusion (figure 1). pretreatment extraoral (a and b) and intraoral (c - g) photographs. initial panoramic x - ray (h) and lateral cephalogram (i) (parents signed informed consent authorizing the publication of these pictures) a panoramic radiograph revealed the presence of all permanent teeth either erupted or in several developing stages. careful evaluation of lateral cephalograms confirmed a class iii malocclusion, with an acute nasolabial angle, and a horizontal growth pattern. following the confirmation of a class iii malocclusion through the cephalometric analysis , clinical differential diagnosis was accomplished by verifying the occlusion pattern at either the intercuspal position (ip) or at the centric relation (cr). the patient showed a crossbite at maximal habitual intercuspation with a forward shift of the mandible and, at cr, a retroposition of the mandible with edge - to - edge contact between the upper and lower incisors. this clinical condition confirmed a functional class iii malocclusion, which greatly favors the orthodontic treatment (figure 2). frontal intraoral view showing the edge - to - edge contact of the incisors - centric relation (b) the patient was at a mixed dentition stage, with great potential of growth, so the main goal of the treatment was to correct the anterior crossbite, while also correcting the functional forward deviation of the mandible, and allowing the maxilla to be in a forward position in relation to the mandible, thus affording a normal development. the proposed treatment protocol comprised two stages: the interceptive and the corrective phases. in the first phase, a chincup was used only at night to maintain mandibular retrusion, and the eschler appliance, also known as " progenic appliance ", was used during the day. the eschler appliance is composed of: a ) retention clasps, e.g. adams clasps for molars, and intermolar auxiliary clasps for deciduous teeth and premolars, b ) an eschler labial bow, made in 0.9-mm wire, and adapted at the labial surface of the lower incisors, c ) an occlusal bite - raising appliance in acrylic resin with a thickness of 2 to 3 mm. if necessary, springs and an expansor screw can be added (figure 3). extraoral (a) and intraoral (b) photographs (parents signed informed consent authorizing the publication of this picture) the chincup was activated by 1/2 inch elastics. these were changed either weekly or whenever necessary, and produced a force of 350 g to 500 g on each side, directed at an angle of 45 in relation to the occlusal plane. activation of the eschler bow was performed by closing the loop sufficiently to make the bow touch the labial surface of the lower incisors, without overpressure, since the 0.9-mm wire exerts intensified force due to its diameter. this appliance was intended to produce an orthopedic forward movement of the maxilla, and an orthodontic lingual movement of the lower incisors. after correcting the crossbite, this was accomplished in order to allow the eruption of the premolars, eliminate the free space caused by the occlusal opening, and avoid a tongue interposition, and a possible posterior open bite. follow - up appointments were scheduled until the complete correction of the anterior crossbite, totalizing a treatment period of 14 months. when the appliances were removed, the patient was seen every six months, up to the complete development of the permanent dentition, characterizing thus the end of the interceptive phase. in this phase, a marked improvement in both facial harmony and occlusion was observed (figure 4). end of interceptive phase: extraoral (a and b) and intraoral (c - e) photographs at the end of the interceptive phase, panoramic x - ray (f) and lateral cephalogram (g) (parents signed informed consent authorizing the publication of these pictures) lateral cephalograms (figure 4) of the end of the interceptive phase showed the successful of an early treatment with an eschler appliance associated with a chincup. a panoramic radiograph showed the end of the mixed dentition, a good root parallelism, and the presence of the third molars (figure 4). during the development of the occlusion, the patient was concerned about the gradual increase of the diastema between the central incisors. however, this condition was expected, since the growing mandible caused the proclination of the incisors, thereby increasing arch length (figure 5). two - year follow up after interceptive phase: extraoral (a) and intraoral (b) photographs presenting the diastema between upper central incisors. lateral cephalogram (c) and panoramic x - ray (d) (parents signed informed consent authorizing the publication of this picture) approximately 2 years after the interceptive phase, and due to the patient's dissatisfaction with the diastema, the second phase of this protocol was initiated with the installation of a fixed appliance (figure 6). the corrective phase aimed to close the diastema and perform small corrections, i.e., axial inclinations. it lasted for about 14 months and the are shown in figure 7. intraoral photographs showing the fixed appliances (a - c) end of corrective phase: extraoral (a, b) and intraoral (c - e) photographs, panoramic x - ray (f) and lateral cephalogram (g) (parents signed informed consent authorizing the publication of these pictures) ten years after the corrective treatment, a new follow - up appointment verified stability of the treatment, and the final can be seen in figure 8. follow - up at 10 years after the treatment: extraoral (a and b) and intraoral (c - g) photographs, panoramic x - ray (h) and lateral cephalogram (i) (patient signed informed consent authorizing the publication of these pictures) superposition of initial, final, and follow - up cephalometric tracings of interceptive phase (a). superposition of initial, final, and follow - up cephalometric tracings of corrective phase (b) in this case, class iii was intercepted, and a fixed appliance was installed only to correct small rotations, the anterior diastema, and to improve axial teeth relationships. after the cephalometric analysis (table 1), it was verified that the sna angle continued to increase, while the snb angle and co - gn were unaltered during the interceptive phase. the measurements representing the vertical position of the mandible, fma, and sn.gogn, were stable. the changes in linear and angular measurements of the upper and lower incisors contributed to obtain a positive overjet. from 9 to 12 years of age a proclination of the upper incisors from 20 to 25, and a retroclination of the lower incisors from 30 to 22 were observed. initial, final, and post - interceptive control cephalometric values corrective orthodontic treatment was initiated 2 years after the finalization of the interceptive phase. table 1 shows the cephalometric values at the initial, final, and 10-year post - corrective follow - up. it was observed that the anb angle remained positive due to the stability of both sna and snb angles, as well as those for fma and sn.gogn. conversely, co - gn showed an increase of 13 mm, from the end of the interceptive phase to the 10-year follow - up after the corrective phase, showing a value similar to normal mandibular growth. the measurements related to incisor inclinations remained stable at the 10-year follow - up, contributing for the maintenance of the positive overjet. the cephalometric analysis of the case under study demonstrated an increase of the anb angle, mandible growth, and mandibular plane stability. the anb was altered to a favorable value in the relationship of the jaws due to the treatment. the linear and angular measurement changes of upper and lower incisors helped to obtain a positive overjet. , in a longitudinal study with skeletal class iii patients, radiographically evaluated the skeletal changes before, during and after chincup therapy. these authors demonstrated that the favorable outcomes obtained were limited to the corrective phase, but returned to the initial features, at the post - treatment period. this demonstrates that the prognosis of cases with great skeletal involvement would not be favorable, unlike the cases described in this report. according to previous studies, there should be proportional values between co - a and co - gn. this case initially demonstrated a co - a measurement of 80 mm, which should be 83 mm, suggesting a maxillary deficiency. however, at the last follow - up appointment, this measurement was 88.5 mm, which has been considered appropriate for a co - gn of 114.5 mm. this study demonstrated the achievement of optimal , and the stability of the correction of a functional class iii malocclusion treated with a progenic appliance associated with a chincup, and followed by corrective orthodontics. in spite of the good outcomes achieved in this case, further long - term clinical investigations are necessary to assure the stability of class iii treatment. this case report shows that that the stability of the correction of a functional class iii malocclusion with minor skeletal involvement is related to both the correct diagnosis and the early intervention. this treatment allowed proper facial growth and development, preventing worsening of the malocclusion, with more severe consequences.

angle class iii malocclusion has been a challenge for researchers concerning diagnosis, prognosis and treatment. it has a prevalence of 5% in the brazilian population, and may have a genetic or environmental etiology. this malocclusion can be classified as dentoalveolar, skeletal or functional, which will determine the prognosis. considering these topics, the aim of this study was to describe and discuss a clinical case with functional class iii malocclusion treated by a two - stage approach (interceptive and corrective), with a long - term follow - up. in this case, the patient was treated with a chincup and an eschler arch, used simultaneously during 14 months, followed by corrective orthodontics. it should be noticed that, in this case, initial diagnosis at the centric relation allowed visualizing the anterior teeth in an edge - to - edge relationship, thereby favoring the prognosis. after completion of the treatment, the patient was followed for a 10-year period, and stability was observed. the clinical treatment showed that it is possible to achieve favorable outcomes with early management in functional class iii malocclusion patients.

on reviewing with a detailed approach to titles and abstracts of articles eliminating duplicates, 40 relevant articles were considered. these articles were found in pubmed, science direct, scopus and google scholar from first reported tugse case till date were evaluated. randomized studies, review articles, case reports and abstracts were included while conference papers and posters were excluded. on reviewing with a detailed approach to titles and abstracts of articles eliminating duplicates, 40 relevant articles were considered. these articles were found in pubmed, science direct, scopus and google scholar from first reported tugse case till date were evaluated. randomized studies, review articles, case reports and abstracts were included while conference papers and posters were excluded. the pathogenesis of tugse still remains controversial. among all etiological factors, mucosal trauma (external or internal physical, chemical ; thermal or electrical) appears to be the major instigating factor of this lesion. clinical and histological presentation is, as an ulcerated lesion with a massive immune response. the inflammatory infiltrate in tugse exhibits sheets of atypical mononuclear cells (predominantly t - cell lymphocyte population), eosinophils, macrophages / histiocytes, and few plasma cells. in earlier studies , immunohistochemistry revealed these atypical mononuclear cells to be belonging to the macrophage and myofibroblast lineage. more recent studies point toward the t lymphocytic function of these cells immunohistochemically, with positivity against all t - cell specific antigens. of late, tugse is agreed upon as a reactive lesion dominated by predominant clonal t - cell population. the exact role of these inflammatory cells to produce such an exaggerated inflammatory response mimicking a malignancy is still unknown. correlating various studies, this appears similar to a hypersensitivity type reaction (predominant t - cell infiltration exhibiting a delayed type of cell - mediated immunity). traumatic ulcerative granuloma with stromal eosinophilia is evoked in most cases, by a traumatic antigen, wherein existing macrophages and histiocytes act as antigen presenting cells. various antigens include toxins, viral, microorganisms, endogeneous degradation products or foreign proteins. on release of cytokines, the predominant cells of eosinophils and lymphocytes are recruited to the submucosal tissue. however, previous studies suggest that the exact presence of eosinophils remains controversial, as most of the traumatic ulcers are devoid of eosinophilic infiltration. on the contrary, certain other authors have concluded that there is increased prevalence of histiocytes and eosinophils than lymphocytes suggesting the aggressiveness of tugse. this process of cyclic reaction continues with increase in proliferating t - cells by release of cytokines / toxic products by the degranulating eosinophils. the accumulation of all these inflammatory cells along with the varied spectrum of cytokines in tissue damage. in addition, studies also depict that a response ing from an exaggerated mast cell - eosinophil reaction may arise similar to that noticed in the pathogenesis of type 1 hypersensitivity reaction. degranulating mast cells releases several mediators like eosinophil chemotactic factor, aryl sulfates arid histamines, and major basic proteins, eventually causing tissue destruction. what is interesting to the researchers, is the uniqueness of tugse in being a slow self - healing ulcer with atypical mononuclear cells and massive eosinophilia. eosinophils appear to be a reactive tissue response to an unknown antigen entering via mucosal trauma. the delayed healing process may be attributed to the decreased synthesis of transforming growth factor, released by the eosinophils in the inflammatory infiltrate of tugse as compared to the eosinophils in other traumatic ulcers. furthermore, eosinophils produces a spectrum of cytokines, like tumor necrosis factor, which induces further tissue damage. the pathogenesis of tugse still remains controversial. among all etiological factors, mucosal trauma (external or internal physical, chemical ; thermal or electrical) appears to be the major instigating factor of this lesion. clinical and histological presentation is, as an ulcerated lesion with a massive immune response. the inflammatory infiltrate in tugse exhibits sheets of atypical mononuclear cells (predominantly t - cell lymphocyte population), eosinophils, macrophages / histiocytes, and few plasma cells. in earlier studies , immunohistochemistry revealed these atypical mononuclear cells to be belonging to the macrophage and myofibroblast lineage. more recent studies point toward the t lymphocytic function of these cells immunohistochemically, with positivity against all t - cell specific antigens. of late, tugse is agreed upon as a reactive lesion dominated by predominant clonal t - cell population. the exact role of these inflammatory cells to produce such an exaggerated inflammatory response mimicking a malignancy is still unknown. correlating various studies, this appears similar to a hypersensitivity type reaction (predominant t - cell infiltration exhibiting a delayed type of cell - mediated immunity). traumatic ulcerative granuloma with stromal eosinophilia is evoked in most cases, by a traumatic antigen, wherein existing macrophages and histiocytes act as antigen presenting cells. various antigens include toxins, viral, microorganisms, endogeneous degradation products or foreign proteins. on release of cytokines, the predominant cells of eosinophils and lymphocytes are recruited to the submucosal tissue. however, previous studies suggest that the exact presence of eosinophils remains controversial, as most of the traumatic ulcers are devoid of eosinophilic infiltration. on the contrary, certain other authors have concluded that there is increased prevalence of histiocytes and eosinophils than lymphocytes suggesting the aggressiveness of tugse. this process of cyclic reaction continues with increase in proliferating t - cells by release of cytokines / toxic products by the degranulating eosinophils. the accumulation of all these inflammatory cells along with the varied spectrum of cytokines in tissue damage. in addition, studies also depict that a response ing from an exaggerated mast cell - eosinophil reaction may arise similar to that noticed in the pathogenesis of type 1 hypersensitivity reaction. degranulating mast cells releases several mediators like eosinophil chemotactic factor, aryl sulfates arid histamines, and major basic proteins, eventually causing tissue destruction. what is interesting to the researchers, is the uniqueness of tugse in being a slow self - healing ulcer with atypical mononuclear cells and massive eosinophilia. eosinophils appear to be a reactive tissue response to an unknown antigen entering via mucosal trauma. the delayed healing process may be attributed to the decreased synthesis of transforming growth factor, released by the eosinophils in the inflammatory infiltrate of tugse as compared to the eosinophils in other traumatic ulcers. furthermore, eosinophils produces a spectrum of cytokines, like tumor necrosis factor, which induces further tissue damage. traumatic ulcerative granuloma with stromal eosinophilia presents as a chronic slow progressive lesion (commonly fourth to sixth decade). predominantly, tugse appears to be more common in males than females (1.6:1) which may exist from 1-week to several weeks. it appears as a red lesion in oral mucosa with rolled, elevated and indurated margins with central areas of fibrinopurulent membrane mimicking squamous cell carcinoma. even though, tongue is the most common site, other regions like buccal mucosa, vestibular mucosa, gingiva, lip, mucobuccal fold, retromolar area, and palate also may exhibit tugse. histopathologically, tugse exhibits ulceration with diffuse polymorphic inflammatory infiltrate (exhibiting epitheliotropism) extending from the superficial epithelium into the deep connective tissue. this dense chronic inflammatory infiltrate includes small round t - lymphocytes, large atypical mononuclear cells, granular macrophages, b - lymphocytes massive eosinophils, plasma cells, and histiocytes in the superficial layer and deep muscle layers. proliferating endothelial cells, blood vessels and focal areas of necrosis are seen scattered throughout the lesion. however, presence of massive eosinophilia is not mandatory in this lesion. a tugse lesion often seen in infants (riga - fede disease) occurs within 1-week to 1-year. mucosal lesions are often caused by repetitive traumatic damage due to backward and forward motions of the tongue over the erupting lower incisors, natal or neonatal teeth. rarely, this lesion has also been termed as atypical histiocytic granuloma as studies have shown diffuse scattered atypical large mononuclear cells (large cells with irregular nuclear contours, fine chromatin, small nucleoli, and abundant cytoplasm) in a of diffuse inflammatory cell infiltrate. immunohistochemically, most of these mononuclear cells show cd30, cd3 and t - cell intracytoplasmic antigen 1 (t - cell lineage markers) positivity for t - cells, while few are positive for cd68 macrophages / histiocytic origin and 1050% cells show ki-67 with high degree of production. literature also previously stated that mononuclear cells show factor xiii positivity suggestive of dendritic cells and vimentin positive suggestive of myofibroblasts. regarding the existence of predominantly cd30 +, what remains unresolved is whether this lesion tugse is just a benign oral counterpart of cd30 + lymphoproliferative disease (lpd) or a reactive benign primary lesion or just a histological simulator of cd30 + lpd. immunohistochemical studies are slightly in favor of tugse being a benign oral counterpart of primary cutaneous cd30 + lpds. confusion however persists, as cd30 + is expressed on b and t lymphocytes, not only in malignancies, but also in many other lymphoproliferative disorders. the polymerase chain reaction of t - cell receptor gene gamma with monoclonality is suggested for a more definitive diagnosis. differential diagnosis includes primary sypilitic chancre, cd30 + lpd, malignant t lymphoma, angiolymphoid hyperplasia with eosinophilia, langerhans cell histiocytosis, pseudolymphoma, metastatic tumors, kimura's disease, salivary gland tumors, lymphomatoid papulosis. the mere presence of eosinophils should differentiate from insects / parasites, granuloma faciale, allergic reactions, angiolymphoid hyperplasia with eosinophilic lymphoid granuloma, eosinophilic fascitis, eosinophilic granuloma, and histiocytosis x. traumatic ulcerative granuloma with stromal eosinophilia lesions are treated with antibiotics like penicillin, removal of traumatic agents, followed by excision if still persistent. appropriate management of the offending tooth is suggested in case of riga - fede disease. other modes of treatment include 0.1% triamcinolone acetonide mouthwash, oral antibiotics, steroids (topical / systemic), electrocoagulation, irradiation, and liquid nitrogen. due to the aggressiveness of the lesion, though a few cases show a retarded healing phase, majority of them show a rapid healing following surgery. prognosis of tugse is normally favorable, yet a long term follow up to at least 2 years is mandatory. this condition likely represents a group of related disorders with overlapping clinical and histopathologic features. we, as clinicians, must have adequate knowledge regarding tugse pathogenesis, and perform biopsy for a histological analysis along with a immunohistochemistry techniques, to rule off other neoplastic lesions at a very early stage.

oral ulcers are a common symptom in clinical practice. among various causative factors, different types of ulcers in oral cavity exist. among this , traumatic ulcerative granuloma with stromal eosinophilia (tugse) appears to be quite neglected by the clinicians due to the limited knowledge and awareness. on reviewing with a detailed approach to titles and abstracts of articles eliminating duplicates, 40 relevant articles were considered. randomized studies, review articles, case reports and abstracts were included while conference papers and posters were excluded. of importance, tugse cases been reported only to a minimal extent in the literature. lack of its awareness tends to lead clinicians to a misconception of cancer. thus, this particular lesion needs to be differentiated from other malignant lesions to provide a proper mode of treatment. the present article reviews various aspects of the tugse with emphasis on the clinical manifestation, pathogenesis, histological, and immunohistochemical study. this study provides the clinician contemporaries, a humble expansion to their knowledge of the disease, based on the searched literature, enabling a more comprehensive management of this rare occurrence.

vasospastic disorder (vd) is a relatively common disorder worldwide population, with a prevalence of 3.3% to 22%. episodic cyanosis, swelling, and pallor on the distal part of the extremities with cold exposure are the main characteristic features of this disorder. this disorder has female dominancy and is especially seen in the 2 and 3 decades of life. if the pathogenesis depends on an underlying disorder, it is called secondary (obstructive) form. the remaining form without any underlying disorders is called primary form. since the first description of this disorder, several etiologic factors have been reported to be responsible for the pathogenesis, but it is still not well known, and every possible etiologic factor is still controversial. in addition, no objective, quantitative diagnostic tool or objective follow - up method has been defined. in this study, we aimed to answer the question can doppler ultrasonography be used for objective follow - up of patients with vasospastic disorder? this is a retrospective study, approved by the local ethics committee of gulhane military academy of medicine. we aimed to describe a quantitative method for the follow - up of patients with vasospastic disorder. between april 2011 and october 2013, data were collected from files of 46 patients with the diagnosis of vasospastic disorder lasting more than 2 years. the diagnostic criteria, which were defined by wigley et al., were used as diagnostic criteria for the enrolled patients. several etiologic disorders such as systemic sclerosis and sle, which can cause secondary vasospastic disorder, were excluded, with detailed physical examination and several laboratory findings. demographic data and additional risk factors of patients were recorded. in our department, the cold stimulation test (cst) is routinely performed to all patients with vasospastic disorders symptomatology due to our national and military regulations. according to our protocol, application of the cst is as follows: 1. hand temperatures of the patients are measured with a probe, which is inserted between the pulp of first and second distal phalanges. at the same time , radial arterial blood flow rate is measured at the level of wrist by doppler usg (detailed below). the most affected hand is immersed into the iced - water at 4c for 20 seconds. 3. after drying the hands, the temperature and the blood flow rate of the radial artery are measured again at the 5, 10, 15 and 20 minutes, with the same technique detailed before. the test is performed after resting at room temperature (approximately 26c) for at least 30 minutes. patients are classified according to their smoking habits, but a standardized protocol about timing of refraining from tobacco prior to testing was not used. in addition to cst, we routinely use a scale (verbal complaint severity scale, vcss), which is used by our department for evaluating the progress in the follow - up period. in this scale , a physician asks all of the patients about their symptoms for determining their progress. the scale is detailed in table 1. simultaneously with cst, patients radial arterial blood flow rate was measured at the level of the wrist. for this measurement, radial arterial blood flow velocity was calculated at the level of the wrist on color doppler ultrasonography (figure 1). logiq book xp scanner (ge medical systems, usa) is used as the doppler usg device. in our clinic this combination includes pentoxifylline 1200 mg / d bid (2600 mg) as a vasodilator molecule, nifedipine 60 mg / d bid (230 mg) as a calcium - channel blocker, and acetylsalicylic acid 300 mg / d (1300 mg) as an antiplatelet agent. after 2 months, we evaluate all patients again to observe their progress. after the second evaluation, patients who do not respond to treatment are referred to interventional treatment methods, or they are not allowed to continue their military duties. demographic data, co - morbid disorders, vcss, and blood flow rates of the included patients at pre - treatment and post - treatment period were all recorded. spss for mac 20.0 package program (spss inc, chicago, il) was used for statistical evaluation. descriptive are expressed as mean standard deviation for normally distributed continuous variables and median values for abnormally distributed continuous variables. comparisons of the parametric values were performed with student t test for normally distributed groups and with mann - whitney u test and wilcoxon signed ranks test with abnormally distributed groups. pearson s chi - square test, fisher s exact test, and mcnemar - bowker test were used for the comparisons of categorical variables. a p value of < 0.05 was considered as statistically significant with a 95% confidence interval. between april 2011 and october 2013, data were collected from files of 46 patients with the diagnosis of vasospastic disorder lasting more than 2 years. the diagnostic criteria, which were defined by wigley et al., were used as diagnostic criteria for the enrolled patients. several etiologic disorders such as systemic sclerosis and sle, which can cause secondary vasospastic disorder, were excluded, with detailed physical examination and several laboratory findings. in our department, the cold stimulation test (cst) is routinely performed to all patients with vasospastic disorders symptomatology due to our national and military regulations. hand temperatures of the patients are measured with a probe, which is inserted between the pulp of first and second distal phalanges. at the same time , radial arterial blood flow rate is measured at the level of wrist by doppler usg (detailed below). the most affected hand is immersed into the iced - water at 4c for 20 seconds. 3. after drying the hands, the temperature and the blood flow rate of the radial artery are measured again at the 5, 10, 15 and 20 minutes, with the same technique detailed before. the test is performed after resting at room temperature (approximately 26c) for at least 30 minutes. patients are classified according to their smoking habits, but a standardized protocol about timing of refraining from tobacco prior to testing was not used. in addition to cst, we routinely use a scale (verbal complaint severity scale, vcss), which is used by our department for evaluating the progress in the follow - up period. in this scale , a physician asks all of the patients about their symptoms for determining their progress. simultaneously with cst, patients radial arterial blood flow rate was measured at the level of the wrist. for this measurement, radial arterial blood flow velocity was calculated at the level of the wrist on color doppler ultrasonography (figure 1). logiq book xp scanner (ge medical systems, usa) is used as the doppler usg device. in our clinic, we routinely use a combination therapy for vasospastic disorders for a 2-month period. this combination includes pentoxifylline 1200 mg / d bid (2600 mg) as a vasodilator molecule, nifedipine 60 mg / d bid (230 mg) as a calcium - channel blocker, and acetylsalicylic acid 300 mg / d (1300 mg) as an antiplatelet agent. after 2 months, we evaluate all patients again to observe their progress. after the second evaluation, patients who do not respond to treatment are referred to interventional treatment methods, or they are not allowed to continue their military duties. demographic data, co - morbid disorders, vcss, and blood flow rates of the included patients at pre - treatment and post - treatment period were all recorded. spss for mac 20.0 package program (spss inc, chicago, il) was used for statistical evaluation. descriptive are expressed as mean standard deviation for normally distributed continuous variables and median values for abnormally distributed continuous variables. comparisons of the parametric values were performed with student t test for normally distributed groups and with mann - whitney u test and wilcoxon signed ranks test with abnormally distributed groups. pearson s chi - square test, fisher s exact test, and mcnemar - bowker test were used for the comparisons of categorical variables. a p value of < 0.05 was considered as statistically significant with a 95% confidence interval. in turkey, military service is mandatory for every male aged 20 years. therefore, all the study population was young males; 32 of the patients (69.5%) were smokers and 14 of these 32 patients were smokers for more than 10 years. none of the patients had co - morbid disorders such as hypertension or diabetes mellitus. the most common complaints were cyanosis (34 patients, 73.9%), numbness (30 patients, 65.2%), and hyperhidrosis (29 patients, 63%). there was a significant decrease in arterial blood flow after cold exposure in both pre - treatment and post - treatment measurements. in the rewarming period , flow rate did not reach its basal values over the test time (20 minutes) in pre - treatment measurements, but it reached its basal value by approximately 10 minute in post - treatment measurements. the changes of arterial blood flow rates during the cst are given in figure 2. there were statistically significant differences between pre - treatment and post - treatment arterial blood flow rates at each measurement time points (p<0.001) except initial measurement (p>0.05). on post - treatment values, there were 10.040.78 cm / s increase in the 5 minute, 6.251.39 cm / s in 10 minute, 6.432.13 cm / s in 15 minute, and 6.381.86 cm / s in 20 minute measurements. all increases at the 5 time points were statistically meaningful when compared to their pre - treatment corresponding time points (p<0.001). in turkey, military service is mandatory for every male aged 20 years. therefore, all the study population was young males; 32 of the patients (69.5%) were smokers and 14 of these 32 patients were smokers for more than 10 years. none of the patients had co - morbid disorders such as hypertension or diabetes mellitus. the most common complaints were cyanosis (34 patients, 73.9%), numbness (30 patients, 65.2%), and hyperhidrosis (29 patients, 63%). patients were categorized into 3 groups according to vcss. there were no statistically significant differences in patient vcss distribution. there was a significant decrease in arterial blood flow after cold exposure in both pre - treatment and post - treatment measurements. in the rewarming period , flow rate did not reach its basal values over the test time (20 minutes) in pre - treatment measurements, but it reached its basal value by approximately 10 minute in post - treatment measurements. the changes of arterial blood flow rates during the cst are given in figure 2. there were statistically significant differences between pre - treatment and post - treatment arterial blood flow rates at each measurement time points (p<0.001) except initial measurement (p>0.05). on post - treatment values , there were 10.040.78 cm / s increase in the 5 minute, 6.251.39 cm / s in 10 minute, 6.432.13 cm / s in 15 minute, and 6.381.86 cm / s in 20 minute measurements. all increases at the 5 time points were statistically meaningful when compared to their pre - treatment corresponding time points (p<0.001). several diagnostic methods and invasive and non - invasive techniques were defined for the diagnosis of vasospastic disorders over time. however, no definitive diagnostic tool or objective and quantitative follow - up method has been defined. the main cause of this failure is the complexity of the etiopathogenesis, which is still unclear. most patients usually complain of many subjective symptoms such as cyanosis, numbness, or painful attacks, which are often aggravated by cold exposure. therefore, objective diagnosis of primary vasospastic disorders is often not possible. to address this diagnostic problem, several tools (e.g., triphasic color changes of hands, finger systolic blood pressure, finger - brachial index, plethysmography, capillaroscopy, and rarely, more invasive techniques) were used in the history of treating this disorder. unfortunately, most of these diagnostic tools require special conditions special technicians, devices, and sometimes laboratory conditions that can not be used in outpatient conditions. it is also too hard to use these techniques in follow - up, even if they could be used for diagnosis. however, for evaluating patient progress, objective, quantitative, and easily performed tests are necessary. cst and its application method are still not standardized worldwide and there are few studies about this method in the literature. we have used our clinical protocol, as detailed above, for approximately 3 years. used 5c water for cold stimulation in their study and they declared that this temperature is better for provoking the vasospasm than water at 10c. therefore, we routinely used 4c ice - water for provoking the vasospasm, as in our present study. in the literature there are few studies about cold stimulation and its effect on ultrasonographic arterial blood flow rates. most of these studies are focused on the differential diagnosis of the primary form vs. secondary form by using doppler ultrasonography. there have been no studies on its usefulness as a marker of treatment efficacy in the follow - up period. we consider that our present study is the first in this area. when we analyzed the ultrasonographic parameters of our study, we observed different patterns of blood flow rates. in pre - treatment measurements, flow rates were not able to reach their basal value throughout the test, which suggests that vasospasm lasts longer. in contrast, in the post - treatment tests, flow rates increased quickly to their baseline values by approximately just 10 minutes. in addition, different from the pre - treatment test, flow rates were above baseline values at the 15 minute measurements, which suggests that vasospasm lasts less than the 15 minutes. in our department , we use the verbal complaint severity scale (vcss) for classification of disease severity. in our study population, despite the given treatment modality, there were no statistically significant changes in patient vcss. when we analyzed these 3 patients and compared their cst independent from the study group, we found that there were no statistically significant differences between ultrasonographic flow rates before and after treatment (p>0.05). in this regard, ultrasonographic flow rate measurements added to cst in our study seem to be helpful for follow - up of patients with vd, both quantitatively and objectively. doppler flowmetry added to standard cold stimulation test for evaluating patients with vasospastic disorders provides better and more objective when compared to patient - oriented subjective scoring systems. this study was performed in a military hospital; therefore, all of the patients were male. the smoking time, smoking cessation time, or the correlation between smoking habits and study protocol were not detailed in this study. the significant cut - off value of ultrasonographic blood flow rate changes (positive or negative) is not well known and was not defined in this study. to define clear - cut values of this parameter this study was performed in a military hospital; therefore, all of the patients were male. the smoking time, smoking cessation time, or the correlation between smoking habits and study protocol were not detailed in this study. the significant cut - off value of ultrasonographic blood flow rate changes (positive or negative) is not well known and was not defined in this study. to define clear - cut values of this parameter

assessing therapeutic efficacy and patient satisfaction objectively and quantitatively has always been a problem in patients with vasospastic disorders. we aimed to present the additive value of ultrasonographic assessment of peripheral arteries secondary to cold stimulation, as a test for treatment efficacy during follow-up.material/methodsarterial blood flow rates were measured from radial artery with doppler usg in patients who presented to our department with vasospastic disorders. ultrasonography was performed at the following intervals; before cold stimulation and at 5th, 10th, 15th, 20th minutes of cold stimulation. patients were controlled by repeat cold stimulation test and doppler us at the 2nd month of the treatment. were analyzed with spss for mac 20.0 package program.we enrolled 46 patients in the study. all patients were male and mean age was 22.32.17 years. most common symptoms were cyanosis and coldness. there were statistically significant differences between pre - treatment and post - treatment arterial blood flow rates at each measurement time point (p<0.001) except initial measurement (p>0.05). on post - treatment values , there were 10.040.78 cm / s increase in 5th minute, 6.251.39 cm / s in 10th minute, 6.432.13 cm / s in 15th minute, and 6.381.86 cm / s in 20th minute measurements. all increases at the 5 time points were statistically meaningful when compared to their pre - treatment corresponding time points (p<0.001).doppler flowmetry added to standard cold stimulation test for evaluating the patients with vasospastic disorders provides better and more objective when compared to the patient - oriented subjective scoring systems.

endophthalmitis caused by filamentous fungi has high ocular morbidity and is associated with a poor visual outcome. recovery of 20/400 or better vision is reported in less than 50% of patients, while up to 60% undergo enucleation.1,2 limitations of antifungals used in the management of filamentous fungal endophthalmitis include the fungistatic nature of the drugs, poor intraocular penetration of topical and systemic antifungals, development of resistance to available antifungal drugs, and lack of routine susceptibility testing of fungal isolates.3,4 thus, current antifungals could fail to eradicate the disease when used as monotherapy. combining drugs like amphotericin b and voriconazole, which have complementary mechanisms of action, has been reported in the successful management of refractory systemic aspergillus infections.5 however, there are reports of disparity in for the in vivo and in vitro effects of combining the two different groups of antifungal drugs.6,7 we report our experience with use of a combination of intravitreal amphotericin b and voriconazole in the management of 12 consecutive cases of filamentous fungal endophthalmitis. this was a retrospective study of 12 consecutive endophthalmitis cases caused by filamentous fungi that were managed at our center between january 2013 and august 2013. medical records were reviewed in accordance with the guidelines laid down by the declaration of helsinki. the data collected consisted of demographic details, affected eye, etiology, duration of symptoms, clinical characteristics, visual acuity and intraocular pressure at presentation and at subsequent follow - up. the collected samples, which included corneal scrapings, vitreous biopsy, and explanted intraocular lens, were plated in both bacterial and fungal culture media. microbiology records were reviewed for intraocular samples tested, direct microscopy , and culture characteristics. the details of the treatment course, including time and number of surgical interventions, along with type, number, route, and duration of use of antifungals, were recorded (table 1). twelve consecutive cases of filamentous fungal endophthalmitis were included in this single - center case series. details of demographic features, clinical characteristics, microbiology , treatment, and clinical outcome for all cases are described in table 1. the patients presented to us at a median 22 (mean 28.4, range 2262) days postoperatively. nine post cataract surgery endophthalmitis cases reported onset of symptoms within one week of surgery. however, patients originating from a remote rural area presented with a delay of 2162 days after surgery. two endophthalmitis cases occurred following open globe injury and one following keratitis after pterygium excision. the presenting complaint was a sudden - onset decrease in visual acuity in eleven eyes (91.6%) associated with pain in eight eyes (66.6%). four eyes had associated corneal or scleral tunnel infiltrate, and fungal hyphae were demonstrated on microscopy for all of these. six eyes (50%) presented with hypopyon or anterior chamber exudates, while all eyes had exudates in the vitreous cavity that were either seen clinically or demonstrated on b - scan ultrasonography. all eyes underwent pars plana vitrectomy with injection of antifungals, comprising daily intravitreal voriconazole (100 g/0.1 ml) and alternate day intravitreal amphotericin b (5 g/0.1 ml), known as the amb - vo regimen, until resolution of vitreous exudates. the decision to inject antifungals was based on detection of fungal hyphae on smears from corneal scrapings or clinical suspicion of fungal etiology. all post cataract surgery endophthalmitis cases underwent iol explantation, seven eyes underwent repeat vitrectomy, and three eyes underwent penetrating keratoplasty. systemic antifungal ketoconazole 200 mg twice daily was administered in all cases with corneoscleral involvement. the mean duration of treatment with the amb - vo regimen was 21.5 (range 1330) days. patients with corneal or scleral involvement also received topical 5% natamycin and 1% voriconazole eye drops. globe salvage was achieved in all cases, with a final visual acuity of light perception or better. visual acuity at last follow - up ranged from 20/60 to light perception, with 20/400 or better in seven of the 12 eyes (58.3%) and 20/60 in 2/12 (16.6%) eyes. all eyes had resolution of vitreous exudates as assessed by indirect ophthalmoscopy or by b - scan ultrasonography where the cornea was hazy. the mean presenting and final intraocular pressures were 9.5 (range 217) mmhg and 6.8 (range 310) mmhg, respectively. clinical pictures of the anterior segments from a few patients at presentation and the final visit are shown in figures 1 and 2. culture - proven fungal endophthalmitis was seen in 11/12 eyes (91.6%), while one post cataract surgery endophthalmitis case did not grow any organisms (table 1, case 3). fungal filaments were seen on direct examination of the vitreous sample (calcofluor white and gram stain) in 4/12 (33.3%) cases and in corneal scrapings in 4/12 (33.3%). significant growth of filamentous fungi was seen in cultures for vitreous samples in 7/12 eyes (58.3%) and explanted intraocular lens plated on chocolate agar in 7/12 eyes (58.3%, table 1). characteristic fungal colonies, either cinnamon brown, powdery, or yellowish green, granular were grown, which were suggestive of aspergillus terreus and aspergillus flavus, respectively. the vitreous from the single case of fusarium endophthalmitis showed a creamy, fluffy pinkish colony and spores characteristic of fusarium on microscopy. fungal endophthalmitis is often refractory to antifungal therapy, with poor functional and anatomic outcomes.5,6 the principal objective of this study was to evaluate the role of combined intravitreal amphotericin b and voriconazole in the management of endophthalmitis caused by filamentous fungi. in the current study , we treated filamentous fungal endophthalmitis with pars plana vitrectomy and a combination of intravitreal antifungal agents. this antifungal combination regimen was based on our clinical experience with case 1 (table 1). this patient had undergone an open globe injury repair followed by pars plana lensectomy and vitrectomy for traumatic cataract and vitreous hemorrhage. two months later, a condensed round mass of anterior vitreous exudates was seen clinically on slit - lamp examination through the pupil behind the iris in the inferotemporal quadrant. fungal hyphae were demonstrated in the vitreous aspirate on microscopy and were later identified as a. flavus from growth on culture. based on initial resolution but re - emergence of exudates during this course, we clinically suspected the isolate to be refractory to voriconazole alone and combined it subsequently with intravitreal amphotericin b given every 48 hours. rapid and complete resolution of vitreous exudates was observed at the end of one week (figure 3). we formulated a regimen comprising a combination of intravitreal voriconazole daily and amphotericin b every 48 hours (the amb - vo regimen). the frequency of injections was based on the half - life of amphotericin b and voriconazole in aphakic vitrectomized eyes being 1.8 days and 2.5 hours, respectively.8,9 the in vivo efficacy and the role of a combination of antifungal agents in life - threatening systemic fungal infections has been established.5 combination of intravitreal antifungals in the management of fungal endophthalmitis with a good visual outcome has been reported previously.10 in vitro studies predicting the interaction of antifungal agents and the of combining drugs have demonstrated variable patterns of drug interactions, like synergism, indifference, and often antagonism between these agents.6 amphotericin b, a polyene, is the most commonly used empirical antifungal agent in systemic mycosis and endophthalmitis caused by filamentous fungi.11 it acts by binding to surface sterols in the cell membrane of the fungi, ing in formation of pores and altered permeability.8 voriconazole, an azole, acts chiefly by depleting ergosterol, the chief bioregulator of membrane integrity.11 steinbach et al hypothesized that azoles may render amphotericin b inactive or be adsorbed onto the fungal cell surface and inhibit binding of amphotericin b to the fungal cell membrane.7 amphotericin b, when combined with lipophilic triazoles like itraconazole, was found to be deleterious or ineffective, while amphotericin b when combined with voriconazole was found to be beneficial in a few but not all experimental studies.57 although filamentous fungus developing resistance to amphotericin b or having inherent resistance has been reported, the frequency has not been described.11,12 a. terreus isolated from the endophthalmitis outbreak in the current series has also been reported to be refractory to amphotericin b in in vitro studies.12 wykoff et al have described fungal isolates, treatment strategies, and clinical outcomes in cases with exogenous fungal endophthalmitis.13 systemic antifungals were required in 83% of patients, with additional intraocular antifungals needed in 95% of eyes. fifty - nine percent of patients were treated with multiple antifungal agents and 20% received anti - fungal agents for 36 months. fifty - four percent of patients achieved a final visual acuity of 20/400 or better while 24% eyes lost perception of light including 20% that underwent enucleation. amongst the various categories, 54% of patients with postoperative fungal endophthalmitis achieved a visual acuity of 20/80 or better, while none of the cases with fungal endophthalmitis following open globe injury achieved vision better than 20/400, and 70% of these underwent enucleation. narang et al reported poor visual and anatomic outcomes in eyes with exogenous fungal endophthalmitis, especially when associated with corneal involvement.2 in the current study, at a mean final follow - up of 12.75 weeks, the best corrected visual acuity achieved was 20/400 or better in 7/12 eyes (58.33%) and 20/60 in 2/12 eyes (16.6%). all of the five eyes with corneal involvement developed hypotony and corneal haze or graft opacification with visual acuity of 20/400 or worse. five of six eyes with persistent hypotony had final visual acuity less than 20/400 at last follow - up, with only one such eye recovering a visual acuity of 20/60. unlike previous reports of poor visual and anatomic outcomes in fungal endophthalmitis following open globe injury,13 patients in the current study recovered a visual acuity of 20/100 or better at the last visit. despite a delayed presentation (mean 27.6 days), 5/9 (55.5%) patients who were part of a postoperative outbreak of endophthalmitis caused by a. terreus finally recovered visual acuity of 20/400 or better. the main limitations of this study are its retrospective design and limited follow - up period. further, all patients could not afford systemic antifungal drugs. due to the paucity of such cases , a prospective randomized controlled trial would be difficult to perform, but a head - to - head comparison between voriconazole alone and a combination of voriconazole and amphotericin b would be useful for establishing which regimen is better. the majority (75%) of isolates in the current series were a. terreus, which has previously been reported to be inherently resistant to amphotericin b.12 it would be useful to study the effect of combining amphotericin b and voriconazole in vitro with regard to possible synergism and correlate it with the in vivo response. in , combination intravitreal amphotericin b and voriconazole injections until complete resolution of vitreous exudates could be a promising modality of treatment in the management of exogenous filamentous fungus endophthalmitis.

purposeto report outcomes of exogenous fungal endophthalmitis treated with combination of intravitreal antifungal agents.designretrospective, non - randomized, interventional, consecutive case series.methodstwelve eyes of twelve consecutive cases of filamentous fungal endophthalmitis were treated with a combination of intravitreal amphotericin - b and intravitreal voriconazole (amb - vo regime) along with pars plana vitrectomy at a single center. clinical characteristics, microbiology , treatment strategy, visual, and anatomical outcomes were analyzed.ten cases out of the twelve were postoperative endophthalmitis of which nine were part of a post cataract surgery cluster. the remaining included endophthalmitis following keratitis post pterygium excision and following open globe injury. the most common fungus was aspergillus terreus, which was isolated in 8/12, followed by a. flavus in 2/12 and fusarium solani in 1/12. the presenting visual acuity ranged from light perception (lp) to counting fingers. the visual acuity at final follow - up was 20/400 or better in 7/12 eyes (58.33%) and 20/60 in 2/12 eyes (range 20/60 to lp). all eyes with corneal involvement had final visual acuity 20/400 or worse. globe salvage was achieved in all cases.combining intravitreal amphotericin - b and voriconazole could be a novel treatment strategy in the management of endophthalmitis caused by filamentous fungus. eyes with corneal involvement had poor visual outcome either with or without therapeutic penetrating keratoplasty.

cerebral watershed (or border zone) infarcts (wi) involve the junction of the distal fields of two non - anastomosing arterial systems, which are hemodynamic risk zones. there are two types of wi: cortical wi, occurring at the junction between cortical territories of the anterior, middle and posterior cerebral arteries; and internal wi, occurring in the white matter between the deep and superficial perfusion systems of the middle cerebral artery. clinical picture of watershed infarctions is often progressive or fluctuating; symptoms may be mild and are therefore probably underestimated. another characteristic feature is occurrence of early - onset partial seizures, which in cortical wi occur more often than in other forms of stroke. due to its sub - acute onset and to the association to partial clonic seizures, wi diagnosis can be challenging for physicians. nevertheless, wis account for up to 10% of all cerebral infarcts and they must be kept under consideration in order to avoid inappropriate treatment in the acute phase. in this phase, diffusion weighted brain mri (dwi) allows a clearer diagnosis than cranial computed tomography (ct). we report on the case of a 69-year - old man who presented to the emergency department of our hospital four days after the sub - acute occurrence of homonymous right hemianopia and right hemiparesis. the patient's medical history disclosed smoking habit (78 pack years beginning at an age of 16 years). one year earlier, a non - small cell lung carcinoma was diagnosed and treated consequently with 6 cycles of palliative chemotherapy (carboplatin and gemcitabin). neurological examination on hospital admission revealed paresis of the right upper limb and a severe motor deficit of the right lower limb with intact sensibility. the patient was admitted with a diagnosis of ischemic stroke. and was started on secondary prevention therapy according to international guidelines for ischemic stroke treatment with acetylsalicylic acid 300 mg daily. a few hours after hospital admission, the patient developed partial epileptic seizures of the right upper limb, which required iv phenytoin infusion. carotid ultarsounds demonstrated occlusion of the right extra - cranial carotid artery and tight stenosis of the contralateral internal carotid artery (figure 1a, b). brain diffusion - weighted magnetic resonance (dwi) revealed multiple hypertintense lesions extending within the cortical watershed area of the left hemisphere, suggesting a large watershed infarct (figure 1c, d, e). figure 1duplex scan showing (a) tight stenosis of the left extra - cranial internal carotid artery and (b) occlusion of the right internal carotid artery. diffusion weighted brain - magnetic resonance imaging (mri) showing watershed infarcts within the left hemisphere (c, d); mr angiography showing hypo - perfusion of the right intra - cranial internal carotid artery (e). duplex scan showing (a) tight stenosis of the left extra - cranial internal carotid artery and (b) occlusion of the right internal carotid artery. diffusion weighted brain - magnetic resonance imaging (mri) showing watershed infarcts within the left hemisphere (c, d); mr angiography showing hypo - perfusion of the right intra - cranial internal carotid artery (e). over the following days , the patient had a nearly full clinical recovery, with a residual slight motor deficit of the distal right upper limb at discharge. surgical or percutaneous treatment of left carotid stenosis was excluded in consideration of his advanced neoplastic disease; he continued adequate medical treatment with antiplatelet therapy acetylsalicylic acid 300 mg daily and statin. we report on a typical neurological picture of cortical wi, which represents a complex clinical entity. our patient sought for medical help only four days after symptoms onset, due to slow progression of the neurological deficit. this was associated to partial epileptic seizures of the upper limb, which are common in cortical wi and display a good response to antiepileptic treatment. another typical feature displayed by the present case was the benign clinical course of the ischemic event, which is often seen in cortical wi. sub - acute onset and association to partial seizures could make the diagnosis of stroke quite challenging. dwi proved to be the most useful imaging technique in order to make a right diagnosis in the acute phase of the disease and to understand its pathogenesis. based on the fact that wi may occur during marked systemic hypoperfusion, the supposed pathological mechanism is hemodynamic failure. this theory is supported by the typical localization of wi in low - perfusion cerebral areas, also called distal or border zone fields. moreover, syncope at wi onset, which is occasionally reported, might support the hemodynamic hypothesis. as opposed to the hemodynamic hypothesis, either cardiac or artery - to artery or microembolism various neuropathological and imaging studies found multiple embolic occlusions in the terminal vascular field distal to atherosclerotic carotid plaques, in particular in the cortical form of wi. anyway, a combination of both theories is likely to be the most realistic mechanism, in terms of impaired washout of emboli in case of decreased perfusion. our patient presented with a cortical wi distal to a tight stenosis of the left internal carotid artery associated to occlusion of the right one. hence, hypoperfusion could definitely play a substantial role in the genesis of the reported case. nevertheless, we suggest that this case could support the hypothesis of an impaired washout of emboli in low - perfusion brain areas. indeed, if hypoperfusion was to be the dominant mechanism of the ischemic lesion, watershed area distal to the occluded carotid artery should to have been at even higher risk. on the other hand , our case keeps in line with the concept that embolic more than hemodynamic mechanism plays a crucial role in the pathogenesis of cortical wi. carotid reperfusion would have been an option in this case, although medical therapy alone was the preferred choice due to the high risk of the procedure in this patient and his global prognosis, due to his underlying disease. wi are a frequent clinical entity, accounting for up to 10% of all cerebral infarcts, although they are often under - recognised due to slow progression of the presenting symptoms and to the frequent association to seizures. our case is a very illustrative example of cortical wi with typical onset, course and imaging appearance. we suggest that it supports the hypothesis of impaired washout of emboli in low - perfusion brain areas in cortical wi; treatment should be chosen accordingly.

watershed infarcts (wi) evolve in hemodynamic risk zones. clinical picture of wi can be associated to partial epileptic seizures. diffusion weighted brain magnetic resonance imaging (mri) allows a clear diagnosis. wi pathogenesis involves either embolic or hemodynamic mechanism. a 69-year old patient presented with sub - acute occurrence of right hemiparesis and partial epileptic seizures of the right arm. carotid ultrasounds demonstrated occlusion of the right extra - cranial internal carotid artery (ica) and tight stenosis of the contralateral ica. brain diffusion - weighted magnetic resonance revealed acute ischemic lesions within the watershed area of the left hemisphere. our case supports the hypothesis of impaired washout of emboli in low - perfusion brain areas as the mechanism underlying cortical wi.

dementia is closely associated with aging, and cognitive decline is common in the elderly. in 2010, the number of people diagnosed with dementia was 35 million, and the population is projected to increase to 115 million by 20501. at present, 8.87% of people in china are older than 65 years of age, and the population is expected to rapidly increase to 22.6% by 2050. in china, 12% of dementia patients are 6069 years old, 48% are 7079 years old, and 1520% are more than 85 years old. the population of elderly people with dementia in china is expected to total 20 million by 20252. in recent years, interventions to reduce cognitive dysfunction have been investigated, including memory therapy3, music therapy4, and exercise therapy5. in china, therapeutic exercise has been used as a community nursing intervention, but it is limited to simple exercises such as walking6. tai chi is a traditional chinese martial art and is widely practiced by chinese people. many studies have shown that tai chi has beneficial effects on balance, falls, and non - vertebral fractures7,8,9. tai chi also reportedly improves renal function in patients with chronic renal disease and cardiac function in cardiovascular disease patients via regulation of lipid metabolism10. many studies have investigated methods to prevent dementia symptoms, but few have investigated the effect of tai chi on dementia11, 12. this study aims to investigate whether tai chi can improve physical and cognitive function in elderly chinese subjects. a total 150 fifty elderly people residing in the local communities were recruited by public announcement. the inclusion criteria were as follows: > 60 years of age; mini - mental state examination (mmse) score > 24, indicating no cognitive impairment; sufficient upper limb mobility to perform requisite finger - pointing tasks, such as flexing and extending the shoulder, elbow, wrist, and fingers; and no neurologic or musculoskeletal disease such as stroke or visual and auditory impairments. subjects meeting the inclusion criteria were randomly allocated to the tai chi group or the control group using a random number table (n = 75 per group, fig . 1fig . 1.study protocol and subject recruitment . a total 150 subjects > 60 years old were randomly divided into a tai chi intervention group and a control group . all participants were investigated at baseline and at 3 and 6 months after beginning intervention .). an independent researcher blinded to the number allocation enrolled and assigned subjects to each group. study protocol and subject recruitment. a total 150 subjects > 60 years old were randomly divided into a tai chi intervention group and a control group. all participants were investigated at baseline and at 3 and 6 months after beginning intervention. subjects in the tai chi group practiced 24-form yang style tai chi for 60 minutes twice weekly for 6 months. the control group participated in other activities such as playing cards or singing at the activity center. however, due to a variety of factors such as hospitalization, the intervention group included 72 people after 3 months and the control group 66 people after 6 months. after completing the study, the control group was provided a similar intervention program. before the study, all subjects were given 10 min to read a document on dementia prevention, and a researcher answered any questions concerning the document. each subject completed a questionnaire concerning gender, age, education, disease status, and the functional capacity tokyo metropolitan institute of gerontology index of competence (tmig, 013 points). the tmig is used to measure the functional capacity in daily life; higher scores indicate greater functional ability13. physical function was assessed using the eyes open / leg standing time, 5-m high walk speed, 10-m normal walk speed, left grip strength, and right grip strength14. the subjects were asked to stand on either leg as long as possible while keeping their eyes open to assess their static balance. the time from when the subject raised a leg until the raised leg touched the ground was measured twice, and the longer time of the two trials was analyzed. grip strength was measured twice in both hands using an adjustable handheld dynamometer to assess muscular strength, and the highest measure for each hand was used. participants were asked to walk 10 m at normal speed or 5 m at maximal speed, and the mean time was measured using a stopwatch. cognitive function was measured using the mmse (030 points) and frontal assessment battery (fab) at bedside (018 points)15, 16. for the mmse, 2324 points indicates mild dementia, while higher scores indicate better cognitive function. for the fab at bedside, data were analyzed using the test before and after intervention in the tai chi group and control group. statistical analysis was performed using spss 13.0, and p < 0.05 was considered statistically significant. of these, 138 subjects completed the 6-month study program, and 12 subjects (tai chi group n = 3, control group n = 9) were lost to follow - up. as a , follow - up data were available for 72 of 75 subjects in the tai chi group and 66 of 75 subjects in the control group. table 1table 1.subjects characteristicsparametertai chi group, n = 72 (%) control group, n = 66 (%) gendermale14 (19.4)20 (30.3)female58 (80.6)46 (69.7)age (years)mean sd68.3 5.970.1 5.7606422 (30.6)12 (18.2)656922 (30.6)20 (30.3)707416 (22.2)18 (27.3)757910 (13.9)14 (21.2)80842 (2.8)2 (3.0)education (years)01132 (44.5)18 (27.2)1240 (55.5)48 (72.8)diseasecardiac16 (22.2)8 (12.1)hyperpiesia12 (16.7)16 (24.2)diabetes mellitus4 (5.6)4 (6.1)other18 (25.0)22 (33.3)none22 (30.6)16 (24.2)physical exerciseyes54 (75.0)54 (81.8)no18 (25.0)12 (18.2)exercise frequency<3/w22 (40.7)20 (47.0)3/w32 (59.3)34 (63.0)exercise time (min)<6038 (70.4)36 (66.6)6016 (29.6)18 (33.4)tmig (413 points)mean sd11.6 1.310.8 2.1physical functionone leg standing time with eyes open (s)28.7 27.123.3 23.05-m high walking speed (s)2.9 0.72.8 0.810-m normal walking speed (s)8.7 2.18.9 2.5left grip strength (kgw)24.7 6.923.6 10.3right grip strength (kgw)25.5 7.023.9 10.5brain functionmmse26.4 2.426.8 1.7fab14.6 2.114.1 2.9tmig; functional capacity tokyo metropolitan institute of gerontology index of competence: mmse; mini - mental state examination: fab; frontal assessment battery at bedside: sd; standard deviation, significant difference between groups; the two groups were well matched at the baseline assessment, and there were no obvious differences in the key outcome variables initially, except the tmig score, which was significantly higher in the tai chi group than in the control group (p < 0.01). tmig; functional capacity tokyo metropolitan institute of gerontology index of competence: mmse; mini - mental state examination: fab; frontal assessment battery at bedside: sd; standard deviation, significant difference between groups; p < 0.01 in the tai chi group, the tmig score increased at both 3 and 6 months. the eyes opened / one leg standing time and fab increased at 6 months, but were unchanged at 3 months. by contrast , the right grip strength increased at 3 months, but not at 6 months. there were no significant differences in the eyes open / one leg standing time, left grip strength, and fab at bedside (p > 0.05) between the tai chi and control groups at 3 or 6 months (tables 2table 2.physical and cognitive functions compared between the tai chi and control groupsparametertai chi ( n = 72) mean (sd)control (n=66) mean (sd)pre - intervention3 months6 monthspre - intervention3 months6 monthstmig11.6 (1.3)12.1 (0.9)12.1 (0.9)10.8 (2.1)11.1 (2.0)11.7 (1.4)physical exerciseone leg standing time with eyes open (s)28.7 (27.1)36.9 (38.3)40.0 (47.1)23.3 (23.0)34.3 (37.5)38.0 (40.7)5-m high walking speed (s)2.9 (0.7)2.8 (0.5)2.8 (0.7)2.8 (0.8)3.2 (0.8)3.4 (0.9)10-m normal walking speed (s)8.7 (2.1)8.5 (2.0)8.6 (1.6)8.9 (2.5)9.7 (2.6)9.6 (2.6)left grip strength (kgw)24.7 (6.9)24.9 (6.5)24.9 (8.8)23.6 (10.3)23.1 (11.5)22.8 (10.3)right grip strength (kgw)25.5 (7.0)26.7 (7.2)25.8 (7.7)23.9 (10.5)23.4 (11.5)23.4 (11.0)brain functionmmse26.4 (2.4)27.8 (1.7)28.0 (2.0)26.8 (1.7)27.1 (1.8)27.3 (1.9)fab14.6 (2.1)14.9 (2.4)15.7 (2.4)14.1 (2.9)14.3 (2.8)14.5 (3.2)tmig; functional capacity tokyo metropolitan institute of gerontology index of competence: mmse; mini - mental state examination: fab; frontal assessment battery at bedside: sd; standard deviation, * significant difference between groups; p < 0.001, p < 0.01, p < 0.05, 3table 3.outcome variables after 3 and 6 months tai chi training3 months after intervention6 months after interventiontai chi group(n = 72)mean (sd)control group(n = 66)mean (sd)tai chi group(n = 72)mean (sd)control group(n = 66)mean (sd)tmig0.5 (1.0)0.5 (1.5)0.5 (1.2)0.9 (1.3)physical exerciseone leg standing time with eyes open (s)8.1 (35.5)11.0 (26.1)11.3 (45.0)14.8 (28.9)5-m high walking speed (s)0.1 (0.5)0.4 (0.5)0.1 (0.7)0.6 (0.7)10-m normal walking speed (s)0.2 (2.2)0.8 (1.3)0.1 (1.6)0.8 (1.7)left grip strength (kgw)0.2 (2.9)0.5 (3.5)0.2 (4.3)0.7 (3.3)right grip strength (kgw)1.2 (3.2)0.5 (4.1)0.2 (3.1)0.5 (3.5)brain functionmmse1.4 (2.2)0.3 (2.2)1.6 (1.9)0.5 (2.6)fab0.3 (1.8)0.3 (2.6)1.1 (2.2)0.5 (2.6)tmig; functional capacity tokyo metropolitan institute of gerontology index of competence: mmse; mini - mental state examination: fab; frontal assessment battery at bedside: sd; standard deviation, significant difference between groups; p < 0.001, p < 0.01, p < 0.05 ). tmig; functional capacity tokyo metropolitan institute of gerontology index of competence: mmse; mini - mental state examination: fab; frontal assessment battery at bedside: sd; standard deviation, * significant difference between groups; p < 0.001, p < 0.01, p < 0.05 tmig; functional capacity tokyo metropolitan institute of gerontology index of competence: mmse; mini - mental state examination: fab; frontal assessment battery at bedside: sd; standard deviation, significant difference between groups; p < 0.001, p < 0.01, p < 0.05 participants in the tai chi group had a significantly decreased 5-m high walking speed (p < 0.001), 10-m normal walking speed (p < 0.01), right grip strength (p < 0.01), and mmse (p < 0.01) compared with the control group at 3 months. furthermore, the tai chi group experienced a significantly decreased 5-m high walking speed (p < 0.001), 10-m normal walking speed (p < 0.001), and mmse (p < 0.01) compared with the control group at 6 months (table 2). the 5-m high walking speed test decreased in the tai chi group at both 3 and 6 months, while the control group showed an increasing trend (tables 2, 3). a similar trend was observed for the 10-m normal walking speed at 3 and 6 months (tables 2, 3). the right grip strength significantly increased after 3 months but decreased at 6 months in the tai chi group. in the control group, the right grip strength decreased at both 3 and 6 months (tables 2, 3). finally, the mmse score increased at both 3 and 6 months in the tai chi group (tables 2, 3). this study shows that tai chi practice improves cognitive and physical functions in the elderly. we found that the mmse score significantly increased in the tai chi group compared with the control group, suggesting that tai chi may improve the mental function of the elderly in china. exercise is known to increase regional cerebral perfusion in the contralateral motor sensory cortex, which may explain why tai chi may increase memory and concentration17. although an increasing number of elderly chinese people practice tai chi, few studies have examined the effects of tai chi on preventing dementia. brown et al.18 randomly divided 135 subjects into a tai chi group and control group for 16 weeks of moderate and low intensity exercise programs, and found that exercise with tai chi was more effective than exercise alone in promoting psychological benefits in both men and women. winter and tse19, 20 showed that tai chi could improve walking speed and grip strength in the elderly. consistent with these previous reports, in this study, we found that walking speed was significantly improved in the tai chi group compared with the control group. in addition, schaller21 reported that tai chi practitioners had significantly better eyes open / leg standing times. however, in this study we found that the eyes open / one leg standing time significantly increased in both the tai chi and control groups after 3 and 6 months, and there was no difference between the two groups. of note, the participants in the control group in schaller s study maintained their current activity level, while the control group in this study participated in non - athletic activities such as playing cards or singing. our also showed a significantly increased tmig in both the tai chi and control groups. however, there was no difference between the two groups, which may be because all of the subjects read a document about dementia prevention before beginning the study. currently, family care and community support systems are not well established in china. therefore, it is particularly important to take precautions for the prevention and early treatment of dementia. presently, there are few intervention studies in china on dementia prevention by tai chi. we conducted a randomized controlled trial to evaluate the potential to prevent dementia using tai chi, and our demonstrate that tai chi can improve cognitive function in elderly people. many studies, especially those evaluating older adults or significantly impaired individuals, have reported functional changes over 3 to 6 months following intervention, but the six - month duration of this study only evaluated the potential effect of tai chi on age - related dementia. tai chi is traditionally considered a life - long skill, and it is believed that with time, both proficiency and health benefits improve. long - term interventional studies and well - designed observational studies comparing long - term tai chi practitioners (e.g. > 10 years) to matched controls are needed to fully evaluate the long - term impact of tai chi on healthy aging22. in , our confirmed that tai chi is potentially useful in preventing dementia in elderly people. tai chi could be an easy and effective preclinical treatment strategy of preventing dementia in elderly people and enhancing the quality of life.

to investigate the effect of tai chi on cognitive and physical function in the elderly. a randomized trial design was used. a total 150 subjects were enrolled and were divided into tai chi and control groups. subjects in the tai chi group participated tai chi for 6 months, and subjects in the control group participated in other non - athletic activities. there were no differences between the groups in the one leg standing time with eyes open, left grip strength, or the frontal assessment battery at bedside after 3 and 6 months of intervention. the mini - mental state examination scores after 3 and 6 months were higher in the tai chi group than in the control group. the right grip strength after 3 months increased more in the tai chi group than in the control group. both the 5-m high walking speed and 10-m normal walking speed were significantly lower after 3 and 6 months of tai chi practice. these suggest that regular tai chi practice may improve cognitive and physical function in the elderly.

during february 2001september 2002, we captured 344 wild mammals in an overflow area in a fauna - rescue program during construction of a hydroelectric plant in lajeado and ipueiras counties (94458s, 482123w) in tocantins state, brazil. during this program, 269 capuchin monkeys (cebus apella), 27 black - howling monkeys (allouata caraya), 12 coatis (nasua nasua), 20 agoutis (dasyprocta sp .), 2 opossums (didelphis albiventris), 5 armadillos (euphractus sexcinctus), 5 collared anteaters (tamandua tetradactila), and 4 gray foxes (cerdocyon thous) were captured. all animals were captured in a sylvatic area and did not have contact with humans and dairy cattle. in field - screening laboratories, animals were then released in areas selected during environmental conservation programs. until 2002, bovine vaccinia had been restricted to southeastern brazil, > 1,400 km from the study area. serum samples were inactivated by heating at 56c for 30 min, and an orthopoxvirus plaque - reduction neutralizing test (prnt) was performed. prnt was used rather than elisa because secondary antibodies required for an elisa for all analyzed species were unavailable. inactivated samples were diluted 1:201:1,640 in minimal essential medium and tested in vero cells by using the vacv - western reserve strain in the prnt as described. human samples positive for antibodies to orthopoxvirus obtained during bovine vaccinia outbreaks were used as positive controls; samples negative for these antibodies were used as negative controls. serum titer was defined as the highest dilution that inhibited > 50% of viral plaques compared with negative controls. orthopoxvirus prnt specificity (97.4%) and sensitivity (93.5%) were confirmed by using receiver - operating characteristic analysis, which compared of prnt, elisa, and clinical symptoms during bovine vaccinia outbreaks. of 269 c. apella samples, 68 (25.3%) had antibodies to orthopoxvirus. of 27 a. caraya samples, 13 (48.1%) had antibodies to orthopoxvirus. seropositivity was detected in 2 (16.6%) coatis and 1 (5.0%) agouti. of 344 animals studied, 84 (24.4%) had antibodies to orthopoxvirus (table). in samples with high neutralizing antibody titers, 55.95% had titers of 80320. only 5 (6.0%) prnt - positive samples had titers < 40 (table). given the serologic cross - reactivity of orthopoxvirus, positive samples could indicate any of > 9 virus species, although it is well established that vacv is endemic to brazil, and infections with other orthopoxviruses are geographically restricted to other continents and have not been identified in brazil. therefore, we performed a molecular investigation to identify orthopoxviruses associated with orthopoxvirus sylvatic circulation. serologic and molecular tests were performed in a blinded fashion and in triplicate. on the basis of previous studies that detected orthopoxvirus dna in serum of infected hosts, a semi - nested pcr was used to amplify the highly conserved orthopoxvirus vaccinia growth factor (vgf) gene (j.s . human vacv dna positive and dna negative serum samples obtained during bovine vaccinia outbreaks were used as positive and negative controls, respectively. eighteen of 344 serum samples were positive in pcr assays (11 from c. apella and 7 from a. caraya ; all were prnt positive). six of the 18 vgf pcr - positive samples were chosen for sequencing and analysis of vgf (4 from c. apella and 2 from a. caraya). , we amplified the hemagglutinin (ha) gene from 2 samples (1 from c. apella and 1 from a. caraya ; both were vgf positive). the vgf and ha pcr products were cloned into the pgemt - easy vector (promega, madison, wi, usa). three clones from distinct pcr amplicons of each sample were sequenced in both orientations by using m13 universal primers and the mega - bace - sequencer (ge healthcare, little chalfont, uk). optimal alignment of the highly conserved vgf gene with clustalw (www.ncbi.nlm.nih.gov/pmc/articles/pmc308517) and mega version 3.1 (www.megasoftware.net) showed 100% identity among all nucleotide and amino acid sequences for monkey serum (appendix figure, panel a). when compared with nucleotide sequences available in genbank, vgf sequences were highly similar (98%100% identity) to the homologous gene from other vacv strains and showed 100% identity. the ha sequences for c. apella and a. caraya showed a signature deletion (appendix figure, panel b) also present in sequences of other vacv isolates from brazil. these ha sequences showed 99.6% identity at the nucleotide level and 99.7% identity at the amino acid level (736 nt of the ha gene were analyzed). phylogenetic trees of the vgf (figure, panel a) or ha (figure, panel b) genes were constructed by using the neighbor - joining method, 1,000 bootstrap replicates, and the tamura 3-parameter model (mega version 3.1). the vgf and ha sequences from monkey samples were deposited in genbank (accession nos . consensus bootstrap phylogenetic trees based on nucleotide sequences of orthopoxvirus vaccinia growth factor ( vgf) (a) and hemagglutinin (ha) (b) genes. trees were constructed with ha or vgf sequences by using the neighbor - joining method with 1,000 bootstrap replicates and the tamura 3-parameter model in mega version 3.1 software (www.megasoftware.net). black dots indicate vaccinia virus (vacv) obtained from cebus apella (vacv - to ca) and allouata caraya (vacv ac). all vgf sequences obtained from monkey serum samples showed 100% and are represented as a unique sequence in the vgf tree (vacv to). hspv, horsepoxvirus; varv, variola virus; cpxv, cowpoxvirus; mpxv, monkeypoxvirus. although vacv strains have been isolated from rodents in forests in brazil (the nearest location, belm, is 750 km from the study area), we detected vacv in wildlife in the brazilian amazon 3 years after reports of exanthematic outbreaks of bovine vaccinia and 40 years after isolation of vacv from forests. our data provide evidence of high prevalence of orthopoxviruses among capuchin and black - howling monkeys in the brazilian amazon. the relationship between infected monkeys and emergence of vacv in rural regions of brazil is unknown. however, transmission of vacv in northeastern brazil has been reported, and outbreaks have been reported in mato grosso, pernambuco (www.amep.org.br/pox.doc), maranho (e.g. kroon et al ., unpub . data), and tocantins, which are in or adjacent to the brazilian amazon. some of these viruses may be related to those isolated in this study because some vacv isolates have the same signature deletion in the ha gene as vacv - to. anthropogenic disturbance of the amazon ecosystem and increases in agricultural and livestock areas increase contact between wildlife and rural populations. however, the effect of vacv in environments in brazil that contain wild animals has not been studied. clinical data for pox lesions in animals tested were not well documented by veterinarians in the study area. ecologic and public health studies should be designed to evaluate risks for infection with vacv during wildlife conservation efforts and determine whether surveillance systems can predict bovine vaccinia outbreaks by monitoring vacv infection in monkeys and other wild animals. amino acid sequences of vaccinia virus (vacv) samples and comparison with homologous genes sequences from several orthopoxviruses, brazil. a ) alignment of vaccinia growth factor gene sequences from 6 monkey serum samples showing 100% identity (horizontal box). vacv - to_ca, sequence from cebus apella; vacv - to_ac, sequence from allouata caraya; hpxv, horsepoxvirus; cpxv, cowpoxvirus; mpxv, monkeypoxvirus; varv, variola virus; ecmv, ectromelia virus. b ) alignment of orthopoxvirus hemagglutinin gene amino acid sequences showing the deletion signature region (vertical box) in vacv - to isolates and several vacv strains isolated during bovine vaccinia outbreaks. arrow indicates polymorphism site in the hemagglutinin amino acid sequences between vacv - to_ca and vacv - to_ac. alignments were made by using clustalw (www.ncbi.nlm.nih.gov/pmc/articles/pmc308517) and mega version 3.1 software (www.megasoftware.net).

to detect orthopoxvirus in the brazilian amazon, we conducted a serosurvey of 344 wild animals. neutralizing antibodies against orthopoxvirus were detected by plaque - reduction neutralizing tests in 84 serum samples. amplicons from 6 monkey samples were sequenced. these amplicons identified vaccinia virus genetically similar to strains from bovine vaccinia outbreaks in brazil.

medicare is a federally administered social insurance program in the united states that guarantees access to health insurance for individuals aged 65 and older and younger individuals with disabilities who have worked and contributed to the system. in 2013, while disability beneficiaries comprise 16.8% of the total medicare population, they account for more than 20% of the total program expenditures. in the last decade, the growth of medicare enrollment from disability beneficiaries has greatly outpaced the overall growth of medicare program. for example, using data from the chronic condition data warehouse medicare 5% sample from centers for medicare and medicaid services, we estimate that from the year 2003 to 2012, the growth of disabled beneficiaries (37%) is much faster than the growth of the aged beneficiaries (21%). as baby boomers age , they are reaching an age of increased likelihood to developing disabilities, which makes it more important to understand the disability beneficiaries under medicare. however, younger medicare beneficiaries with work disabilities have received far less research attention and policy consideration than elderly medicare beneficiaries. the disabled individuals receive medicare coverage through the social security disability insurance (di) program, a primary public transfer program that provides both partial earnings replacement and medicare coverage to workers who lost earnings capacity due to severe and long - term disabilities. the social security definition of disability refers to severe health limitations that prevent individuals from engaging in any substantial gainful activities and are expected to last at least 12 months. as of december 2013, the di program provides cash benefits and medicare coverage to nearly 11 million american workers and their eligible dependents. most di beneficiaries are required to complete a 5-month waiting period before they are entitled to cash benefits and an additional 24-month waiting period before they are entitled to medicare coverage (the 2-year medicare waiting period is waived for those with amyotrophic lateral sclerosis and those with end - stage renal disease). there has been considerable concern that the 2-year medicare waiting period causes significant hardship to new di beneficiaries who lack alternative health insurance coverage and needed health care access to address their health problems. evidence shows that more than 20% of di beneficiaries have no health insurance coverage at all during the medicare waiting period. around 1.8 million di beneficiaries congress has noted the problems with the 2-year waiting period in the ticket to work and work incentives improvement act of 1999. other political effort devoted to changing this policy includes the ending the medicare disability waiting period act of 2007, sponsored by senator jeff bingaman and rep. gene green, which proposes to phase out medicare s 2-year waiting period over a 10-year span and grant more exceptions to people with life - threatening health conditions. studies based on focus groups and interviews with disabled individuals have shown that many of the uninsured lack needed health care to stabilize their health conditions and their situations get worse during the waiting period. yet formal analyses have been rare that identify and quantify any detrimental effect that the 2-year waiting period has imposed on disabled individuals. a handful of previous studies about the medicare waiting period have primarily focused on estimating the fiscal impact of eliminating the 2-year waiting period, which is certainly useful for budgetary consideration of policy changes. a demonstration project (the accelerated benefits demonstration) conducted by the social security administration (ssa) in 2011 provides evidence about effects on health care utilization and estimates the fiscal costs once uninsured di beneficiaries are provided with health - care package during the 2-year waiting period. however, the project does not evaluate the impact of health care access on health outcomes for the uninsured disability beneficiaries during the waiting period probably due to the constraint of the project design such as the narrow time window of the follow - up survey (6 months), which makes it difficult to observe any changes in health outcomes. although the demonstration project provides evidence on some important aspects of providing accelerated health care access to disability beneficiaries, it faces challenges that are endemic to many policy demonstration projects. as the ssa acknowledges, the ab demonstration project is not able to estimate any potential - induced entry effects, which would occur if the accelerated health insurance coverage offered to newly awarded di beneficiaries would make the di program more attractive and induce more applicants to the di program. it is also possible that individuals who expect no health insurance coverage during the medicare waiting period are less likely to apply for di. our study utilizes longitudinal panel data that span over nearly 20 years and allow us to observe disabled workers for a longer period of time and more likely to capture changes in their health and economic status. a lot of research effort has been devoted to examining the impact of medicare on health outcomes. these studies have primarily focused on the medicare impact on elderly beneficiaries rather than disabled beneficiaries. in general, they find that medicare has no discernible effect on health. these studies are helpful for understanding an average effect of medicare on health outcomes among the elderly patients. in the meantime , they may have masked medicare effects on certain subgroups of the population, for example, individuals with poor health. in other words, it is possible that medicare has differential effect on the health distribution. workers younger than 65 who have severe and long - term health impairments and lose earning capacity may qualify for the social security disability benefits through the di program. they are likely more ill and worse off financially on average compared to the elderly beneficiaries who survive age 65 and are entitled to medicare then. although medicare may not show significantly positive health effects for the elderly beneficiaries for their chronic health conditions, as shown in previous literature, it may help improve the health status or at least prevent the health from deteriorating for the disabled beneficiaries for their severe health problems. there is some evidence that for the severely ill patients, acquiring medicare coverage has significant and positive effects on self - reported health, with the largest gains in health improvement among the groups that experience the largest gains in insurance coverage. in this article, we focus on a group of individuals with poor health. they qualify for disability benefits and medicare entitlement through the di program, which adopts the strictest definition of disability compared to the criteria used in most other disability programs in the nation, as well as compared to the disability definition used in disability programs in other developed countries research effort in the past about the di program has mainly focused on understanding the effects of the cash benefits offered by the program. little analysis has been conducted to understanding the effects of the important in - kind benefit, medicare coverage, provided by the di program. the fact that medicare is a federally administered program and its eligibilities are the same for all individuals, limits the extent to which variation in medicare coverage can be used to identify the effects of medicare coverage on individuals in the di program. the medicare waiting period creates a health insurance gap for some disability beneficiaries, while others have alternative health insurance (mostly medicaid) in that period. it provides an opportunity for us to exploit variation in health insurance coverage and identify its effects on disabled individuals. this article is the first to take advantage of the longitudinal health and retirement study (hrs) and estimate health and economic effects of the medicare waiting period for disabled individuals. a previous study has used the same data source to study the health effects of medicare enrollment at age 65 on the near - elderly uninsured. in this article , we explore whether and to what extent the medicare waiting period impacts health and economic status of the di beneficiaries who are uninsured while waiting for medicare to become available. in a quasiexperiment research design, using a difference - in - difference (diff - in - diff) estimator, we compare changes in health and economic outcomes pre-/postentering the di program for the beneficiaries with alternative health insurance and those without during the medicare waiting period. the adjusted diff - in - diff estimates suggest that the disability beneficiaries who are uninsured during the waiting, compared to those who are insured, are 13.6 percentage point more likely to report poor health, 6.3 percentage point less likely to be in excellent health, declare more difficulties in activities of daily living (adl), and about 30% higher medical expenditures from out of pocket. the findings suggest punitive health and economic effects of the medicare waiting period for the uninsured disability beneficiaries. the structure of this article is as follows: the first section introduces the research question. the study uses 10 available waves of the hrs, which cover the 1992 to 2010 period, with some interviews conducted in 2011. the hrs is a biennial national representative survey and it interviews individuals born between 1931 and 1941 and their spouses, as well as additional cohorts that have been added in recent waves of the study. the data provide extensive information on health status, employment history, wealth, income, family structure, and government program participation and transfers for a total of 30 672 respondents. for our analysis, it is crucial to accurately line up the dates pre-/post - di program for disability beneficiaries and identify the medicare waiting period. one is entitled to di benefits 5 months after he becomes technically disabled according to the social security definition of disability assuming he has become di insured by accumulating enough work credits. he is entitled to medicare coverage 24 months after his entitlement to di benefits. to identify the medicare waiting period , we must determine the date 1 is entitled to di benefits, which could be before the receipt of the first di paycheck. in other words, this is because it can sometimes take many months or even years before a di application is approved. the processing times are even longer for claims that are initially denied and then subsequently allowed during the appeal process. also, those who are eventually allowed benefits might not file a disability claim immediately following the disability onset. hence, by the time di beneficiaries are notified that their claim has been allowed, some have completed all or part of the medicare waiting period. linking the hrs files to the social security master beneficiary record file, we are able to identify the official date of disability onset. the linkage of hrs and master beneficiary files is restricted and available from the university michigan hrs for researchers who meet the criteria for access to confidential data. knowing the date of disability onset, we can count 5 months from that date to determine the date for di entitlement, which is when the medicare waiting period starts. the waiting period ends 29 months after the date of disability onset. in the hrs, that is, a 2-year passing is between the adjacent interview waves in the hrs. comparing the interview dates with the di entitlement date , we designate the wave right prior to the di entitlement the pre - di period and the wave right after the di entitlement the post - di period. we exclude in the sample the respondents for whom we do not observe their pre - di period and those for whom we do not observe the post - di period. so we include in the sample the individuals who are awarded di benefits, for whom we can observe both the wave before and the wave after their di entry (waves identified based on the interview date and the di entitlement date if available), and for whom we can determine whether they have public health insurance coverage in the wave after di entry. after applying all the sample restrictions, we are left with a sample of 465 respondents. in the 2-period model aka equation , each respondent is observed twice in the wave before entering di and the wave after. in our analysis, we also expand the specification beyond the 2-period pre-/postcomparison to allow multiple pre- and postperiods aka equation, given the fact that the health effects may not become apparent in a narrow time frame. in the multiperiod model, each respondent contribute up to 4 observations in the waves pre - di entry and up to 4 observations in the waves post - di entry. our goal is to identify the effects of being uninsured during the medicare waiting period for the newly entitled disability beneficiaries. we distinguish the following 2 groups in our analysis: the newly entitled di beneficiaries who do not have any health insurance coverage before medicare becomes available (23% of the sample) and the newly entitled di beneficiaries who have access to alternative public health insurance (primarily medicaid) immediately after entering the di program (77% of the sample). the former group includes the individuals who lose their health insurance after entering the di program (14%) and the individuals who already lose health insurance prior to the di entry (9%). among the latter, when we drop this latter group from our study sample, the diff - in - diff estimates became slightly larger in magnitude and stayed statistically significant. the di beneficiaries with health insurance in the waiting period include the individuals who gain access to public health insurance once entering the di program (57%) and the individuals who get their public health insurance from before entering the di program (19%). as we will show later, the 2 groups appear comparable in a large array of socioeconomic characteristics and health variables at the baseline period. the di beneficiaries who have low income and assets may concurrently receive supplemental security income (ssi) benefits. most of the di - ssi concurrent beneficiaries gain immediate access to medicaid coverage because of their entitlement to ssi benefits. the 2 public disability transfer programs are administered by the ssa and follow the same criteria for determining disability severity. hence, the beneficiaries under the 2 programs are comparable at the baseline in terms of their disability / health status, a key variable in our analysis. table 1 presents preperiod descriptive statistics for the sample of newly entitled di beneficiaries without health insurance coverage and those with alternative public health insurance upon entering the di program. the disability beneficiaries with alternative public health insurance during medicare waiting period include more women, fewer blacks, and more married. they appear a bit less healthy than those beneficiaries with health insurance in the waiting period. the former report more health conditions and more difficulties in adls and instrumental activities of daily living. the major differences between the 2 groups lie in some of the financial variables. the disability beneficiaries with insurance in the waiting period, compared to those without insurance in the waiting period, on average earns 40% less from work before entering the di program. however, the former receives nonlabor income (most likely government transfers) that is 4.5 times the amount received by the latter. these differentials are not surprising as the uninsured beneficiaries consist of disabled individuals who receive di benefits only (rather than di - ssi concurrently) and who are most likely workers with longer worker histories and higher labor earnings compared to the insured disabled individuals who are concurrently receiving the ssi benefits that are means tested. sample means / proportions as of the wave prior to di entry for the di beneficiaries with and without health insurance in the waiting period. abbreviations: adl, activities of daily living; bmi, body mass index; di, disability insurance; iadl, instrumental activities of daily living; prev, previous; unemp ins and workers comp, unemployment insurance and workers compensation. net worth is the sum of assets (primary residence, other real estate, vehicles, businesses, iras, stocks, bonds, checking accounts, cds, and other assets) less liabilities (mortgages, other home loans, and other debt). major health conditions include high blood pressure or hypertension; diabetes or high blood sugar; cancer or a malignant tumor or any kind except skin cancer; chronic lung disease except asthma such as chronic bronchitis or emphysema; heart attack, coronary heart disease, angina, congestive heart failure, or other heart problems; stroke or transient ischemic attack; emotional, nervous, or psychiatric problems; and arthritis or rheumatism. what is worth noting is that half of the disability beneficiaries have an annual household total income of less than us$30 000. it suggests that many beneficiaries could benefit from the recently passed health - care reform, the affordable care act (aca), which makes health insurance more affordable by providing subsidies for families with income below 400% of the federal poverty line to purchase insurance through new health insurance exchanges. we will discuss this aspect further in the last section of this article. the underlying theoretical framework for individuals inherit an initial amount of this stock that depreciates with age and can be increased by investment. changes in health stock over time are determined by increased health stock less health depreciation. health stock is produced by health inputs such as medical care, health knowledge, lifestyle, environment, and so on. so in our reduced - form estimation about health changes over time (pre-/post - di), we control for changes in medical care (health insurance coverage and health - care utilization), changes in health knowledge (proxied by years of schooling, an efficiency parameter in the grossman health production function), changes in wealth and income, and other socioeconomic variables as proxy for other factors that may affect health outcomes and hard to measure empirically such as lifestyle. in a quasiexperiment research design , we use a diff - in - diff estimator, where the first difference is between outcome variables before and after entering the di program and the second difference is between the disability beneficiaries with health insurance and those without health insurance in the waiting period. taking advantage of the panel data, we take the within - person change in outcome variables before and after entering the di program. this is desirable as it guarantees comparability of the groups in the pre- and postperiods, controlling for any compositional changes due to attrition or sampling variation. one identifying assumption in diff - in - diff models is that other unobserved time - varying processes would have similarly impacted the 2 comparison groups. the preperiod differences in some measures between the groups in table 1 suggest a potential vulnerability on this front. we address this by testing for differential changes in a number of potential confounders, including health measures, financial variables, and health service utilization. we then present in addition to unadjusted diff - in - diff estimates, the estimates that account for within - person changes in these and other relevant variables. let yijt denote the change in health and economic outcomes for individual i in group j = 1, 0 (di beneficiaries with and without insurance in the waiting period) between time t and t 2 (2 years between waves in hrs). dj is an indicator for being a di beneficiary with insurance in the waiting period. we begin with a 2-period model in which t 2 denotes the preperiod and t the postperiod, and we estimate a linear equation of the following form:1yijt=+dj+xijt+ijt in this 2-period pre post model, the coefficient is the diff - in - diff estimate of the effect of being insured during the medicare waiting and captures a linear time trend in yijt. we also expand the specification beyond the simple 2-period pre-/postcomparison to allow multiple pre- and postperiods, given the fact that the health effects may not become apparent in a narrow time frame. taking into account the fact that the shape of the time profile could be different for the di beneficiaries with and without insurance in the waiting period, we interact a flexible function of t with the group indicator dj in order to allow the slopes of the time profile to differ before and after di entry and across groups.2yijt=dj+ftdj+ft+xijt+ijt we model f(t) as a sequence of dummy variables for each period t. if we set the reference period to the first postperiod, then the coefficient continues to be the diff - in - diff estimate. an attraction of the first - differenced model shown in equations and is that permanent unobservable differences between the 2 comparison groups are differenced out. this is desirable in light of some preexisting differences between the 2 groups shown in table 1. however, the existence of some preperiod differences also suggests that time - varying processes may differentially impact the 2 groups. for example, health shocks might differentially impact the insured di recipients in the waiting period given their poorer initial health. one way of testing for the presence of confounders is to estimate models like those in equations and for each potential confounder. this is similar in spirit to the test offered in a previous study in the context of regression discontinuity designs. table 3 shows diff - in - diff estimates based on equation for 25 potential confounders, including financial variables, health measures, and health - care utilization measures. statistically significant estimates are evidence that the insured di beneficiaries experienced differential pre-/post - di changes in a given variable. there is little evidence of differential impacts for pre-/post - di changes in most of the variables tested, with the exception of a couple of financial variables such as capital income and income from unemployment insurance and workers compensation (both statistically significant at 10% level). to control for these important time - varying processes, we include these variables in first differences in the vector xijt in equations and. the study uses 10 available waves of the hrs, which cover the 1992 to 2010 period, with some interviews conducted in 2011. the hrs is a biennial national representative survey and it interviews individuals born between 1931 and 1941 and their spouses, as well as additional cohorts that have been added in recent waves of the study. the data provide extensive information on health status, employment history, wealth, income, family structure, and government program participation and transfers for a total of 30 672 respondents. for our analysis, it is crucial to accurately line up the dates pre-/post - di program for disability beneficiaries and identify the medicare waiting period. one is entitled to di benefits 5 months after he becomes technically disabled according to the social security definition of disability assuming he has become di insured by accumulating enough work credits. he is entitled to medicare coverage 24 months after his entitlement to di benefits. to identify the medicare waiting period , we must determine the date 1 is entitled to di benefits, which could be before the receipt of the first di paycheck. in other words, this is because it can sometimes take many months or even years before a di application is approved. the processing times are even longer for claims that are initially denied and then subsequently allowed during the appeal process. also, those who are eventually allowed benefits might not file a disability claim immediately following the disability onset. hence, by the time di beneficiaries are notified that their claim has been allowed, some have completed all or part of the medicare waiting period. linking the hrs files to the social security master beneficiary record file, we are able to identify the official date of disability onset. the linkage of hrs and master beneficiary files is restricted and available from the university michigan hrs for researchers who meet the criteria for access to confidential data. knowing the date of disability onset, we can count 5 months from that date to determine the date for di entitlement, which is when the medicare waiting period starts. the waiting period ends 29 months after the date of disability onset. in the hrs, that is, a 2-year passing is between the adjacent interview waves in the hrs. comparing the interview dates with the di entitlement date , we designate the wave right prior to the di entitlement the pre - di period and the wave right after the di entitlement the post - di period. we exclude in the sample the respondents for whom we do not observe their pre - di period and those for whom we do not observe the post - di period. so we include in the sample the individuals who are awarded di benefits, for whom we can observe both the wave before and the wave after their di entry (waves identified based on the interview date and the di entitlement date if available), and for whom we can determine whether they have public health insurance coverage in the wave after di entry. after applying all the sample restrictions, we are left with a sample of 465 respondents. in the 2-period model aka equation , each respondent is observed twice in the wave before entering di and the wave after. in our analysis, we also expand the specification beyond the 2-period pre-/postcomparison to allow multiple pre- and postperiods aka equation, given the fact that the health effects may not become apparent in a narrow time frame. in the multiperiod model, each respondent contribute up to 4 observations in the waves pre - di entry and up to 4 observations in the waves post - di entry. our goal is to identify the effects of being uninsured during the medicare waiting period for the newly entitled disability beneficiaries. we distinguish the following 2 groups in our analysis: the newly entitled di beneficiaries who do not have any health insurance coverage before medicare becomes available (23% of the sample) and the newly entitled di beneficiaries who have access to alternative public health insurance (primarily medicaid) immediately after entering the di program (77% of the sample). the former group includes the individuals who lose their health insurance after entering the di program (14%) and the individuals who already lose health insurance prior to the di entry (9%). among the latter, when we drop this latter group from our study sample, the diff - in - diff estimates became slightly larger in magnitude and stayed statistically significant. the di beneficiaries with health insurance in the waiting period include the individuals who gain access to public health insurance once entering the di program (57%) and the individuals who get their public health insurance from before entering the di program (19%). as we will show later, the 2 groups appear comparable in a large array of socioeconomic characteristics and health variables at the baseline period. the di beneficiaries who have low income and assets may concurrently receive supplemental security income (ssi) benefits. most of the di - ssi concurrent beneficiaries gain immediate access to medicaid coverage because of their entitlement to ssi benefits. the 2 public disability transfer programs are administered by the ssa and follow the same criteria for determining disability severity. hence, the beneficiaries under the 2 programs are comparable at the baseline in terms of their disability / health status, a key variable in our analysis. table 1 presents preperiod descriptive statistics for the sample of newly entitled di beneficiaries without health insurance coverage and those with alternative public health insurance upon entering the di program. the disability beneficiaries with alternative public health insurance during medicare waiting period include more women, fewer blacks, and more married. they appear a bit less healthy than those beneficiaries with health insurance in the waiting period. the former report more health conditions and more difficulties in adls and instrumental activities of daily living. the major differences between the 2 groups lie in some of the financial variables. the disability beneficiaries with insurance in the waiting period, compared to those without insurance in the waiting period, on average earns 40% less from work before entering the di program. however, the former receives nonlabor income (most likely government transfers) that is 4.5 times the amount received by the latter. these differentials are not surprising as the uninsured beneficiaries consist of disabled individuals who receive di benefits only (rather than di - ssi concurrently) and who are most likely workers with longer worker histories and higher labor earnings compared to the insured disabled individuals who are concurrently receiving the ssi benefits that are means tested. sample means / proportions as of the wave prior to di entry for the di beneficiaries with and without health insurance in the waiting period. abbreviations: adl, activities of daily living; bmi, body mass index; di, disability insurance; iadl, instrumental activities of daily living; prev, previous; unemp ins and workers comp, unemployment insurance and workers compensation. net worth is the sum of assets (primary residence, other real estate, vehicles, businesses, iras, stocks, bonds, checking accounts, cds, and other assets) less liabilities (mortgages, other home loans, and other debt). major health conditions include high blood pressure or hypertension; diabetes or high blood sugar; cancer or a malignant tumor or any kind except skin cancer; chronic lung disease except asthma such as chronic bronchitis or emphysema; heart attack, coronary heart disease, angina, congestive heart failure, or other heart problems; stroke or transient ischemic attack; emotional, nervous, or psychiatric problems; and arthritis or rheumatism. what is worth noting is that half of the disability beneficiaries have an annual household total income of less than us$30 000. it suggests that many beneficiaries could benefit from the recently passed health - care reform, the affordable care act (aca), which makes health insurance more affordable by providing subsidies for families with income below 400% of the federal poverty line to purchase insurance through new health insurance exchanges. the underlying theoretical framework for our research design is a health production function in the 1972 grossman model. individuals inherit an initial amount of this stock that depreciates with age and can be increased by investment. changes in health stock over time are determined by increased health stock less health depreciation. health stock is produced by health inputs such as medical care, health knowledge, lifestyle, environment, and so on. so in our reduced - form estimation about health changes over time (pre-/post - di), we control for changes in medical care (health insurance coverage and health - care utilization), changes in health knowledge (proxied by years of schooling, an efficiency parameter in the grossman health production function), changes in wealth and income, and other socioeconomic variables as proxy for other factors that may affect health outcomes and hard to measure empirically such as lifestyle. in a quasiexperiment research design , we use a diff - in - diff estimator, where the first difference is between outcome variables before and after entering the di program and the second difference is between the disability beneficiaries with health insurance and those without health insurance in the waiting period. taking advantage of the panel data, we take the within - person change in outcome variables before and after entering the di program. this is desirable as it guarantees comparability of the groups in the pre- and postperiods, controlling for any compositional changes due to attrition or sampling variation. one identifying assumption in diff - in - diff models is that other unobserved time - varying processes would have similarly impacted the 2 comparison groups. the preperiod differences in some measures between the groups in table 1 suggest a potential vulnerability on this front. we address this by testing for differential changes in a number of potential confounders, including health measures, financial variables, and health service utilization. we then present in addition to unadjusted diff - in - diff estimates, the estimates that account for within - person changes in these and other relevant variables. let yijt denote the change in health and economic outcomes for individual i in group j = 1, 0 (di beneficiaries with and without insurance in the waiting period) between time t and t 2 (2 years between waves in hrs). dj is an indicator for being a di beneficiary with insurance in the waiting period. we begin with a 2-period model in which t 2 denotes the preperiod and t the postperiod, and we estimate a linear equation of the following form:1yijt=+dj+xijt+ijt in this 2-period pre post model, the coefficient is the diff - in - diff estimate of the effect of being insured during the medicare waiting and captures a linear time trend in yijt. we also expand the specification beyond the simple 2-period pre-/postcomparison to allow multiple pre- and postperiods, given the fact that the health effects may not become apparent in a narrow time frame. taking into account the fact that the shape of the time profile could be different for the di beneficiaries with and without insurance in the waiting period, we interact a flexible function of t with the group indicator dj in order to allow the slopes of the time profile to differ before and after di entry and across groups.2yijt=dj+ftdj+ft+xijt+ijt we model f(t) as a sequence of dummy variables for each period t. if we set the reference period to the first postperiod, then the coefficient continues to be the diff - in - diff estimate. an attraction of the first - differenced model shown in equations and is that permanent unobservable differences between the 2 comparison groups are differenced out. this is desirable in light of some preexisting differences between the 2 groups shown in table 1. however, the existence of some preperiod differences also suggests that time - varying processes may differentially impact the 2 groups. for example, health shocks might differentially impact the insured di recipients in the waiting period given their poorer initial health. one way of testing for the presence of confounders is to estimate models like those in equations and for each potential confounder. this is similar in spirit to the test offered in a previous study in the context of regression discontinuity designs. table 3 shows diff - in - diff estimates based on equation for 25 potential confounders, including financial variables, health measures, and health - care utilization measures. statistically significant estimates are evidence that the insured di beneficiaries experienced differential pre-/post - di changes in a given variable. there is little evidence of differential impacts for pre-/post - di changes in most of the variables tested, with the exception of a couple of financial variables such as capital income and income from unemployment insurance and workers compensation (both statistically significant at 10% level). to control for these important time - varying processes , we include these variables in first differences in the vector xijt in equations and. in table 2, we first present unadjusted diff - in - diff estimates of on several health outcome measures (self - reported poor health, self - reported excellent health, and summary of functional limitations in adl) and out - of - pocket medical expenditures, and then in table 4 we turn to diff - in - diff estimates adjusted by a large array of socioeconomic variables. unadjusted estimates of health and economic outcomes before and after entering the di program for the di beneficiaries with and without health insurance in the waiting period. column 4 is mean within - individual after - minus - before change in the outcome variable. the new disability beneficiaries with access to public health insurance are 2.5 percentage points less likely to report poor health, compared to before entering di, while we observe an increase of 12.1 percentage points in poor health reporting from the new disability recipients who are uninsured. the unadjusted diff - in - diff estimate of , that is, the effect of being insured in the waiting period, on this health measure is 0.145, indicating a 14.5 percentage point drop in reporting poor health after entering the di program among those with alternative public health insurance relative to those without. this unconditional effect is statistically significant and the calculations using across group changes and using within - person changes provide similar (table 2, panel 1). the rate of reporting excellent health dropped right after the di entry for both the di beneficiaries with and without health insurance in the waiting period. the proportion reporting excellent health decreased by about half (from 3.6% to 1.7%) for the new di awardees with access to public health insurance, while that proportion declined much more dramatically from 8.3% to less than 1% for the uninsured disability beneficiaries during the medicare waiting period. the unadjusted diff - in - diff estimate of the effect of having health insurance in the waiting period on this health measure is 0.055 and statistically significant, indicating a 5.5 percentage point larger drop in reporting excellent health after entering the di program among those without alternative public health insurance relative to those with insurance (table 2, panel 2). respondents in the hrs were asked whether they have any difficulty with adl, such as dressing, bathing, walking across a room, eating, getting in and out of bed, and using the toilet. the total number of adls where respondents report any difficulty was close to 1 on average for those insured di beneficiaries before entering di and dropped 8% after entering di. in contrast, the di beneficiaries who are uninsured in the waiting period reported a remarkable increase of about 90% more difficulties in adls when they are uninsured and waiting for the entitlement to medicare. the diff - in - diff estimate on this health measure (0.456) indicates a statistically significant (unadjusted) effect on reducing functional limitations of having alternative health insurance during medicare waiting period (table 2, panel 3). the above diff - in - diff estimates on health outcomes indicate that the health status improves or at least does not deteriorate as much for the insured di beneficiaries in the wave post - di compared to the pre - di period. the effect is somewhat surprising: the insured di beneficiaries consist of mainly ssi recipients who, due to the means - tested nature of the program, are in worse health to begin with (compared to the uninsured di beneficiaries) because of likely lower investment in health through their life, but they see more improvement (or less deterioration) in their health in the waiting period. this evidence likely suggests the positive effect of having alternative health insurance for di beneficiaries in the waiting period. as shown earlier in table 3, except the different health insurance coverage, there is no differential change pre-/post - di between the 2 groups other than in a few financial variables. the worsening of the few financial variables for the insured beneficiaries post - di is likely to have negative or at least nonpositive impact on their health. but we observe overall advantageous health changes among them compared to the uninsured di beneficiaries. the evidence likely underscores the important role played by having access to health insurance in positively affecting health outcomes during the medicare waiting period. given the fact that these disabled workers are usually forced to drop out of the labor force due to the onset of severe work limitation, they not only lose labor earnings but also likely lose employer provided health insurance. for these workers who have catastrophic health problems, health insurance coverage would be valuable. the 2-year medicare waiting period creates additional barrier for the disabled workers who do not have alternative health insurance coverage during the transition period. the evidence we show may suggest negative health effects of discontinuity in health insurance coverage for individuals with serious health impairments. the punitive effect of the lack of health insurance or access to health care would likely be even larger when we account for the disabled beneficiaries who die while waiting for medicare entitlement. about 2% of the sample dies after entering the di program and before being entitled to medicare. this effect needs further investigation when a larger sample for the disabled beneficiaries becomes available. tests for differential pre-/postchanges in potential confounders for di beneficiaries with and without health insurance in the waiting period. abbreviations: bmi, body mass index; di, disability insurance; gov, government; hrs, health and retirement study; iadl, instrumental activities of daily living; psych, psychiatric; unemp ins and workers comp, unemployment insurance and workers compensation. columns are separate regressions based on equation for first - differenced dependent variable listed in column head, where differences are taken between the wave just after di entry and the wave just before di entry. d is a dummy for being a di beneficiary with insurance in the waiting period and thus its coefficient measures the relative change in the dependent variable for the di beneficiaries with and without insurance in the waiting period. we also examine the effect on the out - of - pocket medical expenditures of the medicare waiting period. the medical expenditures in table 2 (panel 4) are in the form of the natural log. we see an increase of about 16% in the out - of - pocket medical expenses for those who are uninsured while they wait for medicare to become available, while the di beneficiaries who have alternative insurance during medicare waiting period lower their out - of - pocket medical expenditures by about 14%. the diff - in - diff estimate of the insurance effect is 0.30, indicating a 30% decrease in out - of - pocket medical expenditures after entering the di program for those with alternative public health insurance relative to those without. in addition, we test the effect of being insured during the medicare waiting period on some other health outcome measure such as self - reported work limitation and the effect on the employment rate. we find the effects statistically significant and with a sign consistent with the hypothesis. however, these unadjusted effects are either primarily driven by the preexisting difference between the 2 groups (table a1, panel 1) or mainly attributable to the changes in 1 group (the uninsured di beneficiaries ; table a1, panel 2). table 4 presents several ols specifications of equation, which contrasts pre-/post - di changes in health and economic outcomes for the di beneficiaries with and without insurance in the waiting period. the estimation sample has 1 first - differenced observation per respondent. to illustrate the impact of controlling for the potential confounders shown in table 3 column 1 shows the unadjusted diff - in - diff estimate of from table 2 for reference. in column 2 , we add first - differenced demographic variables (household size and an indicator for being married). neither of the additional controls have statistically significant effect on any of the outcomes. in column 3 , we add first - differenced financial variables (total wealth, net worth, capital income, total nonlabor income, private pension income, income from unemployment insurance and workers compensation, other government transfer income, and other income). the diff - in - diff estimate of declines slightly in magnitude for the 3 health outcome measures while it goes up a bit in magnitude for the out - of - pocket medical expenditures. among the financial control variables, statistically significant are only the effect of the total wealth on health reporting and the effect of the government transfer income on the out - of - pocket expenditures, indicating that more wealth is associated with more reports of excellent health and less reports of poor health, and receiving government transfer income here is likely associated with receiving medicaid coverage and then lowering self - paid medical expenses. in column 4, we add an extensive set of first - differenced health controls that are listed in tables 1 and 4. again, the diff - in - diff estimates of do not change much for all the outcome measures. among the health control variables, the effects of the onset of cancer, lung disease, heart disease, or high blood pressure on self - reported health status are statistically significant. the onset of high blood pressure or psychiatric problems is shown to have a statistically significant effect on reporting adls difficulties. the onset of high blood pressure, cancer, or stroke has a statistically significant effect on the out - of - pocket medical expenditures. in column 5, we use multiple pre- and postperiods as shown in equation, and the estimates of are not very different. in all these first - differenced specifications, we calculate the standard errors allowing for arbitrary correlation in the outcome measures within each of the 2 comparison groups in a given year. for the 2-period models shown in columns 1 to 4, we use calendar years and obtain 20 group - calendar year clusters. in the multiperiod model shown in column 5, we cluster by group and time period relative to di entry, which gives 16 clusters (= 8 periods 2 groups). adjusted estimates of health and economic outcomes before and after entering di program for the di beneficiaries with and without health insurance in the waiting period. columns 1 to 4 are separate regressions based on equation for first - differenced dependent variable, where differences are taken between the wave just after di entry and the wave just before di entry. column 5 is based on equation for first - differenced dependent variable, where wave - to - wave differences are taken across 4 waves prior to di entry and 4 waves after di entry. d is a dummy for being a di beneficiary with health insurance in the waiting period. income variables include capital income, total nonlabor income, private pension income, income from unemployment insurance and workers compensation, other government transfer income, and other income. we include in the estimation an indicator for missing any health information and an indicator for missing any financial information. standard errors (in parentheses) for 2-period models shown in columns 1 to 4 are clustered by group and calendar year (20 clusters), while standard errors for multiperiod model in column 5 are clustered by group and time period relative to di entry (16 clusters). the di beneficiaries are primarily workers who lose earnings capacity due to severe health limitations. many lose employer provided health insurance when they are struck by catastrophic health shock and have to leave their jobs. the 2-year waiting period for medicare access creates a health insurance gap and great hardship for them when they are having both negative health shocks and financial shocks and when they need affordable health insurance package more than ever. eliminating the medicare waiting period for disabled workers has been discussed among policy makers and researchers. existing studies on the issue have mainly focused on the fiscal impact of the policy. this study is the first to use a longitudinal panel data set, examine a rich set of controls, and analyze the health and economic consequences of the waiting period for uninsured disability beneficiaries. we take advantage of the longitudinal hrs, which covers the period 1992 to 2010 and provides rich information on health status, employment history, wealth, income, family structure, and government program participation and transfers. linking the hrs files with the social security master beneficiary record file , we are able to accurately line up the timing of pre-/post - di entitlement, which is crucial for our study. in the quasiexperimental research design , we use a diff - in - diff estimator, where the first difference is between the health and economic outcome variables before and after a respondent is entitled to di benefits, and the second difference is between the newly entitled di beneficiaries with access to alternative public health insurance and those being uninsured while waiting for medicare. the adjusted diff - in - diff estimates imply a 13.6 percentage point drop in the proportion reporting poor health, a 6.3 percentage point rise in the proportion reporting excellent health, a drop of total adl limitations, and a 30.5% decline in out - of - pocket medical expenditures among the di beneficiaries with health insurance access relative to those without while waiting for the medicare entitlement. the findings highlight adverse health and economic effects of the lack of health insurance in the medicare waiting period for the uninsured disability beneficiaries. the study has its limitations and we should take caution when we interpret the . first, in this study, we hope to estimate the effects of having immediate medicare coverage for the newly awarded di beneficiaries without the 2-year waiting period, that is, the counterfactual effects for di beneficiaries if we eliminate the waiting period. to that end , we examine a group of newly awarded di beneficiaries who have alternative public health insurance (mainly medicaid) in the waiting period and study the changes in their health and economic outcomes. one assumption for applying this research design is that medicaid and medicare are similar coverage, for example, with similar health care services and similar financing arrangements for individuals. but if medicaid has different effects than medicare for di beneficiaries, our estimation using medicaid insured beneficiaries as a counterfactual would likely produce biased for the medicare effects on di beneficiaries. second, the 2 comparison groups, the di beneficiaries with and without health insurance in the medicare waiting period, are similar on demographic variables but do not look quite the same in terms of health status at the baseline (the wave prior to di entry). the insured di beneficiaries appear sicker than the uninsured beneficiaries just before entering the di program. to make the 2 groups more comparable at the baseline this, however, would be difficult to implement, given the small sample size in the study. with the differences between the 2 groups at the baseline, there may be alternative interpretations of the . for example, the health of the insured di beneficiaries does not deteriorate as much or even improve compared to the uninsured di beneficiaries because the former group s initial poorer health may reflect transitory negative shocks, or because the latter group s health may be already on an accelerating downhill trajectory. but these hypotheses are hard to test empirically. third, the 2-year spacing between the hrs waves is not ideal although the hrs is the best available data set for this research. with the 2-year gap between interview waves, it is possible that some of the post - di observations will have become entitled to medicare coverage, even among the designated uninsured group. however, this data limitation unlikely undermines our and even possibly strengthens our findings. group was clear of those observations who were actually insured with medicare, the differential effects in the health and economic outcomes between the insured and uninsured di beneficiaries would have been even larger than what we estimated. the recently passed aca does not directly address the medicare 2-year waiting period associated with the di program. the act does make some changes to help relieve the problems of this health insurance gap. for example, many di beneficiaries can now join a high - risk insurance pool that were created under the aca and receive immediate insurance coverage while they continue to wait for medicare benefits to become available. in addition, starting in 2014 disabled individuals in this waiting period will have access to expanded insurance options through state - based insurance exchanges and expanded medicaid eligibility. although these new options would not completely solve the problems associated with the medicare waiting period, based on the evidence provided in this article, these policy changes will likely provide at least temporary relief for many affected individuals with disabilities. of course long - term effects of these policy changes in the aca for disability beneficiaries will take time to observe. for example, it is possible that expanded insurance coverage possibilities for disability beneficiaries makes the medicare waiting period less of a deterrence and make di more attractive and thus induced more applications to the di program. on the other hand, more accessible health insurance options in general under the aca will possibly make the di program less appealing to some disabled individuals for whom the main driver to apply to di under the status quo is to get medicare coverage. to understand the impact of the aca reforms on the di program, these are interesting and important aspects to study, especially as the relevant data become available with time passing.

purpose: disabled individuals younger than 65 years are entitled to medicare coverage through the social security disability insurance (di) program, but only if they have completed a 2-year waiting period. this is the first study that uses longitudinal panel data, the health and retirement study, and examines whether and to what extent the health and economic status are affected among disability beneficiaries who are uninsured during the medicare waiting period.methods:in a quasiexperiment research design, using a difference - in - difference (diff - in - diff) estimator, we compare changes in health and economic outcomes pre-/postentering the di program for disability beneficiaries with alternative public health insurance and those without.:the adjusted diff - in - diff estimates suggest that disability beneficiaries who are uninsured during the waiting period, compared to those who are insured, are 13.6 percentage point more likely to report poor health, 6.3 percentage point less likely to be in excellent health, declare more difficulties in activities of daily living, and 30% higher medical expenditures from out of pocket.:the findings highlight punitive health and economic effects of the medicare waiting period for uninsured disability beneficiaries. we also discuss the implications of the findings for the affordable care act reform.

an increasing number of patients with chest pain are referred to the casualty department or the coronary emergency room (cer) to prove or exclude a potentially serious disease such as an acute coronary syndrome. the challenge lies in rapidly identifying patients who can safely be discharged from the cer. in addition, it is necessary to identify patients who need an aggressive treatment, as part of an acute coronary syndrome (acs), and to take appropriate action as soon as possible. the diagnosis of chest pain is made for 47 % of all patients who are admitted to the cardiology department. acs is not responsible for symptoms in 40 % of patients presenting with chest pain in the cer. the average period of stay of these patients at the cer in our hospital is currently 11 h. given the increasing pressure on the capacity of beds, a shorter length of stay is warranted. the use of biomarkers in addition to risk scores can be helpful in optimising the discharge policy in the cer, through which a shorter duration of stay might be achieved. this study was designed to investigate and evaluate the opportunities for early discharge created by the availability of new and more sensitive biomarkers such as high - sensitivity troponin t assays (hst). our study focuses on the extent to which the hst in combination with a risk score contributes to the optimisation of earlier decision - making and discharge policy (46 h) in the cer. this study was designed as a prospective cohort study at the cer of the rijnstate hospital in arnhem in the period from february 2011 to july 2011. the study was approved by the local ethics committee and 131 patients initially participated. the clinical cardiologist and the patients for this study, all patients presenting to the cer with chest pain were eligible. patients could be enrolled if the symptoms had been present for less than 6 h. excluded were patients with an indication for percutaneous coronary intervention (pci), as were patients with st - elevation myocardial infarction (stemi) or non - st - elevation myocardial infarction (nstemi ; typical angina with elevated conventional troponin t) diagnosed at admission. finally, patients who had been treated for a cardiovascular complication (e.g. heart failure, unstable angina or myocardial infarction) in the past month were also excluded. all patients were diagnosed and treated based on conventional troponin t. according to the discretion of the cardiologist, non - invasive ischaemia detection could be performed after discharge, during follow - up. myocardial infarction was defined as a rise and/or fall of conventional troponin with at least 1 value above 30 ng / l. patients presenting to the cer underwent an initial clinical evaluation and, as well as the routine laboratory tests including determination of the conventional troponin t, an additional sample was taken to determine the hst (t1). this was repeated at 46 h (t2) and 810 h after symptom onset (t3). if patients presented to the cer within the window of t2, only the t2 sample was taken. as with conventional troponin t, hst was measured on the e170 module of the modular immunoassay (roche diagnostics). we used 14 ng / l as a cut - off value for hst, above which were considered to be increased. for the conventional troponin t assay, this value was set at 30 ng / l. the 10 % coefficient of variation is set at 30 ng / l. the coefficient of the variation of 10 % is set at 13 ng / l. the conventional troponin t does not meet the requirement that the coefficient of variation at the 99 percentile limit is below 10 %. the heart score is a scoring system for patients presenting to the cer with chest pain. by assigning zero, one or two points to an atypical patient history, ecg abnormalities, the patient s age, any risk factors present, and elevated troponin patients who score 0 - 3 points have < 1 % chance of developing a cardiac event, those scoring 46 points have a 12 % chance, while those patients with a score of 7 have on average a 65 % chance of having a myocardial infarction, percutaneous coronary intervention or coronary artery bypass graft, or death within six weeks after presentation (mace). thirty days after discharge, the presence of mace during follow - up was registered retrospectively by checking electronic patient files and personal telephone communication with the patient, with additional information from the general practitioner, when needed. forty - two patients were excluded on basis of the exclusion criteria. finally, 89 patients were included in this study, 52 males and 37 females with an overall mean age of 61 years (range 2090 years). admission time to the cer varied considerably, ranging from 30 min to 6 h after symptom onset. table 2 shows the range and means of the values of hst and conventional troponin t assays measured at the different time points. table 3 shows the of the combination of hst and the heart score at both t2 and t3, with the incidence of mace during follow - up. patients with hst < 14 ng / l at t2 and t3, could have mace during follow - up, whereas no patients with a heart score 3 had mace during follow-up.table 1baseline characteristicsn = 89gender52 male, 37 femaleagemean 61 year (range 2090)duration of symptomsmean 196 min (range 30360)heart score (using ctnt)mean 4.15 (range 18)history of vascular disease39 (44 %) diabetes mellitus17 (19 %) smoking25 (28 %) hypertension29 (32 %) family history of vascular disease30 (34 %) hypercholesterolaemia32 (36 %) table 2descriptive values of the conventional troponin t and the hst assaysconventional troponin (ng / l)nminimummaximummeansdt1 (entry)8916.030.029.61.9t2 (4 h 6 h)8911.0333.032.932.3t3 (8 h 10 h)8913.0661.041.575.0hst (ng / l)nminimummaximummeansdt1 (entry)893.071.910.412.4t2 (4 h 6 h)893.0347.014.737.9t3 (8 h 10 h)893.0675.424.483.8table 3combining hst with heart score at t2 and t3 in order to calculate the risk for macehst (t2)nheart scoremaceno mace 14 ng / l3113031(n = 68)3446331371003 14 ng / l01300(n = 21)1346310871035hst (t3)nheart scoremaceno mace 14 ng / l3113031(n = 65)3146229371003 14 ng / l01300(n = 24)1646412871035 baseline characteristics descriptive values of the conventional troponin t and the hst assays combining hst with heart score at t2 and t3 in order to calculate the risk for mace sixty - eight patients out of 89 (76 %) presented with hst values below 14 ng / l. thirty - one of these patients (46 %) had a heart score between 13. in this group thirty - seven patients (42 %) with hst levels < 14 ng / l at t2 had a heart score above 3. in this group, 3 mace occurred in the one - month follow - up (8 %). these 3 patients all had a pci because of significant coronary stenosis (angiographically > 70 % and/or fractional flow reserve < 0.8) during 30 days of follow - up. in 21 patients (24 %) the probability of a cardiac event in this group was substantially higher (28 %). in this group, there were no patients with a low heart score. in fig. 1 hst levels at t2 and t3 are compared. at t2 (46 h after onset of symptoms), 68 patients had a hst value less than 14 ng / l. at t3 (810 h after start of symptoms), for 3 patients (4 %) hst levels increased to levels above the cut - off value of 14 ng / l (average increase in these 3 patients was 30 %). all 3 patients had a moderately increased heart score between 4 and 6. based on their heart score, they need longer follow - up and repeat hst determination. one patient had occurrence of a mace during follow-up.fig 1eps changes in serum high sensitivity troponin t (hst) in patients with a level 14 ng / l at 4 - 6 h after onset of symptoms eps changes in serum high sensitivity troponin t (hst) in patients with a level 14 ng / l at 4 - 6 h after onset of symptoms we investigated the value of the heart score as risk stratification model, with the use of conventional troponin t or hst in patients with suspected acs. our main findings were: patients with normal hst and a heart score of 3 could safely be discharged at t2, as no mace occurred. with a heart score of 4 or more, further monitoring, serial troponin tests, and/or extra investigation (exercise testing, nuclear imaging, stress echocardiography) may be required, because a small proportion of patients have mace at follow - up. patients with elevated hst at t2 and t3, and/or with a heart score 7 are at high risk of mace. the heart score is a specially validated risk score for patients with chest pain presenting to an emergency department in the netherlands. other risk scores such as the grace or timi risk score are mainly validated in patients with stemi and nstemi and therefore less useful for this category of patients. it should be noted that these risk scores include elevated cardiac markers in their point scoring system. the heart score system makes use of conventional troponin, and this score system should be further validated, especially with the current widespread use of hst in cardiology practice. in our present study, 3 patients (4 %) with initially normal hst at t2 showed a rise above the cut - off value of 14 ng / l at t3. the significance of such an increase is at present unclear, as some advocate that a 100 % rise is associated with acute, thrombotic, myocardial infarction. if clinical decision - making had been based on hst alone, these 3 patients would not be identified as high - risk patients at risk for mace. by also taking the heart score into account the use of a risk score as the heart score can provide additional information for identifying these patients. as mentioned earlier, the heart score has been validated for conventional troponin t and not for hst. it is to be expected that by using a high - sensitivity assay in a new heart score, hst levels will more often be elevated as compared with conventional troponin t, and hereby the new risk score could have a higher sensitivity, at the cost of less specificity. further studies should provide regarding the discriminative value of this new risk score. despite the fact that cardiac troponins are sensitive and are specific biochemical markers of myocardial damage, an increased troponin t level occurs in many diseases in which the heart does not show ischaemia, such as congestive heart failure, pulmonary embolism, renal failure, acute neurological disease, myocarditis or sometimes in apparently healthy persons. furthermore, the availability of new high - sensitive assays for troponin t may allow the detection of small changes in troponin and this test meets the requirements of the universal definition of myocardial infarction. the universal definition of acute myocardial infarct states that there has to be a typical rise and/or fall of biochemical markers (preferably troponin) with at least 1 value above the 99 percentile reference limit with at least one of the following: ischaemic symptoms, ecg changes, pathological q waves or imaging evidence of new loss of viable myocardium motion abnormality. the hst assay does meet the requirement that the coefficient of variation at the 99 percentile limit is below 10 %. the test does not only appear more sensitive, but is able, even 3 h after symptom onset, to differentiate whether there is an acute coronary syndrome or not. early decision - making is therefore possible. a problem that may arise is the possibility of false - positive of acute coronary syndrome. however, it is still unclear whether there should be an absolute or relative increase in troponin values. in addition, at this moment it is also unclear how much the troponin values should increase to speak about an acute coronary syndrome. some advocate that a doubling of the serum hst within 3 h indicates an acute coronary syndrome whereas others (including the guidelines) state that there has to be a rise and/or fall with at least 1 value above 14 ng our study is relatively small; therefore, future studies should be aimed at extending these findings by including more patients. only with evidence from larger study populations, for clinical decision - making can be drawn. , our study suggests that patients with chest pain at the cer, with a negative hst level together with a low heart score, can be safely discharged from the hospital within 4 - 6 h after onset of symptoms.

patients with chest pain have a large impact on available resources in coronary emergency rooms (cer). clinical judgement, ecg, risk scores and biomarkers guide in risk stratification. we investigated if high - sensitivity troponin t (hst) and the heart score could contribute to risk stratification at the cer. all patients with chest pain, without elevated conventional troponin levels at presentation, were included. hst levels were determined at admission (t1), at 46 h (t2) and 810 h after symptom onset (t3). the heart score was calculated as risk score for the occurrence of a major adverse cardiac event (mace). thirty days after discharge, occurrence of mace was registered. eighty - nine patients were included (overall mean age 61 years ( range 2090) ). at presentation, 68 patients (76 %) had a hst below cut - off value of 14 ng / l (mean heart score 3.7, range 19). thirty - one of these 68 patients had a heart score between 13, no mace occurred in this group. for 3 patients (4 %) hst levels increased above 14 ng / l. these 3 patients had a heart score between 46. the majority of patients with chest pain can be safely discharged within 46 h after onset of symptoms using hst and the heart score. in contrast, patients with initially normal hst but a high heart score need longer follow - up and repeat hst determination.

hepatectomy is established as a standard of care for patients with resectable liver - limited metastases of colorectal cancer origin. hepatectomy is also undertaken for a range of other malignant and benign conditions. contemporary experience with techniques such as downsizing of tumours by neoadjuvant chemotherapy and modification of the volume of the future remnant liver by selective portal vein embolization in the ability to offer liver resection to a greater proportion of patients with liver metastases. these changes, coupled with population trends ing in a greater proportion of elderly patients presenting for treatment, mean that, in the 21st century, liver resection for metastatic colorectal cancer is often undertaken in older individuals and/or in patients whose hepatic functional reserve may have been compromised by prior chemotherapy or by preexisting chronic liver disease. although reports outline the feasibility of undertaking liver resection with low perioperative mortality , postoperative morbidity is important as it can influence a range of outcomes including critical care occupancy and in - patient stay, uptake of adjuvant chemotherapy, and quality of life after surgery. during liver resection, ir injury can occur during any of a series of operative steps including liver mobilisation and permanent inflow occlusion of the segment to be resected and during temporary inflow occlusion followed by restoration of blood flow to the future remnant liver (the pringle manoeuvre). ir injury is an important determinant of postoperative morbidity and eventual clinical outcome. strategies employed to minimise ir injury include avoidance of inflow occlusion and ischaemic preconditioning and the administration of pharmacologic agents. at the cellular level, kupffer cells release proinflammatory mediators during liver ir, including oxygen - derived free radicals. the physiologic intracellular defence mechanisms involved in the regulation of oxidative injury include enzymatic pathways catalysed by superoxide dismutase and glutathione peroxidase. glutathione (gsh), a tripeptide composed of glycine, glutamic acid, and cysteine, constitutes the largest component of the endogenous thiol buffer for oxygen - derived free radicals. for tissue gsh synthesis, n - acetylcysteine is a thiol containing synthetic compound. in the isolated, perfused rat liver, administration of nac before reperfusion in concentration - dependent increases in gsh concentrations in bile with corresponding increases in bile flow. currently the most established clinical use for n - acetylcysteine is in the treatment of acetaminophen (paracetamol) poisoning. to date one small clinical trial has assessed the effect of perioperative n - acetylcysteine on prevention of ischemia - reperfusion injury in liver resection. liver function was assessed by postoperative transaminase (alt) rise and this was significantly ameliorated in the treatment group. despite the predominantly favourable of experimental studies there are no substantive clinical trials evaluating the role of n - acetylcysteine or other pharmacological agents to modulate ir injury in patients undergoing hepatectomy. these drugs (in particular n - acetylcysteine) have been available for a long time and anecdotal evidence suggests that they are still used in an off - licence mode as modifiers of liver ir in hepatectomy. a questionnaire survey provides an overview of the contemporary use of pharmacological modulation of ir injury in liver surgical practice. an international target population of clinical units undertaking liver surgery can be obtained through the livermetsurvey. thus the aim of this study is to undertake a questionnaire survey of the use of pharmacological agents to modify liver ir injury in patients undergoing hepatectomy for colorectal liver metastases in units participating in the livermetsurvey. the questionnaire design examined strategies for modulation of liver ir injury during hepatectomy and included questions on a broad overview of techniques for this purpose in addition to pharmacological interventions. specific detail was sought on whether pharmacological agents were used by respondents to treat or reduce liver ir injury with information being sought on the indications for use, choice of agent, use of a loading dose, timing of use in relation to the stage of surgery (preoperative, intraoperative, posttransection, etc .), and dose. as it was anticipated from prior literature searches that n - acetylcysteine would be the most widely used agent, specific focus was given to this drug. a final question sought opinion on whether the respondent supported in concept a randomized trial to evaluate the role of pharmacological ir modifiers in hepatectomy. permission was obtained from the livermetsurvey for their members to be sent an online questionnaire using surveymonkey (https://www.surveymonkey.com/). the livermetsurvey was selected as this is a registry of liver resection outcomes for colorectal hepatic metastases and thus all participants would be likely to be undertaking hepatectomy. response to this questionnaire was discretionary and following the initial email no reminders were sent. the questionnaire was live for a period of six weeks from june 18, 2013, and was sent to 446 participants of the livermetsurvey. there were 85 respondents of whom two were not actively engaged in liver resection practice. these two respondents were excluded to provide a final study population of 83 (19%) respondents. the study was discussed with the hospital's research and development team, who advised that, as there was no patient contact and only email contact with professional colleagues, no ethics committee submission was required. the number of respondents to each individual question is provided and serves as the denominator for the percentage of respondents to that particular question. for several questions, more than one response was permitted and therefore the cumulative total of percentage responses to such questions can exceed 100. there were 72 respondents to this question and more than one response was permitted (figure 1). the most frequently used strategy was avoidance of the pringle manoeuvre by 53 (74%) respondents. six (8%) replied that they used pharmacological agents to modify liver ir injury. fifty - two (77%) stated that they never used pharmacological agents to modify liver ir injury during hepatectomy. ten (56%) stated that a requirement for total vascular exclusion was an indication for the use of pharmacologic agents, 9 (50%) stated that prolonged inflow occlusion was an indication, 9 (50%) used pharmacological agents if there was evidence of deterioration in liver function in the postoperative period, and 8 (44%) used pharmacological agents in the setting of a small - volume future remnant liver, 7 (39%) in major liver resection of more than 3 couinaud segments, 5 (28%) in liver resections undertaken in patients with cirrhosis, and 4 (22%) in patients who had received prior chemotherapy. individual replies were received (one respondent each) for glutamine, desflurane, sevoflurane, bicarbonate, arginine, and a cocktail of methionine with vitamins c and e. there were 22 responses to this question with 14 (64%) replying that they did not use a loading dose. three (17%) used pharmacological intervention prior to surgery in the form of steroids. two (11%) gave nac at induction of anaesthesia, four (22%) during liver mobilisation, two (11%) at commencement of transection, two (11%) postoperatively (routinely), and five (28%) postoperatively (selectively). there were 9 respondents to this from those who used n - acetylcysteine for modulation of liver ir injury. seven (78%) used a standard dose for all patients and 2 (22%) used a body weight - adjusted dose. three (33%) continued the infusion for 48 hours, 3 (33%) until serum transaminases started to fall, 2 (22%) for 72 hours, and 1 (11%) for 24 hours. fifty - six (81%) answered affirmatively and 6 (9%) replied that there should not be a trial. this study reports the of what is thought to be the first survey of the use of pharmacological agents to modify liver ir injury in the perioperative period. first, the do not in any way represent an official position statement of the livermetsurvey. second, given that less than 20% of the target audience responded to this questionnaire, the replies are not indicative of any consensus view. third, it is possible that those who responded had a vested interest in pharmacological modification of liver ir injury and thus the could be skewed by ascertainment bias. the target population was selected as the livermetsurvey participants as by definition these are units undertaking liver surgery for colorectal hepatic metastases. clearly, the views of units and clinicians outwith livermetsurvey will not have been captured by this survey. interpretation of these bearing these caveats in mind produces an interesting overview into pharmacological modification of liver ir injury. first, only a minority of respondents use pharmacological agents to modify liver ir injury during hepatectomy with 6 (8%) of 72 respondents stating that they used this strategy (figure 1). the influence of the phrasing of the question on the answer is seen in that when asked specifically whether pharmacological agents were used to modify liver ir injury in the next question, 13 (19% of 68 respondents) replied that they used these agents selectively in addition to the 3 (4%) who used them routinely. nonetheless it is clear from this small study that pharmacological modification of ir injury during hepatectomy is only used by a minority of respondents. despite the negative of a randomised controlled trial of ischaemic preconditioning to reduce liver ir injury the selection criteria for use of pharmacologic agents are interesting in that those clinicians using these agents employed them for major resections and in individuals with compromised liver parenchyma or function. this observation may be worth bearing in mind if a future randomized trial is to be considered as targeted use may be more appropriate than routine use in all liver resections. in terms of choice of pharmacological agent , it should be emphasised that the questionnaire restricted the responses and that only 18 responses were received to this question with n - acetylcysteine used without a loading dose being the most frequent choice. there is clearly no consensus on the optimum time for commencement of nac or on the duration of use although the majority of respondents favoured a standard dose for all patients without adjustment for body weight. the majority of respondents (56 ( 67% of cohort) ) support a randomised trial and in view of patterns of current use it would appear that the role (if any) to be explored is in patients undergoing extended resection or with compromised functional hepatic reserve or with a small remnant liver after resection. in summary , this is thought to be the first questionnaire survey of a population of clinicians undertaking surgery for colorectal liver metastases to investigate the use of pharmacological agents for modification of liver ir injury. although the response rate was only 19%, the ant population of 83 clinicians is the largest specialist group to contribute data to this question to date. the show that pharmacological modulation of liver ir injury is not a routine component of care after liver resection in contemporary hepatic surgery practice and that it is used routinely by only 3 (4%) respondents within livermetsurvey and selectively by a further 13 (19%) with the indications being major resections or in patients with compromised liver reserve.

objectives. this study is a questionnaire survey on the use of pharmacological agents to modify liver ischaemia - reperfusion (ir) injury in patients undergoing hepatectomy for colorectal liver metastases with the target population being those units participating in the livermetsurvey international registry. methods. members of livermetsurvey were sent an online questionnaire using surveymonkey comprising ten questions on the use of pharmacological agents to modulate hepatic ir injury in the perioperative period after hepatectomy. the questionnaire was sent to 446 clinicians registered with the livermetsurvey. there were 83 (19%) respondents. . fifty - two (77% of 68 respondents to this question) never used pharmacological agents to modify liver ir injury during hepatectomy. thirteen (19%) used pharmacological agents selectively. three (4%) used these routinely. n - acetylcysteine was the most widely used pharmacological agent with equal distribution of use around intraoperative and postoperative periods. . this is believed to be the first survey on the use of pharmacological agents to modify liver ir injury. the target population is clinicians involved in liver resection. the show that pharmacological modulation is used by only a minority of respondents to this questionnaire and that when this treatment is selected, n - acetylcysteine is the most frequently used.

the goal of the present study was to determine if the expression of metallothionein isoform 3 (mt-3) might serve as a biomarker for human bladder cancer. to accomplish this goal, we defined the localization and expression of mt-3 protein and mrna using fresh and archival biopsy specimens obtained from patients undergoing differential diagnosis for a variety of bladder disorders. we used immunohistochemistry, immunoblot, and rt - pcr analysis to define the localization and expression of mt-3 protein and mrna. immunohistochemical analysis disclosed no immunoreactivity for mt-3 in normal bladder cells. the absence of mt-3 expression in the normal bladder was further confirmed by demonstrating that mt-3 mrna could not be detected using reverse transcriptase - polymerase chain reaction (rt - pcr) or mt-3 protein using immunoblot. immunohistochemistry also disclosed no immunoreactivity for mt-3 in archival biopsy specimens from patients with interstitial cystitis and related disorders. immunohistochemical analysis demonstrated that mt-3 was expressed in carcinoma in situ (cis), high - grade bladder cancer, low - grade bladder cancer, and dysplastic lesions. mt-3 immunostaining was intense in both cis and high - grade bladder cancer, and low to moderate in low - grade bladder cancer and dysplastic lesions. we determined mt-3 mrna expression in a subset of these bladder cancer specimens; expression was elevated as compared to that of the housekeeping gene, ss - actin. the cdna from the rt - pcr reaction primed for mt-3 contained a foki restriction site, a site unique for mt-3 as compared to other mt family members. in , this study demonstrates that mt-3 is up - regulated in human bladder cancer and that this up - regulation increases with increasing tumor grade. the finding that mt-3 expression is minimal in normal bladder suggests that mt-3 might be developed into an effective biomarker for bladder cancer.imagesfigure 1figure 2figure 3figure 4

the nevus of ota is a light bluish nevus on the unilateral face, which is congenital or acquired in adolescents, and often seen in asian females. malignant transformations are rarely observed in the orbit 1 and seldom seen among asians 2. as the potential for local invasion or metastasis varies widely, strict prognostic indicators for orbital melanoma have yet to be established 3. animal - type melanoma is a rare type of malignant melanoma that usually arises in skin lesions 4 and shows indolent progression and good prognosis despite metastasis. to the best of our knowledge, this is the first case report of a primary animal - type malignant melanoma in the orbit that developed from the nevus of ota to show a satisfactory clinical course. a 42-year - old woman presented with a right - sided eye swelling and fever. she had been diagnosed with diabetes mellitus 2 years previously, but had received no medication. she had no particular family history, except for the fact that her father had stomach cancer. bluish pigmentations were visible on her right forehead, ear, and eyelid since birth. hertel exophthalmometry showed exophthalmos in her right eye [24 and 18 mm in the right and left eyes, respectively, with a base of 110 mm ( fig . ocular movement of her right eye was restricted in both the horizontal and the upward gaze . intraocular pressure was 19 mmhg in her right eye and 18 mmhg in her left eye . mri showed a right - sided exophthalmos and a 2.53 cm large, well - defined intraconal mass . the tumor was heterogeneous and there was a slightly high intensity observed in the gadolinium - enhanced t1-weighted image ( t1-wi) and a low intensity in the gadolinium - enhanced t2-weighted image (t2-wi) (fig . another 56 mm large mass that was discontinuous with the orbital tumor in the cranial base was also observed and primarily presumed to be a meningioma ( fig . laboratory tests indicated that her diabetes mellitus was uncontrolled ( hba1c, 11.5%) and that she had a normal ldh level (220 u / l). (a) bluish pigmentation was seen on the right eyelid and black pigmentation was seen in the right conjunctiva. (b) t1-weighted image: right - sided exophthalmos was seen and the retrobulbar mass showed a slightly high intensity in the t1-weighted image (indicated by an arrow). (c) t2-weighted image: right - sided exophthalmos was seen and the retrobulbar mass showed low intensity in the t2-weighted image (indicated by an arrow). a 56 mm large mass, which was discontinuous with the orbital tumor in the cranial base, was also enhanced (indicated by an arrowhead). the tumor, which ruptured during the operation because of biopsy for pathological diagnosis, was frozen for later analysis. because the subsequent pathological diagnosis of the frozen specimen during the operation showed it to be a benign lesion, we completed tumor excision without orbital extirpation. (a) macroscopy of the extracted orbital tumor: highly pigmented 62 cm large tumor, which ruptured during the extirpation. (b) hematoxylin and eosin staining of the orbital tumor: melanin - containing cells (melanocytes and melanophages) are abundant in the tumor. (c) hematoxylin and eosin staining of the orbital tumor after using a bleaching method for melanins: diffuse proliferation of the epithelioid cells and spindle cells, and low - grade nuclear atypia were seen. (e) melan - a staining: positive for atypical cells. (f) hematoxylin and eosin staining of the orbital tumor after using a bleaching method for melanins: vast areas of necrosis were seen. after preparation of the sample, the pathological examination of the tumor showed that the lesion was an animal - type malignant melanoma. after using a bleaching method for melanins, hematoxylin and eosin staining was performed (fig . 2c). all the cells had low - grade pleomorphic nuclei, with nucleoli observed in some cells, although they were not found to be prominent. hmb (human melanoma black)-45 (fig . 2d) and melan - a (fig . we performed orbital extirpation with an excision of a 5 mm margin from the nevus of ota ; the orbit was then covered with a skin graft taken from the patient s thigh . these cells were found in the skin, subcutaneous fat, orbital muscle, orbital fat, lacrimal gland, and sclera . also showed that the retina, the uvea, and the cornea were not involved . some parts of the nevus were cellular, with slightly large, round epithelioid melanocytes that contained low - grade pleomorphic nuclei . although some of these were accompanied by small nucleoli, neither necrosis nor mitosis was observed . although no malignant changes were apparent at the time of the procedure, the findings did suggest the possibility that these atypical melanocytes might be an early stage of melanoma . mri performed before the second operation showed that there was a slight enlargement and high intensity in the t1-wi of the cerebral mass that had been considered as a meningioma before . in addition to the second surgery, chemotherapy ( dav therapy, which is a combination of dacarbazine, nimustine, and vincristine), stereotactic radiotherapy (54 gy in 27 fractions), and gamma knife (the marginal dose was 17 gy and the target volume was 0.2 ml) were administered. since then , we have examined her once every 23 months by laboratory tests including lactate dehydrogenase, aspartate aminotransferase, and alanine aminotransferase. we have also taken some biopsies to check the orbital local recurrence and performed annual 18f - fluorodeoxyglucose positron emission tomography to check metastasis. otolaryngologists and dentists have checked the pigmentation of the mouth and physicians have brought her severe diabetes mellitus under control. thirty - three months have passed since the second surgery, with no sign of local recurrence, new metastasis, nor enlargement of the intracranial tumor. the nevus of ota (oculodermal melanocytosis, naevus fusculocoeruleus ophthalmomaxillaris) was described by the japanese dermatologist, ota, in 1939. it is a light bluish nevus on the unilateral face, which is congenital or acquired in adolescents. it has been shown to be more common in east asians (0.21.0%) than in western populations (0.04%) 5. the incidence of malignant change is 0.5% for east asians and about 25% for western populations 2. therefore, the calculated incidence of malignant melanoma associated with the nevus of ota is 0.0010.005% (0.21.0%0.5%) among east asians, whereas it is 0.01% (0.0425%) among western populations. malignant melanoma associated with nevus of ota has been reported to occur in the uveal tract 6, optic nerve head 7, brain, and orbit. as for western populations, rice and brown 8 reported finding six cases of primary orbital melanoma associated with nevus of ota, whereas two other studies have additionally reported two more cases 1,9. however, in terms of east asian populations, ours is the first report of such a case. the original report of the animal - type melanoma was based on a highly pigmented malignant melanoma that was found in a horse and then shown to be a very rare variant of melanoma. it has been reported that this melanoma usually occurs in skin lesions found on the scalp, face, and extremities 10. of the eight reported orbital melanoma cases, pathological diagnosis indicated that five were spindle, two were epithelioid, and one was mixed 1,8,9. the current patient is the first case of the animal - type melanoma that arose in the orbit. histopathologically, animal - type melanoma cells show some nuclear atypia and regular oval to round nuclear shapes, with moderate anisonucleosis, small nucleoli, and distributed chromatin. characteristically, these do not fulfill the cytologic criteria of malignancy. the most important clue to the diagnosis of animal - type melanoma is effacement of the dermal architecture by areas of confluent melanocytic growth. all the cells had low - grade pleomorphic nuclei, with nucleoli seen in some cells. distant metastases of animal - type melanoma are rare and even when they occur, these patients appear to have a better prognosis than those with an ordinary melanoma presenting with metastatic disease 10. it may because of this characteristic that our patient showed neither enlargement of tumor nor neurologic symptoms for long despite an intracranial metastasis. however, even with this low malignant potential, animal - type melanoma is clearly a variant of melanoma 10. therefore, application of the guidelines used for melanoma, clinical investigation of regional and distant metastases, and surgical treatments with safety margins 12 are recommended in these types of cases 13. thirty - three months since the extirpation, the patient is still alive, with careful follow - up examinations, despite having intracranial metastasis. not just the combination therapy but also the low malignancy of animal - type melanoma may have contributed toward this good prognosis. although the possibility is not high that malignant transformation occurs in the nevus of ota, it is important to consider this possibility as it can be seen in any population groups. doctors involved in the treatment of nevus of ota (as in our case, ophthalmologists, pathologists, dermatologists, brain surgeons, otolaryngologists, radiologists, and dentists) should understand that malignant transformation could occur and should cooperate with one another for its treatment. although practical strict prognostic indicators for orbital melanoma are yet to be established 3, this particular case suggests that the characteristic indolent progression of the animal - type melanoma might partly contribute toward the good prognosis.

we present a patient with an animal - type malignant melanoma associated with the nevus of ota in the orbit who showed a good prognosis after a combination of orbital extirpation, chemotherapy, stereotactic radiotherapy, and gamma knife. a 42-year - old japanese woman presented with two tumors, one pathologically diagnosed as right - sided intraconal animal - type malignant melanoma and the other intracranially, presumed to be of the same pathogenesis and both were considered to have arisen from the nevus of ota. she underwent an extirpation of the orbit, chemotherapy (dav therapy, which is a combination of dacarbazine, nimustine, and vincristine), stereotactic radiotherapy (54 gy in 27 fractions), and gamma knife (marginal dose was 17 gy, target volume was 0.2 ml). she has been alive for 33 months since the extirpation, with no sign of local recurrence, new metastasis, nor enlargement of the intracranial tumor. not just combination therapy but also the low malignancy of animal - type melanoma may have contributed toward the good prognosis.

the prevalence of type 2 diabetes (t2d) in rural regions is rising rapidly and has been a great challenge to the local health care programs. in china, large parts of diabetic individuals lived in rural areas, where resources to screen and treat diabetes and its complications were limited. so far, the situations of type 2 diabetes presenting with ketosis prior to admission in the south - west rural region were unclear. ketosis is a symbol of acute disorder of glucose and lipids metabolism in diabetic patients. ketosis or ketoacidosis has been regarded as trait of type 1 diabetes (t1d). because of backward in economy, quite a few patients did not get duly treatment until severe hyperglycemia or diabetic complications occurred. our present study was to investigate the prevalence and characteristics of t2d presenting with ketosis in rural areas aiming to suggest more efforts for the prevention and control of diabetes in rural areas of china. we performed a retrospective analysis of t2d with ketosis in patients admitted to the endocrinology department of baoshan people's hospital of yunnan province between january 2011 and july 2015. overall, data of 391 t2d presenting with ketosis inpatients aged 12 years or older were enrolled. nine patients were excluded because of surgery, pregnancy, trauma, secondary diabetes, or pancreatic exocrine diseases. thus, the other 371 patients (245 males and 126 females) were allocated to the analysis. according to the diabetic duration new - onset group included those with new diagnosed diabetes or no more than 6 months after onset. old - diagnosed group were patients of previously diagnosed t2d presenting with ketosis at this admission. t2d was diagnosed if patients had clinical and metabolic features including overweight or obesity, more than 40 years age at onset, and obvious diabetes family history in combination with preserved -cell function (fasting c - peptide of more than 0.33 t2d with ketosis was diagnosed if patients met the criteria of t2d and had positive urinary ketone body this time on admission . urinary ketone body positive was diagnosed if urinary acetoacetate increased over 15 mg / dl by using sodium nitroprusside method . acidosis was diagnosed if an arterial ph of less than 7.35 and/or blood bicarbonate level of less than 15 overweight and obesity were defined if body mass index ( bmi) 24, which was set forth by chinese guidance of overweight and obesity in adults. data of demographic and clinical characteristics including age, gender, height, weight, bmi, blood pressure, and diabetic family history were collected. laboratory parameters including plasma glucose, hba1c, c - peptide, total cholesterol, and triglycerides were recorded. the chronic diabetic complications including peripheral neuropathy, retinopathy, diabetic foot, and persistent microalbuminuria that were evaluated by trained physicians during the hospitalization. all of the data were analyzed by jmp 9.0 (sas institute, cary, nc). the anova or mann - whitney test analysis was used to evaluate the differences in continuous variables. table 1 showed the clinical characteristics of the new - onset and old - diagnosed t2d with ketosis patients. among the total 371 subjects, 211 of the patients (57%) were classified as new - onset, while 160 (43%) were old - diagnosed t2d with ketosis. the subjects of new - onset group had an average duration of 1.7 months with weight loss of 7.3 kg, while the old - diagnosed group had an average diabetic duration of about 6.3 years with weight loss of 9.2 kg. subjects of new - onset group were younger (47 12 versus 53 13 yr, p = 0.001) and more overweight (50% versus 46%, p = 0.009) than those old - diagnosed t2d with ketosis. family history of diabetes was similar (p = 0.79) between new - onset (35%) and old - diagnosed (34%) group. the blood pressure of the analyzed subjects was not significantly different between two groups. table 2 showed the biochemical characteristics of the new - onset and previously diagnosed t2d patients presenting with ketosis. fasting plasma glucose mmol / l ) and old - diagnosed (16.4 7 mmol / l, p = 0.49) group. 2 h plasma glucose was also similarly elevated (p = 0.86) between new - onset (24.2 8 fasting c - peptide levels were similar ( p = 0.60) between new - onset (0.69 0.11 nmol / l) and old - diagnosed (0.58 0.17 2 h c - peptide was significantly higher ( p = 0.02) in new - onset (2.58 1.1 the hemoglobin a1c ( hba1c) and fructosamine were not different between the two groups. more patients in the old - diagnosed group (19.4%) suffered from acidosis than new - onset group (6.6%). total cholesterol (5.9 1.7 versus 4.9 1.8 mmol / l, resp ., in new - onset and old - diagnosed group, p < 0.0001) and triglyceride (4.4 4.0 versus 3.7 4.2 mmol / l, resp ., in new - onset and old - diagnosed group, p = 0.004) were significantly higher in new - onset than in old - diagnosed group. those new - onset patients showed a lower incidence of predisposing factors than previously diagnosed t2d patients presenting with ketosis (13.8% versus 38.7%, p < 0.001). the potential triggers for subjects in t2d with ketosis were varied. among them, infections were predominant in both groups. the prevalence of respiratory system infection was 10.3% in new - onset and 14.9% in old - diagnosed group. urinary system infection was 1.8% in new - onset and 9.9% in old - diagnosed group. figure 2 showed the prevalence of chronic diabetic complications in new - onset and previously diagnosed t2d with ketosis. the patients with new - onset had higher (p < 0.0001) prevalence of fatty liver disease (26.2%) compared with those of old - diagnosed (15.1%) group, while the prevalence of most chronic complications was higher in old - diagnosed group than new - onset group, including retinopathy (8.1% versus 17.8%, resp . , in new - onset and old - diagnosed group, p < 0.0001), peripheral neuropathy (25.3% versus 47.6%, resp ., in new - onset and old - diagnosed group, p < 0.0001), diabetic foot (13.3% versus 23.2%, resp . , in new - onset and old - diagnosed group, p < 0.0001), and persistent microalbuminuria (8.2% versus 15.1%, resp . , in new - onset and old - diagnosed group, p < 0.0001). no significant differences (p = 0.45) in atherosclerosis were found in new - onset (18.0%) and old - diagnosed (20.1%) group. in this study, we showed the characteristic of t2d with ketosis in rural areas of south - west part of china. it was concluded from the study that the control of t2d was poor in rural area, as reflected not only by extremely elevated hba1c and plasma glucose performed once on admission but also by high prevalence of chronic diabetic complications evaluated during hospitalization. there were limited data about the prevalence of ketosis in type 2 diabetes population. in a study of 134 chinese patients with severe hyperglycemia, there were 24 patients diagnosed with ketosis (17.9%) and 6 patients diagnosed with ketoacidosis (4.5%). subjects of new - onset and old - diagnosed t2d with ketosis group had similarities and differences at clinical manifestations. consistent with previous studies, we found a predominance of male proportion in the study subjects. the underlying mechanisms of male predominance in t2d with ketosis were unknown and needed further investigation. differences of body fat distribution and sex hormone were supposed to be factors contributing to the gender difference. in our study, the prevalence of hyperlipidemia was high in both new - onset and old - diagnosed subject, which were true with many previous studies. some investigators have reported that acute elevation of free fatty acid levels in the circulation increased insulin resistance and impaired -cell function. in our study subjects, the prevalence of hyperlipidemia was 60% in subjects of new - onset group and 29% in old - diagnosed group. we analyzed our data and found that hyperlipidemia was common in the overweight diabetic subjects. bmi of the enrolled patients positively correlated with both triglyceride and total cholesterol level (data not shown). the phenomenon further confirmed the view that hyperlipidemia played substantially roles underlying trigger of ketosis especially in new - onset diabetes. therefore, interventions for prevention and treatment of overweight and hyperlipidemia were essential in t2d patients. infections were the most common precipitating factors for ketosis in t2d patients, which was an important point distinguishing t2d from t1d. in patients with t1d, noncompliance with insulin therapy was considered as first reason for precipitating ketosis. in our old - diagnosed t2d subjects, varied infections accounted for about 40% of the total precipitating factors, indicating that infections were the important precipitating factors leading to ketosis. however, in new - onset ketosis subjects, most cases were nonprovoked ketosis. the discrepancy in prevalence of precipitating factors might indicate the complexity and heterogeneity of t2d with ketosis. more clinical epidemiological surveys need to be conducted to clarify the heterogeneity in t2d with ketosis. in our study subjects, patients had a higher incidence of diabetic chronic complications, especially in those previously diagnosed t2d patients. it was shown by literatures that those who came for regular follow - up diabetic treatment had a lower incidence of retinopathy and nephropathy compared to those who had irregular follow - up. since t2d and most of the chronic complications were silent at first , patients usually did not come to the clinic for routine examination until they developed severe complications of diabetes in rural areas. as a , most patients were complicated with severe chronic complications at their fist diabetic diagnosis. however , late treatments of diabetes and its complications were often associated with higher costs to the affected patient. therefore, it was reasonable to suggest detection and adequate treatment at an earlier stage, by which the excess cost due to treatment of diabetes complications might be minimized. first, we did not perform plasma ketosis test which was more sensitive and specific in detecting ketosis; however, urinary ketone body measurements were widely acceptable in hospitals for diagnosing ketosis. second, we did not measure the body fat distribution which was a potential and important parameter for explaining gender difference in t2d with ketosis. third, the single - center study restricted our ability to assess the overall characteristics of t2d with ketosis in rural areas. further large cohorts and prospective follow - up studies were needed to clarify the characteristics of t2d with ketosis in south - west rural parts of china. in summary, the study shows the characteristics of t2d with ketosis in adults of rural areas in south - west part of china. t2d with ketosis is a severe disorder of glucose and lipids metabolism and prone to acquire chronic complications. thus, there is an urgent and important need for the implementation of effective screening, health knowledge promotion, and control of diabetes.

objectives. type 2 diabetes (t2d) with ketosis was common because of late diagnosis and lacking adequate treatment in rural regions of china. this study aimed to provide the data of t2d with ketosis among inpatients in a south - west border city of china. methods. data of 371 patients of t2d with ketosis who were hospitalized between january 2011 and july 2015 in baoshan people's hospital, yunnan, china, were analyzed. new - onset and old - diagnosed t2d patients presenting with ketosis were compared according to clinical characteristics, laboratory , and chronic diabetic complications. . overall, the blood glucose control was poor in our study subjects. male predominated in both groups (male prevalence was 68% in new - onset and 64% in old - diagnosed groups). overweight and obesity accounted for 50% in new - onset and 46% in old - diagnosed cases. inducements of ketosis were 13.8% in new - onset and 38.7% in old - diagnosed patients. infections were the first inducements in both groups. the prevalence of chronic complications of diabetes was common in both groups. . more medical supports were needed for the early detection and adequate treatment of diabetes in rural areas of china.

adipose tissue, once just believed a passive organ storing excess energy, is now considered a real endocrine organ, with an important role in the regulation of homeostatic systems. it secretes various proteins that can be classified into molecules acting on metabolic processes like glucose homeostasis and insulin sensitivity, and into inflammatory molecules like interleukin 6 (il-6), interleukin 1, tumor necrosis factor-, c - reactive protein (c - rp), leptin, and adiponectin. the adipose tissue may account for 2025% of systemic il-6 circulating levels and seems to be involved in the systemic inflammatory response associated with obesity, insulin resistance, and metabolic syndrome. on this basis, adipose tissue represents a new and fascinating multidisciplinary object of research. we previously found a clear expression of il-6 receptors and c - reactive protein in human adipocytes obtained from adipose tissue fragments of different districts (subcutaneous and omental of noninflamed and inflamed patients), showing that the adipose tissue, and in particular the adipocyte, was not only responsible for generating an inflammatory state per se but it also represents a target, by itself, of systemic inflammation. we observed, in fact, that the adipocyte, stimulated by il-6 through its cell receptor produces c - rp, as it happens in the hepatocyte. adipose tissue is a mesodermallyderived organ that contains, in addition to adipocytes, a stromal population composed by different cell types, such as pre - adipocytes, stem cells, fibroblasts, and macrophages. mature adipocytes account for only 4060% of the whole cell population whatever the type of adipose tissue. pre - adipocytes are present throughout adult life in adipose tissue and can proliferate and differentiate into mature adipocytes, according to the energy balance; they also share some features with macrophages, such as the capacity of phagocytosis in response to different stimuli. another important cell population of the adipose tissue is represented by mesenchymal stem cells (mscs) that are adherent, fibroblast - like, pluripotent, and nonhematopoietic progenitor cells. these cells retain the capacity to undergo into many mesenchymal cell types, including bone cells , neuronal cells, adipose, and muscular tissue in vitro, and of note, into hepatocyte - like cells. thereby, both the pre - adipocyte, with a very restricted potential, and the multipotent mesenchymal cell represent adipocyte precursors. albumin, normally produced by the hepatocyte, is the most abundantly circulating protein in blood, where it plays an important role as binding protein and is involved in the regulation of colloid - osmotic homeostasis. it is also a negative acute phase protein and hypoalbuminemia is the of the combined effects of inflammation, through inhibitory cytokine (especially il-6), and inadequate protein and caloric intake in patients with chronic inflammatory disease, such as chronic renal failure where it represents a cardiovascular risk factor. considered the well - known active role of adipocyte in c - rp production and the presumable link between adipocyte and hepatocyte, we aimed this study at investigating if mature adipocytes express the gene of albumin and at evaluating whether systemic inflammation might regulate this expression, as we previously found for c - rp. furthermore, we speculated whether the adipocyte might contribute to the total circulating levels of albumin, as it happens for il-6. human subcutaneous adipose tissue samples were obtained from twelve subjects divided in two subgroups on the basis of a preliminary evaluation of c - rp circulating levels. according to pepys and hirschfield we assumed a cut - off level of 3 mg / l, in order to discriminate between inflamed and noninflamed patients. on the basis of c - rp values, we obtained a group of six healthy (noninflamed) subjects, without any clinical symptoms or sign of inflammation, who underwent minor surgery procedures and a group of six patients with chronic inflammatory disease who underwent elective surgical procedures: (a) four for cancer and (b) two patients operated for cholecystectomy. all enrolled subjects provided written, informed consent, and the ethics committee of our hospital approved the study. to avoid a possible confounding inflammatory effect exerted by obesity or overweight status, inflamed patients and noninflamed controls were bmi matched in both groups of subjects. the presence of other systemic diseases (vasculitis, rheumatoid arthritis, osteoarthritis, and bowel or lung inflammatory disease) was excluded in all subjects (excepted malignancy); in particular, before the definitive inclusion, the possible existence of any immunological disease, malignancy (in the healthy group), and infectious disease was carefully investigated and excluded. blood samples were collected, before surgical procedures, from all studied subjects to obtain serum aliquots. adipose tissue biopsies were obtained during the surgical procedures in all 12 subjects and for each patient was obtained a sample of subcutaneous total white adipose tissue and a sample of omental adipose tissue. after removal, to get rid of tissue debris the bioptic material was washed twice with warm sterile 0.9% nacl solution; then the sample was immediately frozen in liquid nitrogen and stored at 80c until rna extraction. c - rp circulating levels were determined by a high - sensitivity elisa assay (bender medsystems, vienna, austria) on serum aliquots of all subjects included in the study. the lower detection limit was 3 pg / ml, and the overall intra - assay coefficient of variation has been calculated to be 6.9%. the concentrations of il-6 in plasma samples were analyzed by elisa using a commercially available kit (quantikine ; r&d systems, minneapolis, mn), as described elsewhere. the lower detection limit of il-6 assay was < 0.70 pg / ml, and the coefficient of variation of both inter- and intra - assay was < 5%. adipose tissue fragments obtained from the six noninflamed subjects and from the six inflamed patients were placed in 37c, sterile 0.9% nacl, 5.6 mm glucose, and 25 mm hepes buffer with ph adjusted to 7.4 and containing 50 u penicillin / ml plus 50 mg streptomycin / ml. the adipose tissue fragments were minced under sterile conditions and digested in krebs - ringer bicarbonate buffer supplemented with 5.6 mm glucose, 50 u penicillin / ml, 50 mg streptomycin / ml, and 17 mg type i collagenase/10 g adipose tissue (worthington biochemical, lakewood, nj). digestion was for 75 min at 37c, with rotary agitation at 40 rpm. the isolated cells were filtered through a single layer of chiffon, and the isolated adipocytes were allowed to float for 5 min at 37c. the fluid under the floating adipocytes (containing stromal fraction) was transferred to a conical polypropylene 50-ml centrifuge tube. for the adipocytes, 1 ml of cell suspension of adipose cells was suspended in each tube containing specific adipocyte medium (zen - bio, research triangle park, nc) and incubated in the specific conditions until the cell lysis process. the stromal fraction was centrifuged at 800 g for 10 min. for the isolation of pre - adipocytes, we first centrifuged the cell pellet at 800 g for 10 minutes and then we washed three times with dulbecco's modified eagle medium: nutrient mixture f-12 (ham) in combination 01: 01 (dmem : f12, gibco - brl, carlsbad, california), which has been supplemented to give 15-mm nahco3, penicillin 50-u: ml streptomycin and 50 mg: ml. after washing, the pellet was resuspended by trituration in dmem supplemented pre - adipocyte: f12 containing 10% fetal bovine serum (fbs, gibco - brl, carlsbad, ca). the cells were then plated and incubated in a humidified incubator at 37c in an atmosphere of 5% co2 in air for 24 hours to allow attachment and proliferation of cells. after 24 hours , the medium was removed and replaced by specific pre - adipocyte medium (zen - bio, inc ., research triangle park, nc) and subculture. the genome sequences corresponding to albumin and -actin were obtained from genebank (http://www.ncbi.nlm.nih.gov/) to identify specific primers. we analyzed the exon sequences to establish a pair of primers (sense and antisense) able to generate amplified fragments measuring between 150 and 700 bp in length, with annealing temperature between 55 and 62c. the sense and antisense primers were selected to include at least one intron to prevent genomic dna contamination during amplification. four g of total rna were subjected to cdna synthesis for 1 h at 37c using the ready to go you - primer first - strand beads kit (amersham pharmacia biotech, piscataway, nj, code 27 - 9264 - 01) in a reaction mixture containing 0.5 g oligo - dt (amersham pharmacia biotech cod . pcr amplification of cdna was performed in a reaction mixture containing 4 l of cdna sample and different primer sets ( 20 p / mol each). the amplification of albumin gene and human -actin gene, as an internal control, was achieved using 2 primer sets. after an initial denaturation at 94c for 5 min, pcr reactions were carried out using 30 cycles of 94c for 1 min, the temperature for annealing for 1 min, and 72c for 1 min in a perkin elmer cetus 480 thermal cycler (perkin elmer). pcr products were separated by gel electrophoresis on a 2% agarose gel and stained with ethidium bromide. all signals were normalized to the mrna levels of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase and expressed as a ratio. real - time quantitative pcr analysis for albumin gene was performed on cell lysate (both adipocytes and stromal fraction) using abi prism 7500 (applied biosystems, foster city, ca) and the 5-exonuclease assay (taqman technology). the cdna, synthesized as described above, was used for real - time pcr performed in 96-well optical reaction plates with cdna equivalent of 100 ng of rna in a volume of 25 l reaction containing 1x taqman universal master mix, optimized concentrations of fam - labeled probe, and specific forward and reverse primer for albumin gene from assay on demand (applied biosystems). controls included rna subjected to rt - pcr without reverse transcriptase and pcr with water replacing cdna. the were analyzed using a comparative method, and the values were normalized to the -actin expression and converted into fold change based on a doubling of pcr product in each pcr cycle according to the manufacturer's guidelines, as previously described. homogenates of adipocytes and preadipocytes were prepared in radioimmune precipitation assay buffer (1x pbs, 1% np-40, 0.5% sodium deoxycholate, 0.1% sds containing phenylmethylsulfonyl fluoride, aprotinin, sodium orthovanadate and protease inhibitor tablet) (completetm mini, boehringer - mannheim) containing antipain dihydrochloride (50 mg / ml), bestatin (40 mg / ml), chymostatin (60 mg / ml), e-64 (10 mg / ml), leupeptin (0.5 mg / ml), pepstatin (0.7 mg / ml), phosphoramidon (300 mg / ml), pefabloc sc (1 mg / ml), edta disodium salt (0.5 mg / ml), and aprotinin (2 mg / ml) ]. the cells were centrifuged at 14,000 g for 30 min at 4c and protein concentrations were determined with the bradford assay using bovine serum albumin as a standard. for immunoblotting, protein (100 g) was subjected to 8% sds - polyacrylamide gel electrophoresis. proteins were transferred to polyvinylidene difluoride (pvdf) membranes at a constant voltage of 200 v for 16 h. the pvdf membranes were blocked for 1 h in 5% nonfat dried milk in tbs-0.1% tween buffer (25 mm tris - hcl, 0.2 mm nacl ; 0.1% tween 20 ( v / v) ph 7.6 ) (tbst). the membrane was washed 2x with tbst and then incubated overnight at 4c with the respective primary antibodies. albumin antibody was from cell signaling (beverly, ma); gadph antibodies were obtained from santa cruz biotechnology. after extensive washing of membrane with tbst buffer, anti - rabbit immunoglobulin conjugated with horseradish peroxidase was added at a 1: 5000 dilution and incubated for 1 h at room temperature. an enhanced chemiluminescence kit (amersham, nj) was used to identify protein expression. in sixty - three healthy (noninflamed) subjects and fifty - four microinflamed (c - rp : 5.24 2.26 mg / l) esrd patients undergoing rdt we determined circulating albumin levels (by nephelometry), body weight (bw) to the nearest 50 g (by using a calibrated balance beam scale), body mass index (bmi), calculated as the ratio body weight / height (in kg / m), and body composition, assessed by conventional bioelectrical impedance analysis (bia) and by bioelectrical impedance vector analysis (biva), as previously described. resistance (r) and reactance (xc) were measured by a single - frequency 50 khz bioelectrical impedance analyzer (bia 101 rjl, akern bioresearch, firenze, italy) according to the standard tetrapolar technique, by applying the software provided by the manufacturer, which incorporated validated predictive equations for total body water (tbw), fat mass (fm), fat free mass (ffm), and extracellular water (ecw). the same investigators performed anthropometry and bia measurements. r and xc were normalized by the height of subjects (r / h and xc / h) and the ing vectors were plotted on a graph reporting the gender - specific 50th, 75th, and 95th tolerance ellipses of similar vectors calculated from a reference healthy population. according to the rxc graph method, vectors falling within the reference gender - specific 75th tolerance ellipse indicated normal hydration; short vectors (below the lower pole of the 75th tolerance ellipse) indicated overhydration and long vectors (above the upper pole of the 75th tolerance ellipse) indicated under - hydration. the length of the vector was calculated as |z | = and the phase angle of the vector as the arctan of xc / r. statistical analysis was performed by using unpaired t - test and anova (followed by bonferroni post hoc test) and linear regression analysis. are expressed as means sd; statistical significance was defined as p < 0.05. demographic, anthropometric, and biochemical baseline data of all enrolled subjects are reported in table 2. no significant difference was observed in sex, age, body weight, bmi, waist circumference, and waist - to - hip ratio between the two different groups. on the contrary, plasma c - rp and il-6 levels were much higher in patients with chronic inflammatory diseases than in healthy (noninflamed) subjects (p < 0.01). rt - pcr showed that the adipocytes extracted from all the districts of adipose tissue (omental and subcutaneous) of noninflamed controls and inflamed patients expressed the gene of albumin (figure 1); the mrna for this marker was found in the adipocytes and pre - adipocytes of all fragments of adipose tissue. we did not find any statistically significant difference in albumin gene expression between inflamed and noninflamed patients in adipocytes drawn from both sc and om adipose tissue. as figure 2 shows, we only observed an higher omental adipocyte albumin than subcutaneous one in each group. we concluded that inflammation does not modulate albumin gene expression in the adipocyte, but probably further studies are required to confirm this . figure 3 shows the different albumin gene expression, studied by real - time pcr, in adipocytes and pre - adipocytes obtained from either sc or om fragments of adipose tissue drawn from noninflamed subjects. pre - adipocyte albumin gene expression was higher then the adipocyte one in both om and sc fragments and this difference was statistically significant. we also observed that albumin gene expression in the pre - adipocyte extracted from fragments of om adipose tissue was significantly higher than in preadipocyte from sc fragments. finally, we investigated whether at the protein level adipocytes and pre - adipocyte were also able to express albumin in all the subjects. as shown in figures 4 and 5, western blot analyses showed the presence of albumin in both adipocytes and pre - adipocytes suggesting that both adipocytes and pre - adipocytes can synthesize albumin in sc as well as in visceral adipose tissue, both in healthy noninflamed subjects and inflamed patients. we first investigated the relationship between plasma albumin levels and body fat mass (fm) in 63 healthy subjects who underwent bia excluding from statistical analysis over- and hypohydrated subjects (vectors falling below the lower pole or vectors falling above the upper pole of the 75% tolerance ellipse) evaluated by biva method. as shown in figure 6, a significant negative correlation was observed between plasma albumin levels and fm (r = 0.312, p < 0.05 ( 2-tailed) ). then, we studied the same relationship in 54 microinflamed esrd patients undergoing regular dialysis therapy (rdt). as shown in figure 7, a significant negative correlation was observed between plasma albumin levels and fm (r = 0.391, p < 0.01 ( 2-tailed) ). albumin is the most abundant plasma protein produced by liver cells. to date, no data are present in the literature on albumin expression in mature adipocytes. in the present study , we found, for the first time, a clear albumin expression in human mature adipocytes. in addition, on the basis of our , we can also reasonably affirm that differentiated adipocytes are probably able to synthesize albumin. albumin gene expression ed significantly lower in the adipocytes than in the pre - adipocytes; in particular, it was 42 e 12 times lower in om and sc adipocyte, respectively, as compared with the pre - adipocyte (figure 3). in this way, in fact, yoo and lee investigated the role of albumin in adipocyte differentiation, by using pre - adipocytes cell lines such as 3t3-l1. this is a developmental process by which undifferentiated precursor cells differentiate into mature adipocytes with coordinated changes in cell morphology and gene expression. they found that albumin gene expression was significantly increased at later stages of adipocyte differentiation process and its suppression significantly inhibited lipid droplet formation. the author suggests that albumin could be necessary to stabilize lipid accumulation in mature adipocyte, probably through a direct interaction with fatty acids. however, it is important to underline that these experiments were performed on murine cell lines and their can not correspond to human cell data. in a previous paper we found that not only c - rp gene expression was activated in adipocyte cells, but both il-6 and il-6 receptors gene expressions were found to be higher in inflamed patients than in controls, either in subcutaneous or intra - abdominal adipose tissue. at this regard, in the present study we hypothesized that adipocytes, similarly to hepatocytes, show a different albumin gene expression in inflamed and noninflamed patients, with a lower gene expression in inflamed ones. however, our showed no significant difference in albumin gene expression between inflamed and noninflamed patients when analyzed by real - time pcr (figure 2). on the other hand, figure 2 shows also that albumin gene expression was significantly higher in intra - abdominal than in subcutaneous adipocytes (figure 2). albumin presence, as protein, together with gene expression in adipocytes raises the hypothesis that adipose tissue contribute to circulating albumin levels, as well as it happens for il-6. to verify this hypothesis we evaluated the relationship between serum albumin and fat mass, supposing that higher fat mass corresponds to higher circulating albumin levels however, our did not confirm this hypothesis, but we found a negative significant correlation between albumin and fat mass both in healthy noninflamed subjects and inflamed esrd patients, in contrast with what we expected (figures 6 and 7). in other words, the higher the fat mass the lower was serum albumin concentration was. we suppose that higher fat mass leads to higher production of different proinflammatory cytokines, mainly il-6, that can downregulate albumin, production in hepatocyte via endocrine way, independently of systemic inflammation. we also suppose that il-6 produced by adipocyte could down - regulate, via an autocrine and/or paracrine signaling, albumin gene expression and production in the adipocyte itself. this mechanism could explain both the lack of modulation of albumin gene expression by systemic inflammation in the adipocyte and the negative correlation between fat mass and albumin levels either in noninflamed subjects or in inflamed esrd patients. on the contrary, this mechanism could not operate with inflammatory proteins, such as c - rp. despite the lack of albumin gene modulation by inflammation, in this study something really new was observed: the human adipocyte, once considered a simply depot cell, is now seen as a new and active cell, that in parallel with hepatocyte, is able to produce different proteins, such as c - rp and, as novelty, albumin. the more fascinating hypothesis states the existence of a continuum in adipose tissue cell population that goes from multipotent stem cell to more mature progenitor pools, passing through the pre - adipocyte. this hypothesis might explain why adipocyte and hepatocyte share the expression of some genes, such as albumin gene. as above mentioned, adipose - tissue - derived msc (admsc) displayed the capacity to differentiate into numerous cell types (muscular, neuronal, bone, adipose cells) and, interestingly, into hepatocyte - like cells. a study showed that admsc could be differentiated into functional hepatocyte - like cells by the treatment of cytokine mixtures in vitro, so to become a potential source to hepatocyte regeneration or liver cell transplantation. in another work, the authors showed that the undifferentiated nave admsc were also positive for albumin, g-6-p, and -1-antitrypsin (aat), which are all known to be predominantly expressed in adult liver cells. however, we do suppose that other, more fascinating mechanisms, apart from sharing the same origin, may explain why, to date, adipocyte and hepatocyte produce a so important protein in our organism, like albumin. in , this preliminary study highlights for the first time a new adipocyte activity, among the others already known; however further investigations are needed to confirm and explain our .

aims. our group investigated albumin gene expression in human adipocytes, its regulation by inflammation and the possible contribution of adipose tissue to albumin circulating levels. methods. both inflamed and healthy subjects provided adipose tissue samples. rt - pcr, real - time pcr, and western blot analysis on homogenates of adipocytes and pre - adipocytes were performed. in sixty - three healthy subjects and fifty - four micro - inflamed end stage renal disease (esrd) patients circulating levels of albumin were measured by nephelometry; all subjects were also evaluated for body composition, calculated from bioelectrical measurements and an thropometric data. . a clear gene expression of albumin was showed in pre - adipocytes and, for the first time, in mature adipocytes. albumin gene expression ed significantly higher in pre - adipocytes than in adipocytes. no significant difference in albumin gene expression was showed between healthy controls and inflamed patients. a significant negative correlation was observed between albumin levels and fat mass in both healthy subjects and inflamed esrd patients. . in the present study we found first time evidence that human adipocytes express albumin. our also showed that systemic inflammation does not modulate albumin gene expression. the negative correlation between albumin and fat mass seems to exclude a significant contributing role of adipocyte in plasma albumin.

specimens were collected and stored in 70% ethanol at 80c. dna isolation and amplification of partial fragment of mtcoi gene, purification of amplified pcr products, and sequencing were performed as earlier protocol using non - destructive techniques (buckman et al . voucher specimens have been retrieved after dna isolation and mounted in canada balsam onto glass slides and identified as t. parvispinus using the morphological keys ( mound 2005). the voucher specimens have been submitted to national zoological collections, zoological survey of india, kolkata, india. eleven dna sequences generated in this study were aligned against 80 sequences of t. parvispinus from indonesia as retrieved from national centre for biotechnology information. further, the generated sequences were submitted to genbank database to acquire the accession numbers for t. parvispinus (km485659km485667) and thrips orientalis (km507077km507078). t. orientalis is used as it is a member of the thrips orientalis group. evolutionary genetic divergences with kimura-2-parameter model and neighbor - joining (nj) phylogenetic tree were constructed in mega 6.0 with 1,000 bootstrap replications (tamura et al . haplotyping was carried out in dnasp, and median joining networks were produced in network 4.1 ( bandelt et al . head broader than long, ocellar pair iii arising at the anterior margin of ocellar triangle ; postocular setae i and iv longer than iii ( fig . pronotum with two pairs of posteroangulars setae and two pairs of posteromarginal setae ( fig . metanotum reticulate medially and with faint internal reticules ; median setae long and placed behind the anterior margin ; campaniform sensilla absent ( fig . abdominal sternites iii vi with accessory setae, absent on ii and vii . figs . 13.t . parvispinus, female head and pronotum; meso- and metanotum, fore wing. t. parvispinus, female head and pronotum; meso- and metanotum, fore wing. thirteen females, two males, india: karnataka: bangalore, 10.ii.2014, kamlajayanti (reg . head broader than long, ocellar pair iii arising at the anterior margin of ocellar triangle ; postocular setae i and iv longer than iii ( fig . pronotum with two pairs of posteroangulars setae and two pairs of posteromarginal setae ( fig . metanotum reticulate medially and with faint internal reticules ; median setae long and placed behind the anterior margin ; campaniform sensilla absent ( fig . abdominal sternites iii vi with accessory setae, absent on ii and vii . figs . 13.t . parvispinus, female head and pronotum; meso- and metanotum, fore wing. t. parvispinus, female head and pronotum; meso- and metanotum, fore wing. thirteen females, two males, india: karnataka: bangalore, 10.ii.2014, kamlajayanti (reg . head broader than long, ocellar pair iii arising at the anterior margin of ocellar triangle ; postocular setae i and iv longer than iii ( fig . pronotum with two pairs of posteroangulars setae and two pairs of posteromarginal setae ( fig . metanotum reticulate medially and with faint internal reticules ; median setae long and placed behind the anterior margin ; campaniform sensilla absent ( fig . abdominal sternites iii vi with accessory setae, absent on ii and vii . figs . 13.t . parvispinus, female head and pronotum; meso- and metanotum, fore wing. t. parvispinus, female head and pronotum; meso- and metanotum, fore wing. thirteen females, two males, india: karnataka: bangalore, 10.ii.2014, kamlajayanti (reg . homology search using blast search option ed in 99100% similarity to t. parvispinus sequences from indonesia . we analyzed 91 partial mtcoi sequences of t. parvispinus and t. orientalis in this study . out of which 11 sequences were generated in this study and rest of the 80 sequences were retrieved from national centre for biotechnology . complete dataset after trimming have 604 nucleotides, which shows 89 variable sites with 87 parsimony informative sites . however, the dataset of 89 sequence of t. parvispinus yielded only 14 variable sites out of which 10 were parsimony informative . analysis of 89 sequences of t. parvispinus yielded four haplotypes ( hap_1 to hap_4) (fig . the data show that there is no host - plant or geographical locality specific haplotyping . the total number of segregating sites between four derived haplotypes varies from 1 to 13 ( table 1). further, out of 14 segregating sites, 11 were detected as synonymous changes and 3 as nonsynonymous changes corresponding to nine transitions and five transversions. the analysis of nj tree yielded two major clades with high bootstrap support; clade i includes 89 sequences of t. parvispinus from indonesia and india, while clade ii is represented by two sequences of t. orientalis (fig . nj tree provided here is only to segregate two species based on the reciprocal monophyly criteria and not to interpret phylogeny of genus thrips . fig . table 1.segregating nucleotide sites in the t. parvispinus mtcoi nucleotide sites as depicted in four haplotypeshaplotypes166672104123150165179279360364495537570hap_1 ( indonesia), n = 63catattagatttathap_2 (indonesia, india), n = 18......... c.... hap_3 (indonesia), n = 7tg.. c.g.gccctahap_4 (india), n = 1.. gg.g.t.c.... transversions are shown in bold. f. schultzei is used as an outgroup. segregating nucleotide sites in the t. parvispinus mtcoi nucleotide sites as depicted in four haplotypes transversions both morphological and molecular evidences verify that the specimens collected on papaya represents t. parvispinus. the presence of this pest species on an economically important crop plant like papaya in india raise serious issues and is a concern for quarantine authorities. occurrence of t. parvispinus in other parts of india needs systematic monitoring as it is likely to acquire pest status in future. molecular evidence for shared haplotype (h_2) between indonesia and india indicated that there is a flow of genetic material, and indonesia may be a probable source of invasion of this species to india. however, molecular data on this species from other countries may be helpful to trace exact route of invasion.

south east asia pest thrips species, thrips parvispinus (karny), is a serious pest on a number of agricultural and horticultural crops in a number of plant families. based on an integrated approach of morphology and dna barcoding, invasion of this serious pest is reported first time from india on papaya plantations. molecular data have corroborated with the morphological identification. haplotyping data suggested that the indonesia may be a probable source of invasion of this pest to india.

scorpion envenomation is one of the main problems in the public health system in many countries in the world. this involves 2.3 billion inhabitants in the areas with the scorpion sting threat (chippaux et al . 2008). in 2008, the annual incidence of scorpion stings was 1.200.000 leading to 3250 deaths (chip - paux et al . this number demonstrated a relative growth in a report released in 2012 and exceeded than annually 1,500,000 scorpion stings, however, the mortality rate due to the scorpion stings showed a significant decrease and fell into 2600 deaths per year ( jean - philippe 2012). the highest number of scorpion sting in the world has been allocated to iran after mexico (osnaya - romero et al . the annually 42,500 scorpion stings and 20 following deaths have been reported from 2001 to 2009, in iran ( celis et al . 2007). in the middle east, among the 52 known species of scorpions, the most dangerous scorpions are reported in iran (celis et al . 2007). the scorpion sting has been reported from all of the provinces of iran, however, the most common incidence rates have been detected in khuzestan, with incidences of 541 per 100000 individuals (dehghani et al . hemiscorpius lepturus belongs to the hemiscorpiidae family, and is the most medically important and a dangerous scorpion in khuzestan, iran and the world ( shahbazzadeh et al . hemiscorpius lepturus has been responsible for 15 % of the scorpion sting bite cases, however, it is the leading cause of 89 % of deaths followed by the scorpion sting ( pipelzadeh et al . the lethality arising from this scorpion is approximately 60 times higher than the average for the remaining venomous scorpion stings in the region ( pipelzadeh et al . the venom of h. lepturus leads to acute renal failure, thrombocytopenia and microangiopathic hemolytic anemia, known as the nephrotoxic, hepatotoxic and hemolytic complications of the scorpion venom ( valavi et al . the local signs vary from erythema to necrosis, while the patient feels no pain . on the other hand, the nephrotoxicity is the most important systemic complication that if left untreated could in severe renal, cardiac and pulmonary failure ( pipelzadeh et al . . the venom may induce severe pathological damages in target organs such as skin, blood cells, central nervous system ( cns), and cardiovascular system (seyedian et al . it leads to an increase in liver enzymes aspartate aminotransferase ( ast), alanine aminotransferase (alt) and alkaline phosphatase (alp), indicating the severe hepatic damage (pipelzadeh et al . showed that the h. lepturus causes more rbc lysis and ldh increase, comparing with the complications of envenomation ing from the stings of two other species of scorpions, mesobuthus eupeus and androctonus crassicauda ( khodadadi et al . , it was described that 90 % of patients whom were admitted in hospitals due to the general condition worsening, were stung by h. lepturus ( mir dehghan et al . the immune system cells are distributed throughout the body, from the outer most points to the deepest organs and tissues, such as blood, bone marrow, thymus and spleen . despite this diversity, these cells apply a comprehen - sive supervision on different organs and tissues through the regular circulation from blood to lymph, from inside of the vessels to the outside and from the interior of the organs to the blood, ing in protection of the body against the pathological factors ( vodjgani 2012). the of an experimental study aiming the investigation of the effect of envenomation by h. lepturus on the hematological indices three days after injection of the venom suggested that the leukocyte number has been increased over the normal range, however, there was not any significant difference with the control group (dehghani et al . an increase in the peripheral leukocyte count had been demonstrated during the investigation of the blood among h. lepturus scorpion sting victims ( chitnis et al . 1993). while comparative studies to investigate the effects of h. lepturus venom on hematologic parameters and vital organs of the body have been done, but so far, any experimental research on the effect of the scorpion venom on the immune system cells and its various sub - groups in the early hours after the sting, and further - more determination and investigation of the period and the intensity of the envenomation have not carried out yet. therefore, the aim of the present study was to investigate the effect of h. lepturus envenomation on blood leukocytes and their subgroups in the early hours after the sting. the of this study can help physicians, health officials and the medical staff to fast and accurate treatment of the victims of h. lepturuss sting and prevention of complications of the scorpion venom on important factors of the immune system. sixty male rats from n - mari species (weight range of 300350 grams) purchased from pasteur institute of iran (tehran), were used during the study. the animals were kept in standard cages in animal house at the school of pharmacy of ahvaz jundishapur university of medical sciences. rats were housed in temperature - controlled rooms (2225 c) with constant relative humidity (4070%) and 12h/12h light / dark cycle before doing experimental protocols. the study was performed in accordance with the principles of laboratory care established by the ethics committee of ahvaz jundishapur university of medical sciences, ahvaz, iran. the scorpion electroshocked venom was provided as a lyophylized powder from pasteur institute of iran, (venom and therapeutic biomolecule lab, biotechnology res . the concentration of crude venom protein was determined by using bradford method ( bradford, 1976). the injection volume was 0.1 ml. the animals were randomly divided into three groups of 20 rats in each. the control group did not receive any thing; however, the first and the second groups received h. lepturus venom at the concentrations of 0.1 and 0.01 mg / kg / bw subcutaneously. thereafter, the animals of each group (n= 20) were divided into four subgroups (n= 5), with respect of the four blood sampling time, that were two, six, 24 and 48 hours after the venom injection, the animals were then kept in separate cages. the animals of each subgroup were anesthetized with ketamine and xylazine (alfasan, holland). the blood samples were obtained from the animal s heart amounted to 0.52 ml by syringe. soon after the sampling, the blood was maintained in glasses containing the anticoagulant, edta (ethylene - diamine - tetra - acetic acid), and the leukocytes were counted using the diluent solution, marcanu (rbc lysis buffer) and the neobar slide (hemocytometer) by using the light microscope (olympus, 3h - z - japan), in order to count and determine the leukocyte subgroups (including neutrophils, lymphocytes, monocytes and eosinophils) the appropriate peripheral blood smears were prepared on the microscope slides and then fixed by means of the water - free methanol. then giemsa staining (merck - germany) was carried out by the diluted stain with the rate of 1/10. and finally, the differential counting was performed using a 100x lens microscope (mahbod 2008, mansouri et al . 13 ( version 13, spss inc, chicago, il) and the statistical tests of anova and lsd. 15. the comparison of the meansd of white blood cells (10 per ml) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg and the control group at different times significant difference (p < 0.05) between the experimental and control groups * * significant difference (p < 0.01) between the experimental and control groups the comparison of the meansd of neutrophils (10 per ml) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg and the control group at different times * significant difference (p < 0.05) between the experimental and control groups * * significant difference (p < 0.01) between the experimental and control groups the comparison of the meansd of lymphocytes (10 per ml) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg, and the control group at different times * significant difference (p < 0.05) between the experimental and control groups significant difference (p < 0.01) between the experimental and control groups the comparison of the meansd of monocytes (10 per ml) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg and the control group at different times the comparison of the meansd of eosino - phil (10 per ml) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg and the control group at different times sixty male rats from n - mari species (weight range of 300350 grams) purchased from pasteur institute of iran (tehran), were used during the study. the animals were kept in standard cages in animal house at the school of pharmacy of ahvaz jundishapur university of medical sciences. rats were housed in temperature - controlled rooms (2225 c) with constant relative humidity (4070%) and 12h/12h light / dark cycle before doing experimental protocols. the study was performed in accordance with the principles of laboratory care established by the ethics committee of ahvaz jundishapur university of medical sciences, ahvaz, iran. the scorpion electroshocked venom was provided as a lyophylized powder from pasteur institute of iran, (venom and therapeutic biomolecule lab, biotechnology res . the concentration of crude venom protein was determined by using bradford method ( bradford, 1976). the control group did not receive any thing; however, the first and the second groups received h. lepturus venom at the concentrations of 0.1 and 0.01 mg / kg / bw subcutaneously. thereafter, the animals of each group (n= 20) were divided into four subgroups (n= 5), with respect of the four blood sampling time, that were two, six, 24 and 48 hours after the venom injection, the animals were then kept in separate cages. the animals of each subgroup were anesthetized with ketamine and xylazine (alfasan, holland). the blood samples were obtained from the animal s heart amounted to 0.52 ml by syringe. soon after the sampling, the blood was maintained in glasses containing the anticoagulant, edta (ethylene - diamine - tetra - acetic acid), and the leukocytes were counted using the diluent solution, marcanu (rbc lysis buffer) and the neobar slide (hemocytometer) by using the light microscope (olympus, 3h - z - japan), in order to count and determine the leukocyte subgroups (including neutrophils, lymphocytes, monocytes and eosinophils) the appropriate peripheral blood smears were prepared on the microscope slides and then fixed by means of the water - free methanol. then giemsa staining (merck - germany) was carried out by the diluted stain with the rate of 1/10. and finally, the differential counting was performed using a 100x lens microscope (mahbod 2008, mansouri et al . 13 ( version 13, spss inc, chicago, il) and the statistical tests of anova and lsd. 15. the comparison of the meansd of white blood cells (10 per ml) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg and the control group at different times significant difference (p < 0.05) between the experimental and control groups * * significant difference (p < 0.01) between the experimental and control groups the comparison of the meansd of neutrophils (10 per ml) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg and the control group at different times * significant difference (p < 0.05) between the experimental and control groups * * significant difference (p < 0.01) between the experimental and control groups the comparison of the meansd of lymphocytes (10 per ml) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg, and the control group at different times * significant difference (p < 0.05) between the experimental and control groups significant difference (p < 0.01) between the experimental and control groups the comparison of the meansd of monocytes (10 per ml) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg and the control group at different times the comparison of the meansd of eosino - phil (10 per ml) in peripheral blood of rat between the groups receiving venom with the concentration of 0.01 and 0.1 mg / kg and the control group at different times the scorpion venom has led to a significant reduction of leukocytes in the venom - receiving group with the dose of 0.01 mg / kg at two and 24 hours after the injection, compared with the control group (p < 0.05). this decrease has been maximum of six hours after the injection (p < 0.01). after 24 hours from the injection, the effect of the venom has subsided; and after 48 h there was no any significant difference with the control group (p > 0.05). the toxicity effect of the venom is intensified with the increase of the injected venom concentration, so that in spite of a decrease in venom effect in the group receiving 0.01 mg / kg after 48 hours, the decrease in toxicity effect is still significant in the group receiving 0.1 mg / kg compared with the control group (p < 0.01) (fig . the neutrophil mean count showed a significant decline in the venom receiving group with the dose of 0.01 mg / kg at two, 24 and 48 hours ( p < 0.05), and six hours after the injection, compared with the control group (fig . the mean of the peripheral blood lymphocyte count in the venom receiving group of 0.01 mg / kg at two, six and 24 hours ( p < 0.05), and 48 hours (p < 0.01) after the injection demonstrated a significant decline compared with the control group, respectively. the comparison of the mean and standard deviation of the eosinophil and monocytes of the peripheral blood in the case and control groups showed no significant differences (p > 0.05) (figs . 4, 5). the scorpion venom has led to a significant reduction of leukocytes in the venom - receiving group with the dose of 0.01 mg / kg at two and 24 hours after the injection, compared with the control group (p < 0.05). this decrease has been maximum of six hours after the injection (p < 0.01). after 24 hours from the injection, the effect of the venom has subsided; and after 48 h there was no any significant difference with the control group (p > 0.05). the toxicity effect of the venom is intensified with the increase of the injected venom concentration, so that in spite of a decrease in venom effect in the group receiving 0.01 mg / kg after 48 hours, the decrease in toxicity effect is still significant in the group receiving 0.1 mg / kg compared with the control group (p < 0.01) (fig . the neutrophil mean count showed a significant decline in the venom receiving group with the dose of 0.01 mg / kg at two, 24 and 48 hours ( p < 0.05), and six hours after the injection, compared with the control group (fig . the mean of the peripheral blood lymphocyte count in the venom receiving group of 0.01 mg / kg at two, six and 24 hours ( p < 0.05), and 48 hours (p < 0.01) after the injection demonstrated a significant decline compared with the control group, respectively. the comparison of the mean and standard deviation of the eosinophil and monocytes of the peripheral blood in the case and control groups showed no significant differences (p > 0.05) (figs . 4, 5). the present study was aimed to investigate the effect of h. lepturus venom on leukocytes and their subgroups in peripheral blood of rats. these cells apply a comprehensive supervision on different organs and tissues, through the regular circulation of blood to lymph, from inside of the vessels to the outside and from the interior of the tissues to the blood, ing in a protection of the body against the pathological factors. in general , leukocytes consist of various groups of cells including lymphocytes, monocytes and granulocytes (vodjgani 2012). the clinical syndrome induced by h. lepturus sting is different from the stings by the other scorpions existing in iran and the world, and exhibits more severe manifestations. lack of local pain or being a mild pain after the sting, cutaneous manifestations such as erythema, swelling and necrosis at the sting site, the red blood cell lysis, and nephrotoxicity, including hemoglobinuria, proteinuria and hematuria are some manifestations among scorpion sting victims (radmansh 1990, radmanesh 1998, pipelzadeh et al . the of the present study demonstrated those leukocytes are affected by the venom after two hours, exhibiting sensitivity, decrease in leukocytes continues for six hours after the venom injection . if leukocytes were evaluated after 12 hours, it was possible to notice a decreasing trend, due to prolongation of the presence of the venom into the body . probably these variations in leukocytes are the of cytotoxic effects of h. lepturus venom, which leads to white blood cell lysis and destruction ( shayesteh et al . there is no any considerable experimental or clinical study in this field, and the decrease in leukocytes in the early hours is one of the new findings of this research . lack of the related reports might be due to the difference in evaluation time of the hematological indices in various studies ( dehghani et al 2005, dehghani et al . 2012), it has been the of the use of the anti - venom in human researches, as well (chitnis et al . the leukocyte count trend increased after 24 hours of venom injection, and there was not any significant difference in the venom - receiving group of 0.01 mg / kg with the control group . various studies have reported leukocytosis as well as the major clinical signs of the envenomation by h. lepturus, after other symptom such as hemoglobinuria, hematuria and proteinuria ( chitnis et al . therefore, the reconstruction of leukocytes observed in this study is in agreement with the previous findings ( dehghani et al . 2012). however, the more venom concentration, the more decline in leukocyte count, the slower wbc reconstruction, so that the leukocyte count has not reached to the normal range after 48 hours of envenomation with 0.1 mg / kg. according to the present study, after two hours , the percent of the blood neutrophils has been changed from the normal range of 61 % to 35.2 %. this reduction has been more severe after six hours and has fallen down to 25.5 %. neutrophils make up the most abundant population of white blood cells, and mediate the primary stages of inflammatory response. they are the most effective phagocytes in peripheral blood and have a major role in defense against the extracellular factors (abbas et al . these mature cells migrate to the inflammation site just four hours after the antigen entrance, and have the capability to invade the antigen . phagocytosis of the particles and waste products in neutrophils is associated with a series of biochemical events and morphological changes in the cell ( vodjgani 2012). the majority of the neutrophils at the inflammation site will be wiped out by other cells, such as macrophages, after the phagocytosis of the invader. 2011 aiming to investigate the effect of h. lepturus on the serum levels of cytokines il-1, il-6, il-8 and tnf-, demonstrated a direct relation between the worsening of the patient s general condition and the above - mentioned cytokines. il-8 is a chemotactic protein that is known as nap-1 or the attractive and activator of neutrophils (vodjgani 2012). the increase of this cytokine leads to fever and hyperthermia, which is probably due to neutrophil aggregation, pathogen killing and the death of the two kinds of cells (taraz 2008). tnf- enhances the production of some particular serum proteins such as amyloid a, through affecting hepatocytes. this cytokine suppresses stem cell division, which may lead to neutropenia (vodjgani 2012). therefore, the severe decrease in neutrophils in the group receiving high doses of h. lepturus venom will be reasonable. in the following 24 hours , the neutrophil count especially in the venom - receiving group of 0.01 mg / kg has approached the normal range. regarding the neutrophilia, the neutrophil compensation could be explained. through the neutrophilia the conversion of marginal neutrophils into circulating neutrophils could be noticed. these findings are consistent with the of the research carried out by ghafourian and mohebby on patients with scorpion referred to the hospital bite (ghafourian and mohebbi 2008). in the present study, lymphocytes have decreased during the first hours after the venom injection, and this reduction was significant even after 48 hours. one of the fractions extracted from the venom of h. lepturus may reduce the lymphocyte count (bigdeli et al . lymphocytes make up around 20 to 40 % of the leukocytes, and 99 % of the cells in the lymph, respectively . the process of recognition and processing of antigen by lymphocytes as well as the clonal expansion requires the time, and the precise determination of the mechanism of the effect of the venom of h. lepturus on lymphocytes can not be explained during the first 48 hours, perhaps due to immunological reasons . in the present study, the blood lymphocyte decrease was dose - dependent, which could probably be the of the direct effect of the venom on the lymphocytes, which leads to disruption and lysis of these cells . in fact, it could be concluded that the venom of h. lepturus has a lymphotoxic effect ( ghafourian and mohebbi 2008). in the present research, the peripheral blood eosinophil count in the control group showed no significant difference with the experimental groups. eosino - phil consists of 2 % of leukocytes found in the normal situation in tissues, especially the epithelium of the respiratory tract, gastrointestinal tract and genitourinary tract. these cells have a weak phagocytic ability, and increase mainly in type-1 hypersensitivity responses and parasitic infections, as well. the venom of the scorpion h. lepturus effect on the leukocytes in the early hours, as well as increasing the concentration of the toxin, its destructive power is increased. in other word however, the essential role of these factors is to intensify the inflammation, but recently the natural corticosteroid hormones or similar synthetic substances are used to alleviate the inflammatory reactions against the allograft transplantation and immune system suppression. therefore, the extraction of the useful fractions of h. lepturus venom for natural induction of corticosteroids and decreasing leukocytes may be useful in the treatment of some types of leukemia and the graft surgeries, as well. however, the severe and dose - dependent reduction of the immune cells in the first hours after the injection of h. lepturus venom could be an alarm for health officials and medical staff to perform quick and accurate treatment in the least possible time, and to prevent of complications of the scorpion venom is the body s vital organs.

: the aim of this study was to investigate the effect of hemiscorpius lepturus venom on leukocytes and the leukocyte subgroups in peripheral blood of rat.methods:in this experimental study, sixty n - mari rats were divided into three groups of 20 rats. then the rats in each group were divided into four subgroups based on the blood sampling time that was 2, 6, 24 and 48 hours after the venom injection, respectively. the control group did not receive anything, however, the first and the second experimental groups received 0.1 and 0.01mg / kg of venom, subcutaneously. in accordance with a designated four sampling times, the blood sampling was carried out in three groups. after rbc lysis, the leukocytes and leukocyte sub - populations were determined and counted using appropriate hematological standard methods.:the leukocyte and the neutrophil count at two (p<0.05), six (p<0.01) and 24 (p<0.05) hours after the venom injection showed a significant decline compared with the control group, this decrease was significant at the dose of 0.1 mg / kg until 48 hours after the venom injection (p<0.05). the lymphocyte count showed a significant decline throughout the all hours of the experiment, compared with the control group (p<0.05).: leukocytes are probably affected by the cytotoxicity effect of the h. lepturus venom in a dose - dependent manner. this could be a wakeup call for the medical staff to perform quick and accurate treatment in the least time possible.

solitary plasmacytomas (sp) from monoclonal plasma cell proliferation but lack bone marrow involvement and m protein in serum / urine, seen in multiple myeloma. sp has been classified by world health organisation into solitary osseous plasmacytomas (sop) and solitary extramedullary plasmacytomas (semp). semps are rare and constitute 5% of all plasma cell disorders and have a strong predilection for the upper aerodigestive tract. we hereby describe one such rare case of thoracic epidural semp manifesting as dorsal compressive myelopathy. a 32-year - old female presented to us with a back pain for 2 months and progressive spastic weakness of bilateral lower limbs (b / l lls) for past 8 days. clinical examination revealed spastic weakness (power : 1/5 ; mrc uk) of b / l lls, exaggerated b / l knee and ankle jerks and complete sensory loss at and below l1 level. spine examination revealed no deformity or tenderness. with a clinical diagnosis of thoracic compressive mri revealed a dorsally located epidural lesion at the level of t7-t8 vertebral bodies, which was compressing and pushing the spinal cord anterolaterally. the lesion was isointense on t1-weighted, hypointense on t2-weighted images and enhanced homogenously and extended into the neural foramen. both meningiomas and neurofibromas are isointense to hyper intense and not hypo intense on t2-weighted images. magnetic resonance imaging (mri) showing a epidural lesion at the level of t7-t8 vertebrae (a, sagittal t2-weighted), pushing the cord anterolaterally to right side and extending into the neural foramen (b, axial t2-weighted). postoperative mri (sagittal t1-weighted, parasgittal t2-weighted and axial t1-weighted images) revealed complete excision of the tumor with opening of the subarachnoid space (c - e) at the level of tumor. the spinal cord can be seen to have attained normal shape and position (e) the patient underwent t7 and t8 laminectomy and complete excision of tumor, which was reddish, soft, moderately vascular and located in epidural space with extension into the left neural foramen. as there was no bony involvement and only two level laminectomy was done, no vertebral stabilization was needed. histopathological examination revealed diffuse and dense infiltration by mature and immature plasma cells which were immunohistochemically positive for cd138, with occasional bi - nucleated plasma cell. based on overall histomorphological and immunohistochemical findings, the diagnosis of plasmacytoma was made. (a) microphotograph showing a tumor composed of diffuse infiltration by both mature and immature plasma cells (h and e, 20) (b) plasma cells are highlighted by immunohistochemical staining for cd138 (immunohistochemistry, 40) patient was then evaluated to find a systemic evidence of disease. bone marrow examination revealed < 5% plasma cells and serum electrophoresis and urine examination were negative for m protein. patient received adjuvant radiotherapy (rt) (40 gy in 20 fractions) to operative field. 6 months after surgery patient has started walking (power in bilateral lower limbs : 4/5 mrc uk) and has regained bladder functions. solitary extramedullary plasmacytomas most commonly involve upper aerodigestive (8090% of cases). in 1020% cases other body organs, including skin, testis, ovaries, liver, lungs, spleen etc. however, occurrence of semps as isolated masses in spinal epidural space is very rare. thorough search of the literature revealed only seven such cases reported previously in available english literature. cases of epidural semps in order to make a diagnosis of semp, following criteria must be fulfilled: biopsy from lesion showing monoclonal plasma cellsbone marrow plasma cell < 5%absence of osteolytic bone lesions or involvement of other body tissuesabsence of hypercalcemia and renal failureabsent or low serum m - protein concentration. biopsy from lesion showing monoclonal plasma cells bone marrow plasma cell < 5% absence of osteolytic bone lesions or involvement of other body tissues absence of hypercalcemia and renal failure absent or low serum m - protein concentration. radiologically, the epidural mass can be a diagnostic dilemma. in an endemic country, tuberculosis is the first consideration in the differential diagnosis, and we also considered it as first differential. treating these patients with only antitubercular drugs is not only a futile exercise, it also has adverse consequences in terms of delaying the appropriate treatment for plasmacytoma. it is therefore important to be aware of this rare entity as a differential diagnosis of epidural masses especially when a t2-weighted hypo intense lesion is restricted to epidural space without bony involvement and without associated paravertebral collection. among the 7 cases in literature and the present case , both males and females are equally represented with an average age of 51 years (age range : 4085 years). four patients had impairment of bladder and bowel functions. surgical resection followed by adjuvant therapy (chemotherapy , rt or a combination of ct and rt), surgical resection alone, a combination of plasmapharesis, ct and rt, ct and rt and ct alone were different treatment regimens used in these patients. among the six patients with neurological deficits (paraplegia / quadriplegia) three patients undergoing surgical resection showed improvement in neurological deficits while other three who received ct or combined ct / rt did not show any neurological improvement. according to the guidelines by the british committee for standards in hematology, rt is the treatment of choice for sops and semps. the role of surgery is in providing a tissue diagnosis and in cases of spinal epidural semps, surgical resection relieves the compression on spinal cord thereby leading to neurological improvement. epidural semps are very rare and require a high index of suspicion for diagnosis and appropriate management as both clinical manifestations and radiological features can be quite similar to other more common pathologies in this region. though rt is the treatment of choice for sp, surgical resection helps in providing tissue diagnosis and relieving spinal cord compression.

plasma cell neoplasms from monoclonal proliferation of plasma cells. solitary extramedullary plasmacytomas (semps) are rare and constitute 5% of all plasma cell disorders. semps most commonly involve upper aerodigestive tract. isolated spinal epidural space involvement by semps is extremely rare and to best of our knowledge only 7 such cases have been reported previously in available english literature. we hereby present a rare case of thoracic epidural semp in a 32-year - old female who presented with thoracic compressive myelopathy and discuss the pertinent literature.

the relationship between the gonads (ovaries and testes) and the brain is complicated. it is not surprising that we still do not completely understand all of the effects of hormones on the brain or on seizures. reproductive hormones like estrogen, progesterone, and testosterone have profound effects on the brains of both men and women, even in parts of the brain that are not considered to be critical to reproduction. moreover, these effects occur throughout life, not only in adulthood. during early stages of development, researchers have shown that in laboratory animals, estrogen levels in the fetus are critical to the gender of the fetus.7 after puberty, levels of circulating estrogen and testosterone also affect the brain; the interplay between the early, organizational effects and the postpubertal, activating effects of hormones mediates the net effect of hormones in the adult brain.8,9 besides being synthesized in the gonads, estrogen, progesterone, and testosterone can be synthesized elsewhere, including in the brain itself. such synthesis can potentially occur anywhere in the brain because it occurs in astrocytes, cells that are located in all areas of the brain. astrocytes have historically been considered supportive cells, responsible for housekeeping in the brain (cleaning up debris, removing damaged cells or infection). we now know that astrocytes have many other functions, including the synthesis of hormones, because enzymes in astrocytes metabolize cholesterol, a component of the outer membranes of all cells, into hormones. we do know that both estrogen and testosterone have many effects on the brain, influencing neurotransmitters (which allow neurons to communicate) and neuronal activity.10,11 hormones also have metabolites that have effects on brain activity. for example, allopregnanolone, a metabolite of progesterone, inhibits neuronal activity in many areas of the brain. as a , it has been suggested that progesterone would be help stop seizures.12 because reproductive hormones affect brain activity, the changes in hormones at puberty, pregnancy, or during the menstrual cycle are important to consider when treating women with epilepsy. treatment is complicated, however, because hormones like estrogen and progesterone do not affect all patients in the same way. for example , some women experience changes in the severity and frequency of seizures in relation to estrogen or progesterone treatment, but others do not. similarly, some women have seizures that worsen during part of their menstrual cycle, but some do not. furthermore, some effects of hormones on seizures that have been demonstrated in laboratory experiments are not reproducible. for example, sometimes estrogen appears to promote seizures, but it does nt always.12 besides confusion, this has led to frustration, because clinicians are unsure how to treat patients whose seizures have suddenly worsened, possibly because of changes in reproductive hormone levels. nevertheless, our understanding of effects of estrogen and progesterone on the brain has led to hypotheses that explain why seizures can increase at certain times during the menstrual cycle. as mentioned above, catamenial epilepsy refers to seizures that worsen at particular times during the menstrual cycle. this syndrome has been recognized for more than 100 years, but it has been difficult to study.13 some investigators have even questioned whether catamenial epilepsy exists.14 many clinicians do not find robust sex differences in their patients,1517 and they therefore conclude that syndromes like catamenial epilepsy are not likely to be significant.13 however, clinical research provides strong evidence that catamenial epilepsy is common. andrew herzog, a leader in this field, estimates that catamenial epilepsy occurs in one - third of women with epilepsy.2 a recent epidemiological study in korea estimated that catamenial epilepsy occurs in almost 50 percent of women with epilepsy.18 the accuracy of these estimations is hard to judge, however, because seizures that are unnoticed will be missed, leading to an underestimation of the number of women with catamenial epilepsy. nevertheless, when the condition has been documented, symptoms are often the worst during the days just before the start of menstruation, approximately day 28 of the menstrual cycle, and the days just after the onset of menstruation the perimenstrual period. symptoms also worsen during the periovulatory phase, approximately day 14, when ovulation occurs (see diagram 1, page 9). the rise in circulating estrogen (serum estrogen) during the menstrual cycle affects the brain in many ways, and includes effects on neurons and non - neuronal cells such as astrocytes. in neurons, cell structure spines are important because the spines receive excitatory input from other neurons and help neurons communicate with one another. many scientists believe that a major effect of estrogen in the brain is increased neuronal activity because estrogen increases dendritic spines. circulating progesterone has many effects, because progesterone itself has actions on neurons, and its metabolite allopregnanolone does also. allopregnanolone binds to receptors that are responsible for effects of gamma - aminobutyric acid (gaba), the major inhibitory neurotransmitter in the brain. therefore, when progesterone rises during the menstrual cycle, it is thought that neuronal activity will decrease in many parts of the brain. most of our current understanding of the action of estrogen and progesterone involves the effects of these hormones within the brain, but they also affect many parts of the body that in turn alter brain activity. this complicated interplay between the brain and the rest of the body is one of the reasons epilepsy is hard to treat it may not be a static condition but one that is constantly changing. several hypotheses based on effects of estrogen and progesterone that have been identified in the last decades have been offered to explain catamenial epilepsy. one is that seizures increase when the ratio of estrogen to progesterone is high.5,19,20 this hypothesis explains the worsening of seizures during the periovulatory period and the relative resistance to seizures during the luteal phase (days 1428see diagram 1, page 9), but it does not explain why seizures worsens during the perimenstrual period, because both estrogen and progesterone are low at that time. an alternative view developed after it was shown that falling progesterone levels exert excitatory effects.21 researchers hypothesized that progesterone withdrawal occurs at the end of the menstrual cycle, leading to excitatory effects. literally, there is a decrease in seizure threshold,22 which is the point at which the brain has been sufficiently activated so that a seizure begins. in patients with epilepsy, this threshold is thought to be lower than in other people, so that even mild activation of the brain can lead to a seizure. researchers investigated the notion of progesterone withdrawal by treating laboratory rats or mice with progesterone and then suddenly stopping the treatment. the showed that a decrease in seizure threshold accompanied withdrawal of the treatment.21,22 these data suggest that in a patient with epilepsy, seizures may be more likely after progesterone withdrawal because seizure threshold is lower. subsequent studies, using other methods to simulate progesterone withdrawal, and other means of evaluating seizure susceptibility, confirmed these findings.23,24 progesterone withdrawal therefore provides a potential explanation for perimenstrual seizures. further research into the effects of progesterone withdrawal has suggested that it is not simply a sudden drop in progesterone or its metabolite allopregnanolone that causes the increased susceptibility to seizures. rather, progesterone withdrawal leads to a specific change in gabaa receptors, the neurotransmitter receptors that bind allopregnanolone. the gabaa receptor is normally composed of several components, or subunits. when progesterone withdrawal occurs, increased levels of one of the subunits stops allopregnanolone from having an inhibitory effect on seizure activity,25,26 and other subunit changes lead to the resistance of seizures to some antiepileptic drugs, such as the benzodiazepines.27 progesterone also affects other hormones, called mineralocorticoids. mineralocorticoids normally allow the body to retain sodium, potassium, and, as a , water. this is significant because water balance affects seizures: when neurons in the brain swell, they become closer together, increasing neuronal activity.28 as a , seizure threshold can decrease. during the menstrual cycle, water retention occurs during the end of the luteal phase. during this period, which coincides with the perimenstrual phase, seizure threshold may decrease because water retention increases neuronal excitability. this reasoning has led to the use of diuretics such as acetazolamide in women with catamenial epilepsy, although they have not been completely successful,29 probably because they do not treat the other effects of hormones during the menstrual cycle. recent studies in female laboratory animals suggest that estrogen levels may also contribute to perimenstrual seizures, because estrogen can have effects on the perimenstrual period even if estrogen is elevated only in the preceding phases of the cycle. for example, estrogen increases the concentration of brain - derived neurotrophic factor (bdnf), a molecule that increases excitation in the brain. the increase in bdnf is long - lasting it persists after estrogen levels have returned to normal.30 estrogen can therefore exert long - lasting effects on neuronal activity even if estrogen levels increase only briefly. high levels of bdnf throughout the luteal phase would ; this prediction is supported by clinical studies showing that bdnf is high during the luteal phase.31 during the luteal phase, the actions of bdnf may be inhibited by progesterone and allopregnanolone, but at the end of the menstrual cycle, elevated bdnf may persist after progesterone and allopregnanolone fall. the imbalance between excitatory effects of bdnf and inhibitory effects of progesterone and allopregananolone would explain increased seizures during the perimenstrual period. it is also interesting that bdnf induces the synthesis of neuropeptide y (npy), which is considered to be a potential anticonvulsant.32 the rise in npy could truncate the effects of bdnf, ending the perimenstrual period. there are many neurological and psychiatric conditions in which symptoms worsen in relation to the menstrual cycle. some scientists have suggested that the symptoms are caused by abnormal periods of activity in parts of the central nervous system (cns), and resemble seizures. with regard to migraine headaches, for example , women often report that the most severe episodes follow a perimenstrual pattern. and during a migraine, abnormal patterns of brain activity develop. women who have chronic pain can experience the most severe pain during certain phases of the menstrual cycle. pain may from a period of hyperactivity in the areas of the cns where pain is controlled. the concept of an epilepsy spectrum suggests that these types of hyperactivity of neurons in the cns might be a part of the spectrum, even though pain has not been considered to be a type of epilepsy. supporting this view is the observation that antiepileptic drugs are often successful in the treatment of migraine and pain. why do only some women have catamenial epilepsy, and why, even among those women, is it sometimes difficult to document? the type of epilepsy that is most susceptible to a catamenial pattern appears to involve a part of the brain (the temporal lobe) that does not control movement, so the seizures may not be convulsive.33 there are a variety of nonconvulsive seizures, and they have different causes. different areas of the brain are involved. in temporal lobe epilepsy, for example, seizures are often partial or complex partial: they can arise from one part of the temporal lobe and spread to other areas (but do not always do so), and they are not always accompanied by convulsions. these seizures often start with an aura a sensation of dj vu, for example, or an awareness of a familiar smell and are usually accompanied by a temporary loss of consciousness. after the seizure, the person may be somewhat confused but subsequently resumes normal behavior. because a patient with temporal lobe epilepsy can have seizures without convulsions, the person can be unaware that a seizure occurred he or she would have no memory of it. the person may also not remember the seizure because of the disruption of activity in the temporal lobe at the time of the seizure the temporal lobe is important to normal learning and memory. it is easy to see that the prevalence of catamenial epilepsy could well be underestimated if the type of seizure activity that increases at particular times during the menstrual cycle is nonconvulsive. the occurrence of catamenial epilepsy may also be underestimated because patients may experience nonconvulsive seizures in a catamenial pattern but their physicians do not know they occurred. in addition, physicians can not be sure that all seizures patients report actually occur, because some people appear to imagine seizures these are called pseudoseizures.34 most physicians would use an electroencephalogram (eeg) to document seizures, but some seizures may be missed by eeg if they involve structures deep in the brain, relatively far from the eeg electrodes, which are placed on the scalp, near the brain s surface. may not be diagnosed with catamenial epilepsy if their menstrual cycle is not regular. because chronic seizures can lead to irregular cycles, a woman and her physician may be unable to recognize a pattern. the notion of a spectrum of epilepsy is not only important to women with epilepsy but relevant to research scientists who use laboratory animals to study temporal lobe epilepsy. in these animals, seizures that simulate those in patients with temporal lobe epilepsy are usually defined by five stages of convulsive seizures that were originally described in 1972.35 the stages are easy to see because there are convulsions. it is hard for researchers to agree when an animal has a nonconvulsive seizure, in part because of the difficulty in defining the point at which normal brain activity has increased enough to constitute a seizure. by considering epilepsy as a spectrum ranging from mild to severe, and no longer requiring convulsions to define a seizure, research in epilepsy would broaden in scope, and ultimately many more people with seizures would be helped, such as those people who have seizures that are hard to detect. the more researchers can tell us about the spectrum of seizures in experimental animals, the better equipped we will be to treat the spectrum of seizure disorders that exist in people. in fact, some potential antiepileptic drugs may have been passed over in the last decades of drug screening because drugs are usually tested to see if they stop convulsive seizures in animals. and they are often not used to see if they help animals that are tested for other conditions where nonconvulsive seizures are suspected, such as alzheimer s disease. some candidate antiepileptic drugs might be successful against nonconvulsive conditions but would fail a drug screen because they are unable to ameliorate a convulsive seizure. restricting epilepsy research to the investigation of convulsive seizures is especially limiting for studies of hormones in female animals because convulsive seizures in female rodents stop the ovarian cycle or make it irregular. this effect has been documented for the common animal models of temporal lobe epilepsy.3638 as a , it is hard to conduct research in female rodents to understand how epilepsy influences reproductive function. this is unfortunate, because women with epilepsy often develop reproductive problems, such as infertility or polycystic ovarian syndrome. studying female rats with nonconvulsive seizures would provide more opportunities to understand the adverse effects of seizures on the reproductive system. the idea of the epilepsy spectrum also has implications for the development of new drugs to treat epilepsy. it may be useful to develop drugs that block the consequences of estrogen and progesterone, rather than estrogen and progesterone themselves, because the hormones are very important to normal life. if a particular branch of bdnf signaling could be identified as the most important to seizure threshold, it might be a better endpoint to target. this strategy could be used in concert with progesterone supplementation, or the enhancement of allopregnanolone levels. however, bdnf signaling and gabaa receptors are important to many normal functions. more specific targets than bdnf or the gabaa receptor are needed so that anticonvulsant actions occur without side effects. the more we can clarify the mechanisms underlying catamenial seizure exacerbation, the more likely it is that we can develop treatments without side effects. epilepsy is a devastating illness that has far - reaching effects on patients and their families. patients could be better helped by greater awareness of the epilepsy spectrum because nonconvulsive seizures would be better recognized. deepening the understanding of the epilepsy spectrum could lead to more accurate diagnoses, possibly allowing the detection of epilepsy in individuals who would otherwise not be treated with anticonvulsants. research would benefit by the increasing recognition of the diverse types of seizures, which would lead to a broader focus on drugs that are not necessarily anticonvulsant but antiepileptic.

editor s note: there is not just one type of epilepsy. while some forms of the disease are characterized by convulsive seizures, others involve seizures that are barely noticeable. seizures can occur for many reasons: they can be caused by genetic mutations, injury, or infection early in life. in addition, events in daily life, such as stress, or normal variations in hormones, such as estrogen and testosterone, can influence brain activity and therefore influence seizures. by considering the powerful interactions between the brain and the endocrine system, this influence of hormones on seizures can be understood and new treatment options can be considered.

rsv in and its mutant forms were overexpressed in escherichia coli bl21 (de3) and purified as previously described. the proteins were stored in aliquots at 80c in 20 mm hepes - naoh (ph 7.5), 1.0 m nacl, 20m zncl2, 5 mm 2-mercaptoethanol and 10% (w / v) glycerol. the branched dna substrate mimicking the product of the concerted integration reaction was obtained by annealing three synthetic oligonucleotides (integrated dna technologies). a similar strategy had been used previously to prepare a pfv in - dna complex, which demonstrated that the in - dna complex assembled on the designed integration product has essentially an identical structure to the equivalent complex formed via the forward integration reaction. the viral dna branches carrying the high - affinity gain - of - function mutant rsv u3 long terminal repeat (ltr) sequence (gu3) were attached to the target dna duplex with a palindromic 6 bp spacer to generate a fully symmetrized structure. to prepare the rsv in - dna complexes for crystallization, 30 m half - site dna substrate was mixed with 120 m rsv in in 20 mm hepes - naoh (ph 7.5), 500 mm nacl, 20% (w / v) glycerol, and 1 mm tris-(2-carboxyethyl)phosphine (tcep). the mixture was dialyzed against low salt buffer (20 mm hepes - naoh ph 7.5, 125 mm nacl, 20% ( w / v) glycerol, 1 mm tcep ) at 25c for overnight. at the end of this first dialysis, the mixture was subsequently dialyzed against high salt buffer (20 mm mes - naoh ph 6.0, 1.2 m nacl, 20% ( v / v) dimethyl sulfoxide (dmso), 5% (w / v) glycerol, 1 mm tcep ) at 25c for 24 hours. at the end of this second dialysis, the solubilized rsv in - dna complex (intasome) was purified through size - exclusion chromatography (superdex 200 10/300 gl, ge healthcare) running with 20 mm mes - naoh ph 6.0, 1.2 m nacl, 20% (v / v) dmso, 5% (w / v) glycerol, and 1 mm tcep. the isolated intasome remains stable in the high - salt condition containing 1.2 m nacl that precludes complex formation, suggesting that once the intasome forms, it is kinetically trapped. the solubility - enhancing rsv in mutation f199k completely abolished intasome formation. for the sec - mals analysis , a modified condition was used for the intasome re - solubilization and isolation for better baseline stability (extended data fig . the rsv intasome assembled through dialysis and purified by size - exclusion chromatography was concentrated to 46 mg / ml using centrifugal concentrator ( amicon). various combinations of the lengths of the viral dna (ranging from 16 bp to 25 bp) and flanking target dna (ranging from 14 bp to 22 bp, corresponding to the full target dna lengths of 34 bp to 50 bp) were screened in crystallization trials. the extensive screening yielded only one crystal form with a specific combination of 22-base (length of the non - transferred strand) viral dna branches and 16 base - pair (bp) target dna flanks on either side of the central six bp spacer (fig . dna substrates with two slightly different target sequences were used ( 5-aatgttgtcttatgcaatactc-3/5-gagtattgcataagacaacagtgcacgaatcttgaagacact-3/5-agtgtcttcaagattc-3, or 5-aatgttgtcttatgcaatactc-3/5-gagtattgcataagacaacagtcgaccaaccttcaacttagc-3/5-gctaagttgaaggttg-3), and they produced essentially the same crystals. the rsv intasome crystals were grown through reverse vapor diffusion in hanging drops at 22c, by mixing 1.5 l in - dna complex solution with 1.5 l reservoir solution (3.2 m sodium formate). crystals appeared within 35 days and reached a size of ~150300 m in 3~5 days. even though the rsv intasome crystals initially diffracted x - ray poorly (~ 10), soaking the crystals with a metatungstate cluster compound dramatically improved the resolution (extended data fig . the tungsten cluster was later found to bind between in dimers from separate intasome complexes to mitigate crystal lattice disorder . the crystals were soaked overnight with 0.15 mm metatungstate cluster na6 in a stabilization buffer consisting of 3.2 m sodium formate, 16 mm mes - naoh ph 6.0, 0.8 m nacl, 16% ( v / v) dmso, 4% (w / v) glycerol, and 1 mm tcep. after soaking, the crystals were cryo - protected in 3.2 m sodium formate, 16 mm mes ph 6.0, 0.8 m nacl, 16% (v / v) dmso, 12% (w / v) glycerol, and 1 mm tcep, and were frozen by rapid immersion in liquid nitrogen. the full - length wild - type rsv in, the c - terminally truncated rsv in, and its various mutant forms tested produced essentially the same crystals with indistinguishable x - ray diffraction properties. x - ray diffraction data were collected at the advanced photon source northeastern collaborative access team beamlines (24-id - c / e) and the advanced light source molecular biology consortium (4.2.2) beamline and processed using hkl2000 or xds. the rsv intasome crystals showed varied degrees of pseudo - merohedral twinning with twin operator , owing to the very similar a and c unit cell dimensions of the primitive monoclinic lattice. thus, we screened a large number of crystals to identify ones that diffract to higher resolution and have smaller twin fractions. the structure of rsv intasome was determined by molecular replacement with phaser, using the rsv in ccd, ctd (pdb i d : 4fw1), and a 16 bp b - form dna as search models. 8 copies of ccd, one copy of ctd and three copies of dna molecules were located. refinement of the partial model revealed electron density for two copies of the metatungstate clusters. subsequent iterative model building using coot and refinement with phenix suite allowed placement of the remaining 7 copies of ctd, 8 copies of ntd generated using modeller based on hiv-1 ntd (pdb i d : 1k6y), and building of the inter - domain linkers as well as the remaining parts of the dna molecule guided by the difference electron density maps. a third metatungstate cluster, which is more weakly bound compared to the first two, was positioned manually into residual density. the dna base - pairs and base - stacking restraints were used throughout the refinement. the geometry restraints for protein included the reference - model restraints for ccd and ctd based on the higher resolution rsv in structure (4fw1), and the secondary structure and zinc - coordination restraints for ntd. atomic displacement parameters were refined with grouped b - factors per residue for protein and dna, and a total of 53 tls groups assigned by phenix. the asymmetric unit of the crystal contains one complete rsv intasome, which includes eight in molecules and two viral dna branches emanating from a strongly bent 38 bp target dna. the dataset used for the final refinement was from an rsv intasome crystal grown using selenomethionine - labeled rsv in with the following amino acid substitutions; c23s, l112 m, l135 m, l162 m, l163 m, l188 m, and l189 m, which we confirmed to be active in concerted integration and inhibited by insti similarly to the wild - type rsv in, and a dna substrate carrying a nick at the middle of each target dna branch (the 16-base dna strand shown in olive in fig . the nick occasionally facilitated crystal growth, but it was not necessary for crystallization and did not change the space group or the unit cell parameters compared to crystals grown without the nick . because this nick in the target dna is not biologically relevant, it is not shown in figs . twin refinement protocol was not used as the dataset used for the final refinement had a low ( less than 10%) twin fraction. the summary of data collection and refinement statistics is shown in extended data table 1. the paired - refinement procedure was performed in steps of 0.1 to determine the high - resolution limit (extended data fig . the register of amino acids in the final model was verified by the selenium anomalous difference fourier peaks ( extended data fig . ramachandran analysis shows that 96.0, 3.9, and 0.1 % of the protein residues are in the most favored, allowed, and disallowed region, respectively . the ntd - ccd linker in some of the non - catalytic in molecules and the last four bp at the distal end of one of the viral dna molecules were not built due to poor electron density . rsv in and its mutant forms were overexpressed in escherichia coli bl21 ( de3) and purified as previously described. the proteins were stored in aliquots at 80c in 20 mm hepes - naoh (ph 7.5), 1.0 m nacl, 20m zncl2, 5 mm 2-mercaptoethanol and 10% (w / v) glycerol. the branched dna substrate mimicking the product of the concerted integration reaction was obtained by annealing three synthetic oligonucleotides (integrated dna technologies). a similar strategy had been used previously to prepare a pfv in - dna complex, which demonstrated that the in - dna complex assembled on the designed integration product has essentially an identical structure to the equivalent complex formed via the forward integration reaction. the viral dna branches carrying the high - affinity gain - of - function mutant rsv u3 long terminal repeat (ltr) sequence (gu3) were attached to the target dna duplex with a palindromic 6 bp spacer to generate a fully symmetrized structure. to prepare the rsv in - dna complexes for crystallization, 30 m half - site dna substrate was mixed with 120 m rsv in in 20 mm hepes - naoh (ph 7.5), 500 mm nacl, 20% (w / v) glycerol, and 1 mm tris-(2-carboxyethyl)phosphine (tcep). the mixture was dialyzed against low salt buffer (20 mm hepes - naoh ph 7.5, 125 mm nacl, 20% ( w / v) glycerol, 1 mm tcep ) at 25c for overnight. at the end of this first dialysis, the mixture was subsequently dialyzed against high salt buffer (20 mm mes - naoh ph 6.0, 1.2 m nacl, 20% ( v / v) dimethyl sulfoxide (dmso), 5% (w / v) glycerol, 1 mm tcep ) at 25c for 24 hours. at the end of this second dialysis, the solubilized rsv in - dna complex (intasome) was purified through size - exclusion chromatography (superdex 200 10/300 gl, ge healthcare) running with 20 mm mes - naoh ph 6.0, 1.2 m nacl, 20% (v / v) dmso, 5% (w / v) glycerol, and 1 mm tcep. the isolated intasome remains stable in the high - salt condition containing 1.2 m nacl that precludes complex formation, suggesting that once the intasome forms, it is kinetically trapped. the solubility - enhancing rsv in mutation f199k completely abolished intasome formation. for the sec - mals analysis , a modified condition was used for the intasome re - solubilization and isolation for better baseline stability (extended data fig . for crystallization, the rsv intasome assembled through dialysis and purified by size - exclusion chromatography was concentrated to 46 mg / ml using centrifugal concentrator ( amicon). various combinations of the lengths of the viral dna (ranging from 16 bp to 25 bp) and flanking target dna (ranging from 14 bp to 22 bp, corresponding to the full target dna lengths of 34 bp to 50 bp) were screened in crystallization trials. the extensive screening yielded only one crystal form with a specific combination of 22-base (length of the non - transferred strand) viral dna branches and 16 base - pair (bp) target dna flanks on either side of the central six bp spacer (fig . dna substrates with two slightly different target sequences were used ( 5-aatgttgtcttatgcaatactc-3/5-gagtattgcataagacaacagtgcacgaatcttgaagacact-3/5-agtgtcttcaagattc-3, or 5-aatgttgtcttatgcaatactc-3/5-gagtattgcataagacaacagtcgaccaaccttcaacttagc-3/5-gctaagttgaaggttg-3), and they produced essentially the same crystals. the rsv intasome crystals were grown through reverse vapor diffusion in hanging drops at 22c, by mixing 1.5 l in - dna complex solution with 1.5 l reservoir solution (3.2 m sodium formate). crystals appeared within 35 days and reached a size of ~150300 m in 3~5 days. even though the rsv intasome crystals initially diffracted x - ray poorly (~ 10), soaking the crystals with a metatungstate cluster compound dramatically improved the resolution (extended data fig . the tungsten cluster was later found to bind between in dimers from separate intasome complexes to mitigate crystal lattice disorder . the crystals were soaked overnight with 0.15 mm metatungstate cluster na6 in a stabilization buffer consisting of 3.2 m sodium formate, 16 mm mes - naoh ph 6.0, 0.8 m nacl, 16% ( v / v) dmso, 4% (w / v) glycerol, and 1 mm tcep. after soaking, the crystals were cryo - protected in 3.2 m sodium formate, 16 mm mes ph 6.0, 0.8 m nacl, 16% (v / v) dmso, 12% (w / v) glycerol, and 1 mm tcep, and were frozen by rapid immersion in liquid nitrogen. the full - length wild - type rsv in, the c - terminally truncated rsv in, and its various mutant forms tested produced essentially the same crystals with indistinguishable x - ray diffraction properties. x - ray diffraction data were collected at the advanced photon source northeastern collaborative access team beamlines (24-id - c / e) and the advanced light source molecular biology consortium (4.2.2) beamline and processed using hkl2000 or xds. the rsv intasome crystals showed varied degrees of pseudo - merohedral twinning with twin operator , owing to the very similar a and c unit cell dimensions of the primitive monoclinic lattice. thus, we screened a large number of crystals to identify ones that diffract to higher resolution and have smaller twin fractions. the structure of rsv intasome was determined by molecular replacement with phaser, using the rsv in ccd, ctd (pdb i d : 4fw1), and a 16 bp b - form dna as search models. 8 copies of ccd, one copy of ctd and three copies of dna molecules were located. refinement of the partial model revealed electron density for two copies of the metatungstate clusters. subsequent iterative model building using coot and refinement with phenix suite allowed placement of the remaining 7 copies of ctd, 8 copies of ntd generated using modeller based on hiv-1 ntd (pdb i d : 1k6y), and building of the inter - domain linkers as well as the remaining parts of the dna molecule guided by the difference electron density maps. a third metatungstate cluster, which is more weakly bound compared to the first two, was positioned manually into residual density. the dna base - pairs and base - stacking restraints were used throughout the refinement. the geometry restraints for protein included the reference - model restraints for ccd and ctd based on the higher resolution rsv in structure (4fw1), and the secondary structure and zinc - coordination restraints for ntd. atomic displacement parameters were refined with grouped b - factors per residue for protein and dna, and a total of 53 tls groups assigned by phenix. the asymmetric unit of the crystal contains one complete rsv intasome, which includes eight in molecules and two viral dna branches emanating from a strongly bent 38 bp target dna. the dataset used for the final refinement was from an rsv intasome crystal grown using selenomethionine - labeled rsv in with the following amino acid substitutions; c23s, l112 m, l135 m, l162 m, l163 m, l188 m, and l189 m, which we confirmed to be active in concerted integration and inhibited by insti similarly to the wild - type rsv in, and a dna substrate carrying a nick at the middle of each target dna branch (the 16-base dna strand shown in olive in fig . the nick occasionally facilitated crystal growth, but it was not necessary for crystallization and did not change the space group or the unit cell parameters compared to crystals grown without the nick . because this nick in the target dna is not biologically relevant, it is not shown in figs . twin refinement protocol was not used as the dataset used for the final refinement had a low ( less than 10%) twin fraction. the summary of data collection and refinement statistics is shown in extended data table 1. the paired - refinement procedure was performed in steps of 0.1 to determine the high - resolution limit (extended data fig . the register of amino acids in the final model was verified by the selenium anomalous difference fourier peaks ( extended data fig . ramachandran analysis shows that 96.0, 3.9, and 0.1 % of the protein residues are in the most favored, allowed, and disallowed region, respectively . the ntd - ccd linker in some of the non - catalytic in molecules and the last four bp at the distal end of one of the viral dna molecules were not built due to poor electron density . the secondary structure elements for rsv in are color - coded based on the three in domains similarly to fig . a, the half - site ( gaped duplex) substrate prepared by annealing three oligonucleotides dimerizes via the self - complementary 6-base spacer sequence (underlined) to form a branched structure mimicking the product of the concerted integration reaction (fig . viral dna nucleotides are numbered, and some of the structural elements of rsv in involved in the viral dna interactions are shown . c, x - ray diffraction pattern after the metatungstate - soaking ( see method for details). is perpendicular to the two - fold screw (b) axis of the monoclinic lattice, which lies horizontally. f, paired - refinement analysis to assess the resolution limit of the rsv intasome diffraction data. for each pairwise comparison, model refinements were run at two different resolution limits and the r - factors calculated for a common (lower) resolution cutoff were compared. a, a representative size - exclusion chromatography (sec) profile for rsv intasome, overlaid with that for a mixture of molecular weight markers. b, sec profiles for rsv intasomes formed with in of varying c - termini. c, sec - mals (multi - angle light scattering) analysis of rsv intasome. the intasome formed with rsv in (1269 aa) was separated by sec in a modified condition containing 20 mm hepes ph 7.5, 1.0 m nacl, 5 % glycerol, and 1.0 mm tcep. the absolute molecular mass was determined by light scattering using in - line detectors described previously. a similar sec - mals analysis of the intasome formed with wt full - length rsv in (1286 aa) yielded a molecular mass of 268 kda (n=2, data not shown). the calculated mass of an intasome containing eight rsv in (1269 or 270) molecules is ~288 kda. d, chemical cross - linking analysis of rsv intasome. the rsv intasome and free in (1269 aa) were purified by size - exclusion chromatography in the running buffer: 20 mm hepes (ph 7.5), 1.0 m nacl, 5 % glycerol, and 1.0 mm tcep. the peak fractions of the intasome and in were cross - linked with the indicated amount of ethylene glycol bis - succinimidylsuccinate (egs) as described previously and analyzed by sds - page. the majority of cross - linked species within the intasome were larger than a tetramer. the nupage 412% gradient gel with a mes - based sds - page running buffer was used. anomalous difference fourier maps calculated using the data collected on selemomethionine - labeled rsv intasome, contoured at 3.5 (blue mesh) or 5.0 (orange mesh). b, c, close - up view of a proximal (b) and distal (c) in dimer, respectively. a, b, protein arrangement in the octameric rsv intasome. the inner and outer subunit of one proximal in dimer is colored in green and cyan, respectively, with the catalytic triad (dde) of the inner subunit shown in red. c, a close - up view around the active site of the inner in subunit in the rsv intasome. the dna strands are colored as in fig. 1, and the catalytic triad residues (dde) the color scheme follows that used for the proximal in dimers of rsv in in (a, b). f, a close - up view around the active site of the inner in subunit in the pfv intasome (pdb i d : 3os0). simulated annealing composite omit 2mfodfc density contoured at 1.0 , shown for area within 3.5 from any protein or dna atom in the final model. in a and b, electron densities around protein and dna are colored differently (blue and green, respectively). a, b, dna structure in the rsv (a) or pfv (b) intasome, alternatively referred to as the strand - transfer complex (stc). c, a comparison of dna structures between the rsv and pfv intasomes (stcs). the integration product dnas (rsv in cyan, pfv in red) superimposed at a viral dna terminus are shown in three different view angles. note the significant deviation in overall trajectory of the target dna, and difference in the orientation of the second viral dna molecule. the region spanning the two integration sites on opposing target dna strands is 6 bp for rsv and 4 bp for pfv. the sigma a - weighted 2mfodfc map contoured at 1.5 (a) or 2.5 (b), overlaid with the final model for the central 6 bp region between the two integration sites. data collection and refinement statistics statistics for the highest resolution shell are shown in parentheses. a video showing the overall structure of the rsv intasome and positioning of the three structural domains of in within the intasome.

integration of the reverse - transcribed viral dna into the host genome is an essential step in the lifecycle of retroviruses. retrovirus integrase (in) catalyzes insertions of both ends of the linear viral dna into a host chromosome 1. in from hiv-1 and closely related retroviruses share the three - domain organization, consisting of a catalytic core domain flanked by n- and c - terminal domains essential for the concerted integration reaction. although structures of the tetrameric in - dna complexes have been reported for in from prototype foamy virus (pfv) featuring an additional dna - binding domain and longer interdomain linkers 25, the architecture of a canonical three - domain in bound to dna remained elusive. here we report a crystal structure of the three - domain in from rous sarcoma virus (rsv) in complex with viral and target dnas. the structure shows an octameric assembly of in, in which a pair of in dimers engage viral dna ends for catalysis while another pair of non - catalytic in dimers bridge between the two viral dna molecules and help capture target dna. the individual domains of the eight in molecules play varying roles to hold the complex together, making an extensive network of protein - dna and protein - protein contacts that show both conserved and distinct features compared to those observed for pfv in. our work highlights diversity of retrovirus intasome assembly and provides insights into the mechanisms of integration by hiv-1 and related retroviruses.

a lot of species are vectors of viral, rickettsial, bacterial, and protozoal diseases of birds and mammals, including humans. identification of causative tick is important in making a diagnosis in tick - related dermatoses. in the republic of korea, hard ticks which belong to the family ixodidae are responsible for most tick - related diseases. since the first human case of tick bite was reported in 1982, the total number of cases recorded in the literature reached approximate 40. no amblyomma case has yet been recorded in korea although approximately 100 species of the genus amblyomma were recorded worldwide. here , we report a human case of typical infestation with an amblyomma testudinarium (koch, 1844) adult worm in a korean woman. in july 2009, a 74-year - old korean woman presented to our dermatology clinic with a large mass on her left inguinal region. she was a typical rural housewife frequently worked in the field near her house. on admission she complained of mild discomfort and itching sensation of the skin around the bite area. laboratory examinations were normal or negative for the following tests: hemoglobin 14.1 g / dl, hematocrit 43.3%, white blood cell 8,210/mm (neutrophil 69%, lymphocyte 25%, monocyte 6%), erythrocyte sedimentation rate 6 mm / hr, serum glutamic pyruvic transaminase 30 ten days prior to presentation, the patient began to note the presence of a mass which she thought to be benign. in the clinic, we found a dark green, round, 23 mm organism whose head was partially burrowed under the skin. it was grasped close to the skin and steady pressure applied, pulling the tick straight out perpendicularly to the skin. when the organism was removed by plucking, its mouth part was torn. six months later, the patient was well and then follow - up communication was discontinued. she was probably attacked by the tick while working to collect vegetables and edible sprouts of wild grass in her vegetable garden located in the suburb of suncheon city, jeollanam - do, where she had been residing. she recently had not traveled to any foreign country as well as any domestic area, including jeju island. the isolated tick was fixed with formalin and then stereoscopically examined to determine its species. the body was 23 mm in length, 19 mm in width, and 12 mm in height (fig . the small ornamented scutum was seen as a small shield behind the capitulum on the back . the eyes were located on the lateral edges of the scutum but not projecting beyond scutal contour . although greater part of the mouth including the hypostome was torn off, the left pedipalp only was relatively well preserved and had characteristic appearance ; especially long article 2 with approximately 2.5 times as long as article 3 ( fig . the genital aperture was located at the level between coxa i and coxa ii ( figs . 1, 2b). the anus was located at the level posterior to the spiracular plates and embraced by y - shaped anal groove posteriorly (figs . 1, 2c). the spiracular plate, comma - shaped, was located on the ventrolateral surface posterolaterally to the coxa iv (figs . 1, 2d). the tick had 4 pairs of legs (figs . 1, 3). the coxa i had 2 medium sized subequal spurs, external spur was slightly longer than internal spur (fig . the coxa ii had a single salient ridgelike spur which was much broader than long ( fig . the coxa iii with a single spur was very similar to coxa ii ( fig . the coxa iv had a single spur which was longer than those of the coxae ii and iii ( fig . base upon these morphological features, this tick was identified as a female adult of a. testudinarium ( table 1). ticks are blood - sucking arthropods that parasitize various species of vertebrates in almost every region of the world. in the republic of korea, about 40 human cases have been reported since 1982. the causative ticks reported in korea were identified as ixodes nipponensis, i. ovatus, i. monospinosus, i. persulcatus, haemaphysalis flava, and h. longicornis. the genus amblyomma ticks are large and beautifully ornamented with long mouth parts; possessing eyes and festoons. close examination under a stereomicroscope showed the capitulum of the anterior portion of the body, the scutum of the dorsal portion, and the spiracular plate, genital aperture, anus, and 4 pairs of legs of ventral portion. the pedipalp of the capitulum was slender with the second segment much longer than the third. the scutum was quite ornate in comparison to other parts of the body and had non - protruding eyes at the edge. the coxa of the first leg had subequal 2 spurs (the external one was slightly larger than internal). the spine of coxa on the fourth leg was longer than those on the second and third legs. these characteristics were typical features of the adult female of a. testudinarium. they feed for 7 to 12 days for a full engorgement and reach 20 mm in length after feeding. the genus amblyomma is one of the largest ticks among the hard ticks. in case of a. testudinarium, the size of adult females varies from 5 to 20 mm depending on the stages of engorgement. however, this species may feed for 30 days or longer and reach bigger than 20 mm in length. in the present case, hospital admission was quite delayed. the tick fed over additional 10 days after detected by the patient and was inordinately engorged. it measured 23 mm long, which was bigger than any other human ticks reported in korea. the present tick would have been in situ for at least 1 month. during that time , the patient had some itching and mild pain of the skin around the bite area, but no clinical symptoms of tick - borne infection, including lyme disease and rickettsiosis, occurred. ticks can also cause inflammatory problems in the host without transmitting infection. when the head part was broken off in the removal process, more careful attention her symptoms disappeared gradually after the removal of the tick and the surgical wound was healed in a week. the frequent location involved by a. testudinarium is known to be the axillary, anogenital, or inguinal region, which are rich in apocrine glands. in the present case, tick bite was found on the inguinal region. according to the patient's recall of daily life, she often undressed for urination while working at the vegetable garden nearby her home. it appears that the tick attached itself to her clothing or skin when she was undressed. a. testudinarium is known to be a tropical tick and found mainly in the indian peninsula, south east asia, including myanmar, thailand, malaysia, indonesia, the philippines, taiwan, and japan. in japan, it is especially important in southwestern areas (below about 36 north latitude), including kinki, shikoku, kyushu, and chugoku. the geographical limitation of a. testudinarium in the southwestern part of japan implies that the warm temperature with heavy rainfall and the subtropical vegetation fauna may be a proper environmental condition for this tick. in korea , a. testudinarium has been known to be distributed in jeju island (between 33 to 34 north latitude), which is located in the south sea far from the korean peninsula. however, this species was also collected in a southern coastal area of jeollanam - do during a haemaphysalis phasiana survey using tick drags. in the present case , the patient has been living in a suburban or rural area of suncheon city, approximately 400 km south of seoul, korea. it is an agricultural and industrial city nearby the sea at suncheon bay (35 north latitude) facing the south sea.

a case of tick bite was found in the inguinal region of a 74-year - old korean woman. she was attacked by the tick while working in her vegetable garden in the vicinity of mountain located in suncheon city, the southern coastal area of the korean peninsula. on admission she complained of mild discomfort and itching around the bite area. the causative tick was 23 mm long and had slender pedipalps. the scutum was quite ornate and had eyes at the edge. the genital aperture was located anterior to the level of the coxa ii. the spiracular plate was comma - shaped and the anus was surrounded posteriorly by the anal groove. the coxa i had subequal 2 spurs; the external one slightly larger. the spur of coxa iv was slightly longer than those of coxae ii and iii. the tarsus iv had 2 distinct subapical ventral spurs. it was identified as the fully engorged adult female of amblyomma testudinarium. this is the first human case of amblyomma bite in korea.

hashimoto's encephalopathy is a rare steroid responsive encephalopathy syndrome characterized by persisting or fluctuating neurologic and neuropsychological deficits associated with elevated blood concentrations of anti - thyroid antibodies. the classically described clinical manifestations usually include acute to subacute onset of confusion with alteration of consciousness, but myoclonus is a less publicized manifestation of the disease. we present a case of hashimoto's encephalopathy, presenting as steroid responsive myoclonus in a setting of autoimmune thyroid disease. an 18-year - old male patient was diagnosed to have primary hypothyroidism 6 months back following weight gain, edema and facial puffiness of 3 years duration. his initial thyroid function test (tft) was t4: 2.3 g / dl (5.01 - 12.4) t3: 0.35 ng / ml (0.6 - 1.81) and thyroid stimulating hormone (tsh): 180 iu / ml (0.35 - 5.50) and was started on l thyroxine, with a significant improvement of symptoms. he started developing myoclonus for the past 1 month, for which he was referred to our hospital. on examination, he was conscious, with a cognitive impairment and had memory loss for recent events, with the poor attention span, difficulty in finding words, dyscalculia and dysgraphia. his mini mental status examination (mmse) was 11 suggestive of moderate cognitive impairment. he also had myoclonus, involving both upper limbs and lower limbs. on investigations, blood routine examination and erythrocyte sedimentation rate was normal and liver function test, kidney function test, sodium, potassium, calcium and magnesium were normal and so were the arterial blood gas analysis. his latest tfts show normal t3 and t4 with mild elevation of tsh (tsh-9.1 iu / ml). his electroencephalogram (eeg) showed a diffuse slow wave activity and magnetic resonance images (mri) showed a pituitary hyperplasia with non - specific white matter changes. electroencephalogram showing diffuse slow wave activity magnetic resonance images showing pituitary hyperplasia magnetic resonance images showing non - specific white matter changes antinuclear antibody titer, anti - double - stranded deoxyribonucleic acid, hepatitis b surface antigen, anti - hepatitis c virus, lupus anticoagulant and venereal disease research laboratory (vdrl) test were carried out to evaluate the cause of decreased cognitive function, myoclonus and seizures, which were all negative. csf electrophoresis was normal. in view of these neurologic symptoms, associated with high titers of anti - thyroid antibodies and the exclusion of other possible causes of encephalopathy, he was started on methylprednisolone 1 g / day for 3 days and was shifted over to prednisone 1 mg / kg / day. there was a marked improvement in the symptoms with no further episodes of myoclonus or seizures. one month after discharge, the patient reported a sustained improvement on all parameters, including memory and cognition, making it possible for his to return to an almost normal routine. prednisone dose was tapped over the period of 3 months without the recurrence of symptoms. hashimoto's encephalopathy is a rare steroid responsive neuropsychiatric syndrome, associated with the serologic evidence of anti - thyroid antibodies, when other causes of encephalopathy are excluded. this syndrome was first described by brain et al. in 1966, but it still remains a rare or under diagnosed condition with only about 130 cases reported in the literature until date, predominantly in adult females. the difficulty in diagnosis is due to the varied presentations and subtlety of symptoms, which may be acute or chronic. the pathophysiology of hashimoto encephalopathy is still unclear and the proposed pathogenetic mechanisms include autoimmune cerebral vasculitis, toxic effects of thyroid - stimulating hormone on the central nervous system and neuronal reaction mediated by antibodies. the anti - thyroid antibodies does not appear to be pathogenic and are considered as markers of the disease. based on this observation, it has been proposed to rename hashimoto's encephalopathy (he) as steroid - responsive encephalopathy associated with autoimmune thyroiditis. the clinical manifestations usually include acute to the subacute onset of confusion with alteration of consciousness. two - thirds of patients may experience focal or generalized tonic - clonic seizures and 12% may present with status epileptics. hyperreflexia and other pyramidal tract signs are found in 85% of patients and psychosis, visual hallucinations and paranoid delusions have been reported in 25 - 36% of patients. myoclonus is a less publicized but common manifestation of he, with 52% patients diagnosed to have he, having a history of myoclonus. the diagnosis of he should be considered in patients presenting with the characteristic neuropsychiatric manifestations excluding other causes of encephalopathy. they should have presence of high levels of anti - thyroid antibodies in serum or csf; no alteration in the csf and/or imaging tests compatible with infectious, vascular, or neoplastic etiology; and a good response to immunosuppressive therapy. elevated csf protein is a common and the thyroid profile may be variable, with 23 - 35% of patients having subclinical hypothyroidism, 17 - 20% having hypothyroidism, 7% having hyperthyroidism and 18 - 45% being euthyroid. non - specific eeg abnormalities are seen in 90 - 98% of patients and brain mri may show abnormalities in 49% such as cerebral atrophy, focal cortical abnormality, diffuse subcortical abnormality and non - specific subcortical focal white matter abnormality. the disease is responsive to immunosuppressive therapy and intravenous (iv) methylprednisolone (500 - 1000 mg / day) for 3 - 5 days, followed by an oral dose of prednisone (1 - 2 mg / kg / day), followed by gradual tapering based on the clinical response being the commonly followed protocol. around 2 - 5% of patients may not respond to steroids, in whom azathioprine, iv immunoglobulins or plasmapheresis can be used with good . the diagnosis of he should be considered in patients presenting with the characteristic neuropsychiatric manifestations excluding other causes of encephalopathy. they should have presence of high levels of anti - thyroid antibodies in serum or csf; no alteration in the csf and/or imaging tests compatible with infectious, vascular, or neoplastic etiology; and a good response to immunosuppressive therapy. elevated csf protein is a common and the thyroid profile may be variable, with 23 - 35% of patients having subclinical hypothyroidism, 17 - 20% having hypothyroidism, 7% having hyperthyroidism and 18 - 45% being euthyroid. non - specific eeg abnormalities are seen in 90 - 98% of patients and brain mri may show abnormalities in 49% such as cerebral atrophy, focal cortical abnormality, diffuse subcortical abnormality and non - specific subcortical focal white matter abnormality. the disease is responsive to immunosuppressive therapy and intravenous (iv) methylprednisolone (500 - 1000 mg / day) for 3 - 5 days, followed by an oral dose of prednisone (1 - 2 mg / kg / day), followed by gradual tapering based on the clinical response being the commonly followed protocol. around 2 - 5% of patients may not respond to steroids, in whom azathioprine, iv immunoglobulins or plasmapheresis can be used with good .

hashimoto's encephalopathy (he) is a rare steroid - responsive encephalopathy syndrome, which can have highly variable neuropsychiatric manifestations and can go unrecognized for a long time. he is a diagnosis of exclusion and should be kept in mind when evaluating a patient with a cognitive dysfunction and high titers of anti - thyroid antibodies as it responds dramatically to steroids. steroid responsive myoclonus can be a presentation of he.

blue rubber bleb nevus syndrome (brbns) is an uncommon condition with multiple venous malformations of the skin, gastrointestinal tract, and other visceral sites including the liver, spleen, bladder, spinal cord, lungs, and bones. clinically, the lesions are characteristically blue / purple soft compressible nodules with a rubbery feel. they are dome shaped, almost nipple - like rubber blebs, which may coalesce to form large masses. the gastrointestinal tract is the most common visceral site affected, and lesions share the same pathology as the cutaneous lesions. they are sessile or polypoid and affect all levels of the gastrointestinal tract, in particular, the small intestine. they are prone to bleeding and may cause a significant anemia requiring transfusion and iron replacement therapy. a 21-year - old lady presented to our outpatient department with multiple nontender, soft, rubbery, and compressible swellings distributed over the right side of the chest and right upper limb in a blaschkoid distribution. these lesions first appeared on the right side of the chest during her childhood and they gradually increased in number and size with age. her medical and family history were unremarkable except for the presence of recurrent episodes of painless bleeding per rectum (hematochezia). cutaneous examination revealed multiple blue to purple papulonodules of variable sizes, with diameters ranging from 2 to 15 mm, distributed along right side of chest, the right hand and forearm; which on palpation yielded a feeling of cutaneous herniation. the affected limb was swollen, which when palpated yielded a bag - of - worm like feeling. clinically, the swellings were diagnosed to be blue rubber bleb nevi , which were present in the of cutaneous vascular malformation. bluish papulo - nodule over the right breast multiple skin colored swellings affecting the right hand multiple blue to purple cutaneous swellings affecting the right arm blue rubber bleb nevus on the of swollen right arm photograph depicting the confinement of the lesions to the upper extremity of right side the hematological evaluation revealed iron deficiency anemia. doppler ultrasound hinted the presence of a low - flow lesion, which was suggestive of vascular malformation. computed tomography angiography of right upper extremity and upper chest revealed extensive venous angiomatous malformation in right superior extremity and right axilla, right side of upper chest wall, and right side of the neck with direct filling of contrast from upper part of superior vena cava. the malformation involved subcutaneous fat planes, muscles, and intermuscular portions of the right upper chest wall, right axilla, and right superior extremity. colonoscopy detected the presence of vascular polyps in her sigmoid colon , which presumably led to the recurrent episodes of hematochezia. currently, the patient has been put under oral iron supplements and has been referred to the department of plastic surgery. computed tomography angiogram of showing extensive vascular malformation affecting the right upper limb along with right - upper trunk. ct angiogram showing extensive vascular malformation affecting the right upper limb along with right - upper trunk. blue rubber bleb nevus syndrome is an uncommon condition characterized by multiple venous malformations affecting the skin and internal viscera, gastrointestinal tract involvement being the most common. it was first described by gascoyen in 1806, but in 1958, william bennet bean coined the term blue rubber nevus syndrome for its color and consistency. brbns affects both sexes and is usually sporadic, although autosomal dominant inheritance has been reported. bean described three types of lesions in brbns, which are often noted at birth or during the neonatal period, although they can present later. the first type is the compressible, red - blue, nipple - like lesions. the second type is blue - black nonblanching macular lesions and the third type is subcutaneous, vascular, soft tissue lesions. in our case, the patient presented with lesions akin to the first type. in the english literature, most of the lesions are diffusely distributed all over the body, with blue subcutaneous nodules as the characteristic lesions. however, in our case, the skin lesions are distributed unilaterally, affecting only the right side of the body. the lesions on the upper limb are also arranged in a linear fashion making the case even rarer. to the best of our knowledge , there is only a single report of brbns with linear, unilateral distribution in the world literature, and this happens to be the second case showing such a unique distribution. complications include acute hemorrhage, iron deficiency anemia following chronic bleeding, thrombocytopenia, disseminated intravascular coagulation, intussusception, volvulus, bowel infarction, skeletal bowing, pathologic fracture, bony overgrowth, articular derangement and pain due to phleboliths. the presence of cutaneous lesions should alert clinicians on the possible presence of internal vascular malformations. in our present case , the recurrent episodes of hematochezia possibly occurred due to underlying vascular malformations (polyps) present in her sigmoid colon, which were detected on colonoscopy. these include cryotherapy, sclerotherapy, surgery, and newly - reported long - pulsed neodymium: yttrium - aluminium - garnet 1064 nm laser for cutaneous lesions. we have referred our patient to the department of plastic surgery for possible surgery. while the coexistence of cutaneous and gastrointestinal venous malformations are the hallmark of brbns, venous malformations in additional sites, such as the oral cavity, central nervous system, endocrine glands, musculoskeletal system, kidney, bladder, liver, spleen, heart, and lungs, the extra - cutaneous and gastrointestinal lesions may present with hemoptysis, hematuria and skeletal deformities or be asymptomatic. the differential diagnoses include maffucci syndrome, venous lakes, osler weber rendu syndrome or hereditary hemorrhagic telangiectasia, disseminated hemangiomatosis and kaposi sarcoma. brb nevi can be differentiated from the above conditions based on the latter's characteristic features such as bluish, bleb - like, painful, tender, hyperhidrotic, compressible nodules with feeling of dermal herniation and associated gastrointestinal tract involvement. a complete history and physical evaluation are a must to make a successful diagnosis to help in treatment planning and to prevent fatal potential complications. linear, unilateral distribution of brbns is extremely uncommon; this happens to be the second such case in the english literature to the best of our knowledge. unilateral, linear or blaschkoid distribution of blue rubber bleb nevus syndrome is extremely uncommon; this happens to be the first such case from india.

blue rubber bleb nevus syndrome (brbns) also called bean's syndrome is a rare disorder characterized by multiple cutaneous venous malformations in association with visceral lesions, most commonly affecting the gastrointestinal tract. we report here, a 21-year - old woman patient, who presented with unilateral, blaschkoid distribution of cutaneous venous malformations along with blue rubber bleb nevus and recurrent episodes of hematochezia due to vascular lesions in the sigmoid colon; likely to be a case of brbns. the unusual unilateral, blaschkoid distribution of brbns prompted this present report.

the etoac - soluble fraction obtained from a liquid liquid partition of the etoh extract (100 mg) showed antiplasmodial activity. dereplication as previously described indicated that the extract contained at least one new bioactive compound, so a larger sample was investigated. fractionation of the etoac - soluble fraction of this sample by chromatography on sephadex lh-20, reverse - phase spe, normal - phase silic - gel column chromatography, and c18 hplc yielded compounds 1 and 68, together with fractions that were mixtures of 5,6-dihydro--pyrones and bicyclic tetrahydro--pyrones. purification of these fractions was effected by diol ptlc or hplc to yield compounds 2, 3, 5, and 911. compound 1 was isolated as a clear oil, and its molecular formula was established as c20h36o3 by hresims (m / z 325.2753). its ir spectrum showed absorptions at 3340, 1719, and 1613 cm assigned to a hydroxy group and an ,-unsaturated -lactone moiety, respectively. its h nmr spectroscopic data had signals for a conjugated olefinic moiety at h 6.90 (m, 1h, h4) and 6.03 (dt, j = 9.8, 1.7 hz, 1h, h3), two oxymethine groups at h 4.75 (m, 1h, h6) and 4.00 (brs, 1h, h2), a terminal primary methyl group (h 0.88, t, j = 7.0 hz, 3h, ch315) in the upfield region, and a multiplet at h 2.36 (2h, h5), representing a de - shielded methylene group. in the hmbc spectrum, the methylene protons at h 2.36 (h5) showed correlations both to an olefin group (c 121.3, c3, and 145.0, c4) and to the oxymethine resonance at h 4.75 and c 74.8 (c6). in addition, both the olefinic protons (h 6.90, h4, and h 6.03, h3) and the oxymethine at h 4.75 (h6) correlated with a carbonyl carbon at c 164.6 (c2) (figure 1). these data indicated a six - substituted 5,6-dihydro--pyrone, a common ring system of secondary metabolites found in cryptocarya species. the remaining oxymethine group at h 4.00 (1h, h2) was assigned to c2, which was flanked by two methylenes (c1 and c3) as indicated by hmbc correlations (figure 1) from the methylene protons at h 1.92 (ddd, j = 14.5, 9.6, 2.2 hz, h1) and h 1.65 (m, h1) to c5 (c 30.1), from h2 (h 4.00) to the oxymethine carbon at c 74.8 (c6), and from h2 to the two neighboring methylene carbons at c 41.2 (c1) and c 38.2 (c3). the 6r absolute configuration was determined by the positive cotton effects at 256 nm observed in its ecd spectrum in meoh, arising from the n * transitions of the lactone ring. the h nmr spectra of the (r) and (s) mpa ester derivatives of 1 revealed slight differences in the h chemical shifts of c6 and adjacent protons that allowed the assignment of the r configuration to c2 of 1 (figure s1a). the complete assignment of all protons and carbons of 1 (table 1) was accomplished by further interpretation of its hmbc and hsqc spectra. compound 1 was thus assigned as 6r-(2r - hydroxypentadecyl)-5,6-dihydro-2h - pyran-2-one and named cryptorigidifoliol a. spectra obtained in cdcl3; assignments on the basis of analysis of 2d nmr spectra. data measured at 500 mhz; brs = broad singlet, brd = broad doublet, t = triplet, ddd = doublet of doublets of doublets, dt = doublet of triplets, m = multiplet. j values are in hz and are omitted if the signals overlapped as multiplets. data measured at 125 mhz; ch3, ch2, ch, and c multiplicities were determined by an hsqc experiment. compound 2 was obtained as an oil with the molecular formula c24h44o4 on the basis of its hresims spectrum (m / z 397.3317). the uv, ir, and h nmr spectroscopic data of 2 were comparable to those of 1, suggesting that 2 is also a six - substituted 5,6-dihydro--pyrone. the major difference between the h nmr spectroscopic data of 1 and that of 2 was the presence of an additional signal for an oxymethine proton at h 3.89 (m, 1h, h4) in 2. the hmbc correlation between the oxymethine proton and the carbon signal at c 69.8 (c2) assigned the additional hydroxy group to c4, and this assignment is supported by the hmbc cross peaks between the two adjacent methylene protons (h 1.61, m, 2h, h3 ; 1.47, m, 2h, h5) and the oxymethine carbon at c 74.0. similar to 1, the complete assignments of all protons and carbons of 2 (table 1) were accomplished by interpretation of its hmbc and hsqc spectra. ecd and synthesis and h nmr analysis of its mpa esters were used to assign the configurations of c6, c2, and c4 as r, s, and r, respectively. compound 2 was thus assigned as 6r-(2s,4r - dihydroxynonadecyl)-5,6-dihydro-2h - pyran-2-one and has been named cryptorigidifoliol b. the molecular formula of compound 3 (c22h38o3, hresims m / z 351.2902) and its h nmr spectrum (h 5.34, m, 2h, h10 and h11) indicated that it had an additional disubstituted olefin moiety as compared with compound 1. its uv, ir, and h nmr spectroscopic data indicated the presence of the same -pyrone ring as 1 and 2, and hmbc correlations from h10 and h11 (h 5.34) to the carbons at c 27.0 indicated that the additional olefinic moiety must be located between two methylene groups. long - range correlation from both h9 and h12 (h 2.051.99, m, 4h) and h8 and 13 (h 1.371.31, m, 4h) to the carbons at c10 and 11 (c 130) in the hmbc spectrum assigned the olefinic moiety to c10 and c11. the geometry of the double bond was assigned as z on the basis of the shielded c nmr chemical shift of the methylenes connected to the double bond (c 29.4). the position of the double bond within the alkyl chain was determined unambiguously by analysis of the gc - eims fragmentation of the dimethyldisulfide (dmds) derivative of 3, which showed a major ion at m / z 299 attributable to fragmentation between the two ch3s groups located at the original site of unsaturation. fragment ions at m / z 281 and 145 were also observed and support the assigned structure (figure s2a). the relative configuration of 3 and the assignment of its h and c nmr data were determined by the same methods as for 1 and 2. compound 3 was assigned as 6r-(2r - hydroxy-10z - heptadecenyl)-5,6-dihydro-2h - pyran-2-one and is named cryptorigidifoliol c. the molecular formula of 4 (c24h42o3, hresims m / z 396.3489) differed from the molecular formula of 3 (c22h38o3) only by a c2h4 unit. inspection of the h nmr spectra of 3 and 4 demonstrated that 4 possessed a structure similar to that of 3 but with two additional carbons in the alkenyl chain. the intense fragment ions at m / z 299 and 173 and the additional ion peak at m / z 281 in the eims spectrum of the dmds adduct of 5 indicated that the position of the double bond was between c10 and c11 (figure s2b). the complete nmr data and configurations of all stereogenic centers in 4 were assigned by the same methods as for 13. compound 4 was assigned as 6r-(2s - hydroxy-10z - nonadecenyl)-5,6-dihydro-2h - pyran-2-one and is named cryptorigidifoliol d. the h nmr spectrum of compound 5 (c24h42o4, hresims m / z 377.3060) showed the presence of two oxymethine groups (h 4.63, m, 1h, h2 ; h 4.15, m, 1h, h12) and a double bond (h 5.68, m, 1h, h4 ; h 5.49, dd, j = 15.3, 7.0, 1h, h3) in the alkyl chain, besides the -pyrone signals at h 6.90, 6.03, 4.69, and 2.44. the large coupling constant (15.3 hz) observed for h3 indicated the e geometry of the double bond. in the hmbc spectrum, correlations were observed between the protons at h 1.79 and 1.73 (each m, 1h, h1) and c3 (c 131.4) and between the proton at h 4.63 (m, 1h, h2) and c4 (c 132.6). these observations suggested the connection of the olefinic moiety with the c2 oxymethine functionality. the eims data of 5 showed significant ions at m / z 265 and 247, together with a less intense ion at m / z 111, consistent with assignment of the second hydroxy group to c12 (figure s3). the configurations at c6 and c2 were assigned to be r and s, respectively, by interpretation of the ecd spectroscopic data and by the mpa ester method. an attempt was made to determine the configuration at c12 by using the mpa ester method, but it did not lead to any firm because we could not distinguish the chemical shifts of the protons of the two methylene groups attached to c12. compound 5 was assigned as 6r-(2r,12-dihydroxy-3e - nonadecenyl)-5,6-dihydro-2h - pyran-2-one and named cryptorigidifoliol e. compound 6 had the molecular formula of c24h42o4 on the basis of its hresims (m / z 395.3149). its ir spectrum showed the absorptions characteristic of a -lactone moiety (1719 and 1073 cm). the h nmr spectrum lacked the signals for the vinylic protons of an,-unsaturated lactone unit, and the ir absorption at 1615 cm found in compounds 15 was absent. a new methylene signal was observed at h 2.89 (brd, j = 19.3 hz, 1h, h4a) and 2.78 (dd, j = 19.3, 4.5 hz, 1h, h4b), which showed hmbc correlations with a carbonyl carbon at c 169.7 (c3, figure 2). a signal for a vinylic proton was observed in the h nmr spectrum at h 5.33 (m, 2h, h8 and h9), and the four indices of hydrogen deficiency of 6 thus required a second ring in addition to the lactone and the double bond functionalities. the h nmr spectrum showed the presence of four oxymethine protons (h 4.89 ( m, 1h, h1), 4.36 (t, j = 4.5 hz, 1h, h5), 4.10 (m, 1h, h7), and 3.81 (m, 1h, h2) ) directly attached to c1 (c 73.1), c5 (c 66.0), c6 (c 63.6), and c2 (c 68.3), respectively, assignments that were confirmed by hsqc data. in the hmbc spectrum, cross peaks were observed from the two oxymethine protons at h 4.89 (h1) and 4.36 (h5) to the carbonyl carbon at c 169.7 (c3) and the c7 oxymethine (c 63.6). correlations were also observed between the two oxymethine protons h1 and h5 and c5 (c 66.0) and c1 (c 73.1), respectively, and between the oxymethine proton at h 4.10 (h7) and the oxygenated carbon (c 68.3 ( c2); h 3.81 (m, 1h, h2); figure 2 ). collectively, these correlations permitted assignment of the bicyclic ring system, the alkyl chain substituted at c7, and a hydroxy group at c2 of 6 (figure 2). the long - range cross peaks observed between h8 and h9 (h 5.33, m, 2h) and c 29.6 in the hmbc spectrum indicated that the allylic methylene groups resonated at c 29.6 (c7 and c10) and indicated the z geometry of the sole double bond in the alkyl chain. the position of this double bond was assigned to c8/9 on the basis of the eims fragmentation of the dmds adduct, which had an intense peak at m / z 173 and a peak at m / z 297 corresponding to loss of water from the lactone - containing fragment ion (figure s2c). an attempt to assign the configuration at c2 of 6 by the mpa ester method was not successful because there were no significant (h) differences between its r- and s - mpa derivatives. because bicyclic tetrahydro--pyrones such as 6 are formed from the corresponding 5,6-dihydro--pyrones, as explained below, and all of these 5,6-dihydro--pyrones have the 6r configuration, it is proposed that the orientation of substituents at c1 of 6 (corresponding to c6 in the putative monocyclic precursor) must be the same. because of a change in group priorities, the c1 absolute configuration is s. the preference for the formation of a less - strained cis fused bicyclic system dictates the generation of a 5r - configured center. the same cis fused configuration was demonstrated for some related bicyclic tetrahydro--pyrones by x - ray crystallography. the configuration of 6 at c7 could not be assigned because its 5,6-dihydro--pyrone precursor was not isolated. the complete nmr assignments of 6 (table 2) were facilitated by further interpretation of its hmbc and hsqc spectra. compound 6 was thus assigned as (1s,5r)-7-(2-hydroxy-8z - heptadecenyl)-2,6-dioxabicyclononan-3-one and has been named cryptorigidifoliol f. spectra obtained in cdcl3; assignments are on the basis of analysis of 2d nmr spectra. data measured at 500 mhz; s = singlet, br d = broad doublet, dd = doublet of doublets, ddd = doublet of doublets of doublets, m = multiplet. j values are in hz and are omitted if the signals overlapped as multiplets. data measured at 125 mhz; ch3, ch2, ch, and c multiplicities were determined by an hsqc experiment. compounds 7 (m / z 421.3662) and 8 (m / z 355.2831) also contained a bicyclic tetrahydro--pyrone ring. comparison of their molecular weights and h nmr spectra with those of cryptorigidifoliol f revealed that the only structural differences were in the alkyl chain length and the absence or presence of a double bond or hydroxy group on the alkyl chain. cryptorigidifoliol g, with the molecular formula c27h48o3, was a bicyclic tetrahydro--pyrone similar to 6 but lacked the c2 hydroxy group and had a 20-carbon alkenyl chain at c7. preparation of its dmds derivative permitted assignment of the double bond at c8 (m / z at 299 and 215 ; figure s2c). its configuration at c7 could not be determined because its presumed monocyclic precursor was not isolated. cryptorigidifoliol h was also a bicyclic tetrahydro--pyrone similar to 6 but with a saturated alkyl chain at c7; the chain length was determined to be 14c on the basis of its molecular formula of c21h38o4. as in the case of 7, its configuration at c7 could not be determined. compounds 911 (cryptorigidifoliols i k) are the bicyclic derivatives of 2, 3, and 5, as determined by the conversions described below and by comparison of their h nmr and hresims data with those of their 5,6-dihydro--pyrone precursors and those of compounds 68. because the configurations of the precursors 2, 3, and 5 at the 2 position have been established, the corresponding configurations at c7 of 9, 10, and 11 were assigned as s, r, and r, respectively. compounds 6, 8, 9, and 11 had no significant (h) difference between their r- and s - mpa derivatives, thus precluding assignment of absolute configurations at these centers. the initial purification of the bioactive etoac fraction involved sephadex lh-20 column chromatography, reverse - phase spe, normal - phase silica - gel column chromatography, and c18 hplc. this procedure yielded fractions that in most cases were mixtures of 5,6-dihydro--pyrones and bicyclic tetrahydropyrones. in addition, it was noted that the early - eluting fractions from the open silica - gel column contained either pure 5,6-dihydro--pyrone 1 or mixtures of 5,6-dihydro--pyrones 2, 3, and 5 and bicyclic tetrahydropyrones 911. the later - eluting fractions, however, yielded only the bicyclic tetrahydropyrones 68. this suggested that the 5,6-dihydro--pyrones were cyclized during their exposure to silica gel, with the more polar later - eluting compounds that were exposed for a longer time to silica gel becoming completely converted to cyclized product. pure 5,6-dihydro--pyrone 2 and related compounds were thus prepared by purification by ptlc on diol silica gel, which did not induce intramolecular cyclization. it is worth noting that grkovic and co - workers isolated only 5,6-dihydro--pyrones, without artifacts, by using diol column chromatography during their bioassay - guided fractionation of a papua new guinea species of cryptocarya in order to find compounds that can rescue pdcd4 from tpa - induced degradation. to verify the cyclization hypothesis, silica gel was mixed with compound 2 in meoh / hexanes / etoac and the ing suspension was allowed to stand for 3 h at room temperature. examination of the ing solution showed that only compound 9 was present, confirming that silica gel catalyzed the cyclization of the 5,6-dihydro--pyrone 2 to the bicyclic tetrahydropyrone 9 (scheme 1). compounds 3 and 5 were also treated in the same way, leading to the formation of the corresponding bicyclic tetrahydropyrone derivatives 10 and 11. these observations supported the hypothesis that the bicyclic tetrahydropyrones are formed by intramolecular cyclization of c2-hydroxylated 5,6-dihydro--pyrones. however, this does not rule out the possibility that at least some bicyclic tetrahydropyrones are formed in the plant, and examination of the crude extract by h nmr spectroscopy indicated that it did contain signals consistent with the presence of bicyclic tetrahydropyrones. this could be due to either the presence of the cyclized compounds in the plant or intramolecular cyclization during the extraction of the plant and the processing of the extract in madagascar prior to analysis. regrettably, we did not have access to fresh plant material to test these alternate hypotheses, but the fact that chromatography over silica gel was used in every case where the cyclized compounds are reported strongly suggests that these compounds are indeed formed during the purification process. compounds 111 were evaluated for their antiparasitic activity against the chloroquine / mefloquine - resistant dd2 strain of plasmodium falciparum. compounds 17 exhibited moderate antimalarial activity, with ic50 values of 9.2 0.9, 5.8 1.4, 5.5 0.7, 9.0 3.0, 4.0 2, 7.4 0.6, and 6.0 0.5 m, respectively. compounds 811 were all less active, with ic50 values > 10 m (tables 3 and 4). these data indicate that the presence of the,-unsaturated carbonyl moiety is not essential for antimalarial activity because bicyclic compounds 6 and 7 have activity comparable to that of,-unsaturated carbonyl compounds 15. antimalarial activity against the dd2 strain of plasmodium falciparum antiproliferative activity against human ovarian cancer cells. antimalarial activity against the dd2 strain of plasmodium falciparum antiproliferative activity against human ovarian cancer cells. all of the compounds were also evaluated for their antiproliferative activity against a2780 human ovarian cancer cells, but only compound 3 had an ic50 value less than 10 m (tables 3 and 4). because these compounds have only moderate antiparasitic activities, significant improvement in their potency and therapeutic index will be necessary before they can be used as lead compounds for the development of new antiplasmodial drugs. the formation of compounds 611 serves to highlight the fact that 5,6-dihydro--pyrones containing c2 hydroxy groups in the side chain are susceptible to cyclization in the presence of silica gel at room temperature. it is thus possible that the bicyclic tetrahydropyrones previously reported were also formed by cyclization of the corresponding 5,6-dihydro--pyrones during the isolation process. this finding also provides support for the general belief that silica gel should be avoided for the isolation of most natural products because it will not in general be known whether or not the unknown compounds might be susceptible to similar unwanted reactions. in place of silica gel, diol and c18 ir and uv spectra were measured on an midac m - series ftir and shimadzu uv-1201 spectrophotometers, respectively. 1d and 2d nmr spectra were recorded on a bruker avance 500 spectrometer in cdcl3 or pyridine - d5 (with cdcl3 or pyridine - d5 as reference). high - resolution esi mass spectra were obtained on an agilent 6220 mass spectrometer. open - column chromatography was performed using sephadex lh-20 and silica gel (230 - 400 mesh, silicycle co., usa). semipreparative hplc was performed using shimadzu lc-10at pumps coupled with a semipreparative phenomenex c18 column (5 m, 250 10 mm) and semipreparative varian diol column (250 10.0 mm), a shimadzu spd m10a diode array detector, and a scl-10a system controller. preparative hplc was performed using shimadzu lc-8a pumps coupled with a preparative varian phenomenex c18 column (250 21.4 mm), a shimadzu spd m10a diode array detector, and an scl-10a system controller. preparative tlc was performed using diol plates (500 m / g, sorbtec co., usa). all isolated compounds were purified to 95% purity or better, as judged by hplc (both uv and elsd detection) before determining bioactivity. plant parts of cryptocarya rigidifolia van der werff (lauraceae) were collected and authenticated by f.r. and co - workers at an elevation of about 1000 m from a 13 m tall tree with a diameter of 13 cm at chest height. the tree had round green fruit, with the remains of brown calyx above. collection was made near the towns of imerimandroso and antanandava in the district of ambatondrazaka, north of the zahamena national park, at coordinates 172845 s, 484410 e. the tree was growing in medium - altitude rainforest on the edge of an old area of slash - and - burn agriculture. duplicate voucher specimens (ratovoson 250) were deposited at the centre national dapplication des recherches pharmaceutiques (cnarp), the herbarium of the department of forestry and fishery research (tef), and the missouri botanical garden, st. a ground sample of c. rigidifolia root wood (310 g) was extracted with etoh (1000 ml) at room temperature to yield 16.3 g of crude etoh extract designated mg 0441, and a portion of this extract was made available to virginia tech for bioassay - guided isolation. the crude etoh extract (5.0 g) was dissolved in 90% aqueous meoh (60 ml) and extracted with hexanes. the 90% aqueous meoh layer was evaporated, suspended in h2o (100 ml), and extracted with etoac (100 ml). the antimalarial activities were concentrated in the etoac (ic50 = 2.55 g / ml) and hexanesfractions (ic50 5 g / ml). the etoac fraction (971 mg) was subjected to sephadex lh-20 size chromatography (ch2cl2/meoh = 1:1) to give four fractions, and the most active fraction fr-2 (ic50 = 2.55 g / ml) was fractionated by c18 spe using 70% aqueous meoh, 100% meoh, and ch2cl2 separately. the 100% meoh fraction (ic50 = 1.252.5 g / ml) was divided into eight subfractions by silica - gel column chromatography (hexanes / etoac from 1:1 to 3:7 to 1:9, and a final elution with meoh). the third subfraction (ic50 < 1.25 g / ml) was subjected to c18 hplc with a solvent gradient of h2o / ch3cn, 20:80 to 10:90 from 0.01 to10 min, to 0:100 from 10 to 15 min, and ending with 100% ch3cn to 30 min, furnishing compound 1 (tr = 20.2 min, 1.1 mg, ic50 = 2.98 g / ml) and an active fraction (tr = 20.8 min). further purification of the active fraction by diol ptlc (hexanes / etoac = 4:6) yielded 3 (1.4 mg, ic50 = 1.93 g / ml) and 10 (0.4 mg, ic50 = 5.74 g / ml). the fourth subfraction from silica - gel column chromatography was subjected to c18 hplc with a solvent gradient of h2o / ch3cn, 30:7020:80 from 0.01 to15 min, to 10:90 from 15 to 35 min, to 0:100 from 35 to 50 min, and ending with 100% ch3cn to 60 min. purification by diol ptlc (hexanes / etoac = 4:6) of the mixtures with tr = 37.5 and 40.2 min obtained from this process gave compounds 5 (0.9 mg, ic50 = 3.55 g / ml), 11 (3.1 mg, ic50 > 20 g / ml), 2 (1.3 mg, ic50 = 2.30 g / ml) and 9 (0.5 mg, ic50 = 6.57 g / ml). the fifth subfraction from silica - gel column chromatography was further purified by c18 hplc by using the same profile as the fourth subfraction, giving pure compounds 8 (4.2 mg, tr = 38 min, ic50 = 13.6 g / ml) and 6 (9.6 mg, tr = 40.8 min, ic50 = 1.58 g / ml). the hexanes fraction was subjected to sephadex lh-20 gel chromatography (ch2cl2/meoh = 1:1) to give three fractions, and the most active fraction fr-2 (ic50 2.55 g / ml) was fractionated by preparative c18 hplc with a solvent gradient of h2o / ch3cn, 30:700:100 from 0.01 to 30 min, and ending with 100% ch3cn to 45 min. this process yielded compound 7 (1.6 mg, tr = 39 min, ic50 = 2.50 g / ml) and an active fraction (tr = 32.5 min). further purification of the fraction by diol hplc with a solvent gradient of hexanes / etoac, 90:1080:20 from 0.01 to 10 min, to 70:30 from 10 to 15 min, to 60:40 from 15 to 20 min, to 0:100 from 20 to 25 min, and ending with 100% etoac wash to 35 min, afforded compound 4 (0.9 mg, ic50 2.76 g / ml). colorless oil; d + 6 (c 0.5, meoh); uv (meoh) max (log) 204 (3.63) nm; ir max 3340, 2922, 2849, 1719, 1613, 1089 cm; ecd (c 0.031 mm, meoh) max 203 (+ 8.5), 235 (+ 2.4), 256 (+ 3.8); h and c nmr data, see table 1; hresims m / z 325.2753 (calcd for c20h37o3, 325.2737). colorless oil; d + 9 (c 0.1, meoh); uv (meoh) max (log) 204 (3.83) nm; ir max 3343, 2922, 2850, 1714, 1615, 1092 cm; ecd (c 0.031 mm, meoh) max 209 (+ 9.7), 235 (+ 1.9), 256 (+ 3.5); h and c nmr data, see table 1; hresims m / z 397.3317 (calcd for c24h45o4, 397.3312). colorless oil; d + 19 (c 0.6, meoh); uv (meoh) max (log) 204 (3.91) nm; ir max 3439, 2924, 2855, 1728, 1623, 1044 cm; ecd (c 0.031 mm, meoh) max 207 (+ 9.3), 235 (+ 1.2), 255 (+ 3.1); h and c nmr data, see table 1; hresims m / z 351.2902 (calcd for c22h39o3, 351.2894). colorless oil; d + 18 (c 0.6, meoh); uv (meoh) max (log) 204 (3.50) nm; ir max 3444, 2922, 2855, 1724, 1625, 1034 cm; ecd (c 0.031 mm, meoh) max 208 (+ 10.6), 235 (+ 1.6), 256 (+ 3.0); h and c nmr data, see table 1; hresims m / z 396.3489 (calcd for c24h46no3, 396.3472). colorless oil; d 25 (c 0.4, meoh); uv (meoh) max (log) 204 (3.24) nm; ir max 3382, 2922, 2845, 1729, 1625, 1077 cm; ecd (c 0.031 mm, meoh) max 207 (+ 11.0), 234 (+ 2.3), 256 (+ 3.2); h and c nmr data, see table 1; hresims m / z d 10 (c 0.5, meoh); uv (meoh) max (log) 204 (3.14) nm; ir max 3450, 2932, 2855, 1719, 1073 cm; h and c nmr data, see table 2; hresims m / z 395.3149 (calcd for c24h43o4, 396.3156). colorless oil; d 4 (c 0.5, meoh); uv (meoh) max (log) 204 (3.14) nm; ir max 3450, 2932, 2855, 1719, 1073 cm; h and c nmr data, see table 2; hresims m / z 421.3662 (calcd for c27h49o3, 421.3676). colorless oil; d 11 (c 0.5, meoh); uv (meoh) max (log) 204 (2.66) nm; ir max 3540, 2917, 2845, 1719, 1070 cm; h and c nmr data, see table 2; hresims m / z 355.2831 (calcd for c21h39o4, 355.2843). colorless oil; d 8 (c 0.4, meoh); uv (meoh) max (log) 204 (3.43) nm; ir max 3340, 2943, 2846, 1727, 1068 cm; h and c nmr data, see table 2; hresims m / z 397.3317 (calcd for c24h45o4, 397.3312). colorless oil; d + 4 (c 0.6, meoh); uv (meoh) max (log) 204 (3.62) nm; ir max 3345, 2939, 2844, 1739, 1056 cm; h and c nmr data, see table 2; hresims m / z 351.2902 (calcd for c22h39o3, 351.2894). colorless oil; d 8 (c 0.6, meoh); uv (meoh) max (log) 204 (3.10) nm; ir max 3431, 2956, 2867, 1732, 1036 cm; h and c nmr data, see table 2; hresims m / z 377.3060 (calcd for c24h41o3, 377.3050). pure samples (200 g) dissolved in 200 l of hexanes were treated with dimethyldisulfide (400 l) and 12 drops of iodine solution (6% w / v in ether) in a 6 dram vial at 40 c for 30 h. the mixture was cooled and diluted with hexanes (1 ml), and iodine was removed by extracting with thiosulfate (1 ml, 5% w / v). the organic layer was removed, and the water layer was re - extracted with hexanes / ch2cl2 (1:1). the combined organic layers were dried and subjected to analysis by gc - eims. the in - nmr - tube reaction was carried out to prepare the mpa ester derivatives. the selected 5,6-dihydro--pyrone (0.2 mg) was dissolved in pyridine - d5 in a clean nmr tube, and r---methoxyphenylacetyl (mpa) chloride (0.5 mg) was added to the nmr tube under a n2 gas flow, and the nmr tube was gently shaken. the nmr tube was kept at room temperature overnight and was then analyzed by h nmr spectroscopy. the s - mpa esters of the 5,6-dihydro--pyrones were prepared from s - mpa chloride by the same method. compound 2 (0.2 mg) was dissolved in a mixture of meoh (2 ml), etoac (0.5 ml), and hexanes (0.5 ml). enough silica - gel powder was added to the solution to absorb it, and the mixture was kept at room temperature for 3 h. the ing damp powder was eluted with meoh, and the eluate concentrated on a rotavapor. compounds 3 and 5 (each 0.2 mg) were also treated separately and worked up under the same conditions. the products were identified as 10 and 11, respectively, by h nmr spectroscopy. the a2780 ovarian cancer cell line antiproliferative bioassay was performed at virginia tech as previously reported. paclitaxel was used as the positive control; it had an ic50 value of 73 15 nm. the effect of each compound on parasite growth of the dd2 strain of p. falciparum was measured in a 72 h treatment using the malaria sybr green i - based fluorescence assay as described previously. artemisinin was used as the positive control; it had an ic50 value of 7 1 nm.

antimalarial bioassay - guided fractionation of an etoh extract of the root wood of cryptocarya rigidifolia (lauraceae) led to the isolation of the five new 5,6-dihydro--pyrones cryptorigidifoliols a e and the six bicyclic tetrahydro--pyrone derivatives cryptorigidifoliols f k. the structure elucidations of all compounds were made on the basis of the interpretation of spectroscopic data and chemical derivatization, and the relative and absolute configurations were determined by noesy, electronic circular dichroism (ecd), and 1h nmr analysis of -methoxyphenylacetyl (mpa) derivatives. the bicyclic tetrahydro--pyrone derivatives were identified as products of acid - catalyzed intramolecular michael addition of the 5,6-dihydro--pyrones in the presence of silica gel. a structure activity relationship study suggested that the presence of an,-unsaturated carbonyl moiety is not essential for potent antimalarial activity.

the implication of inflammation in the development of cancers is well recognized. in the 19th century, virchow observed leukocytes in tissues that contained inflammatory infiltrates, which later on was confirmed in several clinical cases. however, these insights were not further examined until data collected from epidemiological studies and experimental models showed a link between chronic inflammation and various types of cancers. chronic inflammation occurs with continuous inflammatory signaling caused by viral, bacterial, and parasitic infections or chemical irritants; exposure to chemical irritants in recruitment of inflammatory cells, increased production of reactive oxygen species (ros), and reduced dna repair. most cancer tissues show specific types of inflammatory response; this subset accounts for approximately 20% of human cancers worldwide and is considered an essential component of tumorigenesis, including tumor initiation, progression, growth, and metastasis. liver cancer is one of the most difficult cancers to treat and is the second leading cause of cancer death worldwide, especially in china, where it accounts for half of total cancer incidence and death. liver cancer is strongly correlated with viral hepatitis infection; more than 90% of liver cancers are associated with chronic hepatitis or liver cirrhosis. however, cell death - regeneration cycles are driven by persistent inflammation that occurs when inflammation - associated signaling can not be inhibited and subsequently in chronic injury. many factors provoke an inflammatory response in the liver and are well known risk factors for liver cancer. liver cancer occurs during the progression of many chronic liver diseases associated with inflammation, such as hepatitis and steatohepatitis. hepatitis b virus (hbv) and hepatitis c virus (hcv) are found in approximately 80%90% of hepatocellular carcinoma (hcc) patients. chronic hbv infection is the dominant risk factor for liver cancers in asia and sub - saharan africa, particularly in china. hcv is a single - stranded rna virus that interacts with numerous proteins in hepatocytes and promotes the progression of hepatocarcinogenesis. chronic hcv infection is the dominant risk factor for liver cancers in japan, united states, and europe. cirrhosis contributes to hepatocarcinogenesis and is observed in most hcc patients with hbv or hcv infection. in addition to these viral infections, acetaldehyde and other metabolites generated by alcohol metabolism increase the amount of ros and reduce dna repair, which leads to hcc. epidemiological analysis revealed that alcohol consumption is positively associated with hcc incidence; diabetes is another risk factor for hcc because epidemiologic studies reported that patients with type 2 diabetes have nearly 2.5-fold increased risk for hcc. recently, obesity was defined as a risk factor with an important role in hcc development. inflammation induced by viral or non - viral infection promotes the initiation and progression of hcc; hcc develops in the of chronic hepatitis. constitutive activation of these inflammatory signaling pathways, such as nf-b and jak / stat signaling pathways, occurs during the process of inflammation and is observed in various cancers. the first link between nf-b and cancer was revealed in a study showing that cancer cells were more sensitive to apoptosis - inducing stimuli after the nf-b signaling pathway was blocked. over the past decades, the diverse roles of nf-b signaling have been discovered in various types of carcinomas, including head and neck squamous cell carcinoma, leukemia, and cancers of breast, liver, prostate, colon, and lung. constitutive nf-b activation was observed in various cancers, where nf-b induces the expression of many pro - survival and anti - apoptotic factors, such as c - iap, xiap, and members of the bcl-2 family of proteins. analogously, the activation of nf-b might downregulate the expression of tumor suppressors, such as phosphatase and tensin homolog (pten); the downregulated expression increases akt signaling and suppresses the apoptosis of cancer cells. in addition to its pro - survival function, nf-b promotes cell cycle progression by inducing cyclin d1 and c - myc expression. nf-b signaling can also induce molecules such as vascular endothelial growth factor (vegf) and matrix metalloproteinases, and nf-b can promote angiogenesis and invasion by driving the expression of these proteins in many cancers. in 2004, a group from israel confirmed that nf-b is an effective therapeutic target to treat hcc. hcc development in a mouse model was prevented through nf-b inhibition by a modified form of ib called super - repressor ib. another well - known signaling pathway that links inflammation and cancer is the janus kinase (jak)/signal transducer and activator of transcription (stat). stat3, one of the most famous stat family members, was identified as a transcription factor that binds to the il-6-responsive element in hepatocytes. numerous studies reported that stat3 can also be activated by oncogenic proteins including ras or src. carcinogens, such as polychlorinated biphenyls, promotes liver cancer and regulates the activity of stat3 in hcc and normal liver cells. likewise, the constitutive activation of stat3 leads to the expression of bcl-2 family proteins essential for survival, such as survivin and ciap2; moreover, the apoptosis of cancer cells occurs when stat3 is downregulated in tumors. stat3 promotes the proliferation of cancer cells by inducing cyclin d1 and c - myc expression. in addition to molecules associated with proliferation and apoptotic inhibition, stat3 regulates the expression of the metalloproteinase family and vegf, consequently promoting angiogenesis and metastasis. stat3 is a key regulator of inflammation and cancer and thus might be an effective therapeutic target in inflammation - induced cancer. in 2006, li et al. inhibited stat3 expression with antisense oligonucleotides in hcc cells and found that stat3 greatly reduced cell proliferation and migratory potential as well as induced apoptosis. mirnas constitute a class of short endogenous non - coding rnas that are highly conserved throughout evolution. the majority of mirnas are transcribed in the nucleus by rna polymerase ii as a primary mirna (pri - mirna); the typical pri - mirna contains stem - loop structures. furthermore, these stem - loop structures are cleaved into precursor mirnas (pre - mirnas) by drosha, an rna - specific ribonuclease enzyme complex. dicer functions in the biogenesis of mirnas as pre - mirnas are transported from the nucleus into the cytoplasm by exportin 5. dicer is an endonuclease that specifically recognizes double - stranded rna complexes and transforms the pre - mirna into a 2022 nt mirna duplex. the mature mirna is incorporated into an rna - induced silencing complex (risc) through its interaction with ago proteins; this mirna recognizes and binds to the 3-utr of target mrnas. the first mirna was discovered in caenorhabditis elegans in 1993 and was identified as an essential regulator of postembryonic developmental events. from that first discovery, currently, mirnas are recognized as key mediators influencing nearly all biological processes, including development, immune response, differentiation, proliferation, apoptosis, invasion, and metastasis. in addition, the expression of numerous mirnas is dysregulated during nearly all pathological processes. mirnas gained a widely accepted role in initiation and progression of various human cancers, including hcc; evidence indicated that these mirnas act as oncogenes or tumor suppressors.. some of these mirnas might be significant to diagnosis or prognosis of hcc patients. accumulating evidence revealed that mirnas are a novel class of molecules linking inflammation and hcc. chronic hepatitis virus infection leads to liver inflammation, damage, fibrosis, cell regeneration, and malignant transformation (hcc). many mirnas play a crucial role in the regulation of hepatitis virus - induced chronic infla - mmation and hcc (table 1). for instance, mir-27 is upregulated by hcv infection and can promote cell migration and invasion by suppressing spry2 in hcc cells. mir-146a expression is consistently increased in hcv - infected cells, primary hepatocytes, and liver tissue from hcv - infected patients. increased mir-146a level promotes viral infection and is associated with the pathogenesis of liver disease. in the following section, we will summarize the key roles of relevant mirnas in hepatitis virus infection and hcc. mirnas associated with hepatitis virus infection and hcc the first mirna discovered in humans is let-7, which is highly conserved across animal species and promotes differentiation. recent studies revealed that let-7 is frequently downregulated in many cancers, such as breast, ovarian, prostate, lung, and liver cancers. the hbv x protein (hbx) encoded by the hbv genome is a multifunctional transactivator that plays crucial roles in hbv - associated hepatocarcinogenesis. hbx lacks a dna binding domain and acts as a trans - coactivator through interaction with other cellular transcription factors, by which hbx might regulate let-7 family members. in addition to hbx induction, hbv pres2 rna can sequester let-7 g, which in turn reduces intrinsic let-7 g functions in hcc. wang et al. concluded that hbx - mediated down - regulation of let-7 might be responsible for reduced let-7 expression in hbv - associated hcc. mirnas from the let-7 family act as tumor suppressors in hcc by inhibiting proliferation and invasion, as well as inducing apoptosis by targeting many oncogenes including high mobility group proteins, bcl - xl, stat3, and c - myc. mir-122 is the most abundant mirna expressed in the liver, which accounts for 70% of the total mirnas in an adult liver. the expression of mir-122 is increased in the liver during embryonic development, and the first identified target of this mirna is cationic amino acid transporter 1. in recent years, increasing evidence suggested that mir-122 plays an important role in initiation and progression of hcc. li et al. found that mir-122 expression is low in patients with chronic hepatitis b and hcc tumors and has negative correlation with fibrotic stage, age, and liver transaminase levels. the downregulation of mir-122 in patients with chronic hepatitis b and hcc tissues is affected by hbv infection. ppar was identified as a transcription factor regulating mir-122 expression; the underlying cause of hbx - induced mir-122 downregulation might be the direct interaction between hbx and ppar, which blocks ppar nuclear translocation. in 2013, li et al. further reported that all four hbv mrnas contain a binding site for mir-122 and act as a mirna sponge sequestering mir-122 in hbv - infected cells. in addition, mir-122 knockout mice will possibly develop hepatitis, fibrosis, and hcc. the reduced mir-122 expression in hcc tissues is characterized by a poor prognosis and consistent with a suppressed hepatic phenotype and an increased metastatic potential. moreover, mir-122 expression can be suppressed by two inflammatory cytokines, il-6 and tnf-, which downregulate the mir-122 transcription factors c / ebp and hnf3 and induce c - myc - mediated c / ebp inhibition in chronic hepatic inflammation. mir-122 downregulation in the upregulation of target genes that promote hepatocarcinogenesis, including pituitary tumor trans - forming gene binding factor (pbf), cyclin g1, pkm2, adam10, adam17, srf, and pkr activator. mir-101 downregulation was reported in various cancers, including hcc, suggesting that mir-101 functions as a tumor suppressor. reduced mir-101 expression in hcc tissues is associated with advanced tumor progression and poor survival in hcc patients; the serum mir-101 level might be a potential hcc biomarker. overexpression of mir-101 sensitizes the hcc cells to apoptosis induced by serum deprivation and chemotherapeutic drugs. mcl-1, a member of the bcl-2 family, is a target of mir-101 and sensitizes cancer cells to apoptosis. mir-101 enhances cisplatin - induced apoptosis by targeting stmn1, rab5a, and mtor, as well as suppresses epithelial - mesenchymal transition (emt) in hepatocytes via the regulation of zeb1 expression, which in e - cadherin down - regulation. in 2010, the first link between mir-155 and hepatitis viruses was based on the observation that mir-155 precursor (termed bic) is responsible for hcv infection. after patients with hepatitis c were treated with interferon alpha and ribavirin, the lowest bic expression was detected in patients who lacked hcv rna in both serum and peripheral blood mononuclear cells. previously, mir-155 was identified as an onco - mir in various cancers, including lung, breast, stomach, and prostate cancers. a mouse model revealed that mir-155 induced by inflammation could promote autoimmune inflammation by enhancing inflammatory t cell development. mir-155 a positive correlation was observed between mir-155 expression and ip-10 levels in the plasma, which is widely considered a marker of liver inflammation degree. upon nf-b activation by hcv - rna infection the upregulation of mir-155 in cases of hepatitis and hcc promotes hepatocyte proliferation and hcc tumor growth by activating the wnt/-catenin signaling pathway. in addition to hcc pathogenesis during chronic viral infections such as hcv, the upregulation of mir-155 was also observed in the pathogenesis of non - alcoholic steatohepatitis (nash)-associated hcc. however, similar to hcv - induced mir-155, the activation of nf-b signaling pathway plays a key role in mir-155 expression in hcc and other hepatocyte - derived cells. in a mouse model of nash, mir-155 downregulated the tumor suppressor target c / ebp, thus promoting liver pathogenesis. mir-21 is a well - established onco - mir that is upregulated in various cancers, including lung, breast, prostate, colon, stomach, pancreatic, and liver cancers. the relationship between mir-21 and inflammation was observed in uv - exposed skin, where mir-21 was activated by ppar via the tgf- signaling pathway. the upregulation of mir-21 was reported in other inflammation - associated tissues and cancers, such as allergy - induced airway inflammation, gastric cancer, pre - b - cell lymphoma, and colorectal cancer. the relevance of mir-21 and inflammation in liver tissues was established by associating biomarkers with hcc diagnosis and prognosis. mir-21 and mir-122 were analyzed in tissues obtained by needle biopsies from both hcv - infected and non - infected patients. the expression of mir-21 was positively correlated with fibrotic stage in patients with chronic hepatitis c; mir-21 promotes liver fibrosis by targeting the fibrosis inhibitor smad7. in serum in addition, one study reported that hbx increases the expression of mir-21 and subsequently promotes the progression of hcc by targeting pten. in another study, hbx - mediated mir-21 upregulation inhibits expression of the tumor suppressor pdcd4 in hcc cells. mir-221 is another onco - mir that is upregulated in cancers such as breast, prostate, glioma, and liver cancers. moreover, the expression of mir-221 in later stages of hcc is higher than that in early stages. therefore, mir-221 is a poor prognosis risk factor because it was increased in mouse models of liver fibrosis and upregulated in the human liver in a fibrosis progression - dependent manner. hbx promotes the proliferation of hcc cells and upregulates the expression of mir-221 during both hbv infection and hcc. several tumor - related genes were reported to be the targets of mir-221; among these genes, p27kip1 is well - recognized. the expression of mir-221/p27kip1 axis was confirmed in glioblastomas, breast, lung, and liver cancers. other than p27kip1, many mir-221 targets were reported, such as hdac6, pten, and tissue inhibitor of metalloproteinase-3 (timp3). in mouse models of hcc, tumor - bearing mice that were treated with chol - anti - mir-221 mir-221 is a potential target for hcc treatment and functions by inhibiting the progression of hcc and promoting survival. chronic inflammation can facilitate the development and progression of hcc by the constitutive activation of inflammation signaling pathways. inhibition of these signaling pathways would be a novel therapeutic strategy to suppress this inflammation - associated process. many mirnas were reported to regulate the inflammatory signaling mediated by nf-b and jak / stat3, among others, in hcc (table 2). mir-195, mir-194, and mir-127 suppressed nf-b activation by directly or indirectly targeting nf-b signaling; these mirnas also function as tumor suppressors in hcc. mir-219 is reduced in hcc where it functions as a tumor suppressor; this mirna might suppress jak / stat3 signaling via direct targeting of structural maintenance of chromosome 4 protein (smc4). mir-637 is a tumor suppressor that inhibits cell growth and induces apoptosis by suppressing stat3 activation in hcc. mirnas as regulators of inflammatory signaling in hcc mir-26a was suggested to play an important role in cirrhosis and inflammation - induced hcc. reduced mir-26a expression was detected in hcc tissues; the decreased expression in hcc tissues might be an independent predictor of poor survival. clinical analysis revealed that the expression of mir-26a is negatively correlated with il-6 mrna and il-6 protein level. as il-6 is a direct target of mir-26a, the mrna and protein levels of this cytokine are significantly reduced by mir-26a overexpression in hcc cells. mir-26a functions as a tumor suppressor that inhibits tumor growth and metastasis, partly through the il - stat3 signaling pathway. m - csf expression was also reported as a predictor of frequent metastasis and poor survival; mir-26a affects macrophage polarization and inhibits macrophage recruitment by down - regulating m - csf expression. in addition, hcc cells transfected with mir-126b exhibit significantly reduced nf-b reporter activity in response to tnf. two well - known upstream regulators of nf-b signaling pathway, tak1 and tab 3, were identified as direct targets of mir-26b. mir-26b prevents doxorubicin - induced nf-b activation and promotes apoptosis in hcc cells. in one study, mir-451 was often downregulated in primary hcc tissues; reduced mir-451 expression was significantly associated with advanced tnm stage, lymph node metastasis, vascular invasion, and poor survival, indicating that mir-451 might act as a tumor suppressor in hcc. mir-451 might inhibit hcc tumorigenesis and metastasis by down - regulating cyclin d1 and c - myc at both mrna and protein levels. further study indicated that mir-451 overexpression affected the nf-b nuclear translocation and significantly decreased nf-b activity by directly targeting ikk. as expected, the inhibition of mir-451 significantly increased nf-b activity in hcc cells. moreover, a recent study revealed that reduced expression of the il-6 receptor was observed in cells that overexpressed mir-451, and that il-6 receptor was a direct target of mir-451. mir-451 reduced vegf production in hcc cells by targeting the il-6/stat3 signaling pathway. mir-124 is a well - known tumor suppressor that is frequently downregulated in various cancers. numerous studies revealed that mir-124 inhibits inflammatory signaling by targeting stat3 in different cancer types including glioblastoma, gastric cancer, endometrial cancer, colon cancer, and hcc. recently, nf-b p65 was reported to be a direct mir-124 target in b - cell lymphomas. the downregulation of mir-124 in b - cell lymphomas enhances the nf-b signaling pathway and upregulates genes, such as myc and bcl-2, associated with proliferation, anti - apoptosis, metastasis, and pro - survival. stat3 was identified as a direct mir-124 target in hcc; one study found that mir-124 inhibited cell proliferation and induced apoptosis in hcc cells by targeting stat3. moreover, an hnf4-nf-b feedback circuit that includes mir-124 was identified in hcc. reduced hnf4 could be a biomarker for poor prognosis, as hnf4 suppresses hcc metastasis by inhibiting nf-b activation. mir-124 also plays an important role in rela suppression by hnf4. mir-124 was identified as an hnf4 target that binds to the 3utr of rela. mir-124 we discovered that mir-125b was significantly downregulated in hcc, could be used as a biomarker for prolonged hcc patient survival, and functions as a tumor suppressor. mir-125b inhibits the proliferation of hcc cells by inducing cell cycle arrest during g1/s transition via its target gene, lin28b. il-6r was characterized as the subunit that binds to il-6; the binding of il-6 to il-6r activates the jak / stat3 signaling pathway. mir-125b can block the il-6-induced phosphory - lation and nuclear translocation of stat3 in hcc cells, which in turn promotes hcc apoptosis. in addition, mir-125b serves as a tumor suppressor that inhibits migration and invasion of hcc cells by targeting the transcriptional co - activator with pdz - binding motif (taz). several studies revealed that mir-370 acts as tumor promoter in gastric cancer, prostate cancer, and aml; whereas other studies reported mir-370 as a tumor suppressor in leukemia and oral squamous carcinoma. xu et al. observed that the down - regulation of mir-370 was associated with hcc development in rats and correlated with poor survival in hcc patients, indicating that mir-370 serves as a tumor suppressor in hcc. the restoration of mir-370 inhibits proliferation, migration, and invasion of hcc cells and suppresses tumorigenesis of hcc by directly targeting lin28a. lin28a is a post - transcriptional modulator of mrnas that can directly bind to p65 mrna. mir-370 represses nf-b activation and inhibits the migration and invasion of hcc cells through downregulation of lin28a. sun et al. confirmed the tumor suppressor role of mir-370 in hcc cells, showing that mir-370 inhibited hcc cell proliferation and colony formation ability by activating the pi3k / akt signaling pathway. in addition to mirnas that inhibit inflammatory signaling, some mirnas, such as mir-155, mir-362, mir-301a, and mir-1180, promote the inflammatory response by repressing negative regulators of inflammatory signaling. the nf-b signaling pathway is activated by mir-1180 overexpression, which occurs through the downregulation of mir-1180 direct targets, otud7b and tnip2. another study confirmed the expression of tnip2, a direct target of mir-1180, in hcc cells. the ectopic expression of mir-1180 promoted the proliferation of hcc cells and inhibited apoptosis because nf-b induces the expression of genes that participate in pro - survival and anti - apoptotic processes. ni et al. demonstrated that mir-362 - 5p, which is frequently upregulated in hcc, promoted nf-b signaling by downregulating cyld; cyld is a protein that binds to nf-b essential modulator and functions as a negative regulator of nf-b signaling. these inflammatory pathways and related transcription factors can affect the development of hcc by regulating mirna expression. several studies confirmed that inflammatory signaling facilitates the development of hcc through the downregulation or upregulation of mirnas in cells (table 3). mirnas as mediators of inflammatory signaling in hcc ning et al. observed feedback regulation of hnf4 and nf-b in hcc. their study revealed that hnf4 dramatically suppresses the nf-b signaling pathway and that rela overexpression or knockdown decreases or increases the expression of hnf4, respectively. during chronic hepatic inflammation, nf-b induced the expression of mir-21, which represses hnf4 expression and contributes to hepatocyte de - differentiation and hepatocarcinogenesis. in addition to direct induction of mir-21 expression by nf-b, mir-21 transcriptional activity is enhanced by high - mobility group box-1 protein (hmgb1), an important participant in inflammation. hmgb1 is a conserved nuclear protein that functions as a chromatin binding factor and allows transcription factors to access specific dna binding sites. hmgb1 enhances p65/p50 or p50/p50 binding to specific dna sites rather than the binding of p65/p65, c - rel / c - rel, p65/c - rel, and p50/c - rel. given its role in the inflammatory cascade, hmgb1 is upregulated in hcc and induces mir-21 expression in hcc cells. the hmgb1/mir-21 signaling axis inhibits the downstream targets reck and timp3 as well as promotes cell proliferation, invasion, and migration. mir-143 is upregulated in hcc tissues, indicating that mir-143 serves as a tumor promoter. mir-143 expression was increased by hbx - mediated nf-b activation, although metastasis will be suppressed by mir-143 inhibition. nf-b activates transcription by specifically binding to the putative mir-143 promoter, as the nf-b binding site identifies 5.06 kb upstream of mir-143. mir-143 promotes cell invasion and tumor metastasis by suppressing the direct target gene fndc3b. moreover, mir-143 inhibition in apoptosis of hcc cells and suppression of hcc tumorigenesis. in addition, mir-143 was upregulated in serum samples from patients with chronic hepatitis and hcc and therefore might be a useful biomarker for both diseases. in addition to the upregulation of mir-221 by hbv, mir-221 can also be induced by hcv infection in an nf-b - dependent manner. however, the increased expression of mir-221 was blocked after treatment with pdtc, an nf-b inhibitor. this finding suggests that the hcv - induced upregulation of mir-221 requires nf-b activation and that nf-b signaling pathway plays an important role in inflammation - associated cancer. the expression of mir-224 was upregulated in both cirrhotic livers and hcc samples, suggesting the use of mir-224 as a biomarker of liver inflammation and hcc. the upregulation of mir-224 begins in the pre - cancerous stage and continues during hcc development. in one study in addition, mir-224 levels were significantly higher in patients with advanced hcc stages than in patients with early stages. mir-224 could be induced by a set of pro - inflammatory factors, such as tnf, lps, and lt, suggesting that nf-b signaling regulates mir-224. inflammation - mediated activation of mir-224 promotes hcc cell migration and invasion by targeting rho gtpase - activating proteins. as an onco - mir , the overexpression of mir-224 also facilitates the migration and invasion of hcc cells by targeting hoxd10 and promotes proliferation by targeting p21. mir-197 is well - studied during the progression of various cancers in which it plays a role in apoptosis, proliferation, angiogenesis, metastasis, and drug resistance. a negative correlation between mir-197 expression and il-6 and stat3 protein levels was observed in hcc tissues, indicating that mir-197 is involved in the stat3 signaling pathway. il-6 is a well - known pro - inflammatory factor that is upregulated during an inflammatory state. the expression of mature mir-197 was reduced by il-6 stimulation, whereas the expression of pri - mir-197 remained unchanged by il-6 stimulation in hcc cells. further investigation revealed that drosha binding to pri - mir-197 decreased after il-6 stimulation, indicating that il-6 downregulated mir-197 in a post - transcriptional manner by inhibiting drosha binding to pri - mir-197. moreover, stat3 was identified as a direct target of mir-197. as a tumor suppressor, mir-197 inhibits proliferation and invasiveness of hcc cells as well as suppresses the tumorigenesis of hcc xenografts. the first evidence of the role of mir-23a in hcc development was revealed in 2008. mir-23a induced by tgf is remarkably increased in hcc tissues and functions as an anti - apoptotic and pro - proliferative factor in hcc cells. later on, wang et al. found that mir-23 can be induced by il-6 and stat3 signaling, as a potential stat3 binding site was identified in the mir-23a promoter region. stat3-activated mir-23a downregulates the target genes g6pc and pcg - a and suppresses gluconeogenesis; this reduction in gluconeogenesis facilitates hcc tumorigenesis. chronic hepatitis virus infection leads to liver inflammation, damage, fibrosis, cell regeneration, and malignant transformation (hcc). many mirnas play a crucial role in the regulation of hepatitis virus - induced chronic infla - mmation and hcc (table 1). for instance, mir-27 is upregulated by hcv infection and can promote cell migration and invasion by suppressing spry2 in hcc cells. mir-146a expression is consistently increased in hcv - infected cells, primary hepatocytes, and liver tissue from hcv - infected patients. increased mir-146a level promotes viral infection and , we will summarize the key roles of relevant mirnas in hepatitis virus infection and hcc. mirnas associated with hepatitis virus infection and hcc the first mirna discovered in humans is let-7, which is highly conserved across animal species and promotes differentiation. recent studies revealed that let-7 is frequently downregulated in many cancers, such as breast, ovarian, prostate, lung, and liver cancers. the hbv x protein (hbx) encoded by the hbv genome is a multifunctional transactivator that plays crucial roles in hbv - associated hepatocarcinogenesis. hbx lacks a dna binding domain and acts as a trans - coactivator through interaction with other cellular transcription factors, by which hbx might regulate let-7 family members. in addition to hbx induction, hbv pres2 rna can sequester let-7 g, which in turn reduces intrinsic let-7 g functions in hcc. wang et al. concluded that hbx - mediated down - regulation of let-7 might be responsible for reduced let-7 expression in hbv - associated hcc. mirnas from the let-7 family act as tumor suppressors in hcc by inhibiting proliferation and invasion, as well as inducing apoptosis by targeting many oncogenes including high mobility group proteins, bcl - xl, stat3, and c - myc. mir-122 is the most abundant mirna expressed in the liver, which accounts for 70% of the total mirnas in an adult liver. the expression of mir-122 is increased in the liver during embryonic development, and the first identified target of this mirna is cationic amino acid transporter 1. in recent years, increasing evidence suggested that mir-122 plays an important role in initiation and progression of hcc. li et al. found that mir-122 expression is low in patients with chronic hepatitis b and hcc tumors and has negative correlation with fibrotic stage, age, and liver transaminase levels. the downregulation of mir-122 in patients with chronic hepatitis b and hcc tissues is affected by hbv infection. ppar was identified as a transcription factor regulating mir-122 expression; the underlying cause of hbx - induced mir-122 downregulation might be the direct interaction between hbx and ppar, which blocks ppar nuclear translocation. in 2013, li et al. further reported that all four hbv mrnas contain a binding site for mir-122 and act as a mirna sponge sequestering mir-122 in hbv - infected cells. in addition, mir-122 knockout mice will possibly develop hepatitis, fibrosis, and hcc. the reduced mir-122 expression in hcc tissues is characterized by a poor prognosis and consistent with a suppressed hepatic phenotype and an increased metastatic potential. moreover, mir-122 expression can be suppressed by two inflammatory cytokines, il-6 and tnf-, which downregulate the mir-122 transcription factors c / ebp and hnf3 and induce c - myc - mediated c / ebp inhibition in chronic hepatic inflammation. mir-122 downregulation in the upregulation of target genes that promote hepatocarcinogenesis, including pituitary tumor trans - forming gene binding factor (pbf), cyclin g1, pkm2, adam10, adam17, srf, and pkr activator. mir-101 downregulation was reported in various cancers, including hcc, suggesting that mir-101 functions as a tumor suppressor. reduced mir-101 expression in hcc tissues is associated with advanced tumor progression and poor survival in hcc patients; the serum mir-101 level might be a potential hcc biomarker. overexpression of mir-101 sensitizes the hcc cells to apoptosis induced by serum deprivation and chemotherapeutic drugs. mcl-1, a member of the bcl-2 family, is a target of mir-101 and sensitizes cancer cells to apoptosis. mir-101 enhances cisplatin - induced apoptosis by targeting stmn1, rab5a, and mtor, as well as suppresses epithelial - mesenchymal transition (emt) in hepatocytes via the regulation of zeb1 expression, which in e - cadherin down - regulation. in 2010, the first link between mir-155 and hepatitis viruses was based on the observation that mir-155 precursor (termed bic) is responsible for hcv infection. after patients with hepatitis c were treated with interferon alpha and ribavirin, the lowest bic expression was detected in patients who lacked hcv rna in both serum and peripheral blood mononuclear cells. previously, mir-155 was identified as an onco - mir in various cancers, including lung, breast, stomach, and prostate cancers. a mouse model revealed that mir-155 induced by inflammation could promote autoimmune inflammation by enhancing inflammatory t cell development. mir-155 a positive correlation was observed between mir-155 expression and ip-10 levels in the plasma, which is widely considered a marker of liver inflammation degree. upon nf-b activation by hcv - rna infection the upregulation of mir-155 in cases of hepatitis and hcc promotes hepatocyte proliferation and hcc tumor growth by activating the wnt/-catenin signaling pathway. in addition to hcc pathogenesis during chronic viral infections such as hcv, the upregulation of mir-155 was also observed in the pathogenesis of non - alcoholic steatohepatitis (nash)-associated hcc. however, similar to hcv - induced mir-155, the activation of nf-b signaling pathway plays a key role in mir-155 expression in hcc and other hepatocyte - derived cells. in a mouse model of nash, mir-155 downregulated the tumor suppressor target c / ebp, thus promoting liver pathogenesis. mir-21 is a well - established onco - mir that is upregulated in various cancers, including lung, breast, prostate, colon, stomach, pancreatic, and liver cancers. the relationship between mir-21 and inflammation was observed in uv - exposed skin, where mir-21 was activated by ppar via the tgf- signaling pathway. the upregulation of mir-21 was reported in other inflammation - associated tissues and cancers, such as allergy - induced airway inflammation, gastric cancer, pre - b - cell lymphoma, and colorectal cancer. the relevance of mir-21 and inflammation in liver tissues was established by associating biomarkers with hcc diagnosis and prognosis. mir-21 and mir-122 were analyzed in tissues obtained by needle biopsies from both hcv - infected and non - infected patients. the expression of mir-21 was positively correlated with fibrotic stage in patients with chronic hepatitis c; mir-21 promotes liver fibrosis by targeting the fibrosis inhibitor smad7. in serum in addition, one study reported that hbx increases the expression of mir-21 and subsequently promotes the progression of hcc by targeting pten. in another study, hbx - mediated mir-21 upregulation inhibits expression of the tumor suppressor pdcd4 in hcc cells. mir-221 is another onco - mir that is upregulated in cancers such as breast, prostate, glioma, and liver cancers. moreover, the expression of mir-221 in later stages of hcc is higher than that in early stages. therefore, mir-221 is a poor prognosis risk factor because it was increased in mouse models of liver fibrosis and upregulated in the human liver in a fibrosis progression - dependent manner. hbx promotes the proliferation of hcc cells and upregulates the expression of mir-221 during both hbv infection and hcc. several tumor - related genes were reported to be the targets of mir-221; among these genes, p27kip1 is well - recognized. the expression of mir-221/p27kip1 axis was confirmed in glioblastomas, breast, lung, and liver cancers. other than p27kip1, many mir-221 targets were reported, such as hdac6, pten, and tissue inhibitor of metalloproteinase-3 (timp3). in mouse models of hcc, tumor - bearing mice that were treated with chol - anti - mir-221 have a survival advantage compared with mice in the control group. mir-221 is a potential target for hcc treatment and functions by inhibiting the progression of hcc and promoting survival. chronic inflammation can facilitate the development and progression of hcc by the constitutive activation of inflammation signaling pathways. inhibition of these signaling pathways would be a novel therapeutic strategy to suppress this inflammation - associated process. many mirnas were reported to regulate the inflammatory signaling mediated by nf-b and jak / stat3, among others, in hcc (table 2). mir-195, mir-194, and mir-127 suppressed nf-b activation by directly or indirectly targeting nf-b signaling; these mirnas also function as tumor suppressors in hcc. mir-219 is reduced in hcc where it functions as a tumor suppressor; this mirna might suppress jak / stat3 signaling via direct targeting of structural maintenance of chromosome 4 protein (smc4). mir-637 is a tumor suppressor that inhibits cell growth and induces apoptosis by suppressing stat3 activation in hcc. mirnas as regulators of inflammatory signaling in hcc mir-26a was suggested to play an important role in cirrhosis and inflammation - induced hcc. reduced mir-26a expression was detected in hcc tissues; the decreased expression in hcc tissues might be an independent predictor of poor survival. clinical analysis revealed that the expression of mir-26a is negatively correlated with il-6 mrna and il-6 protein level. as il-6 is a direct target of mir-26a, the mrna and protein levels of this cytokine are significantly reduced by mir-26a overexpression in hcc cells. mir-26a functions as a tumor suppressor that inhibits tumor growth and metastasis, partly through the il - stat3 signaling pathway. m - csf expression was also reported as a predictor of frequent metastasis and poor survival; mir-26a affects macrophage polarization and inhibits macrophage recruitment by down - regulating m - csf expression. in addition, hcc cells transfected with mir-126b exhibit significantly reduced nf-b reporter activity in response to tnf. two well - known upstream regulators of nf-b signaling pathway, tak1 and tab 3, were identified as direct targets of mir-26b. mir-26b prevents doxorubicin - induced nf-b activation and promotes apoptosis in hcc cells. in one study , mir-451 was often downregulated in primary hcc tissues; reduced mir-451 expression was significantly associated with advanced tnm stage, lymph node metastasis, vascular invasion, and poor survival, indicating that mir-451 might act as a tumor suppressor in hcc. mir-451 might inhibit hcc tumorigenesis and metastasis by down - regulating cyclin d1 and c - myc at both mrna and protein levels. further study indicated that mir-451 overexpression affected the nf-b nuclear translocation and significantly decreased nf-b activity by directly targeting ikk. as expected, the inhibition of mir-451 significantly increased nf-b activity in hcc cells. moreover, a recent study revealed that reduced expression of the il-6 receptor was observed in cells that overexpressed mir-451, and that il-6 receptor was a direct target of mir-451. mir-451 reduced vegf production in hcc cells by targeting the il-6/stat3 signaling pathway. mir-124 is a well - known tumor suppressor that is frequently downregulated in various cancers. numerous studies revealed that mir-124 inhibits inflammatory signaling by targeting stat3 in different cancer types including glioblastoma, gastric cancer, endometrial cancer, colon cancer, and hcc. recently, nf-b p65 was reported to be a direct mir-124 target in b - cell lymphomas. the downregulation of mir-124 in b - cell lymphomas enhances the nf-b signaling pathway and upregulates genes, such as myc and bcl-2, associated with proliferation, anti - apoptosis, metastasis, and pro - survival. stat3 was identified as a direct mir-124 target in hcc; one study found that mir-124 inhibited cell proliferation and induced apoptosis in hcc cells by targeting stat3. moreover, an hnf4-nf-b feedback circuit that includes mir-124 was identified in hcc. reduced hnf4 could be a biomarker for poor prognosis, as hnf4 suppresses hcc metastasis by inhibiting nf-b activation. mir-124 also plays an important role in rela suppression by hnf4. mir-124 was identified as an hnf4 target that binds to the 3utr of rela. mir-124 we discovered that mir-125b was significantly downregulated in hcc, could be used as a biomarker for prolonged hcc patient survival, and functions as a tumor suppressor. mir-125b inhibits the proliferation of hcc cells by inducing cell cycle arrest during g1/s transition via its target gene, lin28b. il-6r was characterized as the subunit that binds to il-6; the binding of il-6 to il-6r activates the jak / stat3 signaling pathway. mir-125b can block the il-6-induced phosphory - lation and nuclear translocation of stat3 in hcc cells, which in turn promotes hcc apoptosis. in addition, mir-125b serves as a tumor suppressor that inhibits migration and invasion of hcc cells by targeting the transcriptional co - activator with pdz - binding motif (taz). several studies revealed that mir-370 acts as tumor promoter in gastric cancer, prostate cancer, and aml; whereas other studies reported mir-370 as a tumor suppressor in leukemia and oral squamous carcinoma. xu et al. observed that the down - regulation of mir-370 was associated with hcc development in rats and correlated with poor survival in hcc patients, indicating that mir-370 serves as a tumor suppressor in hcc. the restoration of mir-370 inhibits proliferation, migration, and invasion of hcc cells and suppresses tumorigenesis of hcc by directly targeting lin28a. lin28a is a post - transcriptional modulator of mrnas that can directly bind to p65 mrna. mir-370 represses nf-b activation and inhibits the migration and invasion of hcc cells through downregulation of lin28a. sun et al. confirmed the tumor suppressor role of mir-370 in hcc cells, showing that mir-370 inhibited hcc cell proliferation and colony formation ability by activating the pi3k / akt signaling pathway. in addition to mirnas that inhibit inflammatory signaling, some mirnas, such as mir-155, mir-362, mir-301a, and mir-1180, promote the inflammatory response by repressing negative regulators of inflammatory signaling. the nf-b signaling pathway is activated by mir-1180 overexpression, which occurs through the downregulation of mir-1180 direct targets, otud7b and tnip2. another study confirmed the expression of tnip2, a direct target of mir-1180, in hcc cells. the ectopic expression of mir-1180 promoted the proliferation of hcc cells and inhibited apoptosis because nf-b induces the expression of genes that participate in pro - survival and anti - apoptotic processes. ni et al. demonstrated that mir-362 - 5p, which is frequently upregulated in hcc, promoted nf-b signaling by downregulating cyld; cyld is a protein that binds to nf-b essential modulator and functions as a negative regulator of nf-b signaling. these inflammatory pathways and related transcription factors can affect the development of hcc by regulating mirna expression. several studies confirmed that inflammatory signaling facilitates the development of hcc through the downregulation or upregulation of mirnas in cells (table 3). mirnas as mediators of inflammatory signaling in hcc ning et al. observed feedback regulation of hnf4 and nf-b in hcc. their study revealed that hnf4 dramatically suppresses the nf-b signaling pathway and that rela overexpression or knockdown decreases or increases the expression of hnf4, respectively. during chronic hepatic inflammation, nf-b induced the expression of mir-21, which represses hnf4 expression and contributes to hepatocyte de - differentiation and hepatocarcinogenesis. in addition to direct induction of mir-21 expression by nf-b, mir-21 transcriptional activity is enhanced by high - mobility group box-1 protein (hmgb1), an important participant in inflammation. hmgb1 is a conserved nuclear protein that functions as a chromatin binding factor and allows transcription factors to access specific dna binding sites. hmgb1 enhances p65/p50 or p50/p50 binding to specific dna sites rather than the binding of p65/p65, c - rel / c - rel, p65/c - rel, and p50/c - rel. given its role in the inflammatory cascade, hmgb1 is upregulated in hcc and induces mir-21 expression in hcc cells. the hmgb1/mir-21 signaling axis inhibits the downstream targets reck and timp3 as well as promotes cell proliferation, invasion, and migration. mir-143 is upregulated in hcc tissues, indicating that mir-143 serves as a tumor promoter. mir-143 expression was increased by hbx - mediated nf-b activation, although metastasis will be suppressed by mir-143 inhibition. nf-b activates transcription by specifically binding to the putative mir-143 promoter, as the nf-b binding site identifies 5.06 kb upstream of mir-143. mir-143 promotes cell invasion and tumor metastasis by suppressing the direct target gene fndc3b. moreover, mir-143 inhibition in apoptosis of hcc cells and suppression of hcc tumorigenesis. in addition, mir-143 was upregulated in serum samples from patients with chronic hepatitis and hcc and therefore might be a useful biomarker for both diseases. in addition to the upregulation of mir-221 by hbv, mir-221 can also be induced by hcv infection in an nf-b - dependent manner. however, the increased expression of mir-221 was blocked after treatment with pdtc, an nf-b inhibitor. this finding suggests that the hcv - induced upregulation of mir-221 requires nf-b activation and that nf-b signaling pathway plays an important role in inflammation - associated cancer. the expression of mir-224 was upregulated in both cirrhotic livers and hcc samples, suggesting the use of mir-224 as a biomarker of liver inflammation and hcc. the upregulation of mir-224 begins in the pre - cancerous stage and continues during hcc development. in one study, patients with lower mir-224 expression exhibited a favorable trend of survival. in addition, mir-224 levels were significantly higher in patients with advanced hcc stages than in patients with early stages. mir-224 could be induced by a set of pro - inflammatory factors, such as tnf, lps, and lt, suggesting that nf-b signaling regulates mir-224. inflammation - mediated activation of mir-224 promotes hcc cell migration and invasion by targeting rho gtpase - activating proteins. as an onco - mir , the overexpression of mir-224 also facilitates the migration and invasion of hcc cells by targeting hoxd10 and promotes proliferation by targeting p21. mir-197 is well - studied during the progression of various cancers in which it plays a role in apoptosis, proliferation, angiogenesis, metastasis, and drug resistance. a negative correlation between mir-197 expression and il-6 and stat3 protein levels was observed in hcc tissues, indicating that mir-197 is involved in the stat3 signaling pathway. il-6 is a well - known pro - inflammatory factor that is upregulated during an inflammatory state. the expression of mature mir-197 was reduced by il-6 stimulation, whereas the expression of pri - mir-197 remained unchanged by il-6 stimulation in hcc cells. further investigation revealed that drosha binding to pri - mir-197 decreased after il-6 stimulation, indicating that il-6 downregulated mir-197 in a post - transcriptional manner by inhibiting drosha binding to pri - mir-197. moreover, stat3 was identified as a direct target of mir-197. as a tumor suppressor , mir-197 inhibits proliferation and invasiveness of hcc cells as well as suppresses the tumorigenesis of hcc xenografts. the first evidence of the role of mir-23a in hcc development was revealed in 2008. mir-23a induced by tgf is remarkably increased in hcc tissues and functions as an anti - apoptotic and pro - proliferative factor in hcc cells. later on, wang et al. found that mir-23 can be induced by il-6 and stat3 signaling, as a potential stat3 binding site was identified in the mir-23a promoter region. stat3-activated mir-23a downregulates the target genes g6pc and pcg - a and suppresses gluconeogenesis; this reduction in gluconeogenesis facilitates hcc tumorigenesis. mirnas have a well - recognized role in liver inflammation and hepatocarcinogenesis; many mirnas are dysregulated during chronic liver inflammation and hcc progression. mirnas were regarded as novel targets for hcc therapy; the first study using mirna - based therapy was conducted in 2005. in that study, mir-122 expression was silenced by antagomirs, which act as efficient and specific silencers of endogenous mirnas. a clinical trial of treating hcv infection by mir-122 inhibition was performed because mir-122 targets hcv genomic rna and promotes hcv viral translation. jong - kook park and colleagues treated tumor - bearing mice with chol - anti - mir-221 or a cholesterol - labeled control oligo. consistent with the in vitro , improved survival was observed in the group of mice treated with chol - anti - mir-221. in addition, mir-21 is a tumor promoter and functions as a prognostic factor, as previously described. mir-21 promotes the proliferation, metastasis, and tumorigenesis of hcc and was validated as an onco - mir involved in drug resistance. mir-21 suppression by lenti - mir-21-i combined with 5-fluorouracil and pirarubicin treatment leads to significant suppression of hcc cell proliferation; 5-fluorouracil and pirarubicin downregulated the mir-21 expression through ap-1 signaling. tumor - bearing nude mice were intra - tumorally injected with either mir-199 adenovirus or pbs as a control. these were confirmed in a transgenic model in which mice were predisposed to hcc development. the group of mice treated with mir-199 adenovirus exhibited a reduced tumor burden and a decreased number of tumor nodules compared with the control group. it can be expected in the future that any mirnas acting as tumor suppressors can be applied as therapeutic oncolytic viruses in a manner similar to mir-199. another mirna - based therapeutic strategy is the design of mirna panels that targets a particular protein to modulate inflammatory response and hcc development. the association between the ikk complex and rap1 is important for nf-b activation because rap1 serves as an adaptor to recruit ikks and aids in phosphorylation of the nf-b p65 subunit. 5-fluorouracil is a widely used chemotherapy drug for advanced hcc patients, but many patients with hcc are highly resistant to this chemotherapy regimen. a set of 4 rap1-mirnas was designed to knock down rap1, leading to a significant increase in the sensitivity of hepg2 cells to 5-fluorouracil - induced apoptosis. a number of mirnas can be induced or inhibited during hepatitis because of viral or non - viral infections. some mirnas are also activated or repressed when inflammatory signaling pathways, such as nf-b and jak / stat3 signaling, can not be turned off. in turn, mirnas that might function as oncogenes or tumor suppressors can promote or suppress both inflammatory cascades in hepatocarcinogenesis. this function indicates that mirnas are novel regulators or mediators of liver inflammation and hepatocarcinogenesis (figure 1). the tumor microenvironment consists of many types of cells including tumor invading immune - cells, fibroblasts, vascular endothelial cells, lymphatic cells, and stromal components. given that numerous components of tumor microenvironment are responsible for the initiation, development, and progression of human cancer, the dysregulated mirnas from tumor specimens might originate from different types of cells in the tumor microenvironment and function in human carcinogenesis by multiple ways. the next investigations should focus on the origin, location, action, and multi - function of the dysregulated mirnas in initiation, development, and progression of hcc. additionally, any study of cell lines might run the risk of false discovery. the from cell lines can not reflect the full aspects of the pathological process of hcc. the next investigations on the exact role of mirnas in inflammation - associated hcc should be verified in patients and reliable hcc models. a simplified sketch illustrates the development of liver inflammation and hcc and shows the key nodes in which aberrantly expressed mirnas participate in inflammation - associated hepatocarcinogenesis. the 5-year overall survival of hcc is lower than 10% worldwide because many patients are diagnosed at advanced stages. therefore, discovering novel viable biomarkers for hcc early diagnosis is urgent. in the last decades, numerous pieces of evidence highlighted the regulatory roles of mirnas in inflammation - associated hcc. evidence also indicated that the dysregulated mirnas might be useful as biomarkers for liver inflammation and hcc progression. in 2015 , mirnas were screened in a large - scale, multicenter, retrospective longitudinal study to explore effective and reliable strategies for monitoring at - risk populations that suffers from chronic inflammation as well as detect hcc at an early stage. serum mir-29a, mir-29c, mir-133a, mir-143, mir-145, mir-192, and mir-505 might act as biomarkers in hcc because they are in significantly higher content in hcc compared with healthy controls or those with chronic inflammation. the classifier composed of these seven mirnas could be more sensitive than -fetoprotein in distinguishing patients with hcc from those who are cancer - free. the distinction requires large - scale, multicenter, independent investigations to confirm its feasibility. several studies illustrated the potential of mirnas as therapeutic targets to prevent or treat hcc. however, additional profiles and detailed mechanisms of mirna functions are required to provide a better understanding of their role in liver inflammation and hepatocarcinogenesis. the additional data will facilitate the application of more mirnas into prevention and therapy for inflammation - associated hcc. this work was supported by grants from the national key basic research program (grant no . 2013cb910504), and the national natural science foundation of china (grant no . 81125016 and 81472565).

liver cancer, primarily hepatocellular carcinoma (hcc), is a major cause of cancer - related death worldwide. hcc is a suitable model of inflammation - induced cancer because more than 90% of hcc cases are caused by liver damage and chronic inflammation. several inflammatory response pathways, such as nf-b and jak / stat3 signaling pathways, play roles in the crosstalk between inflammation and hcc. micrornas (mirnas) are evolutionarily conserved, short endogenous, non - coding single - stranded rnas that are involved in various biological and pathological processes by regulating gene expression and protein translation. evidence showed that mirnas play a pivotal role in hepatitis virus infection and serve as promoters or inhibitors of inflammatory response. aberrant mirna was observed during liver inflammation and hcc. many dysregulated mirnas modulate the initiation and progression of inflammation - induced hcc. this review summarizes the role and functions of mirnas in inflammation - associated hcc, as well as the designed therapeutics targeting mirnas to treat liver inflammation and hcc.

2005 after a detailed polyphasic study and reclassification on the genus thermoactinomyces. at present the genus laceyella has four recognized species namely; laceyella putida , l. sacchari, type species of the genus laceyella , laceyella sediminis and laceyella tengchongensis. cells are aerobic, non - acid - fast, produce endospores and chemo - organotrophic. l. sacchari strain gs 1 - 1, was isolated from hot spring of chumathang, leh, ladakh, india, (n 32 58 e78 15), at the height of 4600 m above sea level. genomic dna was extracted from 36 h old culture using zr fungal / bacterial dna miniprep as per manufacturer's instructions. the genome of l. sacchari strain gs 1 - 1 was sequenced using the illumina - hiseq 1000 paired - end technology that produced a total of 40,874,820 paired - end reads (paired distance ( insert size) ~ 330 bp ) of 101 bp. clc bio workbench v6.0.2 (clc bio, denmark) was employed for preprocessing the data so as to trim and remove low quality sequences. a total of 40,668,128 high quality, vector filtered reads (~ 1194 times coverage) were used for assembly with clc bio workbench (at word size of 45 and bubble size of 98). the final assembly contains 42 contigs of total size 3,324,316 bp with n50 contig length of 249,341 bp; the largest contig assembled measures 698,403 bp. this draft genome comprising 3,324,316 bp was annotated with the help of rast (rapid annotation using subsystem technology) system server. a total of 3429 predicted coding regions (cdss), 6 rrnas and 69 trnas were predicted. rast indicates that strain geobacillus thermodenitrificans strain ng80 - 2 (score 502), geobacillus kaustophilus strain hta426 (score 471) and geobacillus sp. strain g11mc16 (score 436) are the closest neighbors of the strain gs 1 - 1. the strain gs 1 - 1 contains the genes for glycolysis and gluconeogenesis, tca cycle and pentose phosphate pathway. genes of alkaline phosphatase (ec 3.1.3.1), ferroxidase (ec 1.16.3.1), manganese superoxide dismutase (ec 1.15.1.1), shikimate kinase i (ec 2.7.1.7.1), chorismate synthase (ec 4.2.3.5), alcohol dehydrogenase (1.1.1.1) and superoxide dismutase (ec 1.15.1.1) and pathogenicity islands are also present in the genome annotation of strain gs 1 - 1. we have mapped all predicted 3429 cdss to kegg pathways with the help of kass server. the genome has all the essential pathways for dna, rna metabolism, iron, sulfur and phosphorus acquisition and metabolism pathways (fig . the l. sacchari strain gs 1 - 1 whole genome shot gun ( wgs) project has been deposited at ddbj / embl / genbank under the project accession aszu00000000 of the project that has the accession numbers aszu00000000 and consists of sequences aszu01000001aszu01000042. the authors declare that there is no conflict of interest on any work published in this paper.

we report the 3.3-mb draft genome of laceyella sacchari strain gs 1 - 1, isolated from hot spring water sample, chumathang, leh, india. draft genome of strain gs 1 - 1 consists of 3, 324, 316 bp with a g + c content of 48.8% and 3429 predicted protein coding genes and 75 rnas. geobacillus thermodenitrificans strain ng80 - 2, geobacillus kaustophilus strain hta426 and geobacillus sp. strain g11mc16 are the closest neighbors of the strain gs 1 - 1.

the basic fasting protocol is to transfer the fish, at their normal density, to a clean tank, then withhold food. for longer - term fasting that requires more rigorous conditions (e.g., for blood glucose studies), see additional considerations in the discussion. for a 100 ml volume, dissolve the following in distilled water: 725 mg nacl (124.1 mm) 38 mg kcl (5.1 mm) 41 mg na2hpo4 (2.9 mm) 24 mg mgso47h2o (1.9 mm) 16 mg cacl22h2o (1.4 mm) 100 mg nahco3 (11.9 mm) 4 g polyvinylpyrrolidone (pvp) (4%) 1,000 usp units heparin filter, sterilize and store at 4c. cover the microscope base with plastic wrap for protection in case of spills. put a paper towel on top of the plastic wrap. this will eliminate or minimize further focal adjustment once the fish is on the surgical table. cover the microscope base with plastic wrap for protection in case of spills. put a paper towel on top of the plastic wrap. pre - adjust focus by viewing the surgical table and focusing on the sponge. weigh the fish. fill a 500 ml beaker about 1/3 full with fish facility water. wick excess water away from the net and fish by briefly dabbing the net on paper towels. wick excess water away from the net and fish by briefly dabbing the net on paper towels. , we recommend a 35 g beveled steel needle and a 10 l nanofil microsyringe. it is important to remove any bubbles from the syringe and tubing. after filling the syringe and tubing, mount the syringe on the pump, and program the injection volume for the first fish. prepare the surgical table. cut a soft sponge (such as # l800-d, jaece industries) so that it is approximately 20 mm in height. on the flat face, set the petri dish with sponge into a suitably - sized pipette tip box lid. the lid needs to be large enough to hold water to help maintain sponge temperature, but it should be shallow enough to not get in the way. we use a lid from a p200 tip box that is 11.4 cm l x 7.7 cm w x 1.5 cm d. these three items assembled together (sponge in petri dish in box lid) constitute the surgical table. cut a soft sponge (such as # l800-d, jaece industries) so that it is approximately 20 mm in height. on the flat face, make a cut that is 10 - 15 mm deep. set the petri dish with sponge into a suitably - sized pipette tip box lid. the lid needs to be large enough to hold water to help maintain sponge temperature, but it should be shallow enough to not get in the way. we use a lid from a p200 tip box that is 11.4 cm l x 7.7 cm w x 1.5 cm d. prepare the anesthetic. tip: using typical ice cube trays, it will take 3 trays to anesthetize 10 - 12 fish. put the surgical table into a larger container such as a 2.4 liter rubbermaid food storage container. pour some facility water (warm) into the outer container and the surgical table. put the surgical table into a larger container such as a 2.4 liter rubbermaid food storage container. pour some facility water (warm) into the outer container and the surgical table. important: do n't go below 17c for this step. use a net to transfer the fish to the outer container. slowly add ice chips to the container to bring the temperature down to 12c, over the course of several minutes. monitor fish behavior: at 17c or slightly lower, the fish typically will spread its pectoral fins horizontally, gasp, and have rapid operculum movements. as the temperature drops, the fish will swim more slowly and finally stop swimming. as the surgical plane of anesthesia is approached, gasping will stop and operculum movements will slow. the fish is ready for injection when it does not react to being handled. for most fish, as the required temperature is reached (~12c or colder), press on the sponge to saturate it. keep your fingers in the cold water sufficiently so that they will not warm up the fish and bring it out of anesthesia during handling. with cold fingers , gently transfer the fish to the trough of the sponge. position the fish with the abdomen up and the gills in the trough. quickly transfer the surgical table to the microscope stage. working quickly, carefully insert the needle into the midline between the pelvic fins. the needle should point cranially and be inserted closer to the pelvic girdle than to the anus. you should be able to feel when the needle is deep to the body wall. inject the appropriate volume and withdraw the needle. after injection, immediately transfer the fish back to its warm - water (~28.5c) tank for recovery by releasing the fish from the sponge over the tank water. tip: if the fish does not begin swimming immediately, help it to recover by gently swirling water towards the gills. occasionally a scale may be attached and should be removed prior to the next injection. for subsequent injections, use warm facility water to bring the anesthetic chamber water temperature back up to 17c before introducing the next fish. the basic fasting protocol is to transfer the fish, at their normal density, to a clean tank, then withhold food. for longer - term fasting that requires more rigorous conditions (e.g., for blood glucose studies), see additional considerations in the discussion. for a 100 ml volume, dissolve the following in distilled water: 725 mg nacl (124.1 mm) 38 mg kcl (5.1 mm) 41 mg na2hpo4 (2.9 mm) 24 mg mgso47h2o (1.9 mm) 16 mg cacl22h2o (1.4 mm) 100 mg nahco3 (11.9 mm) 4 g polyvinylpyrrolidone (pvp) (4%) 1,000 usp units heparin filter, sterilize and store at 4c. cover the microscope base with plastic wrap for protection in case of spills. put a paper towel on top of the plastic wrap. this will eliminate or minimize further focal adjustment once the fish is on the surgical table. cover the microscope base with plastic wrap for protection in case of spills. put a paper towel on top of the plastic wrap. pre - adjust focus by viewing the surgical table and focusing on the sponge. weigh the fish. fill a 500 ml beaker about 1/3 full with fish facility water. wick excess water away from the net and fish by briefly dabbing the net on paper towels. wick excess water away from the net and fish by briefly dabbing the net on paper towels. , we recommend a 35 g beveled steel needle and a 10 l nanofil microsyringe. it is important to remove any bubbles from the syringe and tubing. after filling the syringe and tubing, mount the syringe on the pump, and program the injection volume for the first fish. prepare the surgical table. cut a soft sponge (such as # l800-d, jaece industries) so that it is approximately 20 mm in height. on the flat face, set the petri dish with sponge into a suitably - sized pipette tip box lid. the lid needs to be large enough to hold water to help maintain sponge temperature, but it should be shallow enough to not get in the way. we use a lid from a p200 tip box that is 11.4 cm l x 7.7 cm w x 1.5 cm d. these three items assembled together (sponge in petri dish in box lid) constitute the surgical table. cut a soft sponge (such as # l800-d, jaece industries) so that it is approximately 20 mm in height. on the flat face, make a cut that is 10 - 15 mm deep. set the petri dish with sponge into a suitably - sized pipette tip box lid. the lid needs to be large enough to hold water to help maintain sponge temperature, but it should be shallow enough to not get in the way. we use a lid from a p200 tip box that is 11.4 cm l x 7.7 cm w x 1.5 cm d. prepare the anesthetic. tip: using typical ice cube trays, it will take 3 trays to anesthetize 10 - 12 fish. put the surgical table into a larger container such as a 2.4 liter rubbermaid food storage container. pour some facility water (warm) into the outer container and the surgical table. put the surgical table into a larger container such as a 2.4 liter rubbermaid food storage container. pour some facility water (warm) into the outer container and the surgical table. important: do n't go below 17c for this step. use a net to transfer the fish to the outer container. slowly add ice chips to the container to bring the temperature down to 12c, over the course of several minutes. monitor fish behavior: at 17c or slightly lower, the fish typically will spread its pectoral fins horizontally, gasp, and have rapid operculum movements. as the temperature drops, the fish will swim more slowly and finally stop swimming. as the surgical plane of anesthesia is approached, gasping will stop and operculum movements will slow. the fish is ready for injection when it does not react to being handled. for most fish,. as the required temperature is reached (~12c or colder), press on the sponge to saturate it. keep your fingers in the cold water sufficiently so that they will not warm up the fish and bring it out of anesthesia during handling. with cold fingers, gently transfer the fish to the trough of the sponge. position the fish with the abdomen up and the gills in the trough. quickly transfer the surgical table to the microscope stage. working quickly, carefully insert the needle into the midline between the pelvic fins. the needle should point cranially and be inserted closer to the pelvic girdle than to the anus. you should be able to feel when the needle is deep to the body wall. after injection, immediately transfer the fish back to its warm - water (~28.5c) tank for recovery by releasing the fish from the sponge over the tank water. tip: if the fish does not begin swimming immediately, help it to recover by gently swirling water towards the gills. occasionally a scale may be attached and should be removed prior to the next injection. for subsequent injections, use warm facility water to bring the anesthetic chamber water temperature back up to 17c before introducing the next fish. intraperitoneal injection involves five steps: fasting, weighing, anesthetizing, injection, and recovery. for each step there this practice is taken from the fish veterinary literature (e.g., brown 1993). longer - term fasting: we have found that a 72-hour fast is required to decrease blood glucose to a baseline level prior to injection (eames et al ., 2010). we have also found that for glucose studies there are several procedures that are required to ensure that the fish are fasted properly. tanks should be offline, clearly labeled as' fasting', and in a location where enthusiastic fish care personnel will not feed them. evaluate the external environment of the tank and take steps to prevent the fish from being stressed from disturbances, as stress is known to raise blood glucose (chavin and young, 1970 ; groff et al . for example, we had a fasting experiment in which a radio was operated daily on the bench that was holding the fish tanks . we found that blood glucose was unusually high and concluded that the fish were stressed by the vibrations . fish should be kept at a density that conforms with good fish husbandry practices . for recommendations, we have had good fasting our fish at a density of 10 - 12 fish in a 9 liter tank ( with 3 layers of marbles taking up some of that volume). separating the sexes may cause stress, so we recommend maintaining a mixed - sex population during the fast. this means that eggs can be laid, and the eggs need to be sequestered so that they will not be eaten. a simple way to sequester eggs is to cover the tank bottom with 2 - 3 layers of marbles. water quality needs to be maintained by removing eggs and waste and by replacing about 10 - 15% of the tank water, daily. for removing eggs and waste, siphoning works well. weighing: when weighing fish that are not anesthetized, care should be taken to minimize water transfer from the net into the beaker, to ensure accurate weighing. if the net (with fish) is blotted on paper towels, the majority of the excess water can be removed, and the weight can be accurately measured. it may be easier to anesthetize the fish prior to weighing, but we have not tested the possible effects of anesthetizing a fish twice in one day. we have tested our technique by weighing the fish first with the netting / blotting method and then re - weighing the fish after it has been anesthetized, and gently blotted dry. we found no significant difference in weight between the methods (p = 0.7927, t - test). additionally, we tested whether this netting / blotting method affected blood glucose, in comparison with simply transferring the fish to the beaker as soon as it is netted (no blotting). we found no significant difference in blood glucose level between the two transfer methods (p = 0.2241, t - test). here we have demonstrated cold water anesthesia as an alternative, because many anesthetics (including tricaine / ms-222 ( brown, 1993) ), raise blood glucose. in previous studies , we have determined that cold water does not raise blood glucose in zebrafish (eames et al . , 2010). for cold water anesthesia, the temperature should be decreased slowly. the rate of decrease seems to depend on the size of the fish, with smaller fish going under faster than larger fish. following injection , you may observe that the fish is recovering too slowly from the anesthetic (see below). this can when either the starting temperature is too low, or when the temperature is decreased too rapidly. the starting temperature is too low if the fish bends laterally upon entering the water. it will rotate its pectoral fins to a horizontal position, gasp, and have rapid operculum movements. , movements will decrease and the fish will lose equilibrium. a surgical plane of anesthesia is reached when the fish can be handled without reacting. to maintain the fish under surgical anesthesia, your fingers must be cold, so keep them in the water prior to handling the fish. the sponge must also be kept cold at the same temperature as the water used for anesthetizing the fish. it is important to saturate the sponge with water that is sufficiently cold to maintain anesthesia once the fish is placed onto it. injection: prior to undertaking injections, you may want to dissect at least one fish to get a sense of body wall thickness. this can help you to judge how far the needle needs to insert to enter the abdominal cavity. additionally, as you insert the needle, you can feel the body wall " give " when the needle enters the abdominal cavity. during the injection, make sure the sponge is saturated with the correct temperature cold water to prevent the fish from reviving during injection. a well - saturated and soft sponge is important for minimizing damage to the scales and mucus covering of the skin. finally, once the fish is anesthetized, work quickly to minimize the time that the fish is under. recovery: the fish should recover from the anesthesia virtually upon entering the warm tank water. if the fish does not begin swimming immediately, gently swirl the water towards its gills to speed recovery. if recovery is slow, then the fish went under too quickly and you should adjust the anesthesia procedure appropriately.

a convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. however, this method makes it difficult to know how much reagent is absorbed or taken up per fish. some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. here we present a method for intraperitoneal (ip) injection into adult zebrafish. injection is into the abdominal cavity, posterior to the pelvic girdle. this procedure is adapted from veterinary methods used for larger fish. it is safe, as we have observed zero mortality. additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. precautions are required to minimize stress throughout the procedure. our precautions include using a small injection volume and a 35 g needle. we use cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water - saturated sponge during the injection itself. we demonstrate the utility of ip injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. as stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle - injected and non - injected adults and show that the procedure does not cause a significant rise in blood glucose.

neuropathic pain, defined as pain arising as a direct consequence of a lesion or a disease affecting the somatosensory system,1 is a disorder that can considerably affect quality of life for patients. even though up to 5% of the general population could be involved, treatment of neuropathic pain continues to be challenging due to the inadequate effectiveness of systemic therapies and their frequent central side effects.2 in the wide area of peripheral neuropathic pain, the identification of specific neuropathic pain syndromes that affect only a circumscribed area of the body may drive a clinical decision to use targeted localized treatment(s). characterized by consistent and circumscribed area(s) of maximum pain associated with negative or positive sensory signs3 as localized neuropathic pain (lnp). according to the relevant guidelines,46 5% lidocaine medicated plaster could be considered as a first - line treatment option for lnp due to its good efficacy and safety profile, especially in elderly patients where comorbidities are quite often present and polypharmacy is poorly tolerated. up to 83% of patients with post - herpetic neuralgia (phn) complain of an lnp syndrome,3 for which 5% lidocaine medicated plaster showed better effectiveness versus placebo7,8 and substantially better pain relief than pregabalin,9,10 with fewer systemic side effects even in long - term treatment.1113 several different mechanisms sustain phn,14 including peripheral inflammation, nerve damage and denervation, and peripheral and central sensitization. lidocaine acts by blocking abnormally functioning (sensitized) nav 1.7 and nav 1.8 sodium channels in dermal nociceptors,15 thereby reducing ectopic discharges, which raise the peripheral ectopic discharge threshold.15 topical 5% lidocaine plaster does not affect a - beta fibers, so does not cause any paresthesia and/or numbness.16 finally, a passive protective action of the plaster itself has been reported.810 in a cohort of patients with lnp due to nerve trauma,17 5% lidocaine medicated plaster showed optimal efficacy in reducing pain and the size of the painful area. to our knowledge, no other structured trial has investigated this possible new endpoint in phn patients. in our first eight phn patients treated with versatis 5% plaster (grunenthal, germany) (the drug was approved in italy in may 2013), we investigated whether it could be helpful both in reducing pain and allodinic (static and/or dynamic) areas. allodynia is a phenomenon in which normally nonpainful stimuli are perceived as painful, and can be a clinical sign of peripheral and central sensitization. the two components can be differentiated using different types of stimuli, ie, peripheral sensitization can be evoked by static mechanical stimulation (static mechanical allodynia) whereas allodynia to dynamic mechanical stimulation is the of central sensitization. while the static component is only found in the injured area, the dynamic component also extends into the halo of undamaged tissue surrounding the injury.18 consistent with the literature and following approval in italy, 5% lidocaine medicated plaster was used in a group of phn patients with untreated lnp, lnp that was inadequately controlled, or side effects from systemic drugs. following our hospital administrative rules, all patients signed an informed consent allowing anonymous use of their clinical data for research purposes. in accordance with our clinical practice , the patients were screened at the first visit and then followed up after 15, 30, and 90 days. patients could telephone and ask for another consultation if they experienced side effects or inadequate analgesia. at all visits , we recorded: daily pain intensity (mean, least, and worst, using the numeric rating scale); paresthesia and dynamic allodynia (scale 0 to 5); and static allodynia with the von frey hair test (caliber from 0 to 18 of von frey monofilaments perceived as painful we used a well established von frey hair set ( touch test sensory evaluators ; north coast medical, inc . morgan hill, ca, usa), with a 5.07 caliper corresponding to 10 g applied for 1.5 seconds, which delivers a clear mechanical suprathreshold nonpainful stimulus. the area of statical tactile allodynic area was measured in cm. during their first visit , patients were instructed regarding how to apply the 5% lidocaine medicated plaster to the most painful areas of intact skin; they were allowed to use a maximum of three plasters for 12 hours a day. depending on the clinical seen during follow - up, we reduced their systemic analgesic medication if good pain relief (of at least 30%) was achieved. eight patients with phn lnp (five men and three women) were observed from may to september 2013. their mean age was 77.757.10 (range 6989) years and mean body mass index (bmi) was 28.15.5 kg / m. the acute episode of herpes zoster had occurred at a mean of 3.73.6 years earlier. the rash affected the trunk in all cases, except for one patient whose left hand was affected. all patients were on polypharmacy, taking a mean of 42 nonanalgesic drugs for comorbid conditions. all patients reported having tried at least one systemic drug for pain relief (antidepressant, anticonvulsant, and/or opioids) with inadequate pain control (five patients) or good pain control but severe side effects (three patients). one patient reported an accidental fall with a rib fracture due to dizziness on anticonvulsant treatment. the patients reported good pain relief (45.00%19.75%) as early as 2 weeks after the start of topical therapy, with further improvement as treatment continued (52.00%23.87% by month 1 and 60.00%18.70% by month 3), and reduction / cessation of systemic treatment in six patients (table 1). paresthesia and dynamic allodynia decreased significantly at 3 months from 2.881.64 to 1.201.10 and from 3.750.71 to 1.801.64, respectively. we observed a similarly consistent reduction in static allodynia from 8.633.58 to 14.000.82 over 3 months. the allodynic (static or dynamic) area was measured in five of eight patients with thoracic phn (figure 1a). the area became progressively smaller from a baseline measurement of 236.38140.34 cm to 128.8095.7 cm after one month (46% reduction, p=0.129) and to 81.3859.19 cm after 3 months (66% reduction, p=0.02). with the reduction in size of the area a more pronounced reduction was observed in the height (50%) than in the length (33%) of the affected area (figure 1b). neuropathic pain continues to be a challenging clinical problem.2 people with neuropathic pain are often elderly and may have several comorbidities and a high risk of drug drug interactions, which presents a serious limitation to therapy.19 to achieve good pain relief, it is often necessary to give a combination of two or more drugs,20 which increases the risk of drug drug interactions and side effects. the mainstay of treatment for neuropathic pain is still adequate personalized therapy based on an understanding of the pain pathophysiology and the patient s clinical features.21 the majority of patients evaluated in our case series complained of the limited effectiveness of pain treatments and/or side effects from systemic treatment for phn, and had had their pain for several months or years. when patient refers a lnp, correctly diagnosed according to validated diagnostic algorithm,22 more recent guidelines4,5 suggest topical products as the first - line therapeutic option since they are better tolerated, have no or few systemic effects and drug drug interactions, and have better patient compliance.9,10,23,24 our case series reflects all these common clinical aspects. the main mechanism of the therapeutic action of lidocaine is blockade of voltage - gated sodium channels.15,25 however, topical lidocaine may have other peripheral actions through a desensitizing effect of trpa1 channels, contributing to its nonanesthetic analgesic effects.26 thus, the reduction in peripheral sensitization could be attributed to both blockade of pathological sodium channel expression and desensitization of trpa1 channels. a characteristic feature of phn is the presence of an area of primary hyperalgesia (static mechanical and thermal allodynia within the damaged area) and secondary hyperalgesia (dynamic mechanical allodynia that surrounds the first area).27 the static mechanical allodynia is mainly mediated by sensitized peripheral nociceptors, while the dynamic component is probably a consequence of altered processing of large diameter primary afferent inputs in the central nervous system. these alterations are at least partially maintained by the barrage of nociceptor activity from both normal and abnormal inputs.27 the importance of ongoing activity to maintain the secondary hyperalgesia has not been completely elucidated, even though in patients with neuropathic pain, secondary hyperalgesia and allodynia have been seen to be critically dependent on continuous afferent input.18 hence, 5% lidocaine medicated plaster could act by adding blockade of pathological sodium and trpv1 channels to the reduction of mechanical noxious / non - noxious inputs via the protective action of the plaster. this reduction of peripheral inputs could contribute to reducing both primary and secondary sensitization, and consequently the size of the painful area,28,29 as also suggested by a healthy volunteer study.30 in our study, the mean size of the painful area was 236.38140.34 cm, which is in agreement with data in the literature.31 regarding the reduction of the static allodynic area, we noticed that there is a much greater reduction of the height (number of metameres involved) rather than of the length (area of the same nerve involved) of the painful area. in phn, the secondary hyperalgesic area is mainly distributed above and below the primary painful area in the dermatome where an overlapping of innervation is anatomically present. it could be arguable that if neural plasticity can be induced by treatment, it may be more evident in peripheral areas of secondary hyperalgesia.18 hence this treatment could have another important clinical effect: the reduction of the allodynic area, as also suggested in a study about lnp with recent onset.17 in fact, a more than 50% reduction in the size of the painful area was recorded, even though the patients had been complaining of phn for several months or years. due to the small number of patients, it was not possible to evaluate if the reduction in size of the painful area was related to the time of onset of phn or to the more pronounced reduction of static rather than dynamic allodynia. nevertheless, our case series highlights a possible role of chronic topical treatment not only in treating lnp but also in reducing the size of the painful area. these data have to be confirmed by appropriately designed controlled trials investigating this endpoint as well as the mechanisms for the activity of 5% lidocaine medicated plaster, ie, whether it acts more on primary rather than secondary hyperalgesia, and whether the time of onset of phn / lnp could be a predictive factor of efficacy.

post - herpetic neuralgia (phn) is neuropathic pain persisting after an acute episode of herpes zoster, and is associated with severe pain and sensory abnormalities that adversely affect the patient s quality of life and increase health care costs. up to 83% of patients with phn describe localized neuropathic pain, defined as a type of neuropathic pain characterized by consistent and circumscribed area(s) of maximum pain. topical treatments have been suggested as a first - line treatment for localized neuropathic pain. use of 5% lidocaine medicated plaster could reduce abnormal nervous peripheral discharge and via the plaster could have a protective function in the affected area. it has been suggested that use of this plaster could reduce pain as well as the size of the painful area. to evaluate this possible outcome , we retrospectively reviewed eight patients with phn, treated using 5% lidocaine medicated plaster. during a follow - up period of 3 months, we observed good pain relief, which was associated with a 46% reduction in size of the painful area after one month (from 236.38140.34 cm2 to 128.8095.7 cm2) and a 66% reduction after 3 months (81.3859.19 cm2). our study cohort was composed mainly of elderly patients taking multiple drugs to treat comorbidities, who have a high risk of drug drug interactions. such patients benefit greatly from topical treatment of phn. our observations confirm the effectiveness of lidocaine plasters in the treatment of phn, indicating that 5% lidocaine medicated plaster could reduce the size of the painful area. this last observation has to be confirmed and the mechanisms clarified in appropriate larger randomized controlled trials.

nephrotic syndrome (ns) in children is usually idiopathic, but may also be triggered by immunological stimuli like vaccination, infection, insect sting, or pollen hypersensitivity. humoral and cellular immune function studies suggest that abnormal immune response is the probable cause in this clinical entity. bee sting has been implicated in the development of ns, but occurrence in children has rarely been reported in literature. these patients need a shorter course of steroids, but show excellent response and have a low risk of relapse. a 2-year - old boy was hospitalized with generalized edema and decreased urine output of 4-day duration. facial swelling developed initially and then the edema extended to the legs, eventually affecting the entire body. there was a history of a bee sting on the dorsal aspect of his right hand 7 days earlier, which had ed in severe pain, redness, and local swelling for about 48 hours. he had no history of atopy or similar episodes in the past. on physical examination, he was afebrile, had facial edema, pitting pedal edema with abdominal distension. his blood pressure was normal (90/60 mm hg), and a bee sting mark was visualized over the dorsum of the right hand. laboratory investigations revealed hemoglobin of 13.7 g / dl, white blood cell count of 25 400/ mm, 35% neutrophils, 58% lymphocytes, 5% eosinophils, 2% monocytes, and platelet count of 331 000/mm. his erythrocyte sedimentation rate was 69 mm / hr and peripheral smear showed normocytic normochromic red blood cells with leukocytosis and adequate platelets. he had decreased serum proteins (total protein 3.9 g / dl, and albumin 1.5 g / dl); elevated serum cholesterol (382 mg / dl); and normal renal parameters (blood urea 34 mg/ dl and creatinine and 0.37 mg/ dl). urine analysis revealed 3 + proteinuria with a 24-hour urinary protein excretion of 0.9 g. complement c3 level was 1.16 g / dl (0.90 - 1.80 g / dl); immunoglobulins profile showed iga - 0.32 g / dl (0.14 - 1.59 g / dl), igm - 0.9 g/ dl (0.43 - 2.07 g / dl), igg - 2.92 g / dl (3.45 - 12.36 g / dl), and ige - 245 iu / l (< 230 chest x - ray was normal and the ultrasound revealed free fluid in the abdomen . bee sting over the dorsum of the right hand on admission, he was managed symptomatically with diuretics and with salt and fluid restriction . after the laboratory , he was started on oral prednisolone ( 2 mg / kg / day). the boy progressively improved and diuresis set in on the fifth day; edema regressed by the seventh day; and urine became albumin - free by the 10 day of admission. corticosteroids were then changed to alternate - day regimen and tapered gradually after 8 weeks and withdrawn. during the regular 1-year follow - up after the end of treatment, the boy remained well without any recurrence. ns in childhood, can be triggered by immunological stimuli including infection, vaccination, insect bites, pollen and drug - induced hypersensitivity. bee stings are usually followed by minor local allergic reactions and rarely anaphylactic or delayed hypersensitivity reactions. other reported systemic complications following multiple bee stings include acute renal failure (arf), myocarditis, myocardial infarction, centrilobular necrosis of liver, acute encephalopathy, guillain - barre syndrome, vasculitis, disseminated intravascular coagulation, and thrombocytopenia. though causal relationship and association between bee stings and development of ns has long been described, no report has demonstrated the presence of bee venom antigens in the glomeruli. ns can develop even after a single bee sting, like in our case which occurred within 7 days. the cause - and - effect relationship between the bee sting and renal disease is speculative, made on the basis of a temporal association. it is postulated that components of the bee venom mediate immunological disturbances, with involvement of t - lymphocytes and their cytokine secretion, influencing the permeability of the glomerular basal membrane with consequent development of proteinuria. pathologic findings of ns following a bee sting are diverse, which include minimal change lesions, mesangial proliferative glomerulonephritis, membranous glomerulonephritis, and glomerulosclerosis. arf following multiple bee stings is attributed to acute tubular necrosis due to hypotension or pigment nephropathy ing from rhabdomyolysis, intravascular hemolysis, and acute interstitial nephritis. most reported cases had a favorable clinical course with corticosteroid treatment. in our case, steroids shortened the duration of disease, and no relapse or chronic renal disease is so far reported. cytotoxic agents may also induce remission in the occasional steroid - resistant cases. in , though rare, children with insect stings, particularly bee stings, must be closely followed up for multiple problems, especially immune - mediated complications such as ns.

the occurrence of nephrotic syndrome following a bee sting is rarely reported in the literature. hypersensitivity is believed to be the precipitating factor for the renal disease. we report a two - year - old boy, who developed generalized edema and decreased urine output, seven days after a bee sting. physical examination and laboratory findings were consistent with nephrotic syndrome; and corticosteroid treatment induced prompt remission with resolution of clinical symptoms and normalization of laboratory findings. there was no relapse of the disease during a one - year follow up.

emergency department (ed) use is common for both children and adults not only in a country without universal health insurance like the united states but also in countries like france where virtually all residents have health insurance. in 2006 the annual rate of ed visits for parisian children less than 2 years old ranged, according to district, from 46.9 to 91.3 per 100 children. many of these visits are not for true emergencies but for what ed physicians, insurers, policy makers, and researchers consider as inappropriate or nonurgent, that is, minor complaints that could have been dealt with in primary care offices on either that or the following day. durand and colleagues found that, in studies in a variety of countries around the world, the estimates of nonurgent visits by adults and children ranged from 4.8% to 90%, with a median of 32%. one reason for the variation was the inconsistency in defining nonurgent, as pointed out also by mistry and colleagues for pediatric ed visits. recent figures on pediatric nonurgent visits include 58% in the united states, 57.1% in italy, and 39.9% in belgium. in many cases, parents do not even try to contact a primary care physician. furthermore, they go to eds even though they often have long waits; their children are likely to undergo more testing and treatment than by their own doctors; the ed physicians do not know their medical histories and usually do not provide follow - up; and in many cases the costs to them will be higher. many researchers have, therefore, tried to understand why parents decide to bring their children to eds. they hope to find out how to reduce unnecessary ed visits and thereby how to improve continuity of care; to lessen overcrowding in eds, distraction from true emergencies, and diversion of ambulances to less crowded but more distant eds; and to decrease health care expenses. reasons given by parents for their decisions to go to pediatric eds (peds), even without referral by their children's primary care physicians, include that they seek a quick and convenient solution to a health issue; that they view the ped as the best place to go because of its better physicians and/or medical equipment; that they are dissatisfied with their primary care physician's office or the physician's diagnosis and/or treatment; that they are very worried about their child's health; that they are in financial straits; that they have a habit of going to the ed; and that they have no access to other care. the methodologies of the above - mentioned studies were not based on a broad theory of human motivation and may, as a , have neglected important motives. in this study, therefore, we examined parental motives by applying michael apter's metamotivational theory (mtt) , which encompasses and systematizes the great diversity of human motives. by including all theoretically possible categories of motives in the questionnaire, we could ensure that it was psychologically complete, that is, that no possible parental motive was missed. objectives of the study were (a) to inventory the motives for going to peds; (b) to order these motives, or categories of motives, as a function of their importance; and (c) to find out how demographic characteristics were associated with these motives. the study was approved by the ethics committee of the children's hospital of toulouse. the participants were parents attending the ped of the children's hospital, a tertiary - level pediatric hospital in toulouse, france. the research assistant invited the parents of every child to participate, informed them about the survey, and obtained oral consent from those who agreed to participate. the first questionnaire contained 69 items referring to motives for going to a ped (table 1). forty - eight items were created by the members of the research team, inspired by mmt (see table 5), by the medical literature (cited above), and by their personal experience as physicians. items were purposely and systematically created to correspond to all of mmt's categories of motives. this questionnaire was then presented to a focus group of parents that reformulated items judged as ambiguous and suggested additional items based on their personal experiences as parents. the new questionnaire was presented to another focus group, and the process was repeated until saturation occurred. the common wording of all items in the questionnaireone of the reasons i came to the ped today with my childwas chosen to acknowledge that several motives could be operating at the same time. the second questionnaire asked about demographics, about previous ed use, and about prior doctor visits during this illness. participants responded individually in the waiting room, before their children were seen. if both parents were present, only one filled out the questionnaires but that one could confer with the other. the research assistant was, in most cases, not present during the process in order not to influence them. they indicated how much they agreed with each statement on the first questionnaire by putting a mark on the 10-point response scale under each one. the treating physician subsequently indicated the severity of each child's illness on a scale of 1 (not severe), 2 (mildly severe), or 3 (severe)and whether the child was hospitalized. the character of this study was both exploratory and confirmatory. through exploratory factor analyses using two - thirds of the sample, exploratory factor analysis is a statistical technique that is used to reduce a number of variables into a smaller set of unobserved factors. the variables within each factor are empirically highly correlated with each other but not with the other variables. if the correlation between items and any factor was lower than 0.30 and their means were very low, the items were removed from the analysis. confirmatory factor analysis is a statistical technique used to test whether a set of factors that has been obtained through exploratory factor analysis of data from one sample fits the data observed in another sample. as the exploratory factor analysis has to be conducted on all the useable items, the confirmatory analysis should only be done on a representative and more manageable subset of items, namely, on the three items with the highest loadings (the highest correlations with the factor) within each of the factors found in the exploratory analysis. therefore, the exploratory factor analysis requires a larger sample size than the confirmatory analysis, calculated in this case to be 2 to 1. the sample size of 500 was determined in accordance with the technical requirements of factor analysis for a maximum expected number of factors of 10. our choice of 10 was based on our analysis of the previous studies of reasons to go to the ed. table 1 shows the mean ratings (on a scale of 1 to 10) of each item in the exploratory factor analysis using the first subsample (n = 332). thirteen of the 69 items did not load on any factor (correlation between item and any factor < .30), and their means were very low. six interpretable and independent factors emerged, namely, being considered by others as responsible parents (which explained 13% of the variance); empathic concern for child's suffering (8% of the variance); seeking quick diagnosis, treatment, and reassurance (12%); dissatisfaction with previous consultation (7%); ped as the best place (7%); and external factors (5%), such as the family physician's absence, the impossibility of borrowing money, and a recommendation from the service that provides urgent home visits. the of the confirmatory factor analysis, conducted on the three items with the highest loadings on each of the six factors using the second subsample (n = 168), are shown in table 3. the mean scores for each factor in the final model on a 10-point scale range from 1.79 (dissatisfaction with previous consultation) to 6.82 (seeking quick diagnosis, treatment, and reassurance). table 4 shows correlations between demographic characteristics, severity indices (as subsequently judged by physicians), and motives. seeking quick diagnosis, treatment, and reassurance was significantly more strongly endorsed by parents of one child than by parents of more than one. considering the ped as the best place to go was given a higher rating by younger fathers than by older ones. empathic concern for the child's suffering was given a higher rating by parents with only one child. being considered by others as responsible parents was a stronger motive for parents of boys than of girls, for younger mothers, for less educated parents, and for parents who were more likely to go to eds without first consulting their regular physician, and a stronger motive in cases judged by the treating physician to be of lesser severity. finally, external factors were more strongly endorsed as motives by parents of older children who had siblings and in cases in which symptoms had more recently appeared. the aim of our study was to make an inventory of the motives of parents in bringing their children to peds using a broad theoretical framework. we found that, as in other studies , parents endorsed a wide variety of motives. finding factors meant the guarantee that parents' responses were meaningful, that is, that their responses were sufficiently coherent such that statistical analysis could reveal a clear structure to them. the emergence of a clear factorial structure also enabled measurement of the strength of each motive. telic in mmt ) seemed to be highly goal - oriented, going to a ped to obtain care they saw as important. this is consistent with findings in other countries. ] described guardians as worried that symptoms represented serious illness and that it was important to seek medical attention. similarly, durand and colleagues, in their study of french adult patients, characterized them as rational consumers (in contrast to the abusive and irresponsible consumers portrayed by the ed health professionals they interviewed). these parents are unlikely, therefore, to agree with the charge that large numbers of visits to peds are unwarranted. the second most highly rated factor was ped as the best place to go (which would be called a conformist motive by mtt). their respect for the ped may have been stimulated by television shows and by reports in the media about the technology and specialization available in hospitals (as well as, in the united states, by advertising). they may not have appreciated that technology and specialized care are not always beneficial to patients. empathic concern for child's suffering received a lower overall rating. those parents who strongly endorsed these motives (labeled as sympathy in mtt) were no longer able to put up with their children's suffering. they tended to be parents of a single child and, therefore, likely to be less experienced in coping with sick children. it is striking, however, that distress at their child's current suffering was much less important than the goal - oriented aim of obtaining the most effective treatment. the other three factors were rated quite low overall but were endorsed by some parents. being considered by others as responsible parents was rated highly by parents who seemed to be concerned about their own image as parents (labeled as these parents tended to be younger ( especially the mothers) and less educated than other parents and to go to eds for themselves more frequently. their child's condition was not likely to be judged as bad by the ed physician. physicians and health services experts, especially in the us, often cite structural factors as important determinants of excessive ed utilization. those few parents who endorsed these motives strongly indicated thereby that going to peds was not a truly personal decision. they followed the instruction of the urgent care service or could not go to another medical facility owing to the absence of a physician or to lack of money. their child's illness tended to be more acute; that is, the child was more likely than others to have experienced pain and/or fever for less than 12 hours. the lowest average rating was for dissatisfaction with previous consultation (a negativist motive in the terminology of mmt). only a few parents were very unhappy with the care provided by their family physicians, in accordance with findings in other countries. it was conducted in a single site in france, and the motives of the 35% of parents who declined to participate are unknown. the study's must, therefore, be generalized with caution to other populations of children and parents and to other sites. furthermore, even if the questionnaire is based on a broad theory of motivation, it has not been validated and should, therefore, be used prospectively again to confirm these . nonetheless, our findings can help to illuminate the current debate about the overuse of eds. access to care is a key issue for parents, as well as for adult patients, and, therefore, is a focus of reform efforts. the 2011 national health interview survey in the united states found that, among adults who went to eds and were not admitted to the hospital (68.9% of those visiting the ed), 78.9% had had at least one access issue for going to the ed (defined as indicating not having another place to go, doctor 's office or clinic being not open, ed being the closest provider ', and/or clinic being not open). it seems possible to reduce ed visits, therefore, by improving access to other sources of care (as in the state of north carolina). without this, as the medicaid experience in the state of oregon showed, expanding insurance will not reduce ed use; it will increase it. accordingly, many reformers have proposed encouraging pediatric practices to expand the availability of urgent visits; france has tried to set up primary care units at or near eds; and italy is requiring its primary care offices to be open 24 hours a day 7 days a week. wang and colleagues tested in the united states a pilot ped diversion program for children with medicaid using extended office hours, multiple access locations, and care coordination that reduced ed visits by 8 visits per 1000 members per month (compared with the control group). because their diversion program was so expensive, however, the overall cost of care was not reduced. our study demonstrates the limits or barriers, from the parents' perspective, to the success of such efforts. we found that parents generally bring their children to peds with serious, goal - oriented motives. similarly, in the united states, the 2011 national health interview survey found that 65.0% of adults not admitted to the hospital cited at least one acuity issue (defined as indicating only hospital could help, advised by health provider to go to ed, and/or problem too serious for doctor 's office or clinic); kubicek and colleagues found that 63% of parents bringing their children for reasons assessed as nonurgent perceived the visit as very or extremely urgent; and kalidindi and colleagues found that parents assessed 94% of their visits as urgent, even though 27% of these urgent visits were assessed as nonurgent by the physicians. thus, even if access issues were greatly reduced in the us and other countries, the acuity issues would persist. because there is no consensus between the population and physicians about what is urgent and what is not , staff members in the eds tend to define many visits as nonurgent, to label them as inappropriate, and to feel frustrated. in general eds in the united states, the presenting complaints of adult patients judged subsequently as primary care treatable do not in fact differ from those judged as needing ed care. furthermore, as cresson ] states, to speak of false urgency' in situations where there is urgency for the patient and not for the doctors is to act as if the latter were the only ones capable of defining urgency. if overworked ed physicians, as well as researchers and policy makers, focus on ultimate diagnoses and wasted resources, they forget that the parents do not have the medical knowledge to make these diagnoses, do not know what the final diagnoses will be, and do not know how the illnesses are going to evolve. as adams concludes, trying to discern low - acuity conditions and putting up barriers to receiving care or denying payment after receiving care will work no better in future generations than in the past and specifically those parents whose own characteristics for example, in a us study, single parents, those who were themselves brought to eds as children, and those with medicaid make them more likely to use the ed. these efforts could involve targeted messaging to high utilizers or more formal instruction, but so far success has been limited. in one study, 130 families were randomly assigned to receive, or not, an educational intervention in the ped that involved reviewing with a research assistant a booklet about how to manage minor illnesses and watching a 10-minute video. at 6 months there was no difference between groups in ped utilization, and most visits were still for minor illnesses. similarly, a 90-minute, interactive educational activity at primary care offices increased 32 parents' knowledge of fever, colds, and minor trauma and increased their after - hours telephone use; but it did not decrease their ed use. education may, of course, have greater effects on other groups of parents than those studied and if done more intensively and repeatedly. yet our findings point to one possible reason for the limited impact of greater knowledge: the importance for many parents of emotional motives of anxiety about and empathetic concern for the child (as would be expected of parents) as well as, for some, of a desire to be seen as a caring parent. the setting able immediately to soothe their anxiety and distress is the hospital and its emergency department. it is not surprising, therefore, that the rate of ed visits has been increasing even in countries, like france, that have extended basic insurance to all residents. when parents in france, as elsewhere, think their children are suffering and might be seriously ill, they are goal - oriented and, often, frightened. they bring their children to the places that are immediately available and that, they think, are most capable of helping their children, to the peds. thus, as hendry, beattie, and heaney in the united kingdom concluded, perhaps it is the service design, rather than patient behavior, that is inappropriate the solution is not only to provide anxious parents with expanded daytime sick visits, 24-hour telephone access, and even night - time home visits, as is currently possible in many places. the solution may also be to expand the peds, that is, to provide urgent visit capacity for minor illnesses and injuries, at lower cost than truly urgent visits, in the places of reassurance and security, the peds themselves.

parents frequently bring their children to general or pediatric emergency departments (eds), even though many of these visits are judged by others to be nonurgent and inappropriate. this study examined the motives behind parents' decisions to take their children to a pediatric emergency department (ped). at a ped in toulouse, france, 497 parents rated their level of agreement with each of 69 possible motives representing all categories of human motivation for coming to the ped that day. exploratory and confirmatory factor analyses found evidence for six separable motives, called (in order of importance) (a) seeking quick diagnosis, treatment, and reassurance; (b) ped as the best place to go; (c) empathic concern for child's suffering; (d) being considered by others as responsible parents; (e) external factors; and (f) dissatisfaction with previous consultation. . parents' motives in bringing their children to the ped are primarily serious and goal - oriented. they are also often emotion based, as would be expected in parents of ill children. the parents would be unlikely to agree that these visits were inappropriate.

south carolina, like many other cities and states that have historically had few latinos, has experienced recent surges in immigration and now has small informal enclaves and social structures. many latinos in these new communities may lack documentation for us residency. consequently, our sampling design took into account the fact that many probability - based sampling designs (e.g., random - digit dialing telephone survey, mail survey from a published list of addresses) would not adequately access this hard - to - reach population. adult (18 years of age) latinos attending 2 clinics in the greater charleston, south carolina, area that serve a primarily latino population were approached for participation in a face - to - face, structured, confidential interview in which no names were recorded. eligible persons included both those seeking care as well as those accompanying patients since the goal was to collect data from latino adults regarding health behavior that was not necessarily linked to a current illness. persons in the waiting rooms of the clinic were approached by an interviewer, who was introduced to the prospective respondent by clinic personnel. if persons did not consider themselves to be latino, they were not included in the study. previously used and validated surveys designed for use with latino populations were accessed from the scientific literature and were consulted for question wording relevant to the present study's aims. an instrument was created in english, pretested initially in english for flow and comprehension, and following modifications was then translated into spanish. once in spanish the first pretest was with medical center personnel who were fluent with spanish and dealt with latino populations. following modifications from the first pretest, the second pretest was conducted with latino persons seen at a clinic different from the ones used in the study to provide pretest experience with the instrument with a similar population. after the second pretest, the instrument was translated back into english so that the wording of the questions could be checked for consistency in both english and spanish. two trained bilingual interviewers administered the survey in a structured format in the participant's preferred language (spanish or english) and reinforced to the potential respondents the anonymous nature of the survey. first, questions were included that assessed acquiring antimicrobial drugs without a prescription while outside the united states and corresponding self - medication. these questions were asked to get an idea of health behavior in their home culture and society. in addition, we asked whether the respondents believed that antimicrobial drugs should be available in the united states without a prescription. second, questions were included that provided an assessment of the importation of nonprescription antimicrobial drugs into the united states and the context and circumstances related to this behavior. third, a set of questions was included that assessed acquisition of nonprescribed antimicrobial drugs within the united states. we were specifically interested in the scope of acquisition of nonprescribed antimicrobial drugs from bodegas, pharmacias, or other stores since this behavior had been suggested in a study in new york city. for both importing nonprescribed antimicrobial drugs and acquiring them within the united states, a series of questions was asked, based on the pretest information, to gain an understanding of why the respondents would engage in these practices. initially, descriptive statistics were computed to gain an understanding of the extent of the acquisition and importation of nonprescribed antimicrobial drugs. we also computed chi - square values to examine bivariate relationships between importation of nonprescribed antimicrobial drugs and acquisition of nonprescribed antimicrobial drugs in the united states by health beliefs and behavior in the home country, access to care in the united states, and demographic characteristics. finally, a stepwise logistic regression model was computed on the dependent variable of acquisition of nonprescribed antimicrobial drugs with the same set of variables (acquired antimicrobial drugs without a prescription outside the united states, believe antimicrobial drugs should be available in the united states without a prescription, health insurance, age, sex, education, time in united states, country of birth, health status) to determine the best predictors of this behavior. because no one born in the united states had acquired nonprescribed antimicrobial drugs in the united states, the category variable of country of birth was not used as a predictor of acquiring nonprescribed antimicrobial drugs. instead, country of birth was coded for inclusion in the regression as 1 ) born in mexico or 2 ) born elsewhere. previously used and validated surveys designed for use with latino populations were accessed from the scientific literature and were consulted for question wording relevant to the present study's aims. an instrument was created in english, pretested initially in english for flow and comprehension, and following modifications was then translated into spanish. once in spanish the first pretest was with medical center personnel who were fluent with spanish and dealt with latino populations. following modifications from the first pretest, the second pretest was conducted with latino persons seen at a clinic different from the ones used in the study to provide pretest experience with the instrument with a similar population. after the second pretest, the instrument was translated back into english so that the wording of the questions could be checked for consistency in both english and spanish. two trained bilingual interviewers administered the survey in a structured format in the participant's preferred language (spanish or english) and reinforced to the potential respondents the anonymous nature of the survey. first, questions were included that assessed acquiring antimicrobial drugs without a prescription while outside the united states and corresponding self - medication. these questions were asked to get an idea of health behavior in their home culture and society. in addition, we asked whether the respondents believed that antimicrobial drugs should be available in the united states without a prescription. second, questions were included that provided an assessment of the importation of nonprescription antimicrobial drugs into the united states and the context and circumstances related to this behavior. third, a set of questions was included that assessed acquisition of nonprescribed antimicrobial drugs within the united states. we were specifically interested in the scope of acquisition of nonprescribed antimicrobial drugs from bodegas, pharmacias, or other stores since this behavior had been suggested in a study in new york city. for both importing nonprescribed antimicrobial drugs and acquiring them within the united states, a series of questions was asked, based on the pretest information, to gain an understanding of why the respondents would engage in these practices. initially, descriptive statistics were computed to gain an understanding of the extent of the acquisition and importation of nonprescribed antimicrobial drugs. we also computed chi - square values to examine bivariate relationships between importation of nonprescribed antimicrobial drugs and acquisition of nonprescribed antimicrobial drugs in the united states by health beliefs and behavior in the home country, access to care in the united states, and demographic characteristics. finally, a stepwise logistic regression model was computed on the dependent variable of acquisition of nonprescribed antimicrobial drugs with the same set of variables (acquired antimicrobial drugs without a prescription outside the united states, believe antimicrobial drugs should be available in the united states without a prescription, health insurance, age, sex, education, time in united states, country of birth, health status) to determine the best predictors of this behavior. because no one born in the united states had acquired nonprescribed antimicrobial drugs in the united states, the category variable of country of birth was not used as a predictor of acquiring nonprescribed antimicrobial drugs. instead, country of birth was coded for inclusion in the regression as 1 ) born in mexico or 2 ) born elsewhere. four persons who were initially approached indicated that they did not consider themselves to be latino. of the remaining 273, 54 refused or provided incomplete information, leaving a sample of 219. a large proportion of the sample (30.6%) believed that antimicrobial drugs should be available in the united states without a prescription. the behavior of acquiring antimicrobial drugs without a prescription while outside of the united states was quite common, with 45.2% indicating that they had done this. a substantial number of persons had transported nonprescribed antimicrobial drugs into the united states (16.4%). the primary illnesses for which they bought the antimicrobial drugs were primarily for what we believe to have been viral respiratory infections. among respondents who reported bringing back antimicrobial drugs that they purchased without seeing a doctor first , the reported conditions they were trying to treat included cough (88.9%), ear infections (88.9%), sore throat (69.4%), and colds (58.3%). when asked whether, on their next trip outside the united states, they would purchase antimicrobial drugs without seeing a doctor first and bring them back into the country, 23.7% of the sample reported " likely " or " very likely. " among persons who transported nonprescribed antimicrobial drugs into the united states, the primary reason reported for doing so was because they had a mistrust of medicines in the united states and were more comfortable with medicines from the home country (30.6%); other reasons included the following: to pay less for medicines bought in the home country than they would for those bought in the united states (19.4%), to avoid going to the doctor while in the united states (16.7%), to avoid the language barrier to care in the united states (13.9%), to prepare for future illness (13.9%), and to treat someone else's medical problem (5.6%). acquiring antimicrobial drugs not prescribed for the respondent within the united states was also a common behavior (19.2%). among those who acquired antimicrobial drugs in the united states without a prescription, 92.9% reported that they had acquired them without prescription at stores in the united states. as with transportation of nonprescribed antimicrobial drugs into the united states, the primary illnesses for which they acquired the drugs without a prescription were what we believe to have been viral respiratory infections. among respondents who reported acquiring antimicrobial drugs without a prescription, they reported attempting to treat " gripe " (flu) (97.6%), ear infections (97.6%), cough (83.3%), and sore throat (80.9%). additionally, 97.6% reported acquiring antimicrobial drugs to treat diarrhea. among persons who had acquired antimicrobial drugs in the united states without a prescription, 64.3% suggested that doing so was preferable to going to the doctor, while 26.2% reported that it was cheaper than paying for the doctor visit in addition to paying for the prescription. only 7.1% of this group reported acquiring antimicrobial drugs in this way because of language barriers. tables 2 and table 3 show the relationship between home country behaviors, health beliefs, access to care variables, and demographic characteristics to importing nonprescribed antimicrobial drugs and acquiring nonprescribed antimicrobial drugs in the united states. persons who acquired nonprescribed antimicrobial drugs in the united states had beliefs and practices consistent with limited regulations on antimicrobial drugs. the best predictors of acquiring antimicrobial drugs in the united states without a prescription are shown in table 4. the strongest predictors were health beliefs and practices consistent with limited regulations on antimicrobial drugs. this study confirms the existence of a large reservoir of nonprescription antimicrobial drugs in the united states that are used for likely inappropriate self - medication in the latino community. besides being imported, many of these nonprescription drugs are acquired from small stores, showing an organized system of nonprescription antimicrobial drug distribution within the latino community in the united states the cultural beliefs and practice of obtaining antimicrobial drugs without prescriptions, particularly for what are likely viral respiratory infections, is reflected in the antimicrobial drug use patterns of the latino community in this united states. health beliefs and practices that were instilled in their countries of origin appear to be maintained even after living in the united states, as the relatively high frequency of acquisition of nonprescription antimicrobial drugs in the united states demonstrates. as previous research has shown, persons born in the united states are less likely to acquire antimicrobial drugs without a prescription within the united states. in fact, none of the latino respondents born in the united states had acquired antimicrobial drugs without a prescription in the united states. thus, a special emphasis with patient education should be made to target this population to instill health beliefs that are more consistent with those proposed by the us medical and public health communities. an additional issue that may play a role in the ability to encourage appropriate use of antimicrobial drugs in this community are problems associated with access to health care. although health insurance was not a distinguishing variable, common reasons given by the respondents for both importation and acquisition of nonprescribed antimicrobial drugs in the united states revolved around the economics of doctor visits and costs of medication in the united states. a lack of health insurance may encourage latinos to self - medicate with antimicrobial drugs. first, the sample was recruited from a single mid - sized community, thereby limiting the generalizability of the . however , many areas in the united states have seen recent large increases in the latino population. these new communities may reflect different practices than communities that have had generations of latino immigrants (e.g., miami, new york, san antonio). a second limitation concerns the location of data collection. by focusing on persons who sought treatment at a health clinic, latinos who may not access care in the formal health care system would not be represented in this study. a direction for future research would be to investigate these issues about antimicrobial drug use in a broader community sampling frame. third, the are based on self - reports of behavior, some of which may be somewhat threatening to relate to an interviewer. thus, the reports of antimicrobial drug importation and acquisition of nonprescribed antimicrobial drugs may be an underreport of actual behavior. several strategies were used to try and obtain valid reports, including multiple pretests and having the clinic staff introduce the interviewers to the potential participants. following the pretests, the interview was modified to contain several instances of descriptions of antimicrobial drugs and names that would be recognizable to the latino community. moreover, each time a respondent reported acquiring antimicrobial drugs, the respondent was asked by the interviewer to name the drug to make sure that the respondent had actually acquired antimicrobial drugs. if in fact the represent underreporting, the findings are even more dramatic because nearly 20% acknowledged getting antimicrobial drugs without a prescription in the united states. however, because this study is one of the first to document the problem and the first, to our knowledge, to specifically focus on both importation and us acquisition of nonprescribed antimicrobial drugs, government agencies have not yet responded to this problem. the south carolina department of health and environmental control has a judicious antibiotic use initiative (south carolina careful antibiotic use, http://www.scdhec.gov/health/disease/sccause). their initiatives are similar to many other initiatives that focus on both physician and patient education, but thus far have not focused on latinos and nonprescription antimicrobial drug importation and local acquisition. a substantial number of persons within the us latino community self - medicate with antimicrobial drugs obtained without a prescription both inside and outside the united states, which adds to the reservoir of these drugs in the united states. the public health system and clinicians should be aware of the different health belief systems and practices in ethnic minority communities. patient education materials should communicate in a culturally competent way the dangers of self - medication and antimicrobial misuse to both recent immigrants and others in the latino community. us health policies also may need to be revised, with consideration given to tightening regulations to reduce the presence of nonprescribed antimicrobial drugs and to increasing access to care for many latinos.

we investigated in a sample of latinos the practices of antimicrobial drug importation and use of nonprescribed antimicrobial drugs. in interviews conducted with 219 adults, we assessed health beliefs and past and present behaviors consistent with acquiring antimicrobial drugs without a prescription in the united states. many (30.6%) believed that antimicrobial drugs should be available in the united states without a prescription. furthermore, 16.4% had transported nonprescribed antimicrobial drugs into the united states, and 19.2% had acquired antimicrobial agents in the united states without a prescription. a stepwise logistic regression analysis showed that the best predictors of having acquired nonprescribed antimicrobial drugs in the united states were beliefs and behavior consistent with limited regulations on such drugs. many persons within the latino community self - medicate with antimicrobial drugs obtained without a prescription both inside and outside the united states, which adds to the reservoir of antimicrobial drugs in the united states.

the author receives funding from the swedish research council, the ragnar sderbergs stiftelse, the novonordisk foundation and lund university. the author is also part of the following strategic research consortia in diabetes: the linnaeus lund university diabetes center (ludc) funded by the swedish research council and the excellence of diabetes research in sweden (exodiab) supported by the swedish government. the author was responsible for the conception, design and drafting of the manuscript and approved the final version for publication. this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited.

common genetic variations in the gene encoding transcription factor 7-like 2 (tcf7l2) reveal the strongest association with type 2-diabetes known to date. these lead to impaired insulin production and output, but the mechanisms of disease remain incompletely known. in this issue of diabetologia, two publications provide new insights into tcf7l2-dependent diabetes.

acquired retinal astrocytoma is a benign glial intraretinal tumor.1 it is considered the retinal equivalent of low - grade brain astrocytomas. despite its benign cytology, it can show progressive growth and behave as a locally aggressive malignant tumor. the clinical course can be complicated with exudative retinal detachment, vitreous hemorrhage, neovascular glaucoma and a blind, painful eye, ultimately leading to enucleation.23 ] the management of acquired astrocytoma remains controversial. external beam radiotherapy,2 laser photocoagulation,4 cryotherapy, plaque radiotherapy and pars plana vitrectomy and endoresection5 were previously proposed as the treatment options to avoid enucleation.3 however, these treatments have been ineffective in many patients.234 over time, some studies revealed success with verteporfin photodynamic therapy (pdt) in the treatment of acquired retinal astrocytomas.678 in this case report, we present a patient with symptomatic, presumed acquired retinal astrocytoma - without evidence of tuberous sclerosis - that showed dramatic regression after a single session of pdt. a 42-year - old female noted decreased visual acuity in her right eye for 1 month. the visual acuity was counting fingers at one meter in the right eye and 20/20 in the left eye. , there was a juxtapapillary, amelanotic, yellow - white lesion with circinate exudation, exudative inferior hemiretinal (including fovea) detachment and overlying marginal retinal hemorrhages. the lesion was 3.6 mm in thickness and had a basal diameter of 3 mm. optical coherence tomography (oct) imaging through the fovea demonstrated a serous retinal detachment. oct through the lesion showed preservation of the linear configuration of the retinal pigment epithelial layer with deep optical shadowing, suggesting a tumor of retinal and not choroidal origin. fluorescein angiography revealed early hypofluorescence with scarce intrinsic vascularity and diffuse late - phase hyperfluorescence with subretinal fluorescein leakage. ultrasonography disclosed a solid, pedunculated lesion inferior to the optic disc with medium internal reflectivity and inferior exudative retinal detachment. there was no systemic evidence of tuberous sclerosis or neurofibromatosis. before photodynamic therapy: (a) color fundus photograph shows a juxtapapillary, yellow - white retinal lesion with circinate exudation and exudative retinal detachment. (b) optical coherence tomography (oct) through the fovea demonstrates serous foveal detachment. (c) oct through lesion shows preservation of the linear configuration of the retinal pigment epithelial layer with deep optical shadowing, suggesting a tumor of retinal origin. (e) diffuse late - phase hyperfluorescence with subretinal fluorescein leakage and note the lack of typical small, well - defined fine blood vessels over the tumor in the early vascular filling phases. (f) ultrasonography reveals a non - calcified, pedunculated retinal mass with acoustic solidity and inferior retinal detachment patient was diagnosed with presumed acquired retinal astrocytoma. with informed consent from the patient, the tumor was treated with a single session of pdt using standard treatment parameters (50 j / cm light dose, 600 mw / cm power and 83 s duration). an intravenous injection of 6 mg / m body surface area verteporfin over 10 min was performed then laser with emission wavelength of 689 nm was delivered to the entire tumor in one spot (5400 microns), avoiding the optic disc as much as possible. following pdt, the visual acuity decreased to hand motion due to dense vitreous hemorrhage that developed during the 1 week after treatment. thereafter, the visual acuity gradually improved to 20/40 with complete regression of the tumor into a fibrotic scar - like tissue at 6 months, 20/25 with complete resolution of lipid exudation and exudative retinal detachment at 14 months and 20/20 at 2 years. at 51 months follow - up, visual acuity remained stable with complete tumor atrophy and no recurrence. regression of tumor after photodynamic therapy at: (a) 1 week, (b) 1 months, (c) 2 months and (d) 3 months 2 years after photodynamic therapy: (a) marked regression of the tumor. (b) optical coherence tomography (oct)(fovea) shows disappearance of the serous retinal detachment. (c) oct (lesion) precisely demonstrates the intraretinal location of the tumor with overlying epiretinal membrane. (e) late phase angiography showing hyperfluorescence with staining of the residual, intraretinal fibrotic tissue benign retinal astrocytic lesions include reactive retinal gliosis, astrocytic hamartoma and acquired astrocytoma.17 retinal gliosis usually occurs after trauma, inflammation, or infection. astrocytic hamartoma is typically stable, diagnosed in early childhood and is often associated with tuberous sclerosis or neurofibromatosis.1 in contrast, acquired astrocytoma is a sporadic tumor that occurs in relatively older individuals, is not associated with tuberous sclerosis and is often symptomatic and progressive.13 thus, any delay in the diagnosis and treatment may in poor visual outcomes and increased ocular morbidity. the diagnosis of acquired retinal astrocytoma depends on the clinical finding and imaging .1 it appears as a yellow - white nodular lesion within the sensory retina. fluorescein angiography typically shows small, well - defined fine blood vessels in the tumor in the early vascular filling phases and rather intense, diffuse late staining. oct can be used to document the intraretinal location of the tumor and its highly reflective features. fine needle aspiration may be required in establishing the correct diagnosis in atypical cases.7 the differential diagnosis of acquired retinal astrocytoma includes choroidal melanoma, solitary idiopathic choroiditis, retinoblastoma and retinal hemangioblastoma.1 the tumor reported in the current case was located in the sensory retina. the confirmation of the retinal location with oct (especially images from follow - up such as figure 2c) aided in differentiate it from a tumor of choroidal origin. furthermore, the color of the tumor was yellow - white and it was hypofluorescent without any evidence of small, well - defined capillaries in the early phase of angiography. in contrast, retinal hemangioblastoma is typically a reddish - pink tumor of the fundus and shows rapid hyperfluorescence in the arterial phase of the fluorescein angiography. finally, retinoblastoma of comparable size to our case does not usually cause such lipid exudation and exudative retinal detachment. the pedunculated architecture with acoustic solidity and lack of calcification in b - scan ultrasonography also favors the diagnosis of presumed acquired astrocytoma. despite its atypical features in fluorescein angiography and lack of histopathologic confirmation , our case presumably represents an example for an acquired retinal astrocytoma.138 we believe that the presence of lipid exudation and exudative retinal detachment is consistent with the aggressive behaviour.8 pdt has been used for treatment of various intraocular tumors, including choroidal hemangioma,9 osteoma,10 melanoma,11 exudative changes over nevus / combined hamartoma,1213 retinal capillary hemangioma14 and retinal vasoproliferative tumors.15 due to the poor response of acquired astrocytoma to laser photocoagulation and radiotherapy, recent studies have focused on pdt to avoid enucleation in the management of this lesion.678 in 2006, mennel et al.6 reported the first successful treatment with pdt of a symptomatic retinal astrocytic hamartoma associated with tuberous sclerosis. they demonstrated complete resolution of subretinal fluid, disappearance of tumor vessels and improved vision after one session (166 s) of pdt. in 2008, shields et al.7 described the use of pdt for a pigmented variant of acquired astrocytoma in an 18-year - old female with no history of tuberous sclerosis. they showed minimal involution of the tumor, but complete resolution of macular exudation, edema and subretinal fluid with improvement of visual acuity following one session of pdt with standard parameters. eskelin et al.8 reported similar outcome in two cases with symptomatic, locally aggressive astrocytomas. they used pdt successfully as a secondary treatment (after failure with prior laser photocoagulation) for a juxtapapillary astrocytoma in a 34-year - old male with tuberous sclerosis and as a primary treatment for a macular astrocytoma in a 68-year - old man without tuberous sclerosis. following one session of pdt with standard parameters, they observed regression of tumors and complete resolution of exudative retinal detachment in both patients. in our case, a pronounced effect on the symptomatic, juxtapapillary, presumed acquired retinal astrocytoma was obtained with a single session of pdt. the vitreous hemorrhage after pdt presumably occurred due to the rupture of superficial vessels over the tumor. our paper demonstrates dramatic regression of a symptomatic, juxtapapillary, presumed acquired retinal astrocytoma with complete resolution of exudative retinal detachment and lipid exudation after one session of pdt. thus, pdt can be considered as a first - line treatment in cases with acquired retinal astrocytoma.

photodynamic therapy (pdt) has been used for treatment of various intraocular tumors including choroidal hemangioma, vasoproliferative tumor, amelanotic choroidal melanoma and choroidal neovascular membrane due to choroidal osteoma. this case report documents the effect of pdt for a presumed acquired retinal astrocytoma. a 42-year - old female with a juxtapapillary acquired astrocytoma was treated with a single session of pdt using standard parameters. the tumor showed dramatic regression over 6 months into a fibrotic scar. it remained regressed and stable with 20/20 vision after 51 months of follow - up. we believe that pdt can be used as a primary treatment for acquired retinal astrocytoma.

hemangioblastoma (hb) of the central nervous system is a benign neoplasm of the central nervous system that usually involves the brain and spinal cord. it may occur sporadically or in association with von hippel - lindau (vhl) disease. in sporadic, non - vhl cases, the tumors are usually solitary, and local recurrences are seen occasionally after surgery. in contrast, hbs associated with vhl disease are usually multiple and continue to arise over the course of a patient's life, which necessitates lifelong surveillance. disseminated hb is an extremely unusual type of recurrence, and it has recently been called hemangioblastomatosis. we report a case of hemangioblastomatosis in a patient without vhl and discuss the biological and clinical features associated with this disease. in 1996, a 41-yr - old man underwent surgical excision of a solitary cerebellar mass with the pathological diagnosis of hb (fig . he had no family history or clinical stigmata to suggest the presence of vhl disease . gross total removal of the tumor was possible, and no evidence of residual or recurrent disease was observed on magnetic resonance imaging ( mri) performed 1 yr after the surgery (fig . ten years after the surgery, he returned to the hospital complaining of low back pain and hypesthesia in his right posterior thigh . mri of the brain showed multiple mass lesions in the suprasellar area and in the anterior aspect of the upper spinal cord accompanied by diffuse leptomeningeal enhancement ( fig . whole - spine mri revealed numerous tiny enhancing nodules suggestive of leptomeningeal metastasis along the spinal cord and cauda equina ( fig . genomic dna was isolated from peripheral blood leukocytes using a wizard genomic dna purification kit according to the manufacturer 's instructions ( promega, madison, wi, u.s.a .). the three exons of the vhl gene as well as their flanking introns were amplified and sequenced by an abi 3730 sequencer (ame bioscience, toroed, norway). the patient underwent surgical resection of the cauda equina lesion and subtotal removal of the tumor. histopathological examination confirmed the diagnosis of hb, and the microscopic findings were identical to those of the cerebellar hb operated on 10 yr earlier (fig . 2c). he subsequently received fractionated radiotherapy (a total dose of 3,600 cgy) for a residual lesion in the cauda equina. brain mri scans obtained 6 months after the diagnosis of recurrence revealed interval growth of the suprasellar lesion, and gamma knife radiosurgery was performed. although the treated lesions showed no further growth, his condition gradually deteriorated with general weakness, anorexia, and frequent vomiting. the patient died of septic shock and respiratory failure at 1 yr after the diagnosis of disseminated recurrence. hbs account for 1 to 2% of primary intracranial tumors and 5 to 15% of posterior fossa tumors in adults. approximately 62% of hbs arise sporadically, and 38% occur in association with vhl disease. the average age at presentation of sporadic cerebellar hbs is approximately 35 yr, and these tumors are more common in men. hb is thought to be a benign tumor curable by microsurgery; however, several previous studies have reported that the recurrence rate after surgical excision is 15 - 27%. vhl disease is an autosomal - dominant neoplasia syndrome caused by a germline mutation or deletion on the short arm of chromosome 3. patients with vhl tend to present with neurological symptoms and signs at a younger age than those with sporadic disease. the syndrome is characterized by the development of multiple visceral and central nervous system (cns) lesions. tumors of the cns include hbs, which affect 48 - 80% of patients with predisposing sites in the cerebellum (44 - 73%), brainstem (10 - 25%), spinal cord (13 - 50%), and retina (25 - 60%), as well as endolymphatic sac tumors, which affect 10 - 15% of patients. the visceral lesions include renal cell carcinoma, renal cyst, pheochromocytoma, pancreatic cyst, and pancreatic neuroendocrine tumor. only 12 cases of disseminated hb have been reported previously. mohan et al. first reported two cases of disseminated hb that developed several years after complete excision. bakshi et al. reported a case of diffuse spinal leptomeningeal enhancement on mri associated with multiple hbs and termed this condition hemangioblastomatosis. all 12 previously reported cases of disseminated hb arose in patients who had solid cerebellar hb as a primary lesion, and 9 of the patients had no association with vhl disease. all of the patients underwent surgical treatment of the primary lesion, and hemangiomatosis developed after variable intervals ranging from 6 months to 22 yr. in reported cases, biopsy specimens from the primary and secondary lesions were similar in histopathology to that in our case. tumor infiltration to leptomeninges was identified in five cases by mri demonstrating diffuse leptomeningeal enhancement on the surface of the brainstem and spinal cord. the outcomes after recurrence were very poor, and most patients died within 3 yr. the most common cause of death was respiratory failure due to pontomedullary or cervical cord compression. the currently available treatment did not have a significant effect on the progression of the disease. eight patients with disseminated hb received from 13 to 56 gy of irradiation to the posterior fossa or spinal cord; however, long - term tumor control was not achieved. advanced radiation techniques, such as radiosurgery, may be more effective than conventional fractionated radiotherapy, but it is difficult to perform high - dose radiation therapy or radiosurgery for numerous lesions scattered throughout the entire neuraxis. the use of interferon-, thalidomide or a vascular endothelial growth factor receptor antagonist to inhibit angiogenesis did not in remarkable tumor response. though vhl gene mutation was not detected in our case, the vhl gene may be mutated in sporadic cases of hb of the cns. in a molecular genetic analysis of four cases of hemangioblastomatosis without vhl, weil et al. found no evidence of germline alterations in the vhl gene, but the somatic deletion of one copy of the vhl gene. these findings implicate a monoclonal origin of multiple, separate deposits of tumors in patients with sporadic hb. the of comparative genomic hybridization studies suggest that other genes, in addition to the vhl gene, may be involved in the dissemination of hbs. because no case of de novo development of disseminated hb without previous surgery has been reported, it is strongly suggested that the spillage and spread of tumor cells through the csf space may be an origin of hemangioblastomatosis in patients with a genetic predisposition to the condition. all previously reported cases showed multiple mass lesions exclusively in the infratentorial area and spinal cord except only one case. paucity of supratentorial lesions may reflect csf flow and effect of gravity supporting the hypothesis of tumor spread through csf pathway. preventive measures to reduce tumor cell spillage should be considered during operation, and further study to determine the biological risk factors of disseminated recurrence is needed.

we report a very rare case of hemangioblastomatosis that developed after surgical removal of a solitary cerebellar hemangioblastoma (hb). a 51-yr - old man presented with back pain 10 yr after undergoing surgery for cerebellar hb. magnetic resonance imaging showed numerous mass lesions along the entire neuraxis accompanied by prominent leptomeningeal enhancement. genomic dna analysis showed no mutation in the von hippel - lindau (vhl) genes. a surgical specimen obtained from a lesion in the cauda equina showed pathological findings identical to those of the cerebellar hb that had been resected 10 yr earlier. external beam radiation therapy and radiosurgery were subsequently performed; however, the patient succumbed one year after receiving the diagnosis of hemangioblastomatosis. the reduction of tumor cell spillage during surgery and regular long - term follow - up are recommended for patients with hbs.

in the recent years, the identification of genes involved in the modulation of inflammatory and apoptotic processes and the improved understanding of mechanisms linked to the aberrant activation of the inflammasome, amultiprotein intracytoplasmatic scaffold complex synthesizing the biologically active interleukin- (il-1), the prototypic master cytokine affecting nearly all cell types, have allowed the delineation of a new group of diseases called monogenic autoinflammatory syndromes (maiss). from the etiopathogenetic point of view, in spite of the heterogeneity of genes responsible for the various maiss (table 1), the inflammasome represents an ideal point of convergence of most of these diseases, that is, the cell structure crucial to the regulation of innate immunity: its proper assembly allows for regular activation of caspase-1 and physiological production of proinflammatory cytokines, in primis il-1, necessary to respond to a heap of different danger signals, as bacterial peptidoglycans, genotoxic stress, and crystals. in the pathogenesis of many maiss, the erroneous assembly of the inflammasome leads to an exaggerated conversion of pro - il-1 to its active form and subsequent disproportionate overwhelming inflammatory response. autoimmune, was intended to highlight the spontaneous nature of the inflammatory attacks, which occur in the absence of any pathogenetic role of autoantibodies or autoreactive t lymphocytes. therefore, the contribution of as - yet unidentified environmental factors as potential triggers of abnormal inflammatory processes might be likely. clinically speaking, a few characteristics common to all maiss have been identified, such as the recurrent nature of inflammatory episodes, presence of fever, and frequent involvement of the skin, serous membranes, eyes, joints, lymph nodes, gastrointestinal tract, and nervous system. each of these syndromes may manifest itself with more or less severe inflammatory signs and symptoms of varying frequency and duration, associated, from the laboratory point of view, with increased phlogistic parameters (table 2). to date, there are twelve known maiss: familial mediterranean fever (fmf); tumor necrosis factor receptor - associated periodic syndrome (traps); cryopyrin - associated periodic syndrome (caps), a group which includes familial cold urticaria syndrome (fcas), muckle - wells syndrome (mws), and chronic infantile neurological cutaneous articular (cinca) syndrome; mevalonate kinase deficiency (mkd); nlrp12-associated autoinflammatory disorder (nlrp12ad); granulomatous maiss which include blau syndrome (bs) and early - onset sarcoidosis (eos); and, finally, the hereditary pyogenic disorders including papa (pyogenic arthritis, pyoderma gangrenosum, and acne) syndrome, majeed syndrome (ms), and deficiency of the il-1 receptor antagonist (dira). maiss are generally characterized by early onset (in the first year of life or early childhood), but, in more than a few cases, in particular for fmf and traps, adult onset has also been described. in such cases, the utilization of a highly sensitive and specific score can be useful in guiding diagnosis. type aa amyloidosis is the most serious complication of most maiss, due to excessive production of serum amyloid - a (saa), synthesized in the liver following stimulation by certain proinflammatory cytokines, such as il-1, and also il-6 and tumor necrosis factor- (tnf-). due to persistent activation of the chronic inflammatory process, whether clinically manifested orsubclinically, excess saa is deposited in the form of fibrils in various organs, particularly the kidneys, with the consequent progressive development of severe proteinuria, leading to nephrotic syndrome and kidney failure. other areas that may be involved include the autonomous nervous system (with orthostatic hypotension, impotence, and altered intestinal motility), liver and spleen (with hepatosplenomegaly), muscles, heart (with contractility and circulation abnormalities), and gastrointestinal tube (with diarrhea and malabsorption). therefore, close monitoring of serum saa levels even during healthy periods is necessary to prevent or promptly treat a secondary amyloidosis or to verify the efficacy of treatment. from a therapeutic point of view , colchicine has been proven to be the treatment of choice for patients with fmf, while nonsteroidal anti - inflammatory drugs (nsaids) and corticosteroids are utilized above all to treat symptoms in most of the other maiss, with varying . the introduction of biological agents, such as anti - tnf (etanercept, infliximab, and adalimumab) and anti - il-1 (anakinra, canakinumab, and rilonacept), has nonetheless opened up new interesting possibilities for the management of these heterogeneous disorders. the purpose of this paper is to describe the main genetic, clinical, and therapeutic aspects of maiss, focusing on their current classification and general details, shown in tables 1 and 2, with the ultimate goal of increasing awareness of these conditions among various specialties of internal medicine. familial mediterranean fever (omim 249100) is transmitted by autosomal recessive inheritance and, in its most frequent and classic phenotype, is characterized by recurrent acute fever episodes, polyserositis, arthritis, and erysipelas - like erythema: it is due to the presence of mutations (among the 200 identified to date) in the mefv (from mediterranean fever) gene which encodes the protein pyrin, also known by its european name marenostrin (table 1). this protein is made up of 781 amino acids and is expressed mainly in neutrophil and eosinophil granulocytes, monocytes / macrophages, and fibroblasts of the skin, peritoneum, and synovia. pyrin mutations cause altered inflammasome function, which leads to increased synthesis of proinflammatory cytokines (mainly il-1), activation of transcription factor nf-b, and altered inhibition of apoptosis, all demonstrated in subjects with fmf. in the past , fmf was believed to pertain almost exclusively to populations living near the mediterranean basin, but today it is widely held that other populations can also be affected. still, the most affected populations continue to be armenians, turks, arabs, and non - ashkenazi jews with a rate of occurrence that oscillates between 1: 400 and 1: 1.000 in turkey and that is around 1: 1000 in israel and 1: 500 in armenia. onset of fmf usually occurs in the first two decades of life, with a relatively small number of adult - onset cases. from the clinical point of view , three different fmf phenotypes have been identified: phenotype 1 is characterized by recurrent inflammatory episodes lasting 1272 hours, sometimes triggered by stress, physical exercise, or infections and preceded by nonspecific symptoms like lack of appetite, malaise, and irritability. during these episodes, fever and abdominal pain abdominal pain due to sterile peritonitis may sometimes last a few days after the fever has ceased. about half of patients present thoracic pain with acute pleuritis, almost always monolateral, and/or pericarditis. inflammation of the tunica vaginalis of the testis may also occur, leading to recurrent episodes of acute orchiepididymitis in these subjects. skin manifestations, also of brief duration (usually 1248 hours) and generally associated with fever, are characterized by the periodic appearance of erysipelas - like lesions, approximately 10 cm in diameter, usually localized on the surface of the legs between the hip and knee and/or on the tops of the feet. muscular - skeletal involvement is frequent, often in the form of arthralgia and myalgia, which may be prolonged and crippling, significantly reducing patients' quality of life. about 30% of fmf patients have also arthritis, especially affecting the large joints, which may last for several days after fever has resolved: arthritis is only rarely erosive and is generally mono- or oligoarticular. possible, but rare, is aseptic meningitis, accompanied by headache and possibly by electroencephalographic alterations and convulsions. in addition, fmf has also been associated with other rheumatological diseases, such as spondyloarthritis, rheumatoid arthritis, systemic lupus erythematosus, vasculitis of small and medium vessels (like henoch - schenlein purpura, polyarteritis nodosa, and behet 's disease), and vasculitis of large vessels (like takayasu 's arteritis). acute episodes are associated with increased laboratory phlogosis indicators, particularly erythrosedimentation rate (esr), c - reactive protein (crp), saa, and fibrinogen; other laboratory findings may include neutrophil leukocytosis, thrombocytosis, anemia, and, less frequently, an increase in immunoglobulins, particularly classes a and d. the increase in saa during fmf attacks, which is also possible during asymptomatic periods, is a clue to the progression towards amyloidosis: saa measurement is thus a useful parameter for highlighting a state of subclinical inflammation and revealing a potential secondary systemic amyloidosis. phenotype 2 refers to fmf patients with proteinuria or kidney failure ing from amyloidosis, in whom the inflammatory attacks typical of fmf occur only afterwards. this phenotype also includes subjects belonging to the families of fmf patients who evolve towards systemic amyloidosis as the sole manifestation of the disease. phenotype 3 includes subjects carrying one of two mutations (homozygous or heterozygous) of the mefv gene without presenting with any of the known clinical manifestations. diagnosis of fmf is primarily clinical and based on the use of the tel - hashomer diagnostic criteria, divided into major and minor signs, as shown in table 2: the presence of two major criteria, or one major and two minor criteria, allows for a definitive fmf diagnosis, while the presence of a single major criterion and one minor one may point towards a probable diagnosis, which can be confirmed thereafter by the presence of mutations in the mefv gene. the most common of the mefv gene mutations is m694v (in exon 10), which, in homozygous cases, is correlated with an earlier disease onset and, more frequent, joint involvement, and occurrence of amyloidosis. from a therapeutic point of view, colchicine is now recognized as the drug of choice in the treatment of fmf, as it is effective in almost 95% and completely prevents the acute episodes in 60% of the patients. in addition, colchicine has also been proven to be effective in preventing secondary complications of amyloidosis. unlike the case of gout, colchicine is not effective in aborting an established acute episode and should be used for prophylaxis only. initial dose is usually 11.2 mg daily, to increase every 1 - 2 months (depending on the frequency of the acute attacks) until an effective response is obtained, up to a maximal dose of 2.02.4 mg per day, if tolerated. optimal dosage should be determined on case - by - case basis to achieve maximal efficacy with minimal side effects. in children with fmf, 0.02 - 0.03 mg / kg / day of colchicine should be given, up to a maximum daily dosage of 1.82.0 mg / day. since colchicine treatment is often complicated by frequent gastrointestinal side effects, some experts recommend lactose - free diet in order to improve colchicine tolerance. colchicine therapy for fmf during pregnancy has not been reported to harm either the mother or her fetus. contraindications to colchicine will include hypersensitivity to any component of its formulation and severe renal or hepatic impairments, requiring a cautious use in the elderly with renal, liver, or biliary disease. nsaids and corticosteroids, sometimes at high doses, rarely achieve satisfying clinical to control the disease. valid therapeutic alternatives in patients who fail to respond to colchicine include il-1 inhibitors (anakinra, canakinumab, and rilonacept) and anti - tnf- agents (adalimumab, etanercept, and infliximab). traps (omim 142680) is an autosomal dominant disease caused by prevalently missense mutations in the tnfrsf1a gene, made up of 10 exons encoding for the p55 1a receptor of tnf (tnfr1a): the vast majority of mutations are found on exons 2, 3, 4, and 6 , and they can be distinguished as high- or low - penetrance ones. the former are located in cysteine - rich n - terminal domains, fundamental for the assembly of the receptor's three - dimensional structure , and they are characterized by early disease onset and more severe clinical manifestations; the low - penetrance mutations, such as r92q and p46l, tend to be associated with onset of disease in adulthood and less pronounced or atypical clinical characteristics. although the biological alteration involves the tnf receptor, the pathogenesis of traps also seems to be associated with a dysregulation in the secretion of il-1 and il-6, as well as oxidative damage correlated with the mitochondrial production of free radicals. clinically speaking (table 2), patients complain of inflammatory attacks of extremely variable duration and intensity (from 1 - 2 days to 3 - 4 weeks), characterized by fever episodes accompanied more or less constantly by sterile peritonitis with abdominal pain, diarrhea or constipation, nausea, and vomiting. mono- or bilateral periorbital edema is a very characteristic and almost pathognomonic sign of the disease, often associated with conjunctivitis and periorbital pain. also very frequent are arthralgias, muscle cramps, and/or centrifugally spreading migratory myalgias and chronic fasciitis. muscular symptoms may include edema and swelling of the muscular group involved, usually localized, while skin symptoms mostly include a serpiginous rash consisting of migratory and painful patches, histologically characterized by the presence of perivascular lymphocytic and monocytic infiltrates. serous membrane inflammation is also common, usually in the form of polyserositis. pericardial or myocardial involvement has also been reported as the only clinical manifestation of traps. during acute episodes, and sometimes also in asymptomatic periods, there is a marked increase in phlogosis indicators (esr, crp, and saa), as well as neutrophil leukocytes, aptoglobin, fibrinogen, and platelets. in up to 25% of patients carrying mutations involving cysteine residues and in about 2% of those carrying low - penetrance mutations, therefore, proteinuria and saa serum levels must be constantly monitored to avoid overlooking an occult subclinical amyloidosis and its progression towards end - stage kidney damage, which is the most dreaded complication of the disease. diagnosis requires the identification of a mutation in the tnfrsf1a: thus, for patients with clinical symptoms that lead to the suspicion of traps, genetic tests are indispensible. from a therapeutic point of view , high doses of nsaids and corticosteroids may prove useful during acute phases, though they do not reduce the frequency of attacks and furthermore do not prevent amyloidosis. in addition, when administered for long periods of time, high - dose corticosteroids can cause serious systemic side effects. colchicine and immunomodulating or immunosuppressant agents have also been proven to have very little efficacy in traps. due to the genetic defect at the origin of the pathology, it was clear that the use of anti - tnf agents could have an important effect in these patients. in fact, etanercept has been proven to be useful in reducing the intensity and duration of acute attacks, although in some cases it gradually loses efficacy. infliximab and adalimumab, by contrast, for reasons only partially understood, may, paradoxically, evoke typical acute inflammatory attacks of the disease. treatment with anti - il-1 agents, on the other hand, has been proven to be particularly efficacious in preventing attacks and inducing a rapid and long - lasting remission of the disease, as well as in the prevention and even regression of amyloidosis. recently , the il-6 receptor antagonist tocilizumab has been used in etanercept- and anakinra - resistant patients with good , suggesting a possible role of il-6 in the pathogenesis of traps. cryopyrin - associated periodic syndromes are a group of autoinflammatory diseases transmitted by autosomal dominant inheritance caused by mutations in the nlrp3 gene (also called cias1 or pypaf) encoding for cryopyrin, a crucial inflammasome protein that directly activates il-1. to date, more than 90 nlrp3 gene mutations have been identified, most of them in exon 3. these mutations induce an imbalance in il-1 production, leading to fever attacks associated with other multiple inflammatory symptoms (table 1). the least severe is familial cold autoinflammatory syndrome (fcas) (omim 120100). muckle - wells syndrome (mws) (omim 191900) is the clinical phenotype of medium severity. finally, chronic infantile neurological cutaneous articular (cinca) syndrome (omim 607115), also known as nomid (neonatal - onset multisystem inflammatory disease), presents a decidedly more severe overall clinical picture. while fcas and mws may be family associated, cinca syndrome due to the seriousness of the clinical phenotype is usually associated only with sporadic mutations. fcas generally appears during the first few months of life and is characterized by brief recurrent inflammatory episodes, usually triggered by generalized exposition to low temperatures or sudden changes in temperature. recently, the possible emergence of a fcas - like phenotype in adult patients or carriers of low - penetrance mutations has been described. symptoms include fever, urticaria - like rash that responds poorly to antihistamines, conjunctivitis, headache, arthralgia and/or arthritis, and fatigue. generally, inflammatory attacks in fcas decrease spontaneously, and an increase in acute - phase phlogistic indicators is usually seen during acute episodes. progress to amyloidosis is rather rare in patients with fcas, in contrast with other caps. mws is characterized by a variable clinical progression, with an episodic - recurrent or chronic pattern, and early childhood onset, usually in the first months of life. in addition to the symptoms typical of fcas, patients often also manifest episcleritis, neurosensorial deafness, and secondary amyloidosis in up to 25% of cases. finally, cinca syndrome, the most severe of the caps, appears in the first weeks of life, being characterized by widespread nonpruritic urticaria - like skin rash. in addition to the manifestations seen in fcas and mws, cinca syndrome may also manifest with uveitis, papilledema, optic nerve atrophy leading to blindness, cerebral atrophy, mental retardation, increased intracranial pressure, ventriculomegaly, chronic aseptic meningitis, and, finally, a characteristic deforming osteoarthropathy of the large joints and hypertrophy of growth plates. many patients present a typical facies characterized by prominent frontal eminences, saddle nose, and hypoplasia of facial bones. also in cinca syndrome, there is a risk of amyloidosis with frequent progressive kidney involvement. in addition, from the laboratory point of view, all caps are characterized by persistent elevated neutrophil leukocytosis, increased acute - phase proteins, and chronic anemia. on the basis of etiopathogenetic mechanisms rooted in overproduction of il-1, caps have been treated with anti - il-1 agents: anakinra was the first drug utilized in these patients, with exciting from the neurological point of view as well. the safety and tolerability of rilonacept have been demonstrated in a group of pediatric and adult caps patients, while canakinumab has been shown to be safe and effective both in controlling clinical / laboratory indicators of disease activity and in controlling amyloidosis - related complications. also known as hyperimmunoglobulinemia d syndrome, mkd (omim 260920) is an autosomal recessive disease caused by mutations in the mvk gene (table 1), encoding for the enzyme mevalonate kinase, involved in the atp - dependent phosphorylation of mevalonic acid into 5-phosphomevalonate. the most frequently found mutations are v377i, i268 t, h20p / n, and p167l, at least one of which is found in 71.5% of patients: they are responsible for reduced mevalonate kinase activity, which leads to overproduction of proinflammatory isoprenoids, reduced synthesis of cholesterol, and accumulation of mevalonic acid in plasma and urine. the disease onset usually occurs during early childhood, generally within the first year of life, or in any case within the first 5 years. the emergence of symptoms after 5 years of age automatically excludes a diagnosis of mkd. acute episodes generally occur every 46 weeks and last about 37 days on average, with asymptomatic periods between attacks. the main clinical manifestations are recurrent fever (above 38.5c), headache, mouth ulcers, abdominal pain, vomiting, and/or diarrhea (table 2). more than 60% of patients may present with joint involvement in the form of arthralgia and/or arthritis, especially affecting large joints. during acute episodes, a nonspecific maculopapular rash may appear, while urticaria, erythema nodosum, and purpura are less frequently reported. attacks are generally more frequent during childhood and adolescence, but the disease may persist into adulthood in more than half of patients. amyloidosis may be present in a smaller number of patients in comparison with other maiss, estimated at around 3% of cases. the possibility of macrophage activation syndrome during the course of an inflammatory attack has been observed in a patient with mkd. a closely related disease is mevalonic aciduria (omim 610.377), which is due to a near - total inactivity of the enzyme mevalonate kinase: in this condition, recurrent fever episodes appear in association with serious systemic signs, such as delayed growth, cranial - facial dysmorphism, microcephalia, cerebellar atrophy, ataxia, psychomotor retardation, retinal dystrophy, and cataracts. in terms of laboratory findings , mkd is invariably marked by leukocytosis and elevated phlogosis indicators during fever attacks, while many patients show increased serum igd concentration (with levels above 100 iu / ml) and, less frequently, serum iga between fever attacks. urinary concentration of mevalonic acid may be increased during acute febrile flares and may thus sometimes be useful to the diagnosis. however, genetic testing to evaluate the mvk gene remains essential for a definite confirmation of mkd. in terms of therapy , nsaids and corticosteroids may bring about partial relief of symptoms. statins, in particular, simvastatin, seem efficacious in reducing the duration of acute episodes. the rationale behind their utilization is based on an attempt to reduce production of mevalonic acid by blocking the enzyme 3-hydroxy-3-methylglutaryl - coenzyme a reductase. in resistant cases, treatment with anti - il-1 and anti - tnf drugs has been proven to reduce both the frequency and the intensity of inflammatory attacks. this is an autosomal dominant disease caused by mutations in the nlrp12 gene encoding for the protein nlrp12 (or monarch-1), which plays a crucial role in immune system mechanisms against pathogenic agents (table 1). as in the case of caps , it can be induced by generalized exposure to cold and is characterized by recurrent fever episodes lasting for 510 days accompanied by skin rash, headache, lymphadenopathy, mouth ulcers, and abdominal pain. treatment choice depends on the seriousness of the overall clinical picture and is based on the use of antihistamines, nsaids, and corticosteroids in less serious cases or the administration of anakinra in more serious ones. however, loss of efficacy of anakinra has been described in a few patients , raising the possibility of using anti - tnf- and anti - il-6 agents. granulomatous autoinflammatory diseases include blau syndrome (bs, omim 186580) and early - onset sarcoidosis (eos, omim 609464); both are caused by mutations in the nod2/card15 gene, with subsequent dysregulation of the inflammatory response and formation of noncaseous granulomas (table 1). blau syndrome is an autosomal dominant granulomatous inflammatory disease caused by mutations in the region encoding for the nucleotide - binding domain region of the nod2/card15 gene (table 1): the protein nod2 is mainly expressed in monocytes and plays a crucial role in the clearance of bacteria, particularly, mycobacterium tuberculosis, as it is capable of interacting with peptidoglycan and activating the nf-b signal route. the most frequently observed mutations are missense substitutions involving arginine residue at position 334 within the nod2/card15 gene (r334w or r334q). the onset is generally in childhood, around the age of 5, and the disease affects joints, skin, and eyes: the most common manifestation is a symmetrical polyarthritis of hands, feet, wrists, elbows, and ankles, which can also lead to joint ankylosis. skin lesions may be in the form of dark red macular - papular - nodular rash or lichenoid - like lesions, which are generally symmetrical and appear on the trunk and/or limbs. under histological examination, the skin lesions present noncaseous granulomas with gigantic multinuclear cells. eye involvement is the most serious complication of bs and is manifested in the form of recurrent bilateral anterior uveitis or bilateral granulomatous panuveitis, associated with eye pain, photophobia, and blurred vision. ocular inflammation often leads to chorioretinitis, keratopathy, cataracts, glaucoma, or retinal detachment and may also involve other ocular structures such as conjunctiva, tear ducts, retina, and optic nerve. additionally, bs has been described in association with persistent or intermittent fevers, granulomatous arteritis, cranial neuropathies, and hearing loss. the familial form of bs can be differentiated from eos, a multiorgan sporadic disease characterized by onset in the first 4 years of life, joint, skin, eye, lymph node involvement, and recurrent fevers, with possible abdominal or central nervous system involvement. from the histological point of view, the presence of noncaseous epithelioid granulomas is observed in the involved tissues. pulmonary parenchyma, involved in more than 90% of patients with adult sarcoidosis, is usually spared in eos. in spite of the notable clinical similarities, later, genetic analyses demonstrated that many patients with eos also presented with mutations in the nod2/card15 gene. for this reason, some authors have proposed that bs and eos are, respectively, familial and sporadic forms of the same disease. sporadic bs in that they are carriers of mutations in the nod2/card15 gene and limiting diagnosis of eos to those pediatric patients with sarcoidosis but no mutations. high doses of corticosteroids may be utilized with variable success. the use of anti - tnf- and anti - il-1 biological agents also seems encouraging. the hereditary pyogenic disorders include papa (pyogenic arthritis, pyoderma gangrenosum, and acne) syndrome, majeed syndrome (ms), and deficiency of the il-1 receptor antagonist (dira): all of these disorders are characterized by the presence of sterile abscesses that mainly affect the skin, joints, and bones. papa syndrome (omim 604416) is an autosomal dominant disease caused by mutations in the pstpip1 gene encoding for cd2-binding protein-1 (cd2bp1), involved in the proper assembly of the cytoskeleton, which normally inhibits pyrin - mediated inflammatory signals and the activation of caspase-1. it appears in early childhood and is characterized by joint involvement, manifested with severe self - limiting pyogenic arthritis. in terms of skin involvement, the appearance of pyoderma gangrenosum and nodular - cystic acne is described in early childhood. arthritic episodes usually respond readily to treatment with corticosteroids, while pyoderma gangrenosum is treated with topical immunosuppressant drugs. in a few reports, patients with papa syndrome responded wonderfully to treatment with anti - tnf- and anti - il-1 agents. majeed syndrome (omim 609628) is a very rare autosomal recessive disease described for the first and only time in 1989 in two brothers and a cousin with childhood - onset recurrent chronic multifocal osteomyelitis and congenital dyserythropoietic anemia; neutrophilic dermatosis was also reported in the two brothers. the disease is caused by homozygous mutations in the lpin2 gene (table 1) encoding for lipin 2, the role of which has not yet been clarified. clinically, this syndrome is characterized by recurrent fever attacks associated with multifocal sterile osteomyelitis, dyserythropoietic anemia, and chronic diffuse neutrophilic dermatosis with onset in early childhood. its treatment is empirically based on the use of nsaids and corticosteroids, although excellent have recently been described with administration of anakinra and canakinumab. dira (omim 612852) is an autosomal recessive disease caused by missense mutations in the il1rn gene encoding for the il-1 receptor antagonist 1; there is a lack of endogenous self - regulation of il-1 activity, with consequent excessive proinflammatory action of il-1 itself (table 1). disease onset is in the first weeks of life, and, in the initial phases, it may mimic neonatal sepsis, with multifocal osteomyelitis, periostitis, pustular skin lesions of various sizes, skin, ungueal alterations, hepatosplenomegaly, and the risk of multiorgan failure. radiological findings may include signs of osteolytic lesions, bone sclerosis, enlargement of the epiphysis of the long bones, and periosteal reaction. due to the absence of endogenous il-1 receptor antagonist, treatment is based on the use of anakinra, bringing about an excellent clinical improvement in a few days or weeks. thus, in , the elucidation of the molecular basis of maiss has helped us recognize the consequences of excessive il-1 signaling, proinflammatory isoprenoid production, or aberrant nk-b activation (figure 1). future studies will hopefully also evaluate the clinical benefit of different highly selective biologicals for each of the maiss: the availability of these new therapeutic options for patients who have previously failed to respond to conventional treatments (nsaids, corticosteroids, colchicines, or immunomodulating agents) and the promise of patient - centered treatment strategies are doubtlessly the start of a new era in the management of these rare complex disorders.

monogenic autoinflammatory syndromes (maiss) are caused by innate immune system dysregulation leading to aberrant inflammasome activation and episodes of fever and involvement of skin, serous membranes, eyes, joints, gastrointestinal tract, and nervous system, predominantly with a childhood onset. to date, there are twelve known maiss: familial mediterranean fever, tumor necrosis factor receptor - associated periodic syndrome, familial cold urticaria syndrome, muckle - wells syndrome, cinca syndrome, mevalonate kinase deficiency, nlrp12-associated autoinflammatory disorder, blau syndrome, early - onset sarcoidosis, papa syndrome, majeed syndrome, and deficiency of the interleukin-1 receptor antagonist. each of these conditions may manifest itself with more or less severe inflammatory symptoms of variable duration and frequency, associated with findings of increased inflammatory parameters in laboratory investigation. the purpose of this paper is to describe the main genetic, clinical, and therapeutic aspects of maiss and their most recent classification with the ultimate goal of increasing awareness of autoinflammation among various internal medicine specialists.

brain tumors located in the suprasellar region are frequently found in childhood, and include craniopharyngioma, germinoma, optic nerve glioma and histiocytosis. following the extension of the tumor or intervening procedures, such as surgery, chemotherapy and radiation, considerable damage to the hypothalamic pituitary axis is almost inevitable. accordingly, most children with suprasellar tumors suffer from endocrinological sequelae, even after the original lesion has been cured. in addition, these children have been shown to have a high risk of developing morbid obesity, which has recently been referred to as hypothalamic obesity. in this report , we studied the prevalence of obesity among children with suprasellar tumors who had been treated at our institute. additionally, we evaluated metabolic aberrations in these patients, and tried to correlate them with clinical features and endocrinological alterations of the patients. patients with remitted suprasellar tumors, who had been diagnosed and treated at our institute and had attended our endocrinological unit more than once from april 2005 to april 2006, were enrolled in this study. those with rathke s cleft cyst or hypophysitis were excluded. in addition, a 28-yr - old male with craniopharyngioma, who had been subjected to long - term bed rest because of a serious neurological sequela, was also excluded. as depicted in table 1table 1 profiles of 23 patients with remitted suprasellar tumors, a total of 23 patients (10 males and 13 females) met the enrollment criteria, including 10 patients with craniopharyngioma (43%), 7 patients with germinoma (30%), 4 with optic nerve glioma (17%), and single cases of infundibuloma and langerhans cell histiocytosis. the current ages of the patients were distributed from 3 to 22 yr old, the median of which was 14 yr old. all craniopharyngioma patients had near - total or total tumor removal for the first line treatment, followed by additional radiotherapy or chemotherapy in cases of incomplete resection or tumor recurrence. some cases were forced to undergo repetitive operations. in patients with germinoma and optic nerve glioma, radiotherapy and chemotherapy were the mainstay of the treatment, following biopsy or partial resection. one case of germinoma with syncytiotrophoblastic giant cells underwent autologous peripheral stem cell transplantation. with each patient, a retrospective review regarding the contents of therapies and the extent of endocrinological dysfunction was carried out. for the assessment of metabolic alteration, the degree of overweight (% -overweight) was determined and body fat mass was estimated for each patient. the percent overweight was expressed as the quotient of the excess weight to the ideal weight. in female patients less than 15 yr of age and males less than 18, ideal weight was based on the auxological data from normal japanese children. for the remaining patients, the weight compatible with a body mass index of 22 body fat mass was measured by whole - body bioelectrical impedance analysis (tbf-310, tanita corp . in addition, blood pressure, total cholesterol level, triglyceride ( tg) level and the homeostatic model assessment of insulin resistance (homa - r : fasting iri ( u / ml) fasting glucose (mg / dl)/405 ) was determined. the diagnosis of gh deficiency followed the consensus guideline of the growth hormone research society as well as the national guidelines for the diagnosis of gh deficiency. in brief, the gh responses to exogenous gh secretagogues less than 3 ng / ml indicated severe gh deficiency (sghd) whereas gh responses between 36 ng / ml were considered to be moderate gh deficiency (mghd). obesity was defined according to the criteria made by national experts as follows: more than 20% overweight and/or % -body fat exceeding 25% in males, 30% in females younger than 11 yr, and 35% in females older than 11 yr. hyperlipemia was defined as either an elevated fasting total cholesterol level above 220 mg / dl or fasting tg level above 140 mg / dl. presence of insulin resistance was suggested when homa - r exceeded 2.5, according to the guidelines from the japan diabetes society. statistical analysis was performed using ystat2000.xls. for the comparison among groups according to tumor pathology, the yates -test and non - parametric statistical methods (kruskal wallis - h - test) were applied because of the relatively small samples and skewed distribution of the data. for the comparison between groups with or without gh administration, the yates -test or fisher s test was applied as necessary. table 2table 2 endocrinological status of 23 patients shows the endocrinological status of the patients at the time of study. all patients with craniopharyngioma suffered from panhypopituitarism with diabetes insipidus except case 1, in whom the tumor was identified by chance through evaluation for head injury. all patients with germinoma also had more than one defect of the pituitary hormones. on the other hand, none of the patients with optic nerve glioma showed adrenal insufficiency or diabetes insipidus, but they had been treated for precocious puberty. the condition of so - called growth without gh, normal or accelerated linear growth despite gh deficiency, was observed in 6 patients. of these , 3 patients had chosen to take gh supplementation, expecting the effect to improve their metabolic aspects. in table 3table 3 metabolic status of 23 patients, the of investigation for the metabolic aspects of the patients are summarized. obesity, judged from either the % -overweight or % -body fat, was found in 12 out of 23 patients (7 males, 5 females). fasting blood samples in the morning could be obtained in all but one patient. in those samples, an elevated total cholesterol level (> 220 mg / dl) was identified in 4 patients. elevated tg (> 140 mg / dl) was found in 6 patients, including 2 patients (cases 5 & 13) who exhibited strikingly elevated postprandial tg levels of more than 1,200 mg / dl. in addition, another patient (case 16) also had postprandial hypertriglyceridemia, as much as 1,029 mg / dl, despite a normal fasting tg level. homa - r exceeding 2.5 was found in 7 patients (3 males, 4 females) including one (case 9) whose fasting glucose was 101 mg / dl. collectively, out of 23 patients, 16 patients had at least one metabolic aberration, such as obesity, hyperlipemia and insulin resistance, which accounted for two thirds of all the patients. of these, obesity was most frequently found, and 9 out of 12 obese patients had either accompanying hyperlipemia or insulin resistance. the relationship between obesity, hyperlipemia and insulin resistance is summarized in table 4table 4 summary of the relationship between obesity, hyperlipemia and insulin resistance (n=22), with categorization according to the tumor pathology. among the 3 major groups of tumor pathology (craniopharyngioma, germinoma and optic nerve glioma), the incidence of obesity was not significantly different: 60% craniopharyngioma, 43% germinoma and 75% in optic nerve glioma (p=0.90). in addition, the % -overweight in each group did not differ significantly (p=0.66). hyperlipemia was most frequently observed in germinoma (71%), followed by craniopharyngioma (30%), and optic nerve glioma (0%). this order was reversed in insulin resistance: optic nerve glioma 75%, craniopharyngioma 30% and germinoma 14%. these differences showed no statistical significance (p=0.22 and p=0.38, respectively). among the gh deficient patients, 11 patients were on gh replacement therapy, whereas 11 patients were not at the time of evaluation. in each group, likewise, the incidences of hyperlipemia and insulin resistance were not significantly different (3/11 vs. 6/11, p=0.39 ; 4/11 vs. 2/11, p=0.64, respectively). these did not change fundamentally, when 2 patients with previous gh usage were added to the gh treatment group. the dose of supplemented hydrocortisone (mg / m / day) did not correlate with the degree of overweight, as shown in the fig. 1 the relationship between supplemented hydrocortisone doses (mg / m / day) and the degree of overweight (%) in patients with acth deficiency.. the relationship between supplemented hydrocortisone doses (mg / m / day) and the degree of overweight (%) in patients with acth deficiency. all 6 patients with growth without gh had at least one metabolic aberration: obesity in 4, hyperlipemia in 5 and insulin resistance in 3. conversely, all 3 patients with combined obesity, hyperlipemia and insulin resistance had accompanying growth without gh. table 2table 2 endocrinological status of 23 patients shows the endocrinological status of the patients at the time of study. all patients with craniopharyngioma suffered from panhypopituitarism with diabetes insipidus except case 1, in whom the tumor was identified by chance through evaluation for head injury. all patients with germinoma also had more than one defect of the pituitary hormones. on the other hand, none of the patients with optic nerve glioma showed adrenal insufficiency or diabetes insipidus, but they had been treated for precocious puberty. the condition of so - called growth without gh, normal or accelerated linear growth despite gh deficiency, was observed in 6 patients. of these , 3 patients had chosen to take gh supplementation, expecting the effect to improve their metabolic aspects. in table 3table 3 metabolic status of 23 patients, the of investigation for the metabolic aspects of the patients are summarized. obesity, judged from either the % -overweight or % -body fat, was found in 12 out of 23 patients (7 males, 5 females). no patients were hypertensive at the time of investigation. fasting blood samples in the morning an elevated total cholesterol level (> 220 mg / dl) was identified in 4 patients. elevated tg (> 140 mg / dl) was found in 6 patients, including 2 patients (cases 5 & 13) who exhibited strikingly elevated postprandial tg levels of more than 1,200 mg / dl. in addition, another patient (case 16) also had postprandial hypertriglyceridemia, as much as 1,029 mg / dl, despite a normal fasting tg level. homa - r exceeding 2.5 was found in 7 patients (3 males, 4 females) including one (case 9) whose fasting glucose was 101 mg / dl. collectively, out of 23 patients, 16 patients had at least one metabolic aberration, such as obesity, hyperlipemia and insulin resistance, which accounted for two thirds of all the patients. of these, obesity was most frequently found, and 9 out of 12 obese patients had either accompanying hyperlipemia or insulin resistance. the relationship between obesity, hyperlipemia and insulin resistance is summarized in table 4table 4 summary of the relationship between obesity, hyperlipemia and insulin resistance (n=22), with categorization according to the tumor pathology. among the 3 major groups of tumor pathology (craniopharyngioma, germinoma and optic nerve glioma), the incidence of obesity was not significantly different: 60% craniopharyngioma, 43% germinoma and 75% in optic nerve glioma (p=0.90). in addition, the % -overweight in each group did not differ significantly (p=0.66). hyperlipemia was most frequently observed in germinoma (71%), followed by craniopharyngioma (30%), and optic nerve glioma (0%). this order was reversed in insulin resistance: optic nerve glioma 75%, craniopharyngioma 30% and germinoma 14%. these differences showed no statistical significance (p=0.22 and p=0.38, respectively). among the gh deficient patients, 11 patients were on gh replacement therapy, whereas 11 patients were not at the time of evaluation. in each group, likewise, the incidences of hyperlipemia and insulin resistance were not significantly different (3/11 vs. 6/11, p=0.39 ; 4/11 vs. 2/11, p=0.64, respectively). these did not change fundamentally, when 2 patients with previous gh usage were added to the gh treatment group. the dose of supplemented hydrocortisone (mg / m / day) did not correlate with the degree of overweight, as shown in the fig. 1 the relationship between supplemented hydrocortisone doses (mg / m / day) and the degree of overweight (%) in patients with acth deficiency.. the relationship between supplemented hydrocortisone doses (mg / m / day) and the degree of overweight (%) in patients with acth deficiency. all 6 patients with growth without gh had at least one metabolic aberration: obesity in 4, hyperlipemia in 5 and insulin resistance in 3. conversely, all 3 patients with combined obesity, hyperlipemia and insulin resistance had accompanying growth without gh. hypothalamic obesity refers to intractable weight gain following hypothalamic damage, which has been described in both children and adults. this complication has been most frequently observed after treatment for craniopharyngioma, accounting for 50 to 70% of children with craniopharyngioma. dietary modification tends to fail, leaving these patients at high risk of developing morbid obesity. because obesity is a primary risk factor for morbidity in the general population, hypothalamic obesity may be a serious complication both in physical fitness and in psychological aspects. excessive hunger due to a disturbed satiety center was once postulated. however, it was demonstrated that the energy intake in children with hypothalamic obesity is not always increased. insulin hypersecretion from pancreatic beta cells ing from diminished vagal tone due to a damaged ventromedical hypothalamus is a promising hypothesis. in addition, increased 11-beta - hydroxysteroid dehydrogenase activity was demonstrated in hypothalamic obesity patients, which may play an additional role in its pathogenesis. in assessing obesity, three patients (case 8, 14, 19) showed adequate weight despite their increased % -body fat, highlighting the importance of measuring the fat mass in addition to anthropometric data. it may be more appropriate to evaluate the visceral fat mass directly by ct scan. the incidence of obesity was as high as 52%, a figure that is in accordance with previous reports. the identified risk factors for developing hypothalamic obesity include the presence of any endocrinopathy, tumor infiltration into the hypothalamus, irradiation to the hypothalamus greater than 51 gy, and a younger age at diagnosis (4, 18). in our series, most of the craniopharyngiomas extended to the hypothalamus, making post - surgical panhypopituitarism inevitable. on the other hand, many patients with germinoma or optic nerve glioma received high - dose irradiation, which caused multiple pituitary hormone deficiency. these factors may have contributed to the high incidence of obesity in our series. of note, obese patients with optic nerve glioma lacked diabetes insipidus, suggesting that the presence of diabetes insipidus is not always necessary for the development of hypothalamic obesity. gh supplementation, either previous or current, did not decrease the incidence of obesity. this finding is in accordance with the report from the kigs database in which gh treatment for 3 yr did not influence the body mass index of craniopharyngioma patients. the hydrocortisone dosage, given to patients with acth deficiency, was merely physiological, and it showed no relationship with obesity. these findings imply that endocrinological management (gh supplementation or hydrocortisone reduction) is not capable of ameliorating obesity. about one third of the patients were estimated to have insulin resistance by homa - r. visceral fat accumulation and an elevated leptin level have been demonstrated in hypothalamic obesity, which elucidates the high prevalence of insulin resistance in our series. as mentioned above, insulin hypersecretion ing from hypothalamic damage was thought to be the main cause of hypothalamic obesity. however, it has not been clarified whether insulin hypersecretion precedes and causes visceral fat accumulation or whether hypothalamic damage directly increases the visceral fat mass. in any case, insulin secretion must be enhanced to compensate for insulin resistance, which would further increases the visceral fat accumulation. therefore, once this cycle is established, progressive deterioration in metabolism may be inevitable. the effectiveness of somatostatin - analogue, which inhibits insulin secretion, has been reported in the reduction of body weight in hypothalamic obesity children. considering the prevalence of insulin resistance, this agent seems promising in our series, especially those with high iri levels. hyperlipemia, either hypercholesterolemia or hypertriglyceridemia, was found in 9 patients out of 21 tested. in particular, inordinately elevated postprandial tg was found in 3 patients. the contribution of a genetic factor may be minimal in them, because preoperative examinations showed normal tg levels, and neither of their parents had elevated tg levels. reported that 6 craniopharyngioma patients had higher tg levels than the control obese group . in their report our finding underlines the importance of evaluating the lipid metabolism in hypothalamic obesity, in particular a high tg level. gh deficiency must be responsible, because untreated children, adolescents and adults with gh deficiency have been found to have elevated tg levels. however, gh deficiency alone seems insufficient to elucidate the extremely elevated postprandial tg values. effort should be focused on delineating tg metabolism in hypothalamic obesity, unraveling the mechanism and establishing a pertinent treatment. in our series, hyperlipemia was frequently found in germinoma patients, while the incidence of insulin resistance was high in optic nerve glioma. we could not explain this difference, and it may be due to the small sample size. another notable issue we found was that patients with growth without gh tended to have profound metabolic alteration. this condition has been encountered mainly in children following surgery for suprasellar tumors. in addition, insulin hypersecretion has been considered to be a major cause of growth without gh. thus, both hypothalamic obesity and growth without gh arise following the hypothalamic damage, and both conditions are closely related to insulin metabolism. we showed that the patients with growth without gh had increased incidences of obesity (67%), hyperlipemia (83%) and insulin resistance (50%), compared to the whole study group, and patients presenting with growth without gh may be regarded as at particularly increased risk for metabolic aberration. it has been demonstrated that hyperinsulinemia is not always present in growth without gh patients, which was also true in our series. this finding indicates that some additional factor(s) other than insulin hypersecretion must be involved in the development of growth without gh. this unknown factor(s) may provide the reason for the high prevalence of metabolic aberration in growth without gh. half of the patients with suprasellar tumors manifested obesity after the completion of therapy, irrespective of its pathohistology and gh treatment. a high incidence of insulin resistance and/or hyperlipemia, including inordinately elevated postprandial tg, was also found. in growth without gh cases,

weight gain is a common sequela of suprasellar tumors, referred to as hypothalamic obesity. we undertook an evaluation of obesity and metabolic aberrations among patients treated at our institute. during the 12 mo from apr. 2005, 23 patients (10 males and 13 females) with remitted suprasellar tumors attended our clinic: 10 patients with craniopharyngioma, 7 with germinoma, 4 with optic nerve glioma and others. of these, 12 patients (52%) were found to have obesity on the basis of percent overweight and/or percent body fat. elevated cholesterol and/or triglyceride (tg) was found in 9 patients (39%), and insulin resistance was suspected in 7 patients (30%). three patients exhibited strikingly elevated postprandial tg levels. all 6 patients with the growth without gh phenomenon had at least one metabolic aberration. in , the prevalence of hypothalamic obesity was nearly half in our series, and hyperlipemia and insulin resistance were also frequently found. the increased risk for metabolic aberration in growth without gh patients was suggested.

mechanical valve thrombosis (mvt) may cause valve dysfunction, and its onset may be acute or gradual, according to the nature of thrombi (rapid or slow growth) and involvement of hinge point (abrupt or insidious). edmunds defined mechanical valve thrombosis as a complication attributable to any type of thrombus, without evidence of infection, attached around a mechanical valve, deranging hemodynamics, or interfering with valvular function. the risk of valve thrombosis is highest in the tricuspid position, with reported rates as high as 20%, while the incidence of left side valve thrombosis varies from 4% to 8.6% within 5 yr of initial valve replacement. eleven - year probability of prosthetic valve thrombosis has been reported to be 0.01% to 0.02% in the mitral or aortic position, and tilting disc valves are most commonly reported in association with this complication. detection of valve thrombosis may be easy or difficult, and if valve thrombosis is suspected, transthoracic echocardiographic evaluation can be used to confirm the diagnosis. transesophageal echocardiographic examination is often necessary in the mitral position because of the thrombi involvement along the left atrial side of the sewing ring. several factors such as inadequate anticoagulation, drug interaction, pannus overgrowth, and pregnancy have been suggested to promote valve thrombosis. this analysis was undertaken to evaluate the risk factors and outcome of mechanical valve thrombosis in patients who had undergone valve replacement. between january 1981 and march 2006, 2,908 mechanical valve replacements were performed in 2,298 patients. among these hospital records were reviewed retrospectively, including; preoperative clinical findings, initial valve procedures and etiologies, any type of event during follow - up or anticoagulation, diagnostic methods and characteristics of valve thrombosis, perioperative management, and outcomes. several factors were evaluated to identify risk factors of mvt development; including sex, valve location, and number of valves replaced. twenty patients presented with mvt, 6 (30%) men and 14 (70%) women. valve replacement involved a single valve in 13 patients (65%), and double valve replacement was done in 7 patients (35%) (table 1). one patient had a history of cerebral embolism and another patient had a history of unilateral lower extremity edema. left atrial appendage resection was performed in 4 (20%), and the cox - maze procedure in 4 (20%). the mean postoperative left ventricular ejection fraction was 579.3%, and the mean initial hospital stay was 16.96.7 days. anticoagulation (heparin and warfarin) was started 24 to 48 hr after all valvular procedures. patients were discharged with an international normalized ratio (inr) level of 2.0 to 2.5 for mitral and aortic valve replacement cases and of 2.5 to 3.0 for tricuspid valve replacement cases. the mean age at mvt diagnosis was 42.014.0 yr. the distribution of mvts according to the site of valve implantation was as follows: 14 (70%) mitral, 3 (15%) tricuspid, 2 (10%) aortic, and 1 (5%) tricuspid / aortic. among double - valve prosthesis recipients, mvts were in a mitral prosthesis in 3, an aortic prosthesis in 3, and in a tricuspid prosthesis in 2 the mean time from first valve replacement to prosthetic valve thrombosis was 121.875.4 (0.9 - 284.7, median 96.0)months. the mean time between the diagnosis ofmvt and surgical treatment was 22.342.5 days (range,0 - 180 days). symptoms at presentation were dyspnea in 12 (60%) patients, cardiogenic shock in 1 (5%) patient, and miscellaneous symptoms (myalgia, cough, or fever) in 2 (10%) patients (table 2). in five patients, the majority of patients (11, 55%) with mvt were of nyha functional class iii or iv at the time of diagnosis. the mean duration between symptom onset and surgical treatment was 87.8121.7 days (range, 0 - 365 days). thromboembolism (stroke, limb or organ ischemia) occurred between initial valve replacement and valve thrombosis in 2 (10%) patients. mvt was diagnosed by transthoracic echocardiography (tte) in 13 (65%) patients, and by transesophageal echocardiography (tee) in 7 (35%) patients. inr profiles when thrombosis was diagnosed were 1.660.64 (1.02 - 2.68) for left - sided valves and 1.420.53 (1.07 - 2.03) for tricuspid valves. mvt occurred during pregnancy in 7 (35%) patients, and of these, two patients had a history of denying warfarin for fear of possible embryopathy. one patient experienced thrombosis at full term and underwent valve replacement, 2 days later she had a normal full term baby without any adverse event. poor compliance with anticoagulant treatment leading to a lower inr than recommended was responsible for mvt in another 5 (25%) patients. adding two patients with pregnancy, poor warfarin compliance was responsible for 7 patients (35%). these patients skipped warfarin for no distinct reason, and their compliances with treatment and medication were poor. in 2 (10%) patients mvt was thought to be related to a drug interaction, i.e., one received medication for a common cold 1 week before thrombosis detection and the other patient had consumed nutritional supplements with or without medication. one patient with a bjork - shiley convexo - concave tilting disc valve did not take any warfarin probably due to poor compliance. he did not take medications for 285 months, and we could not follow him up. he presented at out clinic due to cold sweating and dyspnea 3 weeks before a diagnosis of mvt. pannus formation was present with thrombus at the time of operation in 45% (9/20) of the patients. two (10%) of the 20 received streptokinase - based thrombolytic therapy, but it was not successful. one patient had acute mitral valve thrombosis, and due to hemodynamic instability, the patient received thrombolysis. both were treated by surgical intervention and have been in good physical condition until now. all patients underwent surgery under redomedian sternotomy, consisting of mitral valve replacement in 14 patients (70.0%), tricuspid valve replacement in 3 patients (15.0%), aortic valve replacement in 2 (10%), and tricuspid/ aortic valve replacement in one (5%). thrombi were found around valve hinge points and sewing rings, and all valves concerned were replaced other than thrombectomy. only one patient received a tissue valve; she was 65 yr at the time of diagnosis, and she was non - compliant to warfarin with a frequent bleeding history. the aortic cross clamping time averaged 104.942.8 min (range, 52 - 191 min), and the cardiopulmonary bypass time was 164.662.7 min (range, 79 - 286 min). emergent or urgent operation was done in 8 cases (40%) due to heart failure and hemodynamic compromise. early complications were atrial fibrillation, bleeding, vocal cord paralysis, cerebral infarction, atrioventricular conduction block, and pneumonia. oral anticoagulation after surgical treatment consisted of warfarin alone in 16 (16/19, 83%) patients and a combination of warfarin and plavix in 3 (3/19, 17%) patients. the mean hospital stay was 18.518.0 (range, 8 - 89) days, and the mean intensive care unit stay was 3.32.4 days (range, 1 - 9) days. no late mortality or mvt recurrence occurred over 63.349.9 (0.5 - 165.1) months of mean follow - up. our data show that mvt is a very rare complication after valve replacement and that its clinical manifestations are various. the time between initial valve replacement and diagnosis of mvt was 121.875.4 months, and the median was 96.0 months. in most cases, thrombosis occurred insidiously; only one patient presented with acute valve thrombosis one month after valve replacement. reported 39 cases of prosthetic valve thrombosis with a 39.0 42.0-month mean interval of occurrence from initial operation and an in - hospital mortality of 41%. the difference between our series and durrleman's data might be due to different patient populations. some patients might have been lost, and possible unknown sudden death can not be ruled out. clinical course might depend on the type of replaced valves; patients with bileaflet mechanical valves might have had stable hemodynamics due to a well - functioning single leaflet or have had unstable vital signs due to bileaflet involvement. the fact is that the clinical manifestations of mvt are various. during follow - up, we usually suspect valve thrombosis in cases with valve click loss, skipped warfarin, newly appeared symptoms, and so on. in the present study, tte detected valve thrombosis in 75% of our series, and we found incidental valve thrombosis using this modality in five patients during routine echocardiographic follow - ups. in those cases, none had symptoms, and we could not follow with urgent operations. detection of valve thrombosis may be difficult, and some series have reported that as many as 50% of cases are diagnosed postmortem. in a recent study, the delay between first onset of symptoms and hospitalization was found (from 1 to 45 days). previous studies have shown that a delay in diagnosis and a long duration of symptoms in a higher mortality. however, physical examination is an inadequate diagnostic tool and strong clinical suspicion is the most important to detect these complications. any new or worsening symptoms in a patient with mechanical valve warrant a thorough investigation to exclude valve thrombosis. in the present series, mvt was diagnosed by tte in 13 (65%) patients and by transesophageal echocardiography in 7 (35%) patients. during follow - up, we suggest a periodical echocardiographic examination. early detection and diagnosis may often be limited by a progressive or insidious course. the difference of valve thrombosis incidence and the pattern of onset between tilting disc and bileaflet valve is still uncertain. another remarkable point of the present study is that pregnancy and inadequate anticoagulation influenced the occurrence of this complication. buttard and colleagues found inadequate anticoagulation at the time of diagnosis in 45% of their patients: 27% for medical reasons (surgical intervention, neurologic events, or pregnancy) and 17% because of poor drug compliance. a definite relationship has already been established between inadequate anticoagulation and valve thrombosis. anticoagulation during pregnancy is a difficult problem to solve, as pregnancy induces various biophysiologic changes and a hypercoagulable status. the risk of warfarinization during pregnancy is teratogenicity, particularly between the 6th and 12th weeks of gestation. therefore, it is obvious and mandatory to use anticoagulation extremely carefully to prevent valve thrombosis or thromboembolism. our data also demonstrate difficulties in maintaining adequate anticoagulation during pregnancy; however, no safe and reliable protocol for anticoagulation therapy during pregnancy has been established. pannus formation, defined as excessive fibrosis around a prosthetic valve, may occur in 25% of patients as early as the first postoperative month. it plays an important role in the mechanism of obstruction and could be the sole cause of mechanical valve thrombosis occurrence. in the present series, the precise role of pannus in the pathogenesis of valve thrombosis remains unknown. however, whether it caused valve thrombosis or not was still uncertain, so we did not include it as a cause of valve thrombosis. recently, roudaut et al. conducted a retrospective study of 110 patients treated with fibrinolytic therapy and concluded that this treatment should be reserved for selected patients (those with tricuspid thrombosis, critically ill patients, and patients with contraindications to surgical intervention). thrombolysis appears to be useful as a medical bridge to operation and as a major curative modality when a patient's medical condition or hemodynamics is poor. in the present series the present study shows that pregnancy and inadequate anticoagulation probably influenced the occurrence of this rare complication. however, our knowledge of mvt is still inadequate, and more efficient ways of eradicating this complication should be suggested.

in the present study, the authors investigated the management of mechanical valve thrombosis (mvt). from january 1981 through march 2006, 2,908 mechanical valve replacements were performed in 2,298 patients at our institution. twenty (0.87%) patients presented with mvt, 14 (70.0%) were women, and the mean age of the patients was 42.014.0 yr. thrombosis involved mitral in 14 (70.0%), aortic in 2 (10.0%), tricuspid / aortic in 1 (5%), and tricuspid in 3 (15%). the interval from first operation to valve thrombosis was 121.875.4 (0.9 - 284.7) months. the most frequent clinical presentation was heart failure (13/20, 65%), and predisposing causes of mvt were: poor compliance with warfarin, pregnancy, drug interaction, and unknown. all 20 patients underwent valve replacement: mitral (14, 70.0%), tricuspid (3, 15.0%), aortic (2, 10%) and tricuspid / aortic (1, 5%). one early death occurred due to left ventricular failure, but no late mortality occurred during 63.349.9 (0.5 - 165.1) months of follow - up. mvt was treated successfully, and pregnancy and inadequate anticoagulation were found to influence the occurrence of this rare complication.

squamous cell carcinoma (scc) of the thyroid is an extremely rare disease accounting for less than 1% of all thyroid tumors. in the world health organization classification , squamous cell carcinoma thyroid is defined as scc of the thyroid should be composed entirely of tumor cells with squamous differentiation. ,, it is thought to arise from undifferentiated follicular cells, metaplastic follicular epithelium or remnant tissue of the thyroglossal duct. primary scc of the thyroid affects older patients (fifth to sixth decade of life) with rapidly increasing thyroid mass with or without cervical lymphadenopathy. other symptoms include dysphagia, dyspnea and hoarseness of voice due to infiltration of adjacent structures. at the time of diagnosis , these tumors are usually locally advanced with invasion into trachea, esophagus or major vessels. although the outcome is dismal, aggressive surgery along with adjuvant radiotherapy are recommended in the management of this rare and aggressive cancer for optimum outcome. here, we are reporting such a rare case of scc of thyroid who has been treated with palliative radiotherapy. a 70-year - old male patient noticed a rapidly increasing painless mass at the anterior aspect of the left side of neck for 1 month. it was also associated with dysphagia and stridor for 2 weeks. he was a heavy smoker who used to smoke 1 pack of cigarettes / day. there was no history of any previous radiation exposure in the neck. on physical examination, an 11 cm 7 cm firm lobulated mass was found in the left side of the anterior aspect of the neck. contrast enhanced computed tomography scan showed evidence of well - defined large, lobulated heterogeneously enhancing solid cystic lesion measuring 11 cm 8.6 cm 7.4 cm in relation to the left lobe of thyroid gland. the lesion showed multiple thin intervening septae along with few specks of calcification within it. posteriorly lesion was extending up to the vertebral column and inferiorly retrosternally up to the brachiocephalic trunk. mass effect of the lesion was seen in the form of compression and displacement of the trachea toward right and there was also compression and displacement of the left subclavian and carotid vessels, left internal jugular vein and left sternocleidomastoid. on positron emission tomography scan, a large multi lobulated heterogeneously enhancing solid - cystic mass (7.8 cm 7.7 cm 5.8 cm) was seen in the left side of the neck arising from the left lobe of the thyroid gland with intense fluorodeoxyglucose (fdg) avidity (standardized uptake value max 17.7) in the solid component and along the peripheral margin of the cystic component. the mass was extending into the anterior mediastinum until the level of d4 vertebra and posteriorly until the prevertebral fascia. it was seen to displace the trachea to the right and compressing it. contrast enhanced computed tomography scan showed evidence of well - defined large, lobulated heterogeneously enhancing solid cystic lesion measuring 11 cm 8.6 cm 7.4 cm in relation to the left lobe of thyroid gland. the lesion showed multiple thin intervening septae along with few specks of calcification within it. mass effect of the lesion was seen in the form of compression and displacement of the trachea toward right and there was also compression and displacement of the left subclavian and carotid vessels, left internal jugular vein and left sternocleidomastoid positron emission tomography scan revealed a large multi lobulated heterogeneously enhancing solid - cystic mass (7.8 cm 7.7 cm 5.8 cm) was seen in the left side of the neck arising from the left lobe of the thyroid gland with intense fluorodeoxyglucose avidity (standardized uptake value max 17.7) in the solid component and along the peripheral margin of the cystic component fine - needle aspiration cytology (fnac) revealed scattered malignant epithelial cells which showed moderate pleomorphism, high nuclear cytoplasmic ratio, hyperchromatic nuclei and mild to moderate cytoplasm. aggressive treatment in the form of radical surgery was not possible in this particular patient because of its unresectability due to encasement of major vessels. hence, we delivered palliative radiotherapy 30 gy in 10 fractions over 2 week's period. though there was not much regression of the disease, but his pain and shortness of breath was improved reasonably and patient is leading a good quality - of - life for last 1 year. (a) fine needle aspiration cytology smear shows scattered and cluster of tumor cells with moderately pleomorphic, hyperchromatic nuclei and inconspicuous nucleoli, moderate to abundant cytoplasm. (b) photomicrograph showing fiber cell with elongated cytoplasm and hyperchromatic nucleus, characteristic of squamous cell carcinoma most of the cases were reported in elderly patients with the presence of local invasion at the time of presentation. the overall prognosis of the tumor is poor with a survival rate of less than a year after the diagnosis. ,, the etiology of this condition is not clear, but hypothesis include the metaplasia theory (squamous cells originating from remnant ultimobranchial duct or thyroglossal duct) as a of metaplasia of papillary or follicular cells of the thyroid or from the embryonic remnants with metaplasia of follicular epithelium (thyroglobulin positive variety) (follicular variable). it may also arise as de novo appearance from the follicular cells without metaplasia (thyroglobulin negative variety), which has a worse prognosis.,, fnac confirms the diagnosis of scc, but it is mandatory to exclude scc that has spread from adjacent upper aero digestive tract sites, as well as metastasis from distant sites, such as the lung, kidney or gastrointestinal tract. the evaluation therefore usually involves a combination of endoscopy and radiographic examination by computed tomography to rule out the neighboring or metastatic carcinoma. , in our patient, we could not perform biopsy because there was encasement of major vessels and the diagnosis was confirmed by fnac only. we have excluded the possibility of the tumor arising from an adjacent structure such as the esophagus or representing a metastasis from a primary growth elsewhere, by different investigative procedures. in several reports based on small series of patients with scc of the thyroid, the recommended treatment was an early diagnosis and combining the best available therapeutic modalities with extensive surgery. , however, the success rate of radical surgery was compromised by the invasive nature of the tumor and a poor definition of surgical treatment. described optimal surgical therapy as hemithyroidectomy or total thyroidectomy, depending upon the multifocality, followed by radiotherapy. the addition of post - operative radiotherapy could lead to improved loco - regional control. until date simpson and carruthers noted no benefit in two patients treated with adriamycin nor in another treated with 5-fluorouracil and mitomycin, while shimaoka and tsukada failed to achieve a response in three patients treated with nitrogen mustard, vincristine and ab-132, respectively. cisplatin has been shown to have benefited patients suffering from other squamous cell cancers of the head and neck but, to date, no information is available on its use in scc of thyroid. even after optimum treatment, this aggressive malignancy has very dire prognosis with dismal outcome. in this kind of deadly disease, where survival is not expected to be more than 1 year, quality - of - life is very important. in this particular situation, though we were not able to deliver optimum treatment to the patient, but we could achieve effective palliation with radiotherapy alone and gift patient a good quality - of - life.

primary squamous cell carcinoma of the thyroid is an extremely rare neoplasm with aggressive behavior. until date, only around 60 cases have been reported in the literature. primary treatment of the patient is radical surgery. with optimum treatment survival is not more than 6 months in this aggressive malignancy. however in our patient surgery it was not possible because of unresectability of the mass due to encroachment of major vessels. hence, we have delivered radiotherapy alone, with which effective palliation could be achieved and patient is leading a good quality - of - life for last 1 year.

although levodopa (ld) still remains the gold standard for symptomatic efficacy in reducing the motor symptoms of parkinson s disease (pd),1 its long - term use is associated with the development of potentially disabling motor complications, including response oscillations as well as drug - induced dyskinesias (ld - induced dyskinesias), affecting approximately 30% of patients after only 2 years of ld exposure.2 the precise underlying pathophysiology of ld - induced motor complications is still incompletely understood; however, it is believed that pulsatile dopamine - receptor stimulation, leading to neuroplastic changes in the basal ganglia circuitry, plays a pivotal role.1,35 dopamine agonists (das) act directly on striatal dopamine receptors, with preferential effects on the d2 over d1 subfamily, and generally have considerably longer half - lives than ld (table 1).6 while the first agents of this class were ergolinic compounds with affinities to multiple non - da - receptor types, the newer agents are nonergolinic and seem to lack the risk for cardiovalvular fibrosis, recently reported for ergolinic agonists,7 which have since been withdrawn from many markets. in clinical trials involving patients with early pd, initial monotherapy with das was consistently associated with a significantly reduced risk of motor complications compared with ld, over double - blind follow - up periods of up to 5 years (table 2),810 such that das are currently recommended as first - line therapies particularly for patients with younger age at onset.11,12 in addition, das are established first - line therapies to reduce motor fluctuations in ld - treated patients.11,12 both types of clinical benefit from das are likely to be related to their longer half - life, ing in more continuous striatal da - receptor stimulation. continuous 24-hour delivery with once - daily dosing has become possible with a transdermal formulation of the short half - life agonist rotigotine and, recently, novel extended - release (er) formulations have been developed for the nonergolinic oral compounds ropinirole and pramipexole. the latter principle is likely to increase convenience and possibly adherence, particularly for patients with early disease that can be managed with monotherapy. the aminobenzothiazole compound is highly selective for the d2 receptor family and, intragroup, has preferential affinity for the d3 receptor subtype.13 the agent lacks affinity for dopamine receptors d1 and d5 and displays only little affinity for d2 and d4 receptor subtypes.14 pramipexole er was designed as a prolonged - release tablet with pramipexole dihydrochloride monohydrate dispersed homogeneously throughout the matrix. the active substance is released proportional to the square - root of time15 by two different mechanisms: diffusion and erosion,15 reaching its maximum plasma concentration (cmax) approximately 6 hours after oral administration (immediate release : cmax 13 hours). in other respects the pharmacokinetic profile mostly coincides with the well - known ir formulation. following oral administration , the agent shows a bioavailability of > 90%.15 in fact, pharmacokinetic studies observed an increase in cmax with concomitant intake of a high - fat meal (24% after a single dose administration and about 20% after multiple dose administrations), but there was no significant change in overall area under the curve (auc024 hours).15,16 protein binding is < 20% and the agent is metabolized only to a small extent and predominantly eliminated by renal excretion (~90%). with a renal clearance of approximately 400 ml/ min, the elimination half - life (t) varies from 8 hours in the young to 12 hours in the elderly. pramipexole er is available in five dosage strengths (0.26 mg, 0.52 mg, 1.05 mg, 2.1 mg, and 3.15 mg) and is approved for use in early pd as well as adjunct therapy in advanced pd with motor complications. two large randomized double - blind placebo - controlled phase iii trials were conducted to evaluate the clinical efficacy of pramipexole er in early pd patients.17,18 these are summarized as follows. two hundred and fifty - nine patients with early pd at hoehn and yahr stage 1 to 3, diagnosed within the preceding 5 years, were randomized (2:2:1) to receive pramipexole er (0.263.15 mg qd), pramipexole ir (0.0881.1 mg tid), or placebo. following a 7-week - flexible up - titration phase, drug doses were maintained for an additional 26 weeks, during which, to maximize patient retention in the trial, open - label ld rescue medication was permitted for subjects experiencing insufficient control of parkinsonism. post - ld - rescue data were censored for the primary analysis. at 18 weeks an interim analysis on a subset of 253 patients was planned to evaluate superiority of efficacy of pramipexole er to placebo on the unified parkinson s disease rating scale (updrs) ii + iii (primary endpoint).18 at 33 weeks, the study aimed to demonstrate noninferiority of pramipexole er to pramipexole ir in the combined score on updrs ii + iii (primary endpoint).17 noninferiority was predefined as a treatment - group difference for which the lower bound of the 95% confidence interval (ci) did not exceed 3 points. this margin had been chosen conservatively to be well outside the minimally clinically relevant difference on the updrs, which has been suggested to be 7 points for parts ii and iii combined.19 at 18 weeks, the adjusted mean change in updrs ii + iii combined scores, censoring post - ld - rescue data, was 7.4 (1.1) in the pramipexole er group, compared with 2.7 (1.3) in the placebo group (p = 0.0010 vs placebo) and 7.5 (1.1) in the pramipexole ir group (p = 0.0006 vs placebo).18 including data from subjects receiving ld rescue medication, the adjusted mean change was 5.1 (1.3) for placebo, 8.1 (1.1) for pramipexole er (p = 0.0282 vs placebo), and 8.4 (1.1) for pramipexole ir (p = 0.0153 vs placebo).18 hence, using either approach, a statistically significant difference between pramipexole groups and placebo could be demonstrated from week 4 onward (er p = 0.0111, ir p = 0.0042 vs placebo at week 4 by either approach).18 at 33 weeks, updrs ii + iii change censoring post - ld - rescue data was 8.2 for er and 8.7 for ir. the ant treatment difference was 0.5 (95% ci : 2.3 to + 1.3), thereby establishing noninferiority of the er formulation. including ld - rescue data, the adjusted mean decrease was 8.5 versus 9.4, revealing a difference of 0.9 (95% ci : 2.7, + 0.9), which is still well within the predefined margin.17 as already demonstrated at 18 weeks, the 33-week analysis confirmed the superiority of pramipexole er over placebo (adjusted mean change in updrs ii + iii score : 8.2 er vs 1.2 placebo).17 consistent with the symptomatic efficacy of pramipexole, fewer patients on active treatment required ld - rescue medication (7.0% er, 4.3% ir, 21.4% placebo).17 superiority of both pramipexole formulations could also be shown for secondary outcome measures, including global impression of improvement responder rates (41.4% er, 45.1% ir vs 20.6% placebo), patient global impression of improvement responder rates (34.4% er, 32.4% ir vs 16.5% placebo), and updrs ii + iii responder rates (66.7% er, 63.8% ir vs 35.0% placebo).17 a summary of the primary and key secondary endpoints at week 33 is given in table 3. rascol et al conducted a randomized, double - blind, double - dummy, parallel - group study to assess efficacy, safety, and tolerability of an overnight switch from pramipexole ir to er in early pd patients.20 after a 24-week open - label run - in on pramipexole ir tid, 156 patients were switched overnight either to er or to ir at an unchanged daily dosage (randomized 2:1). subjects were allowed a one - step dose adjustment as required for efficacy and/or tolerability 4 and 5 weeks after switching. the primary efficacy endpoint was defined as the proportion of patients successfully switched (with or without any dosage adjustment) at the end of week 9, determined as no worsening from baseline updrs ii + iii score > 15% and no withdrawal due to drug - related adverse events (aes). noninferiority was predefined as a 95% ci with a lower bound not exceeding 15%. at week 9, 84.5% of patients in the er versus 94.2% in the ir group were considered successfully switched (table 4). the absolute difference between groups was 9.76% (95% ci : 18.81%, + 1.66%), hence noninferiority of pramipexole er was not formally demonstrated. however, after 9 weeks, 80.6% of er and 84.6% of ir recipients were successfully switched without requiring any dose adjustments. in the er group, 16.5% had increased and 2.9% had decreased their dosage, while corresponding percentages in the ir group were 13.5% versus 1.9% (table 5). the between - group difference for increased versus unchanged / decreased dosage was not significant (p = 0.6190). the mean pramipexole dosage at 9 weeks was 2.75 (0.95) mg / day (+ 0.12 mg / day from baseline) in the er, compared with 2.83 (0.86) mg / day (+ 0.09 mg / day from baseline) in the ir group. pooled safety data of the clinical trials in early and advanced pd, comprising information from 803 pd patients exposed to clinically effective doses of pramipexole er, show a slightly higher rate of aes for both pramipexole ir and er compared with placebo. however, no significant difference in ae profiles has been found between the two pramipexole formulations. a summary of the most common side effects is given in table 6.15 safety evaluation of the 33-week trial in early pd17 also identified somnolence, gastrointestinal complaints, and dizziness as the most frequent aes. in this study, a small numerical increase in epworth sleepiness scale score was observed in both pramipexole groups but mean values remained below the cut - off for excessive daytime sleepiness. impulse control disorders were also slightly more common in both active treatment groups (four patients in the er, three in the ir, and one in the placebo group). taken as a whole, pramipexole er showed the same safety and tolerability profile as pramipexole ir. poor compliance has been identified as a major issue in several disease areas2124 including pd.25,26 the irregular intake of prescribed medication affects health care on many levels, including poor symptom control and reduced quality of life,27 distortion of treatment effectiveness28 as well as increased health care expenditure.29,30 the reasons for noncompliance are manifold and include fear of side effects, complex drug regimens incompatible with everyday life, and dementia. in one study, poorer compliance was identified to be more likely among younger patients and those with complex drug regimens, depression, and lower quality of life.26 grosset et al demonstrated an inverse correlation of compliance and drug doses per day in pd patients31 and showed that low therapy adherence was significantly associated with poor motor scores (updrs), more days absent from work, and worse mobility (pdq39).31 in this study, all measures of therapy adherence (total number of days adherent, timing adherence, and total therapy adherence) were significantly higher for once - daily medications, including das taken once versus three times daily.31 therefore, the availability of once - daily formulations of das with established efficacy in pd represents an advantage even if there is no added benefit of enhanced clinical efficacy or safety. in addition, from the patient perspective there is the obvious advantage in terms of convenience and ease of use, particularly in early disease where monotherapy with a single dose per day is possible. due to blinding purposes, the possible beneficial effects of a once - daily formulation on convenience and adherence have not been possible to assess in the phase iii trials.17,18,20,32 er formulations of a long - acting da such as pramipexole may also contribute to improved symptom control during the night, although this has not been formally studied to date and is more relevant to advanced rather than early pd. furthermore, there is a possibility that the pharmacokinetic profile of a slow - release formulation may reduce the risk of peripheral dopaminergic side effects such as nausea and vomiting as well as central adverse reactions, including somnolence and daytime sleepiness, by avoiding rapid plasma level increases and high peak concentrations as compared with their ir counterparts. this has, however, not been demonstrated in the pivotal clinical trials with pramipexole er. pramipexole is widely established as a symptomatic treatment in early as well as advanced pd. the development of an er formulation, with stable pramipexole plasma concentration over 24 hours, now offers a bioequivalent once - daily alternative. double - blind randomized controlled trials in early as well as advanced pd have established the noninferiority of pramipexole er compared with ir as well as the superiority of both formulations over placebo. the overnight switch from the standard to the once - daily formulation was shown to be successful in > 80% of patients without requiring any dose adjustments. potential benefits of prolonged - release once - daily da formulations include improved compliance and a potential for better symptomatic control over the day as well as during the night. however, the latter, along with reduced risk of dopaminergic side effects, has not been formally demonstrated in the pivotal trial program. from a patient perspective there is little doubt that once - daily drugs offer major advantages in terms of convenience, especially for initial monotherapy in early pd.

the aim of this article is to provide a short review of the most relevant pharmacological and clinical data on pramipexole extended release (er) as well as to address the clinical utility and potential advantages of a once - daily formulation especially in the treatment of early parkinson s disease (pd). pramipexole is widely established as a symptomatic treatment in early as well as advanced pd. the development of an er formulation, with stable pramipexole plasma concentration over 24 hours, now offers a bioequivalent once - daily alternative. double - blind randomized controlled trials in early and advanced pd, have established noninferiority of pramipexole er compared with immediate release as well as superiority of both formulations over placebo. the overnight switch from the standard to the once - daily formulation was shown to be successful in > 80% of patients without requiring any dose adjustments. potential benefits of the prolonged - release design, which have not yet been formally demonstrated in the pivotal trial program, include improved compliance and a potential for better symptomatic control, particularly in patients with early disease that can be managed with monotherapy.

glutaric aciduria type i is caused by deficiency of the enzyme glutaryl coa - dehydrogenase (gcdh) in the metabolic pathway of lysine, hydroxylysine, and tryptophan, leading to an accumulation of glutaric acid (ga) and its derivative 3-hydroxy glutaric acid (3ohga) (funk et al . 2005 ; harting et al . 2-hydoxyglutaric ( 2ohga) acidurias are characterized by the presence of elevated concentrations of either l-2ohga or its enantiomer d-2ohga and of -ketoglutarate (kg) in body fluids (read et al . 2007). whereas l-2ohga aciduria is caused by mutations of the fad - dependent l-2ohga dehydrogenase (rzem et al . 2004), the underlying metabolic defects of d-2ohga aciduria are due to mutations of the enzyme d-2-hydroxyglutarate dehydrogenase (struys et al . 2005). we have recently identified the sodium - dependent dicarboxylate transporter 3 (nadc3) and the organic anion transporter 1 (oat1) to be responsible for the uptake of ga and its derivatives from the blood into renal proximal tubular cells (hagos et al . whereas oat1 is involved in the efflux of various neurotransmitter metabolites from the cerebrospinal fluid to the blood across the choroid plexus ( alebouyeh et al . 2006) and possibly the choroid plexus (pajor et al . 2001). besides nadc3, another electrogenic sodium - dependent di- or tricarboxylate transporter, hnact, has been identified in the brain. nact is preferentially located in neurons, especially in the hippocampus, cerebellum, cerebral cortex and olfactory bulb (inoue et al . since neuropathological findings observed in patients suffering from glutaric aciduria type 1 as well as from d- and l-2ohga aciduria occur in neurons, we investigated the impact of ga derivatives on hnact expressed in xenopus laevis oocytes . in vitro transcription of hnact- and hnadc3-crna capped crna from the human nact ( genbank accession no . plasmids were linearized with not i and in vitro crna transcription was performed using the t7 mmessage mmaschine kit ( ambion, austin, tx, usa) according to the manufactor s instructions. the ing crna was suspended in purified, rnase - free water to a final concentration of 1 g/l. solutions a standard oocyte ringer solution (ori) was used for oocyte preparation, storage, and for the uptake as well as for the electrophysiologic measurements. ori contained (in mm): 110 nacl, 3 kcl, 2 cacl2, 5 hepes / tris, adjusted to ph 7.5. citrate, succinate, glutarate (ga), its derivatives, 3-hydroxyglutarate (3ohga), d- (d-2ohga), and l-2-hydroxyglutarate (l-2ohga), and glutamate were added to ori in the concentrations indicated in the figure legends and ph was adjusted to 7.5. all chemicals, including those for ori, for oocyte preparation and storage, as well as for the uptake and electrophysiologic experiments were purchased from sigma - aldrich (taufkirchen, germany). 3ohga was obtained from c. mhlhausen (uke, hamburg, germany). oocyte preparation and storage stage v and vi oocytes from xenopus laevis (nasco, fort atkinson, wi, usa) were separated by an overnight treatment with collagenase (typ cls ii ; biochrom, berlin, germany), subsequent washings in calcium - free ori and maintained at 1618c in ori containing a calcium concentration of 2 mm. one day after removal from the frog, oocytes were injected with 23 nl crna coding either for hnact or hnadc3, or an equivalent amount of water (mocks) and maintained at 1618c in ori supplemented with 50 m gentamycin and 2.5 mm sodium pyruvate. after 34 days of incubation with daily medium changes, oocytes were used for tracer uptake studies. transport experiments uptake of citrate (ccitric acid ; ge health care, freiburg, germany) or glutarate (cglutaric acid ; mp biomedicals, heidelberg, germany) in hnact- or of succinate (csuccinic acid ; perkin elmer, rodgau, germany) in hnadc3-expressing oocytes was assayed at room temperature. inhibition of citrate uptake was determined by simultaneous application of 12 m c - citrate and ori containing 1 mm of ga, 3ohga, d- or l-2ohga, respectively, for 30 min. c - glutarate was used at a concentration of 20 m. inhibition of succinate uptake was determined by simultaneous application of 18 m c - succinate plus 40 m sodium succinate and ori containing 1 mm glutamate. after incubation in the respective solutions, radioactivity was aspirated and the oocytes were washed twice in ice - cold ori. oocytes were dissolved by gently shaking for 2 h in 100 l 1 n naoh, neutralized with 100 l 1 n hcl, and their c - contents were determined by liquid scintillation counting (tricarb 2900tr ; perkin elmer). inhibition experiments were performed at least in duplicate with 810 oocytes for each experimental condition. electrophysiologic analysis these studies were carried out 34 days after crna injection at room temperature. oocytes were placed into a 0.5-ml chamber on the stage of a microscope and impaled under direct view with borosilicate glass microelectrodes filled with 3 m kcl (biomedical instruments, zllnitz, germany). current - voltage (i - v) recordings were performed using a two - electrode voltage clamp device (oc725a ; warner, hambden, ct, usa) in the voltage clamp mode. substrate - associated currents were obtained by subtraction of the currents in the presence from those in the absence of the substrate of interest. paired student s t test was used to show statistically significant difference of citrate uptake in the absence and presence of the ga derivatives or of succinate uptake in the absence and presence of glutamate. michaelis - menten constants (km) for 3ohga, d- and l-2ohga were determined by sigmaplot software (systat software, point richmond, ca, usa) using the michaelis - menten equation i = imax /(km +), where i is the current, imax is the maximum current observed at saturating substrate concentrations, km is the substrate concentration at half - maximal current, and s is the substrate concentration. in hnact - expressing oocytes (5 oocytes from 4 donors), application of 1 mm citrate led to potential - dependent inward currents showing larger current amplitudes at 90 mv than at more depolarizing potentials (fig . the inward currents were abolished upon substitution of all sodium by n - methyl - d - glucamine ( data not shown). in contrast to citrate, the ga derivatives 3ohga, d-2ohga, l-2ohga, and ga itself, did not evoke any potential - dependent inward currents. the individual currents induced by these compounds were similar in hnact - expressing oocytes (fig . did not change this ( data not shown), indicating that ga and ga derivatives are either not transported by hnact or transport is electroneutral. to discriminate between these possibilities, the uptake of citrate was compared to the uptake of glutarate within 30 min in the same batch of hnact - expressing oocytes (fig . 1c). whereas citrate uptake increased by a factor of 3.76 0.63 (three independent experiments) as compared to mocks, the uptake of ga in hnact - expressing oocytes and in mocks was virtually identical. current - voltage (i - v) relations of substrate - associated currents in hnact - expressing oocytes are shown in (a), (c) and mocks (b). oocytes were superfused first with ori and subsequently with ori to which 1 mm of the respective ga derivative was added. subtraction of the currents obtained in the presence from those in the absence of the respective substrate revealed the substrate - associated currents, i. oocytes were first tested for the current induced by the prototypical substrate, citrate, to test for successful expression and only oocytes showing citrate - associated currents > 20 na at 90 mv were used in this study. afterwards, 3ohga, d-, l-2ohga, kg, and ga were applied at random order. a, b means sem of 5 oocytes from 4 donors and of 3 oocytes from 3 donors, respectively. c comparison of the uptake of labeled citrate and glutarate in the same batch of oocytes. whereas the hnact - expressing oocytes (3 independent experiments with 10 oocytes for each experimental condition) took up citrate (12 m), current - voltage (i - v) relations of substrate - associated currents in hnact - expressing oocytes are shown in (a), (c) and mocks (b). oocytes were superfused first with ori and subsequently with ori to which 1 mm of the respective ga derivative was added. subtraction of the currents obtained in the presence from those in the absence of the respective substrate revealed the substrate - associated currents, i. oocytes were first tested for the current induced by the prototypical substrate, citrate, to test for successful expression and only oocytes showing citrate - associated currents > 20 na at 90 mv were used in this study. afterwards, 3ohga, d-, l-2ohga, kg, and ga were applied at random order. a, b means sem of 5 oocytes from 4 donors and of 3 oocytes from 3 donors, respectively. c comparison of the uptake of labeled citrate and glutarate in the same batch of oocytes. whereas the hnact - expressing oocytes (3 independent experiments with 10 oocytes for each experimental condition) took up citrate (12 m), no uptake of glutarate (20 m) was observed. in hnadc3-expressing oocytes (4 oocytes from four donors), succinate (1 mm) evoked substrate - dependent inward currents at all potentials tested (fig . 2a, closed circles). subsequent application of glutamate (1 mm) did not give rise to such currents (fig . 2a, open circles). neither succinate- nor glutamate - associated currents were observed in mocks (data not shown). in addition, succinate uptake was only marginally inhibited by 1 mm glutamate during an incubation time of 15 min (fig . 2effect of succinate and glutamate on hnadc3 . a current - voltage ( i - v) relations as a function of membrane potential (vc) in hnadc3-expressing oocytes were obtained by substraction of the currents in the presence of 1 mm succinate in ori or of 1 mm glutamate in ori from those measured in ori alone. data present mean values sem of 4 oocytes from 4 donors. b succinate uptake in the absence and presence of 1 mm glutamate in hnadc3-expressing oocytes (black columns) and mocks (grey columns) as obtained in two independent experiments with 810 oocytes for each experimental condition. effect of succinate and glutamate on hnadc3. a current - voltage (i - v) relations as a function of membrane potential (vc) in hnadc3-expressing oocytes were obtained by substraction of the currents in the presence of 1 mm succinate in ori or of 1 mm glutamate in ori from those measured in ori alone. b succinate uptake in the absence and presence of 1 mm glutamate in hnadc3-expressing oocytes (black columns) and mocks (grey columns) as obtained in two independent experiments with 810 oocytes for each experimental condition. uptake of succinate in the absence and presence of glutamate was not significantly different. at 60 mv, hnadc3 mediates translocation of 3ohga, d-, and l-2ohga with low affinity (hagos et al . these kinetic measurements were now extended to a broader potential range ( 90 to 0 mv), because, in the disease, astrocytes might have lower membrane potentials and the transporter may change its affinity to the ga derivatives. at all potentials tested, the km for 3ohga was larger than the km for d- and l-2ohga. whereas the km values for d- and l-2ohga were similar and decreased with depolarizing membrane potential, the km for 3ohga increased at depolarization (fig . the largest current amplitudes were observed for d-2ohga ( 3 oocytes from 3 donors); the amplitudes measured for 3ohga (7 oocytes from 3 donors) and l-2ohga (6 oocytes from 5 donors) were similar in magnitude at all potentials tested (fig . 3michaelis - menten constants ( km ( mm) ) (a) and maximal substrate - inducible currents (imax ( na) ) (b) of the ga derivatives as a function of membrane potential (vc). hnadc3-expressing oocytes were subsequently superfused with increasing substrate concentrations of 3ohga: 0.1, 0.2, 0.5, 1, 2, and 5 mm; d- and l-2ohga: 0.01, 0.05, 0.1, 0.5, and 1 mm in ori at ph 7.5. data were obtained at the end of a 10-s perfusion with the respective solution at the indicated vc and were calculated from 7 oocytes of 3 donors for 3ohga, for d-2ohga from 3 oocytes from 3 donors, and for l-2ohga from 5 oocytes from 4 donors. michaelis - menten constants (km ( mm) ) (a) and maximal substrate - inducible currents (imax ( na) ) (b) of the ga derivatives as a function of membrane potential (vc). hnadc3-expressing oocytes were subsequently superfused with increasing substrate concentrations of 3ohga: 0.1, 0.2, 0.5, 1, 2, and 5 mm; d- and l-2ohga: 0.01, 0.05, 0.1, 0.5, and 1 mm in ori at ph 7.5. data were obtained at the end of a 10-s perfusion with the respective solution at the indicated vc and were calculated from 7 oocytes of 3 donors for 3ohga, for d-2ohga from 3 oocytes from 3 donors, and for l-2ohga from 5 oocytes from 4 donors. to date, the brain is the only mammalian tissue from which nact has been cloned (inoue et al . subsequent studies have shown that its expression is restricted to neurons ( wada et al . therefore, we reasoned that hnact might be able to accept ga derivatives which were suggested to induce the neuronal damage observed in patients suffering from glutaric aciduria type 1 and d, l - hydroxyglutaric aciduria . however, these compounds neither induced substrate - associated currents nor did they inhibit the uptake of citrate, the prototypical substrate of hnact, in hnact - expressing oocytes . in addition, we found no interaction of hnact with kg, a compound recently described to be taken up by neurons ( shank and bennett 1993). consequently, uptake of the ga derivatives into neurons by nact is not the cause of the observed neurodegeneration. particularly for glutaric aciduria type 1 , it is suggested that glutaryl - coa, ga, and 3ohga accumulating in the synaptic cleft is involved in the pathogenesis of the disease (klker et al . because these compounds do not readily cross the blood - brain barrier, they accumulate within the brain ( klker et al . 2004 ; wajner et al . 2004 ; gerstner et al . 2005 ; sauer et al . 2006), where they may inhibit the delivery of neurometabolic precursors. due to the absence of pyruvate carboxylase (cesar and hamprecht 1995 ; hassel 2001), neurons lack the capacity to perform de novo synthesis of tricarboxylic cycle constitutents (tca) and therefore depend on extracellular sources of tca intermediates to replenish intracellular pools of neurotransmitters, such as glutamate and -aminoisobutyrate (gaba). astrocytes, which in contrast to neurons, express pyruvate carboxylase (shank et al . 1985), play a major role in feeding tca constituents to the neurons. since nact is localized in neurons, we assumed that nact is responsible not only for the uptake of citrate but also for the uptake of other tri- and dicarboxylic acid derivatives into neurons. this, however, was not the case: ga and kg were not accepted by nact. the synaptic action of glutamate is terminated by its uptake into astrocytes and or into presynaptic neurons. uptake of glutamate into astrocytes occurs by distinct excitatory amino acid transporters (eaats) and possibly by low affinity sodium - dependent transport systems (danboldt 2001 ; holten et al . one such candidate transporter may be nadc3, recently localized to astrocytes ( yodoya et al . however, glutamate neither induced inward currents nor inhibited succinate uptake in hnadc3-expressing oocytes, and 1 mm glutamate reduced succinate uptake only by 19.9 8.5 % ( n.s .). therefore, hnadc3 can be excluded as a transporter facilitating glutamate uptake into astrocytes as opposed to recent speculation (frizzo et al . 2008). at 60 mv, hnadc3 carries kg and ga with high and the other ga derivatives with moderate affinity (hagos et al . 2008). in the present study, we found that the affinities for d- and l-2ohga increased whereas that of 3ohga decreased at depolarized potentials. besides km the different ga derivatives also differ in their imax being largest for d-2ohga, indicating that d-2ohga can be taken up into astrocytes at high rates and may interfere with the uptake of kg which is needed by astrocytes for the synthesis of glutamine (peng et al . although our findings did not elucidate the mechanisms by which the ga derivatives induce the neuronal damage observed in the affected patients, we can definitely exclude nact as a transporter facilitating the uptake of d- and l-2ohga as well as 3ohga into neurons . the impaired uptake of kg due to the efficient interactions of d- and l-2ohga with nadc3 could interfere with the astrocytes ability to produce tca derivatives and to feed neurons with these compounds needed for synthesis of neurotransmitters . as, during brain development, energy requirements are increasing ( harting et al . 2009), the metabolic support of astrocytes may be limited due to accumulating ga derivatives in the synaptic cleft. this in turn may induce the observed neuronal damage due to shortage of fuels when the brain is in a most vulnerable phase as recently discussed by strauss et al.

concentrations of glutarate (ga) and its derivatives such as 3-hydroxyglutarate (3ohga), d- (d-2ohga) and l-2-hydroxyglutarate (l-2ohga) are increased in plasma, cerebrospinal fluid (csf) and urine of patients suffering from different forms of organic acidurias. it has been proposed that these derivatives cause neuronal damage in these patients, leading to dystonic and dyskinetic movement disorders. we have recently shown that these compounds are eliminated by the kidneys via the human organic anion transporters, oat1 and oat4, and the sodium - dependent dicarboxylate transporter 3, nadc3. in neurons, where most of the damage occurs, a sodium - dependent citrate transporter, nact, has been identified. therefore, we investigated the impact of ga derivatives on hnact by two - electrode voltage clamp and tracer uptake studies. none of these compounds induced substrate - associated currents in hnact - expressing xenopus laevis oocytes nor did ga derivatives inhibit the uptake of citrate, the prototypical substrate of hnact. in contrast, d- and l-2ohga, but not 3ohga, showed affinities to nadc3, indicating that d- and l-2ohga impair the uptake of dicarboxylates into astrocytes thereby possibly interfering with their feeding of tricarboxylic acid cycle intermediates to neurons.

fibromyalgia is a chronic pain syndrome of unknown origin, estimated to affect 25% in all populations studied (raspe, 1992 ; wolfe et al ., 1995 ; fibromyalgia is characterized by widespread pain, fatigue, poor sleep, and dyscognition ( wolfe et al ., 2010) and is often also associated with mood disorders such as depressive episodes and anxiety. although the underlying pathophysiology is not entirely understood, fibromyalgia has been associated with augmented central nervous system (cns) processing of nociceptive stimuli using both quantitative sensory testing (qst) and functional neuroimaging. (gracely et al ., 2002 ; desmeules et al ., 2003 ; petzke et al ., 2003 ; smith et al ., context the notion of dysfunctional endogenous pain inhibition has been proposed to play a pivotal role in the genesis of chronic widespread pain . this is supported by studies demonstrating impaired or absent conditioned pain modulation ( cpm) in these patients (lautenbacher and rollman, 1997 ; vierck et al ., 2001 ; julien et al ., 2005 ; chalaye et al ., functional ( gracely et al ., 2002 ; giesecke et al ., 2004 ; jensen et al ., 2012) and structural (kuchinad et al ., 2007 ; schmidt - wilcke et al ., 2007 ; 2013) brain imaging studies have shed some light on possible central mechanisms that might play a role in the genesis of chronic pain in fibromyalgia. one such approach is the investigation of fluctuations in blood oxygenation level dependent (bold) signals at rest, termed resting state functional connectivity (rs - fc). only recently have changes in rs - fc been demonstrated in fibromyalgia, such as a hyper - connectivity between the default mode network (dmn), a constellation of brain regions involved in self - referential thought, and the insular cortex (ic), a brain region known to play a pivotal role in pain perception (napadow et al ., 2010). interestingly this hyper - connectivity may be a marker for chronic pain intensity as two independent trials have shown that decreases in connectivity between the dmn and ic following treatment were associated with reductions in clinical pain (napadow et al ., 2012 ; harris et al ., 2013). treatment of fibromyalgia in terms of a clinically relevant reduction in widespread pain is often challenging and both pharmacological and non - pharmacological approaches (bernardy et al ., 2013). food and drug administration for the treatment of pain in fibromyalgia: pregabalin, a compound that binds to the 2 subunit of a voltage dependent presynaptic calcium channel, and two selective serotonin (5-ht) and norepinephrine (ne) reuptake inhibitors, milnacipran (mln) and duloxetine (goldenberg et al ., 2010 ; schmidt - wilcke and clauw, 2010 ; schmidt - wilcke and clauw, 2011). other drugs, such as tricyclic compounds (tca, e.g. amitryptiline), viewed as non - selective 5-ht and ne reuptake inhibitors, have also repeatedly been shown to be efficacious in the treatment of fibromyalgia and are frequently used in pharmacological treatment regimens. the way 5-ht and ne reuptake inhibitors act to reduce pain is still a matter of debate as spinal, subcortical, and cortical mechanisms have all been proposed. however the overall effect of any of these individual treatments has been modest (huser et al ., 2013). moreover a key limitation in the treatment of fibromyalgia, and chronic pain in general, is that there are no reliable tools to guide treatment assignment for individual patients. as such recently our group has shown that chemical and functional imaging in fibromyalgia can be used to predict treatment response to pregabalin in fibromyalgia (harris et al ., 2013). here we sought to identify rs - fc patterns that might predict treatment response in fibromyalgia to the selective 5-ht and ne reuptake inhibitor mln. since preclinical studies have indicated that dual reuptake inhibitors are thought to have a favorable effect on endogenous pain inhibition which is believed to be dysfunctional in fibromyalgia (lautenbacher and rollman, 1997 ; julien et al ., 2005) , we specifically focused on rs - fc to brain regions involved in antinociception and pain modulation such as: the periaqueductal gray (pag), the rostral part of the anterior cingulate cortex (acc), the dorsolateral prefrontal cortex (dlpfc) and the amygdala. inclusion criteria were: meeting 1990 american college of rheumatology criteria for fm with chronic widespread pain for at least 6 months; 1870 years of age; non - lactating and non - pregnant; right handed; score between 40 and 90 mm (inclusive) on a 100 mm pain visual analogue scale (vas); willing to withdraw from cns - active therapies marketed as antidepressants (monoamine oxidase inhibitors, tricyclics, tetracyclics, selective serotonin reuptake inhibitors, norepinephrine reuptake inhibitors, and snris); willing to withdraw from stimulant medications such as those used to treat attention deficit disorder and attention deficit hyperactivity disorder (e.g. mixed amphetamine salts, methylphenidate, dextroamphetamine) or fatigue associated sleep apnea or shift work (e.g. modafinil); willing to withdraw from anorectic agents such as diethylpropion, sibutramine, and phentermine; and if currently taking pregabalin and/or gabapentin, to remain on a stable dosage throughout the duration of the study. major exclusion criteria were: significant risk of suicide; medical conditions including cardiac diseases, glaucoma, autoimmune disease, systemic infections (e.g. human immunodeficiency virus, hepatitis), active cancer, pulmonary disease or dysfunction, unstable endocrine disease (must be stable at least 3 months prior to study enrollment), unstable diabetes, unstable thyroid disease; pregnant or lactating; any other severe, acute, or chronic medical or psychiatric conditions that could increase risk or interfere with trial ; body mass index greater than 36; treatment with any experimental agent, including mln, within 30 days before screening; and contraindications with mri procedures. all study participants gave written informed consent. the study protocol and informed consent documents were approved by the university of michigan institutional review board (ann arbor, michigan) and forest laboratories (new york, ny). all imaging data were stored, validated, analyzed, and assessed for quality at the university of michigan independent of forest personnel. patient demographics, medications, and identification of inclusion for analysis are listed in table 1. all patients were randomized in a double - blind, two - period crossover study of mln versus placebo (fig . potential participants underwent an initial visit, prior to the first neuroimaging session, wherein they were evaluated for study criteria . after meeting inclusion / exclusion criteria, consenting patients were randomized to either mln first or placebo first for period 1, and which followed 14 week washout period to withdraw from all excluded medications that could interfere with efficacy and neuroimaging measures . this washout period included a 1-week single - blind placebo run - in period to reduce the possibility of placebo effects during the first double - blind treatment period . following the placebo run - in period, all participants underwent their first neuroimaging scan ( pretreatment for period 1) which involved functional connectivity magnetic resonance imaging (fcmri). following this initial scan, subjects randomized to receive mln in the first period, underwent dose escalation of mln up to 200 mg / day over the course of 2 weeks, with a maintained fixed dose for 4 weeks, at which time an identical post - treatment fcmri session was conducted. those subjects randomized to placebo for period 1 took matching placebo pills over the course of 6 weeks before undergoing an identical post - treatment fcmri session. all participants then entered a 1 week taper and 2 weeks of a placebo washout, during which time a placebo sugar pill was consumed daily. once the washout period was completed, all patients crossed over to the other study drug for period 2 (i.e. those who had mln for period 1 received placebo for period 2 and vice versa .). placebo data included in all analyses came from either period 1 or period 2 (depending on treatment order), and were included for all subjects regardless of treatment order. all subjects were informed that they would be dosed with mln or placebo at various times throughout the study, but were not told when they were transferred from one treatment to the other. we assessed treatment response for both clinical pain as well as evoked experimental pain in all participants prior to and following each study period. clinical pain was assessed with the short form of the brief pain inventory (bpi) which captures both pain severity (bpi sev) and interference due to pain (bpi int) (cleeland and ryan, 1994) measured over the course of the previous week. changes in these components (post minus pre) were used as measures of treatment response for clinical pain. changes in pain both following mln and placebo were assessed in spss, version 20, by performing paired t - tests. further, we specifically investigated whether changes following mln treatment differed significantly from changes following placebo treatment by performing paired samples t - tests of the change scores for all behavioral measures, also in spss, version 20. when assessing for responders compared to non - responders to a treatment period, we defined a patient to be a responder if she had 30% improvement for a behavioral measure after a treatment period. patients with < 30% improvement after a treatment period were categorized as non - responders. evoked experimental pain data was also collected in and outside the mri scanner before and after each treatment period. pressure - pain was administered to the left thumbnail with three distinct conditions using the multi - modal automated sensory testing (mast) system (harte et al ., 2013): 1 ) an equal pressure condition of 1.5 kg / cm, 2 ) the amount of pressure required to elicit pain50 on the 0100 nrs, and 3 ) a faint touch rest condition. both behavioral and bold fmri response data will be analyzed and reported in a subsequent manuscript. in this investigation we were primarily interested in identifying baseline neuroimaging parameters that could predict changes in clinical and evoked pain specifically following treatment with mln. resting state fmri data were acquired using a t2 * -weighted spiral sequence (tr = 2.0 s, te = 30 ms, fa = 90, matrix size 64 64 with 43 slices, fov = 20 cm and 3.12 3.12 3 mm voxels), using a 3 tesla general electric, signa scanner 9.0, vh3 with 16 rod birdcage transmit receive radio frequency coil. during the 6 min resting state fmri acquisition period (180 scans), subjects were asked to remain awake with their eyes open and to stare at a motionless cross presented on the screen. minimal cognitive tasks such as staring at a cross are thought not to disrupt resting state networks (greicius et al ., 2003). the first 6 images of the resting state scan were discarded from the data set and not analyzed in order to avoid equilibration effects. a t-1 weighted structural gradient echo data set (tr 1400 ms, te 1.8 ms, flip angle 15, fov 256 256, yielding 124 sagittal slices with a defined voxel size of 1 1 1.2 mm) was also acquired for each subject. data were pre - processed and analyzed using fsl (http://www.fmrib.ox.ac.uk/fsl) and statistical parametric mapping software packages (spm, version 8, functional imaging laboratories, london, uk), as well as the functional connectivity toolbox conn (cognitive and affective neuroscience laboratory, massachusetts institute of technology, cambridge, usa) running under matlab 7.5b (mathworks, sherborn, ma, usa). upon collection of the functional data, cardiorespiratory artifacts were corrected for using the retroicor (hu et al . pre - processing steps included motion correction ( realignment to the first image of the time series), normalization to the standard spm epi template (generating 2 2 2 mm resolution images) and smoothing (convolution with an 8 mm fwhm gaussian kernel). subject head motion was assessed by evaluating three translations and three rotations for each scan. translational thresholds were set to 2 mm, while rotational thresholds were limited to 1. a subject was to be excluded from the analysis if head motion exceeded either of the thresholds in one of the six dimensions. as snris are thought to augment antinociceptive mechanisms, we were particularly interested in rs - fc to brain regions known to be involved in the descending antinociceptive / pain modulatory system. the following regions were chosen based on the current literature suggesting involvement in antinociception / pain modulation (bingel, 2010): three seeds covering the rostral acc, the ventral (vacc), pre - genual (pgacc) and subgenual (sgacc) regions, and bilateral seeds in: the dorsolateral prefrontal cortex (dlpfc), the amygdala, and the periaqueductal gray (pag); seed regions were created using the spm extension tool marsbar. all seed regions were created as spheres. for further information including size and location, , seed regions' time - series were extracted; white matter, cerebrospinal fluid, and realignment parameters were entered into the analysis as covariates of no interest. a band - pass filter (frequency window : 0.010.1 hz) first level analyses were performed correlating seed region signal with voxel signal throughout the whole brain, thereby creating seed region to voxel connectivity maps (one map per seed per individual). for group analyses we focused on pre- mln rs - fc as a predictor of subsequent change in pain ing from the drug. our primary approach was to enter pre - treatment connectivity maps in a multiple regression analysis with changes in pain (post - mln minus pre - mln) in spm. were deemed significant using a family wise error (fwe) cluster corrected threshold of p < 0.05. within a priori brain regions, including: the ic, posterior cingulate cortex (pcc), precuneus, inferior parietal lobule (ipl), and dlpfc, a small volume correction using a sphere with a radius of 5 mm was performed, and were deemed significant at a (fwe) cluster level corrected threshold of p < 0.05. these regions had been selected as a priori regions because we were specifically interested in rs - fc changes to regions known to be involved in pain processing, which allowed for small volume correction analysis based on previously published . significant were then extracted and entered into spss, version 20, to assess for outliers. to determine if these were specific to mln, identical analyses were performed for the placebo period, using the significant regions identified as predicting response to mln. in order to rule out chance differences by treatment period at baseline, we compared the mln and placebo correlations for the two cross - over periods separately (i.e. those that had mln first and those that had placebo first). correlations were performed in spss, version 20, and were found to be significant at p < 0.05. finally, we sought to incorporate multiple functional connectivity measures in a regression analysis to explore for collinearity amongst our rs - fc outcomes and to further improve prediction of treatment response, while simultaneously controlling for pre - treatment pain levels (harris et al ., a multiple linear regression model was created with pre - treatment pain measures and rs - fc correlation values serving as independent variables and post - treatment pain as the dependent variable . a model was first constructed with each rs - fc measure individually and then in combination ( when multiple rs - fc values predicted the same pain outcome) by way of forward selection. eight were excluded from the analysis for the following reasons: six withdrew prior to completing all neuroimaging sessions (two prematurely stopped taking mln, two had side effects from the drug that prevented participation in the trial, and two terminated due to personal reasons), and two additional participants were excluded because they did not reach the pre - specified dose of mln before imaging. the drop - out rate due to adverse events of the medication was 9% (2 of 23 participants). thus fifteen subjects (mean age : 40.7 10.2) were included in the present analysis. multiple dimensions of clinical pain were found to show significant decreases and trends towards decreased pain after treatment with mln but not placebo (bpi sev change ; mln : mean = 0.88 1.8, p = 0.076 ; pbo : mean = 0.17 2.3, p = 0.78 ; bpi int change ; mln : mean = 1.1 1.7, p = 0.03 ; pbo : mean = 0.56 2.1, p = 0.31). when comparing the effects of treatment periods to each other, no significant differences between mln and placebo treatment were observed (bpi sev : p = 0.39, bpi int : p = 0.50). according to our a priori definition of responders: 5 of 15 patients (33%) were responders to mln and 3 of 15 patients (20%) were responders to placebo treatment for bpi sev, whereas 7 of 15 patients (47%) were responders to mln treatment and 4 of 15 patients (27%) were responders to placebo for bpi int scores (table 2). we found strong associations between baseline rs - fc values (i.e. rs - fc correlation values between our pre - specified antinociceptive brain regions and other brain regions involved in pain processing / modulation) and changes in clinical and experimental pain measures after treatment periods (table 3). a significant association was found between rs - fc of the right pag seed and the right mid - ic, and subsequent reduction in clinical pain severity (bpi sev ; mln : r = 0.885, p < 0.001 ; placebo : r = 0.216, p = 0.440 ; table 3 and fig . less rs - fc activity was associated with greater reductions in clinical pain following mln treatment but not placebo . we also observed a significant association between connectivity of the right dlpfc and the left ipl, and changes in bpi sev during mln but not placebo ( mln : r = 0.873, p < 0.001 ; placebo : r = 0.030, p = 0.917 ; table 3 and fig . 3b). with respect to the limbic system, connectivity between the left amygdala and the precuneus / posterior cingulate cortex (pcc) was found to have a negative correlation with change in bpi sev in response to mln but not placebo (mln : r = 0.916, p < 0.001 ; placebo : r = 0.389, p = 0.152 ; table 3 and fig . low levels of connectivity between the pgacc and the right posterior ic were found to be associated with a greater reduction in clinical pain interference ( bpi int) following mln but not placebo (mln : r = 0.900, p < 0.001 ; placebo : r = 0.082, p = 0.771 ; table 3 and fig . 4a). for placebo, lower levels of connectivity between the pgacc and the left dlpfc were associated with greater pain reduction, i.e. patients with less connectivity of these two structures at baseline would showed a greater response in clinical pain interference (bpi int) during placebo treatment (placebo : r = 0.869, p < 0.001, mln : r = 0.050, p = 0.859 ; table 3, fig . 5). when assessing each treatment period separately, it was found that order of treatment (mln or placebo administered in the first period) did not impact the . two linear regression models predicting changes in bpi sev during the mln period were constructed. the first model for this measure included pre - mln bpi sev values (adjusted r square = 0.34, p = 0.014), right pag to right mid - ic connectivity (adjusted r square = 0.50, p < 0.001), and right dlpfc to left ipl connectivity (adjusted r square = 0.10, p = 0.001) as independent predictors and explained 94% of the variance of post - mln pain severity. a second model explained 95% of the variance of post - mln bpi sev scores which included pre - mln bpi sev values (adjusted r square = 0.34, p = 0.014), right pag to right mid - ic connectivity (adjusted r square = 0.51, p < 0.001), and left amygdala to right pcc / precuneus (adjusted r square = 0.10, p < 0.001) as independent predictors (table 4). independent predictors including pre - mln bpi int scores (adjusted r square = 0.58, p = 0.001) and pre - mln connectivity between the pgacc and right posterior ic (adjusted r square = 0.32, p < 0.001) explained 90% of the variance of post - mln pain interference (table 4). we investigated rs - fc of cortical and subcortical structures involved in pain modulation to determine parameters that would predict treatment response to treatment with mln in patients with fibromyalgia. importantly, we find that acc ic as well as pag ic connectivity at baseline were predictive of treatment response: patients that displayed lower pgacc ic connectivity or pag ic connectivity, respectively, showed greater reductions in clinical pain following mln treatment. other rs - fc measures were also predictive of clinical response to mln treatment, such as dlpfc ipl and amygdala precuneus / pcc connectivity, while less pgacc dlpfc connectivity was predictive of placebo response. we hypothesize that a subgroup of fibromyalgia patients with poor connectivity between pro- and antinociceptive brain regions, profits from snri treatment, and this pattern reflects a dysfunctional endogenous antinociceptive system or can be viewed as a biomarker thereof. a dysfunctional endogenous antinociceptive system has been suggested to be a contributor to the genesis of pain in fm (lautenbacher and rollman, 1997 ; vierck et al . there is indeed evidence that cns levels of the two key neurotransmitters within the antinociceptive system, 5-ht and ne, are lowered in fm as indicated by decreased levels of the corresponding metabolites in the cerebrospinal fluid ( russell et al ., 1992 ; snris are thought to support the antinociceptive system by increasing synaptic 5-ht and ne levels that in turn reduce the nociceptive input to the brain . snris are well characterized with respect to their molecular mechanisms, targeting presynaptic transporter proteins ( e.g. sert), thereby raising synaptic 5-ht and ne levels. however, since 5-ht and ne can bind to different receptor subtypes with different effects and concentrations at different cns sites, the overall effect of 5-ht and ne reuptake inhibition is highly complex. in the context of pain and pain modulation however predominantly spinal mechanisms are discussed to account for the analgesic effects (yoshimura and furue, 2006 ; burnham and dickenson, 2013). for 5-ht this is via binding to 5-ht1a, 5-ht1b and 5-ht1d receptors that lead to pre- and post - synaptic hyperpolarization of pain transmitting neurons. furthermore 5-ht increases inhibitory transmitter release (e.g. gaba) from interneurons via 5-ht3 and possibly 5-ht2. ne, on the other hand is thought to act via presynaptic 1 receptors leading to a suppression of glutamate release from both a and c afferent fibers, as well as via 2 receptors increasing inhibitory transmitter release from interneurons. importantly these mechanisms seem to play a beneficial role in a variety of chronic pain conditions, such as diabetic neuropathy (boyle et al ., 2012), osteoarthritis (chappell et al ., 2009) as well as fibromyalgia (goldenberg et al ., 2010 ; huser et al , 2012b) and are not thought to be specific to one particular pain condition. interestingly for fibromyalgia a recently published study investigating spinal effects of mln via the assessment of the nociceptive flexion reflex (r iii), came to the that mln must also have supraspinal effects, based on the observation that there was a dose dependent analgesic effect in the absence of an mln associated modulation of the nociceptive flexion reflex (matthey et al ., 2013). the only study to date that we are aware of to directly test whether diminished descending analgesic activity is predictive of treatment response with these classes of drugs is a study by yarnitsky and colleagues showing that those neuropathic pain patients with impaired descending activity at baseline on qst were more likely to respond to duloxetine (yarnitsky et al ., 2012). our choice of seed regions for the rs - fc connectivity analyses was based on the current literature highlighting the role of some key structures in pain inhibition and pain modulation, i.e. the pag, the rostral acc, the dlpfc and the amygdala (petrovic et al . , 2002 ; wager et al ., 2004 ; bingel et al ., 2007). our strongest findings, which are also well in line with our a priori hypotheses is, that acc ic connectivity and pag ic connectivity were predictive of treatment response. within the endogenous antinociceptive system the pag plays a central role coordinating via the rostroventromedial medulla (rvm) (moreau and fields, 1986 ; heinricher et al ., 2009) activity in the descending 5-ht and ne pathways that project to the spinal cord to decrease nociceptive routing from the periphery. the pag itself receives input from both spinal nociceptive neurons a variety of cortical structures. it has been hypothesized that there are two cortical systems that mediate cortical top down modulation; one pathway involves descending input from the rostral acc (to the prefrontal cortex and then to the pag); a second pathway arrives at the pag from the ic via the amygdala (schweinhardt and bushnell, 2010). that said, it needs to be acknowledged that most evidence stems from animal research (fields, 2004) and that the precise mapping of these pathways in humans is currently unknown. however, the notion of the rostral acc pag connectivity (hardy and leichnetz, 1981), interacting in this regard, has recently been supported by both functional (petrovic et al ., 2002 ; bingel, 2010 ; kong et al ., 2010) and structural (stein et al ., 2012) imaging studies. the interaction of the rostral acc and ic in antinociception and pain modulation on the other hand remains to be further elucidated. both structures are part of a cortical opioidergic network (petrovic et al ., 2002) , as such the rostral acc might exert direct antinociceptive activity on the ic cortex, or both structures modulate the pag either interactively or independently, and rs - fc reflects coordinated activity of the two systems. when looking at resting state networks the bilateral ic together with the dorsal acc, mcc and supplementary motor area make up the so called salience network and connectivity within this network influences perceptual decisions on pain. the vacc, pgacc and sgacc as such are not part of the salience network, but rather belong to the executive control network (vacc) and the dmn (pgacc and sgacc) (beckmann et al ., as such one would not expect highly correlated bold activity between the rostral acc and ic even in healthy subjects ; intriguingly a negative correlation in resting state activity between these two regions is predictive of mln response . in a recently performed study we found an increased rostral acc ic rs - fc in young patients with temporomandibular disorder that we interpreted as an early compensatory mechanism in a group of young patients developing a chronic pain condition ( ichesco et al ., 2012). effort , it is tempting to hypothesize that a break down in this mechanism is then associated with further pain chronification, based on an increasingly dysfunctional antinociceptive system. the other rs - fc patterns that predicted response to mln treatment were the right dlpfc and the left ipl as well as amygdala and the posterior cingulate cortex. the ipl as well as the posterior cingulate cortex are both part of the dmn. the role of the dmn in chronic pain is beginning to be investigated, as in our previous work in fibromyalgia suggesting that ic connectivity to the dmn is associated with increased pain (napadow et al ., 2010). interestingly in this present work, we show that lowered dmn connectivity to antinociceptive regions, such as the dlpfc, are predictive of response to mln. other dmn regions such as the pcc have also been highlighted as being involved in pain in other functional and structural imaging studies (erpelding et al ., another interesting finding of our study is that pgacc left dlpfc connectivity predicted response to placebo treatment . fibromyalgia patients with a lower pgacc left dlpfc connectivity showed greater pain reductions while undergoing placebo treatment . the placebo effect in fibromyalgia patients has recently been investigated in meta - analyses based on randomized, placebo - controlled trials . in these studies, 1830% of patients have been shown to be placebo responders ; as such the magnitude of placebo responders in drug trials of fibromyalgia is similar to that seen in other chronic pain conditions ( huser et al ., 2011 the neural mechanisms underlying the clinical observation that cognitive factors such as beliefs and expectations can modulate pain perception have been investigated using various brain imaging methods ( petrovic et al ., 2002 ; bingel, 2010), mostly using short term interventions. the role of the dlpfc cortex in placebo research has been underlined by both fmri studies (wager et al ., 2004 ; wager et al ., 2011) as well as transcranial magnetic stimulation (tms) studies describing a significant impairment of placebo analgesia associated with tms induced functional lesions of the left dlpfc (krummenacher et al ., 2010). as such the dlpfc is viewed as key region for initiating placebo related changes in pain perception, the implementation of which then requires the interaction of other cortical and subcortical brain regions, such as the rostral acc and the pag. while increases in the rostral acc activity and acc pag connectivity during placebo analgesia are most likely to be mediated by opioidergic transmission (petrovic et al ., 2002) dlpfc playing a key role in placebo analgesia, they also extend it to a clinical setting, where baseline rs - fc predicts placebo effects visible 6 weeks after treatment initiation. despite the significant progress that has been made in understanding the pathophysiology of fibromyalgia, these advances have not yet been translated into pharmacological treatment. it is generally thought that the underlying pathophysiology of fibromyalgia is heterogeneous leading to a similar phenotype of chronic widespread pain, with only a subgroup of patients with diminished synaptic 5-ht and ne levels responding to drugs that augment this activity. despite our small sample size the rates of both drug and placebo responders are well in line with larger, randomized studies. consistent with this idea, the effect of any one of the united states food and drug administration's approved drugs examined in isolation is modest with a 30% improvement in pain occurring in only 3040% of patients (huser et al ., 2012b), which is also the case in other chronic pain states; nsaids and opioids for example have modest efficacy in conditions such as osteoarthritis or chronic low back pain (clauw, 2010). this stresses the need to develop tools that predict treatment response, in order to then design individually tailored therapies (woolf, 2010). only very recently have studies indicated that brain imaging might be suited to perform such predictions (harris et al ., 2013 ; resting state fmri might be a particularly interesting tool, as it is easy and safe to perform with only little demands on patients ' cognition and cooperation . first of all our study is based on a rather small study sample, i.e. our findings might not be representative of the larger fibromyalgia population and may only apply to a subgroup of fibromyalgia patients . also while our drop - out rates, as well as treatment response to mln and placebo, were comparable to those seen in larger randomized, placebo - controlled trials, our need to be reproduced in a larger study sample . as rs - fc analyses allow no assumptions on causality, or on the directedness of influence, it is conceivable that connectivity between two regions could be driven by a third region, not identified in the analysis . furthermore there is no necessity for a direct causal relationship between brain connectivity patterns identified in this study and clinical response . dual reuptake inhibitors are likely to act first and foremost on the spinal level ( in the context of pain inhibition), and the connectivity measures of forebrain structures might thus relate only indirectly to the site of action. in this scenario connectivity measures would need to be viewed as surrogate markers, however this would not detract from their potential clinical usefulness. finally, there are four snris used in clinical practice, including venlafaxine, desvenlafaxine, duloxetine and mln. mln blocks 5-ht and ne reuptake to an equal extent whereas greater selectivity at 5-ht sites has been described for venlafaxine and duloxetine. in this regard it is uncertain whether our can be generalized to other snris or even tcas. besides confirmation by studies with larger sample sizes further research is needed to extent our findings to other snri and non - selective reuptake inhibitors (e.g. tcas .). overall we were able to show that rs - fc patterns of brain structures involved in antinociception and pain modulation might be useful parameters for the prediction of treatment response to the snri mln in fibromyalgia patients. as in clinical practice only a subset of patients respond to pharmacological treatment, such approaches might turn out useful tools to identify subgroups of patients likely to respond to one or the other approach moving towards an individualized medicine.

fibromyalgia is a chronic pain syndrome characterized by widespread pain, fatigue, and memory and mood disturbances. despite advances in our understanding of the underlying pathophysiology , treatment is often challenging. new research indicates that changes in functional connectivity between brain regions, as can be measured by magnetic resonance imaging (fcmri) of the resting state, may underlie the pathogenesis of this and other chronic pain states. as such, this parameter may be able to be used to monitor changes in brain function associated with pharmacological treatment, and might also be able to predict treatment response.we performed a resting state fcmri trial using a randomized, placebo - controlled, cross - over design to investigate mechanisms of action of milnacipran (mln), a selective serotonin and norepinephrine reuptake inhibitor (snri), in fibromyalgia patients. our aim was to identify functional connectivity patterns at baseline that would differentially predict treatment response to mln as compared to placebo. since preclinical studies of mln suggest that this medication works by augmenting antinociceptive processes, we specifically investigated brain regions known to be involved in pain inhibition.15 fibromyalgia patients completed the study, consisting of 6 weeks of drug and placebo intake (order counterbalanced) with an interspersed 2 week wash out period. as a main finding we report that reductions in clinical pain scores during mln were associated with decreased functional connectivity between pro - nociceptive regions and antinociceptive pain regions at baseline, specifically between the rostral part of the anterior cingulate cortex (acc) and the insular cortex (ic), as well as between the periaqueductal gray (pag) and the ic: patients with lower preexisting functional connectivity had the greatest reduction in clinical pain. this pattern was not observed for the placebo period. however a more robust placebo response was associated with lower baseline functional connectivity between the acc and the dorsolateral prefrontal cortex.this study indicates that acc ic connectivity might play a role in the mechanism of action of mln, and perhaps more importantly fcmri might be a useful tool to predict pharmacological treatment response.

several malignant tumours can metastasize to the colon, including those of the stomach, breast, ovary, cervix, kidney, lung, prostate and skin. clinical symptoms and signs may suggest the diagnosis, but one third of such patients are asymptomatic and the diagnosis may be an incidental finding at autopsy. therapeutic options include resection with intent to cure (primary and secondary tumours resection), palliative interventions (colonic resection or colostomy), or non - surgical treatment. if all metastases can be resected, the prognosis is reportedly the same as that of the primary tumour. two years previously, a 49-year - old male caucasian patient, a chronic smoker, was diagnosed with small cell carcinoma of the lung. a ct scan of the thorax revealed mediastinal lymphadenopathy, and ct scans of the abdomen, pelvis and brain were essentially normal. the patient received chemotherapy for 3 months, with a partial response in the thoracic changes on ct, followed by three months of radiotherapy, including prophylactic radiotherapy to the brain. one year later there was no residual tumour on a ct scan of the thorax or at bronchoscopy. follow - up by the chest department a further year later showed significant progression of the lung cancer, with lung and lymphatic metastases on a follow - up chest ct scan (the abdomen was not scanned). further chemotherapy was given but the patient's health deteriorated; he received several blood transfusions for normochromic normocyctic anaemia. six months later he attended the emergency department with anorexia, asthenia and generalized abdominal pain for 3 days, with obstipation for 8 days, without nausea or vomiting. he looked pale and was tender in the right lower abdomen with rebound tenderness over mcburney's point (blumberg 's sign); he had normal bowel sounds. abdominal ultrasound (us) revealed an area of liquid in the right lower quadrant containing an elongated structure suggestive of acute appendicitis. through a mcburney incision surgical exploration revealed a mass in the right colon. a midline incision was then made, and two tumours were identified: the one in the right colon was mobile, and a second one in the sigmoid colon was fixed to the sacrum and deemed irresectable. a right hemicolectomy with an ileocolic side - to - side stapled anastomosis was performed together with a sigmoid loop colostomy. the appendix was normal macroscopically and also on histology of the resected bowel which confirmed that the right colonic tumour was a secondary from small cell carcinoma of the lung (immunohistochemistry showed immunoreactivity of tumour cells for cytokeratin cam 5,2 dot - like, for chromogranin, for synaptofisin and cd56) with secondary cancer confirmed by intense and diffuse immunoreactivity for thyroid transcription factor-1 (ttf-1) consistent with metastatic lung cancer. he was discharged on the 11th post - operative day, and died 12 weeks after surgery. colonic metastases are rare, and usually secondary to malignant tumours of the stomach, breast, ovary, cervix, kidney, lung, prostate, or skin. the few published reports suggest that colon metastases from lung cancer tend to be from giant cell carcinoma and squamous cell carcinoma. although a third of such colonic metastases are asymptomatic or found only at autopsy, more often they present clinically. ochsner and debakey found gastrointestinal involvement in 4.3% of 3047 autopsies. usually colonic metastases are diagnosed later than the primary tumour; however there are cases of synchronous or prior diagnosis. exuberant symptoms are rare, but there can be signs and symptoms of bowel obstruction, lower gastrointestinal haemorrhage (macroscopic or occult), intestinal perforation, gastrointestinal fistula, anaemia, and weight loss. transfusion - dependent anaemia has been described in the literature, and normochromic / normocytic anaemia is frequent in chronic oncologic diseases. with the current availability of positron emission tomography (pet scanning), colonic metastases may be diagnosed more frequently than previously, even in patients without mediastinal lymph node involvement. pet scans may come to have a major role in the evaluation of lung cancer. a pathological diagnosis is crucial. in our case confirmation that the right colonic lesion was metastatic from the lung cancer came from the intense diffuse immunoreactivity for ttf-1, which is not expressed by primary colonic cancer, and there was immunoreactivity of tumour cells for cytokeratin cam 5,2 dot - like, for chromogranin, for synaptofisin and cd56. the prognosis of lung cancer with intestinal metastases is poor, with a mean survival of 4 to 8 weeks and a maximum of 16 weeks, as we found in our patient (who survived 12 weeks post - operatively). if resection of the colonic metastases is possible, the prognosis is that of the primary tumour. cases complicated by bowel obstruction, haemorrhage or intestinal perforation managed by emergency laparotomy with resection of metastases have a reported mean survival of 6 months, with a maximum of 13 months. chemotherapy in patients with both primary and secondary non - resectable lesions may prolong survival (23 weeks has been reported) but chemotherapy can induce intestinal perforation in patients with known intestinal metastases. therapy must be individualized, and there are three surgical options: resection with curative intent (of both primary and secondary lesions), palliative therapy (colon resection or colostomy), or no intervention at all. in selected cases, given the poor general health of our patient and the non - resectability of both the primary and secondary lesions, the management decisions appear to have been appropriate. metastatic lesions of the colon are rare, and may raise difficult problems of management. survival reports and treatment options vary. the dilemma as to which lesion to treat first (primary or secondary) needs to be considered case - by - case. gastrointestinal metastatic disease should be considered in the differential diagnosis of patients with lung cancer presenting with an acute abdomen. aggressive surgical management provides the best chance of palliation, can decrease the duration of hospitalization, and improves quality of life. resecting both the secondary colonic and lung primary tumours may increase survival, as can chemotherapy though treatment must be individualised. written informed consent was obtained from the patient for publication of this case report.a copy of the written consent is available for review by the editor - in - chief of this journal on request. ceca data collection, manuscript writing, manuscript review, lsr data collection, cmca manuscript review

introductioncolonic metastases are rare, and usually secondary from malignant tumours of the stomach, breast, ovarian, cervix, kidney, lung, prostate, or skin. around one third are asymptomatic or found only at autopsy.case reporta middle - aged male smoker, who had a small cell carcinoma of the lung diagnosed two years previously and treated with radiotherapy and chemotherapy, was admitted to the emergency room with intense abdominal pain and constipation. with the suspicion of an acute appendicitis he was submitted to surgery. at laparotomy he was found to have a normal appendix but two hard colonic lesions: a mobile one in the right colon and the other fixing the sigmoid colon to the sacrum. a right hemicolectomy and a sigmoid loop colostomy were performed. pathology showed those lesions to be colonic metastases from small cell carcinoma of the lung.discussioncolonic secondaries are most frequently diagnosed in patients who have had a known primary tumour, and may present with bowel obstruction, lower gastrointestinal haemorrhage, gastrointestinal fistula, or intestinal perforation. presentation with acute abdomen is rare, and survival is usually limited.colonic metastatic disease should be considered in any patient presenting with an acute abdomen and past history of lung malignancy.

the use of dental implants has revolutionized prosthodontics and the fixed treatment options that can be offered to patients. high survival rates and long - term predictability for clinically loaded endosseous implants have been consistently reported ing in one of the most successful treatment modalities in dentistry. however, despite the early characterization of factors related to implant fixture success, it is noticeable that the understanding of prosthetic - related failures has been less explored. typically, an implant - supported rehabilitation is comprised by an endosseous implant that connects to a transmucosal abutment (2-piece), which receives the single or multiple unit prosthetic restoration. the location of this connection can be either submerged or at bone crest level or nonsubmerged. regardless of location and type of connection (internal or external), it is important that the best implant - abutment interface fit is achieved in order to favor the stress distribution between connecting components and biological response, hindering microorganism colonization at this interface. the most commonly used internal or external connections involve the use of a screw to clamp implant fixture and abutment. the stability of this connection is secured through a clamping force which is challenged by unclamping forces derived from occlusal function. according to bolted joint mechanics, to achieve and maintain the stability of the screw - type connection, it is important that the gap size is minimum, which will decrease the likelihood of screw loosening. it has been demonstrated that when gaps were minimized, the chances of screw loosening also decreased , thus showing the positive relationship between gap size and screw loosening. clinically, the absence of a gap - free implant - abutment interface can induce biological and mechanical complications, jeopardizing the implant long - term success. an intense host immunological response (acute inflammatory process) has been found at or near to gaps around implant - abutment interface, leading to a potential bone loss. the bacterially contaminated interface may elicit and maintain an inflammatory process (peri - implantitis). such bacterial colonization may initiate during the surgical placement of the implant, the reopening, and installation of an intermediary or through the misfit of the prosthetic connection. thus, as the interface gap allows fluid and bacterial microleakage , the implant well may serve as a bacterial reservoir that allows microorganisms to seep in and out, perpetuating a peri - implantitis disease. however, implant design junctions have changed over time, providing a more predictability of bone stability as displayed by one - piece implant and by switching platform concept. these positive reports may intensify the clinical negative impact provided by marginal misfit, when the implant - abutment interface is located at or below the alveolar crest. from a mechanical perspective it has been identified in a systematic review that the most common technical complication of a single - unit implant - supported reconstruction is abutment or occlusal screw loosening, with a cumulative incidence of 12.7% after 5 years of followup. one potential reason for this type of failure could be an ill - fitted implant - abutment interface destabilizing the implant - abutment connection. despite the outstanding success rates of modern implantology, one crucial factor is the maintenance of bone level on the long term, which can be negatively affected by mechanical and biological complications. since the establishment of peri - implant bone level after the healing period is somewhat predictable, its stability over time can be affected by inherent issues in the implant - abutment connection from a mechanical and biological perspective. studies have most commonly investigated the sealing capability to bacterial or color marker migration towards or from the implant well. direct observations of the implant - abutment interface have also been performed by x - ray, scanning electron microscopy (sem) , and optical microscopy. another possibility is the cross - sectional analysis and evaluation of the misfit made as a function of implant radius, which allows a more comprehensive observation of the adaptation along the implant - abutment interface. however, the evaluation of implant - abutment sealing capability followed by sem direct observation has not been addressed to date. this study sought to compare the sealing capability and marginal fit of two external hexagon implant systems by spectrophotometric quantification of microleakage at several incubation times followed by sem observation of the implant - abutment interface. two external hexagon implant systems (4.1 mm diameter) were used for this study (n = 10 per system : tryon - sin, sistema de implantes nacional, so paulo, sp, brazil and osseotite - biomet 3i, palm beach, fla, usa). the implants and their proprietary abutments were first subjected to the sealing capability testing and then to direct sem observation of the interface. in order to quantify the amount of the color marker (1% acid - red in propylene glycol hydrosoluble pigment) (caries detector, kuraray medical incorporation, okayama, japan) dissolved in the distilled water, a calibration curve was determined through linear regression (best line fit) using a fraction of a color marker volume in water. seven color marker increments of 0.1 l (to 0.7 l) were added using an automated pipette (eppendorf research pro, westbury, usa) to 1.3 ml of distilled water placed in 2.5 ml vials. the absorbance of these color marker increments dissolved in water (figure 1) was quantified with a spectrophotometer calibrated to a wavelength of 560 nm (fluostar optima the maximum amount of 0.7 l was determined from a pilot study which indicated that this volume was enough to fill the implant well and remained free of contact with the torqued abutment screw most apical portion . samples from each increment ( n = 5 per increment) were analyzed in the spectrophotometer calibrated to a wavelength of 560 nm to acquire the absorbance values, which were used to compose the absorbance curve. the starting point to formulate the absorbance curve was pure distilled water without color marker. in the most apical portion of the implant well 0.7 l amount of color marker was dispensed by means of an automated pipette. subsequently, the implants were held by a vise connected to a bench in a vertical position where the abutments were assembled onto the implants and torqued to 20 ncm (as per manufacturers recommendations) using a hand torque wrench (tmec, sin sistema de implantes, so paulo, brazil). the connected implants were placed into 2.5 ml vials (eppendorf research pro, westbury, usa) filled with 1.3 ml of distilled water assuring that the implant - abutment interface remained immersed, but not the interface between abutment and screw. the capped vials containing the implants and water were then kept at room temperature. using an automated pipette, samples of 100 l (n = 3 for each implant) were acquired at 1, 3, 6, 24, 48, 72, 96, and 144 hrs incubation time at room temperature. each sample was transferred from the respective vial to a microplate (costar 96, costar the arithmetic average of the three absorbance values was determined and used for statistical analyses . two - way anova at 95% level of significance and tukey 's test for multiple comparisons were utilized . specimens were subjected to marginal fit evaluation in the sem ( model 3500s, hitachi ltd ., osaka, japan) at a 15 kv acceleration voltage and 750x magnification. the calibration curve generated by the 0.1 l increments of the color marker (up to 0.7 l) dissolved in water was linear presenting a r of 0.9974 (figure 2). the color marker release quantification showed no statistical difference (p > .05) between groups. however, both groups increased the amount of color marker release as a function of incubation time (figure 3). the highest amount of color marker release was observed at 144 hours relative to all previous incubation times (p < .000001) for both groups. although our showed the presence of gaps during the sem marginal observation of the interface, caution must be taken when only this technique is considered as a method to evaluate the fit of the joint, since variations in gap sizes have been shown to occur along the implant radius in cross - sectional observations of this interface. in addition, whereas knowledge of the interface size can allow the understanding of the potential of the bacterial colonization, it is limited in providing more insight into the possibility of fluid passage through the implant - abutment interface. when compared to the of sealing capability testing of internal connecting systems, the present data shows leakage also occurring in the investigated external connection systems. the external hexagon connection was chosen for its long history of use and the plethora of data concerning its application. although efforts to reduce leakage by the use of polymeric components in the interface have hindered but not eliminated bacterial colonization, only a screw less interference - fit implant - abutment connection has shown to restrain bacterial passage along its interface. besides the alteration in connection designs, positive outcomes in bone level maintenance, when compared with matching implant - abutment dimensions, have been noticed in clinical prospective studies by two distinct approaches. the first involves the positioning of the implant - abutment interface inward and away from the outer edge of the implant (platform switching concept) and the second comprises the absence of the connection by the use of one - piece implants. the for the first may be mainly attributed to distance increase between abutment interface and bone level, perhaps decreasing the bone response and consequently the bone loss. for one - piece implants, however, the application of the platform switching concept seems to be limited to larger - diameter implants (5.0 or 6.0 mm) of the prosthetic platform diameter. considering that a relationship between extension of bone loss and the magnitude of the inflammatory process has been suggested, likely associated with the presence of microorganism in the implant - abutment interface , there seems to be a direct relationship between peri - implant disease and interface gap. therefore, alterations in connection designs have always gained attention in implant - supported prosthodontics where considerable effort has been devoted to improve the stability and minimize the implant - abutment interface gap. imperfections related to machining of implants components, excessive torque during abutment placement (which may allow the distortion of its parts), and in addition the misfit between implant - abutment are factors that have been related to interface gap origin. therefore, new studies involving the sealing capability of different connection systems combined with fatigue testing to evaluate the effect of misfit on systems mechanical performance may bring insight for the development of new connection designs. evaluation of the sealing capability of two different systems showed the passage of fluids in both groups, and both groups presented implant - abutment gaps in the sem micrographs.

to evaluate the sealing capability of external hexagon implant systems and assess the marginal fit, two groups (n = 10 each) were employed: sin (sistema de implantes nacional, brazil) and osseotite, (biomet 3i, usa). sealing capability was determined by placing 0.7 l of 1% acid - red solution in the implant wells before the torque of their respective abutments. specimens were then placed into 2.5 ml vials filled with 1.3 ml of distilled water with the implant - abutment interface submerged. three samples of 100 l water were collected at previously determinate times. the absorbance was measured with a spectrophotometer, and the data were analyzed by two - way anova (p < .05) and tukey's test. marginal fit was determined using sem. leakage was observed for both groups at all times and was significantly higher at 144 hrs. sem analysis depicted gaps in the implant - abutment interface of both groups. gaps in the implant - abutment interface were observed along with leakage increased at the 144 hrs evaluation period.

postmenopausal osteoporosis is a systemic skeletal condition that affects many millions of women around the world. the national institutes of health consensus conference defined osteoporosis as a disease of increased skeletal fragility accompanied by microarchitectural deterioration and low bone mineral density (a t score for bone mineral density below 2.5). osteoporosis prevention and treatment have relied on hormonal treatments such as estrogens and selective estrogen - receptor modulators, and on anticatabolic drugs and bone resorption inhibitors including bisphosphonates.. these compounds are potent suppressor of osteoclast activity, improve trabecular and cortical architecture, and increase bone mineral density thereby reducing the risk of fracture in women osteoporosis. since 2003, numerous reports proposed an association between bisphosphonate use and osteonecrosis of the jaw bone as a long - term side effect of this class of agents. according to the american association of oral and maxillofacial surgeons' position paper , patients may be considered to have bisphosphonates - related osteonecrosis of the jaw (bronj) if all of the following three characteristics are present: current or previous treatment with a bisphosphonate, exposed, necrotic bone in the maxillofacial region that has persisted for more than eight weeks, and no history of radiation therapy to the jaws. although bronj is a dose - related side effect and it is more common in cancer patients, a recent paper showed that the frequency of osteoporosis patients on oral bps affected by bronj was higher than previously reported (n = 470, 7.8%). the purpose of this longitudinal study is to present data from 76 female patients treated with bisphosphonates for postmenopausal osteoporosis and referred to the unit of oral diagnosis and day surgery of the university of milano for diagnosis and treatment. starting in 2005, all female osteoporotic patients treated with bps were referred to the unit of oral diagnosis and day surgery for evaluation and management. before the visit and treatment, this study was approved by the director of the clinic in accordance with the declaration of helsinki. each tooth was probed at four sites (three buccally and one lingually) using a north carolina probe to measure probing pocket depth and recession. periodontitis was based on measures of the presence and extent of cal in at least 20% of the sites probed, cal > 4 mm. relevant clinical data regarding bps treatment, comorbidities, oral findings, dental treatment plan, and past and present dental therapies, were assessed. patients were followed up every three months for routine clinical examination, oral hygiene instructions and debridement; restorative care, periodontal and dentoalveolar procedures were provided when needed according to the american association of oral and maxillofacial surgeons guidelines. individuals were included in the stage i group if they had asymptomatic exposed bone without any evidence of infection. finally, patients were included in the stage iii group if they had exposed necrotic bone with evidence of infection, pain, and one or more of the following: pathologic fracture, extraoral fistula, or osteolysis extending to the inferior border of the mandible or sinus floor. the management of bronj aimed to reduce lesion size, soft and hard tissue inflammation and to alleviate pain. nonsurgical treatment included wide spectrum antibiotics, antifungal agents, and mouthwashes with an antimicrobial solution. surgical treatment included debridement without mucosal elevation and removal of loose segments of bony sequestra. we described the distribution of participants' characteristics, including demographics, smoking status, medical history, and the prevalence and clinical features of bronj. we performed a logistic regression analysis to estimate odds ratios (ors) and 95% ci for exposures of interest such as diabetes, hypertension, dental or periodontal abscess, multiple dental decays, periodontitis, dental extraction, and the presence of onj. a total of 76 caucasian women were included in this analysis. at the time of enrollment, patients ranged in age from 51 to 91 years, with a median age of 69 years (interquartile range 6274) (table 1). in addition, 34.2% suffered from hypertension, 21.1% had cardiovascular disease, 5.3% of patients reported having diabetes, 5.3% were in treatment with an immunosuppressant agent, and 3.9% previously underwent chemo- and radiotherapy. multiple dental decays and periodontitis were present in 61.8% and 77.6% of the individuals, respectively. 61.8% of patients were receiving alendronate, 21.1% clodronate, 3.9% ibandronate, or 13.2% risedronate. patients had received bps consecutively for a mean duration of time of 191 weeks (95% ci, 150.9230.7). of these 76 patients, seven (9.2%) had active onj at first visit and were being treated in our clinics (table 1). three patients were classified as being stage i, and four individuals were stage ii. among these, the majority (85.7%) was in the mandible while 14.3% had onj in the maxilla. the triggering events for onj were identified as previous dentoalveolar surgery (n = 2), local trauma from dentures (n all bronj patients received wide spectrum antibiotic therapy, and four patients underwent surgical debridement . closure of the exposure and complete remission was obtained in 4 cases out of 7 . at present none of the non - onj patients submitted to invasive dental treatments developed onj signs and/or symptoms . individuals with dental or periodontal abscess were significantly more likely to develop onj ( table 2). women with a positive history for diabetes had a significant increase in the odds of having onj (or : 3.7, 95% ci 0.340.9). patients who underwent dental extraction while receiving bps therapy were three times more likely to develop onj (or : 2.9, 95% ci 0.515.9). no significant associations were observed for the following variables: age, smoking status, type of bps used, hypertension, cardiovascular disease, immunosuppressant, previous radio- and chemotherapy, and multiple dental decays. bisphosphonates are the most widely prescribed drugs for the treatment of osteoporosis, with more than 190 million prescriptions dispensed worldwide. the of our analysis showed that the incidence of onj attributable to the use of bisphosphonates was 9%. our findings are in agreement with those of otto et al., who conducted a large multicenter trial on the relationship between onj and the use of bps and showed that 7.8% of cases (n = 470) were associated with oral bps therapy due to osteoporosis. the risk increased also for patients who underwent dental - alveolar procedures and for those women who had periodontal disease and dental or periodontal abscess. our are also consistent with those from a paper by the m. d. anderson cancer center group, which reported that patients who had a dental or periodontal abscess and were taking bisphosphonates, were at a seven - fold increased risk for developing bronj. individuals who had dental extractions were three times more likely to be diagnosed with bronj.. showed that cancer patients with a positive history of dental extractions were associated with a significant increase in the odds of detecting onj (or : 13.2 ; 95% ci 3.747.3 ; p < .0001). however, our odds ratios are lower than those reported by hoff and colleagues. it may be suggested that cancer patients have a slower healing process than cancer - free but osteoporotic patients; both radiation therapy and chemotherapy can affect the ability of cells to reproduce, which slows the healing process in the mouth. in addition, chemotherapy may reduce the number of white blood cells and weaken the immune system, making it easier for the patient to develop an infection. though small numbers limited our ability to fully evaluate the risk factors for onj, no associations were observed for tobacco use, presence of multiple decays, previous radiotherapy and/or chemotherapy, or concomitant use of immunosuppressant medications. another limitation to note for this study is that our study population was hospital based. therefore, our may not be generalizable to the population at large. in order to overcome these sample - size issues, our findings indicate that patients with osteoporosis receiving bps may develop osteonecrosis of the mandible, especially in the presence of an active infectious process in the mouth such as periodontal disease or suppuration. in addition, management of this condition requires the use of prolonged medical treatment and may require oral surgical procedures. as such, there is an urgent need to fill a knowledge gap in better characterizing this condition, identifying the main cause, and determining individual susceptibility for the intervention and prevention of bronj. to follow up on our findings, additional large clinical trials that aim to find how to overcome bisphosphonate - associated onj and to predict who may benefit from bisphosphonate treatment without accompanying risk of onj are warranted. in the meantime , patients receiving bisphosphonates therapy should be followed carefully to avoid the occurrence of extended osteonecrotic lesions.

the aim of this longitudinal study is to present data from 76 female patients treated with bisphosphonates (bps) for postmenopausal osteoporosis and referred to the unit of oral diagnosis and day surgery of the university of milano for diagnosis and treatment. all patients received a thorough oral examination. the diagnosis of osteonecrosis of the jaw bone (onj) was made from radiographic and clinical findings. 9% of individuals had bronj at first visit. patients with dental or periodontal abscess were significantly more likely to develop bronj (or : 2.9, 95% ci 0.515.9). patients with osteoporosis receiving bps may develop bronj, especially in the presence of an active infectious process in the mouth. clinicians should carefully follow up on individuals receiving bisphosphonates therapy to avoid the occurrence of osteonecrotic lesions.

eukaryotic cells depend on vesicle - mediated endocytosis and secretion to interact and communicate with their environment. such membrane - based cellular processes are dynamic as well as closely interconnected and thus require complex cellular logistics. small vesicles, for example, cycle between the endoplasmic reticulum (er), golgi and larger spherical compartments termed endosomes. these endosomes are actively transported along polarized microtubules by the action of kinesin and dynein motor proteins to cover long distances. kinesins mainly transport their cargo to the plus ends of microtubules whereas dynein mediates transport in the opposite direction. in simple eukaryotes such as membrane trafficking is necessary to support fast expansion of growth cones at the hyphal tip. at the apex, a particular active zone of secretion is visible as spitzenkrper, which is proposed to act as a vesicle supply center. adjacent to this structure the polarisome assembles actin cables and, most likely, anchors microtubules to the membrane thereby directing the cytoskeleton - dependent membrane traffic toward the tip. they also deliver biosynthetic enzymes such as chitin and glucan synthases to sustain the formation of cell walls as well as hydrolytic enzymes for nutrient acquisition and cell wall remodelling. in addition, pathogenic fungi secrete a battery of effector proteins to interact with their animal or plant host. trafficking in the reverse direction by endocytosis is required for the uptake of membranes and membrane - associated proteins such as trans - membrane receptors. protein cargo can be either recycled to the plasma membrane or directed to the vacuole for degradation. in fungi, the basidiomycete ustilago maydis has been established as a fungal model system that is particularly well - suited to study the mechanism and biological function of microtubule - dependent membrane dynamics. important for infection is a developmental switch from saprophytic yeast cells, which proliferate by budding (fig . the key transcriptional regulator that triggers filamentous growth is the heterodimeric transcription factor bw / be . to constitute an active heterodimer subunits thus, in wild type cells plasmogamy of the mates is a prerequisite for generating the active transcription factor . however, monokaryotic lab strains have been established that express an active bw / be heterodimer under control of regulated promoters ( induced by either arabinose or nitrate). in these haploid strains hyphal growth, which closely resembles that of wild type filaments, can be elicited synchronously and reproducibly by switching the carbon or nitrogen source of the medium (fig . ( a) yeast and (b) filamentous form of strain ab33rab5ag expressing an active bw2/be1 variant under the control of a nitrogen - source regulated promoter and the green fluorescent protein (gfp) fused to the n - terminus of rab5a (filamentous growth was induced by changing the nitrogen source of the medium ; size bars, 10 m). rab5ag - positive endosomes (arrowheads in the inverted image detecting gfp fluorescence) shuttle bi - directionally along microtubules (kymograph in the lower panel). in the kymograph time thus, motion of rab5ag is visible as defined tracks (note the highly processive movement and the reversal of shuttling at the poles). (c) model depicting the motor - dependent mechanism (three motor system) of endosome transport (endosomes carry the small g protein rab5a and the snare yup1 ; symbols are explained in the inlay). in u. maydis, microtubule - dependent transport of endosomes is important for various steps of the life cycle: triggering cell separation during budding, receptor recycling during pheromone signaling and maintenance of polarity during filamentous growth. here, we review recent advances on the detailed transport mechanism as well as on the biological function of endosomes in signaling, endocytosis and mrna transport. for further information on microtubule transport and u. maydis biology as mentioned above, the processes involving membrane trafficking are tightly interwoven and highly complex. a common concept in investigating endosomal compartments is the identification of characteristic marker proteins that differentiate endosomes. at present, two different classes of endosomes are known in u. maydis: mcs1- and rab5a - positive endosomes. mcs1, the fungal class 17 myosin, consists of two domains: an n - terminal myosin motor domain and a c - terminal chitin synthase domain. transport of mcs1-positive endosomes does not depend on the myosin motor domain of mcs1 and is mediated by other actin- and microtubule - dependent motors. the integral motor domain of mcs1 is thought to tether the membrane units to the hyphal tip. membrane - based mcs1 trafficking is needed for its secretion by exocytosis, a process apparently required for cell wall remodelling and virulence. rab5a is a small g protein belonging to the family of rab proteins, which serve as specific markers of distinct classes of endosomes. in u. maydis, rab5a - positive endosomes were suggested to be early endosomes because they can also be stained with the styryl dye fm464, which has been used to study the endocytic pathway. however, fm464 is not specific for endocytosis because it also stains secretory vesicles of the fungal spitzenkrper as well as secretory vesicles downstream of the trans - golgi network in plant cells. moreover, it has not so far been reported that rab5a - positive endosomes maturate into late endosomes in u. maydis, and there is a second rab5 protein, rab5b, encoded in the genome. therefore, we use the more generic term rab5a - positive endosomes throughout this review. an important component of rab5a - positive endosomes is the t - snare - like protein yup1 (related to vam7p from s. cerevisiae) that has been proposed to function in fusing endocytic vesicles with endosomes. its precise role is yet unclear but loss of yup1 in altered cell morphology and reduced endocytic recycling of the pheromone receptor pra1 (see below). moreover, yup1 is important for endosome movement, because their motility is drastically impaired in temperature - sensitive yup1 mutants under restrictive conditions. in the last decade, endosomal transport in u. maydis has been studied in great detail, both in the yeast form and in filaments. rab5a - positive endosomes shuttle along microtubules throughout the cell moving in a highly processive fashion. at the poles they change direction without extensive pause (fig . each cell contains two to four microtubule bundles that consist of single microtubules oriented in an antiparallel fashion . near the poles of filaments ( i.e., about 10 m from both ends) microtubules are not arranged in antiparallel bundles but form single unidirectional extensions with plus ends facing to the poles (fig . - end directed movement of endosomes along antiparallel bundled as well as single microtubules is mediated by kin3 ( fig . 1c), a kinesin-3 type motor that is related to unc104 and kif1a from nematodes and mammals, respectively. the motor contains a c - terminal pleckstrin homology (ph) domain for interaction with phosphatidylinositol lipid containing membranes. the ph domain is essential for unc104-dependent transport in neurons of c. elegans and kin3-driven movements of endosomes in filaments of u. maydis. each endosome contains about three to six kin3 and these are responsible for the highly processive bidirectional movement along the antipolar bundles in filaments (fig . minus - end directed transport of endosomes is mediated by the dynein motor dyn1/2 ( fig . this becomes important at the poles of filaments because in the unidirectional region of the microtubule cytoskeleton, minus end - directed transport is the only option for endosomes to return to the antiparallel array . this can nicely be seen in temperature - sensitive mutants of dyn2 where rab5a - positive endosomes accumulate at the poles under restrictive conditions . dyn1/2 can also move in the minus - end direction independently of rab5a - positive endosomes . if such a minus - end directed dyn1/2 motor meets an oncoming endosome, dyn1/2 is able to reverse the direction of the moving endosome toward the minus end . this process has been proposed to protect endosomes from falling off the track . how are motors recycled once they reach the ends of microtubules ? kin3 associates with rab5a - positive endosomes in both directions of transport . this holds true, even at the poles, where unidirectional microtubules and no longer antiparallel bundles are present . in this area dyn1/2 mediates minus - end directed transport of endosomes . here, kin3 is a cargo and no specific recycling system for kin3 is needed . in contrast, dynein is actively transported back to the plus - ends of microtubules by conventional kinesin . therefore endosomes accumulate at the poles in the absence of conventional kinesin because dynein required for minus - end directed transport is missing at the poles . in summary, a tripartite motor system consisting of kin3, dynein and conventional kinesin manages the complex logistics of long - distance transport of endosomes along a highly organized array of microtubules ( fig . interestingly, rab5a - positive endosomes exhibit rather diverse functions throughout the life cycle of u. maydis . in yeast cells they play an important role in septum formation and cell separation . 2a) reminiscent of the mutants don1 and don3 that were originally identified owing to their striking colony morphology. the explanation for this mutant phenotype is the failure to form a secondary septum, which is essential for cell separation after cytokinesis (fig . don1 encodes a guanine nucleotide exchange factor ( gef) specific for the small gtp binding protein cdc42 and don3 is a ste20-like protein kinase acting parallel to this signaling module. don1 carries a c - terminal fyve domain that is specific for the recognition of phosphatidylinositol-3-phosphate lipids and thus targets don1 to rab5a - positive endosomes. kin3-mediated transport of these don1-loaded endosomes is critical for cell separation in order to synchronize cdc42-triggered secondary septum formation with the accumulation of vesicles involved in lysis of the fragmentation zone (fig . 2c and d). (a) colony and (b) yeast cell morphology of strain ab33kin3 in comparison to the progenitor strain (size bar, 10 m). (c, d) model depicting the function of rab5a - positive endosomes in the delivery of don1 to the site of septation, a process essential for formation of the secondary septum. the red rectangle shown in the middle panel of c is enlarged in d. during mating rab5a - positive endosomes are involved in endocytic recycling of the pheromone receptor pra1. pra1 accumulates in endocytic vesicles inside the cell in temperature - sensitive yup1 mutants under restrictive conditions and thus is missing at the plasma membrane of conjugation tubes. this correlates with a reduced recognition of compatible mating partners and in a fusion defect of conjugation tubes. thus, endocytic recycling of pra1 mediated by rab5a - positive endosomes appears to be important for polarized accumulation of the receptor at the growth cones of mating hyphae that orient their growth toward pheromone sources. during filamentous growth microtubule - dependent motor function is important for maintaining unidirectional growth of the tip cell and the concomitant formation of retraction septa at the distal end of the hyphae. 3a ) and predominantly bipolar filaments lacking retraction septa, indicating that unipolar growth is disturbed (fig . 3b and c). similar observations were made in the absence of functional dynein and conventional kinesin or in filaments treated with the microtubule inhibitor benomyl. (a) colony and (b, c) cell morphology of kin3 filaments. the edges of colonies growing under filament - inducing conditions are shown in (a). monokaryotic filament carrying wild type allele of kin3 (b) is compared with the bipolarly growing filament of kin3 (c) strain grown under filament - inducing conditions (size bars, 10 m). (d, e) model depicting the function of rab5a - positive endosomes during transport of mrnps. red rectangle shown at the growth cone is enlarged in e (symbols are explained in the inlay). it is noteworthy that loss of rna - binding protein rrm4 causes a similar growth defect: mutant filaments grow mainly bipolar and fail to insert retraction septa. this rrm - type (rna recognition motif) rna - binding protein constitutes an integral part of a transport machinery mediating microtubule - dependent long - distance transport of mrnas. rrm4 binds to distinct sets of target mrnas encoding cytotopically related proteins including polarity factors such as rho3 and ribosomal proteins such as ubi1, a natural fusion protein of ubiquitin, and rpl40 of the large ribosomal subunit. rrm4-mediated mrna transport is believed to promote subcellular accumulation of rho3 at the retraction septum by local translation (fig . co - transport is observed throughout the whole filament and co - localization with these endosomes does not depend on the presence of kin3 . in the absence of rrm4, endosomal movement is not drastically impaired suggesting that rab5a - positive endosomes shuttle independently of rrm4 and its mrna cargo . nevertheless, loss of rrm4 causes defects in filamentous growth resembling mutants with defects in microtubule function . thus, mrna transport appears to be an important function of trafficking rab5a - positive endosomes, particularly during filamentous growth ( fig . this novel function of endosomes has broad implications for mrna transport in general . in u. maydis we could demonstrate unambiguously that microtubule - dependent mrna transport is mainly endosome dependent ( fig . it has been observed before that mrna transport can be tightly linked to membrane trafficking . in s. cerevisiae actin - dependent transport of mrnas can be achieved by hitchhiking on the er . during development of d. melanogaster, the endosomal marker rab11 as well as components of escrt - ii ( endosomal sorting complex required for transport) are important for correct subcellular localization of mrnas encoding morphogens. therefore, it is speculated that endosomes might be directly involved in short range mrna transport. research in recent years has revealed that endosome action is a lot more complex than previously anticipated. this is well supported by recent findings on the variety of endosomal functions in the fungal model system u. maydis: along with their function in endocytosis, rab5a - positive endosomes are important for cell separation, pheromone signaling and mrna transport. the connection to mrna transport, in particular, adds a novel aspect to the growing list of endosomal functions. thus substantiating current views that these endomembrane compartments function as multipurpose platforms if, for example, mrnas encoding proteins involved in endocytosis are themselves transported by endosomes, alterations of endosome function might disturb endocytosis not only directly but also indirectly via mrna transport. in the future, joint efforts of scientists focusing on mrna and membrane trafficking are needed to explain both the mechanistic and functional aspects of this complex and interlaced molecular sorting machinery.

long - distance trafficking of membranous structures along the cytoskeleton is crucial for secretion and endocytosis in eukaryotes. molecular motors are transporting both secretory and endocytic vesicles along polarized microtubules. here , we review the transport mechanism and biological function of a distinct subset of large vesicles marked by the g - protein rab5a in the model microorganism ustilago maydis. these rab5a - positive endosomes shuttle bi - directionally along microtubules mediated by the unc104/kif1a - related motor kin3 and dynein dyn1/2. rab5a - positive endosomes exhibit diverse functions during the life cycle of u. maydis. in haploid budding cells they are involved in cytokinesis and pheromone signaling. during filamentous growth endosomes are used for long - distance transport of mrna, a prerequisite to maintain polarity most likely via local translation of specific proteins at both the apical and distal ends of filaments. endosomal co - transport of mrna constitutes a novel function of these membrane compartments supporting the view that endosomes function as multipurpose platforms.

fiber post systems are routinely used in restorative dentistry. in the absence of solid consensus as regards the selection criteria of the post system, clinicians often succumb to marketing tactics as opposed to clinical evidence. this study attempts to bridge this gap by using finite element analysis model to analyze the effect of oblique loading forces and their stress concentration in the entire assembly when incorporated into the tooth structure. the likelihood of survival of a pulpless tooth is directly related to the quantity and quality of the remaining dental tissue. a post is commonly placed in an attempt to strengthen the tooth. however, in vitro and in vivo studies have demonstrated that a post does not reinforce endodontically treated teeth, despite improving retention when less than 3 to 4 mm of vertical height or around 25 to 50% of clinical crown is remaining. recent developments in esthetic dentistry have supported the use of a fiber post to restore endodontically treated teeth. the need for light translucent composite resins and ceramics to mimic natural tooth requires the use of translucent posts to replace metal posts in the esthetic zone of the oral cavity. the presence of a metal post can cause shadowing of the soft tissues adjacent to the root surface; this adversely affects the esthetic required of bonded resin and ceramic restorations in the anterior region. when a fiber - reinforced post is bonded within the root canal, it dissipates functional and parafunctional forces, reducing the stress on the root. acute consequences of such forces in the assembly are not anticipated, but there will be a gradual build - up of destructive stresses that finally cause the failure of the assembly. when a catastrophic force is placed on the crown of the tooth, the crown will fracture instead of the post, transmitting the energy of force down the root and creating a vertical root fracture. consequently, a post system should be able to dissipate the function of energy and even overcome moderate trauma. fiber - reinforced posts have demonstrated the ability to fracture at the coronal portion of a tooth restoration with the presence of catastrophic forces without root fracture, permitting scope of retreatment of the remaining root structure. thus, the structural integrity and clinical longevity of post- and core - restored teeth are therefore strongly dependent on the state of stress created in their different regions due to occlusal loads. in addition to the magnitude and direction of such loads, the stress state at a given point within a restored tooth is also influenced by factors like the design and material of the post and the quantity and quality of the remaining root tissue. as the extensive loss of root dentin increases the risk of radicular fracture, appropriate mechanical behavior of the post is considered, in this case, to be fundamental to the success of rehabilitating restored teeth. earlier, various methods have been used to study stress concentration in the tooth structures namely, photo - elastic studies (photomechanical investigations), strain gauge studies, etc. finite element analysis (fea) is one of the more commonly used techniques for stress analysis. this basic concept of this technique is the visualization of actual structure as an assembly of a finite number of elements. three post systems from different manufacturers were used in maxillary central incisors (virtual model) and were subjected to forces so as to elicit data regarding their tensile strength. varied biomechanical behavior under different stresses is expected as the post systems used are significantly dissimilar in their design, taper, and modulus of elasticity. a maxillary central incisor was created virtually using computer - aided designing (cad) in a 2-dimensional model. this design has all parts of an endodontically treated tooth, i.e., enamel, dentin, cementum, alveolar bone, gutta - percha, fiber - post, luting cement, and full coverage crown. material properties like young's modulus and poisson's ratio were provided to the design. the design had only one variation which was shape of three brands, group - a (dt light post, rdt, bisco, france), group - b (luscent anchor, dentatus, usa), and group - c (relyx, 3 m espe, germany) according to their taper (due to design patent) and modulus of elasticity. dt light post is double tapered; luscent anchor in comparison has parallel shape with taper in last 1.8 mm of the apical end and in contrast relyx has uni - taper (8%) in middle third of the post shape. (a) shows the virtual model, (b) shows meshed assembly, (c) shows comparative with lowest value of force generated, (d) shows parallelism of glass fibers in relyx post modulus of elasticity of the post systems used for study elastic properties of the materials involved in the fem calculation virtual model was rendered to meshing , which represents division of the virtual model into smaller elements constrained by four nodes and they show dimensional changes as individual units; the entire design assembly was subjected to finite element analysis software (ansys) for oblique loading forces (25 n, 80 n, and 125 n) at 60 degrees to the lingual surface of the incisor. a maxillary central incisor was created virtually using computer - aided designing (cad) in a 2-dimensional model. this design has all parts of an endodontically treated tooth, i.e., enamel, dentin, cementum, alveolar bone, gutta - percha, fiber - post, luting cement, and full coverage crown. material properties like young's modulus and poisson's ratio were provided to the design. the design had only one variation which was shape of three brands, group - a (dt light post, rdt, bisco, france), group - b (luscent anchor, dentatus, usa), and group - c (relyx, 3 m espe, germany) according to their taper (due to design patent) and modulus of elasticity. dt light post is double tapered; luscent anchor in comparison has parallel shape with taper in last 1.8 mm of the apical end and in contrast relyx has uni - taper (8%) in middle third of the post shape. (a) shows the virtual model, (b) shows meshed assembly, (c) shows comparative with lowest value of force generated, (d) shows parallelism of glass fibers in relyx post modulus of elasticity of the post systems used for study elastic properties of the materials involved in the fem calculation virtual model was rendered to meshing , which represents division of the virtual model into smaller elements constrained by four nodes and they show dimensional changes as individual units; the entire design assembly was subjected to finite element analysis software (ansys) for oblique loading forces (25 n, 80 n, and 125 n) at 60 degrees to the lingual surface of the incisor. when all three groups were subjected to analysis, group c showed maximum stress distribution in the entire structure and least forces generated (stress concentration) at apical third of the tooth model, which was significantly different from other two groups. the in each model were presented in terms of the von misses stress values which decides the likelihood of failure of structure and material analyzed. when oblique forces were applied to the structure, the design showed movement in the direction of force applied, i.e., buccal flexion transferring compression at the interface of crown - core - dentin (coronal) near the cej (cemento - enamel - junction). similar change of shades of colors occurred at gutta - percha - post - dentin (radicular) interface confirming stress concentrations at this junction that can act as potential site of bond failure. colored bar at the bottom of the analysis showed maximum force (red) and minimum force (blue) generated when the analysis was conducted in ansys software. minimal changes in colors were observed in apical third of the tooth structure that signifies complete dissipation of occlusal load and predicting potential absence of vertical fractures that were a of using metal post in in vivo and in vitro experiments conducted in the past. the structure of the tooth which remains after endodontic treatment is usually undermined by previous episodes of caries, fracture, and tooth preparation. it is established that many detrimental effects produced during rehabilitative procedures are due to lack of understanding biomechanical principles underlying the treatment. the purpose of using products that have mechanical properties closer to dentin and enamel has always been to obtain best resistance and retention of the restoration done during application of dislodging or occluding forces, thus protecting the remaining tooth structure. when occlusal forces act on tooth surface, they divide into two components (vectors). such forces create stresses as dentin is a dynamic structure and such stress escapes into dentin in the form of crack propagation, ultimately ing in crazing. fiber post systems are a popular choice of modern dentists for replacing and restoring missing tooth structure in grossly mutilated teeth. this vogue of fiber post is due to their ease of handling, manipulation, and impeccable esthetic value, especially for anterior teeth. fea is a mathematical analysis of designs which can demonstrate the amount of stress generated on application of forces and site of fracture and application of this technology in dentistry is very important which can reduce operator's bias during sample preparation for in vitro studies. all variables were eliminated due to cad and only the product desired for study acted as a variable. for conducting fem study, poisson's ratio and young's modulus of elasticity of all components were taken from previous studies. variety of products in the same genre always adds to dilemma of choosing rightly. anterior teeth subjected to oblique forces angulated at 60 degrees form the long axis on the lingual surface as mandibular incisors contact the maxillary teeth at this angulation. three different amounts of forces were chosen to observe behavior of post system from mild forces (25 n) for biting to heavy bruxism forces (125 n) and a near mean average of the forces (80 n). relyx, when subjected to forces within the parameters of the study, ed in least amount of forces (stress concentration) that reached the gutta - percha - cement - post interface at the apical third of endodontically treated tooth. this can be attributed to the parallel fibers present in the post and unique modulus of elasticity, which provided better resistance to applied forces. for superior mechanical properties, the glass fibers have parallel orientation and are distributed equally over the surface area. additionally, during the manufacturing process, the glass fibers are pretensioned for enhanced post stability. therefore, during the clinical application, relyx fiber posts have to be cut with a diamond disc (according to manufacturer 's specifications provided in product brochure). in a restored tooth, when the loading angle is kept fixed, increasing the load was found to increase the von misses stress. applying this data to a real model , one can suggest that the loading at which the tooth is being subjected may not create immediate failure in the tooth, but may create cracks in the tooth assembly which may, over a duration of time, cause catastrophic failure. a virtual model can not completely mimic an in vivo model that is subjected to all types of occlusal forces. a 2-d model can never represent an actual 3-dimensional tooth structure. also, the accuracy of the entire analysis depends on software, model created, and elemental values given to the study. incorrect values as inputs and inaccurate design can lead to varying which are vulnerable to wrong . an accurate estimation of can be obtained which can increase anticipation for a better clinical performance, when mechanical testing for failure will be conducted, and improves their predictability. data available in the literature indicating better performance with fiber post are probably the of the use of adhesive cementation. because the longevity of bonding procedures involving radicular dentin are uncertain, a long - term clinical follow - up is necessary to confirm the effectiveness of glass fiber posts as a restorative solution for endodontically treated teeth. disclaimer: as the author of the abovementioned article, affirm that i neither have financial affiliation (e.g., employment, direct payment, stock holdings, retainers, consultant - ships, patent licensing arrangements, or honoraria) or involvement with any commercial organization with direct financial interest in the subject or materials discussed in this manuscript, nor have any such arrangements existed in the past three years. within the confined limitations of fea, in the given framework, fiber post relyx (3 m) is the best choice among other competitive products available, ing in longevity of the restoration that is the final goal of the grossly decayed teeth. it is conclusive from the of the study that fiber post system with components of higher modulus will enable better stress distribution. additional observations suggest that when oblique load (tensile) is applied on a tooth restored with fiber post, there is high stress concentration at gutta - percha - cement - post interface. also, the stress concentration was present in the immediate vicinity of apical end of the post and was found to be inversely proportional to the young's modulus value of the post system used. mechanical testing of these products with larger sample sizes are required to support the of studies conducted using fea.

aims and objectives: to study the stress concentrations in endodontically treated maxillary central incisor teeth restored with 3 different fiber post systems subjected to various oblique occlusal loads.materials and methods: fem analysis was used to analyze stress concentrations generated in maxillary anterior teeth. computer aided designing was used to create a 2-d model of an upper central incisor. post systems analyzed were the dt light post (rdt, bisco), luscent anchor (dentatus) & relyx (3m - espe). the entire design assembly was subjected to analysis by ansys for oblique loading forces of 25n, 80n & 125 n: the ant data showed that the relyx generated the least amount of stress concentration.:minimal stress buildups contribute to the longevity of the restorations. thus relyx by virtue of judicious stress distribution is the better option for restoration of grossly decayed teeth.

in most of the peer review publications in the late 17 century, authorship of papers generally used to be autonomous and was attributed to the sponsors. however, now the readers wish to know who paid for research and who did the work. problems with authorship persist everywhere despite the international committee of medical journal editors (icmje) criteria. authorship is considered as currency in the field of biomedical sciences and most of the researchers open their first publication account either during their undergraduate or during postgraduate studies and then continue to add further when they acquire faculty positions. someone with impressive research publications curriculum vitae (cv) has much better chances of selection and it also helps in further academic promotions. strong publication record also leads to further publications, providing great career opportunities; they are preferred to be considered for tenure status appointments, grants and funding. in addition they also earn respect and admiration in the community of research scientists. publish or perish has been very popular in the west now for many years. in the developing third world countries, recognition and credit for published research work for academic appointments in medical institutions started only about two decades ago with the that now faculty members are under compulsion to write and publish, hence at times the quality of their research is not very good. there is also a temptation to get gift authorship and this menace has been spreading everywhere. it is generally felt that manuscripts having too many authors certainly include a few whose names have been added without any intellectual contribution and they are the recipients of gift authorship. in order to overcome this problem, renie proposed contributor - ship system many years ago. listing contributor - ship european medical writers association (emwa) guidelines state that medical writes and statistician do not qualify for authorship but their role should be acknowledged. issues related to authorship consist of almost 25% of the cases discussed at cope meetings and this issue is discussed at almost every peer review congress and medical editors conferences held in different parts of the world. just like everywhere else, in pakistan too, medical editors have been faced with this issue of authorship and many times even the details of contribution which is now demanded by some journals, do not enable those listed in the bylines as authors but the editors can not do much in this regard. the objective of this study was to assess the knowledge and ascertain views of researchers on icmje criteria for authorship, their current practice of choosing authors of scientific papers, views on gift authorship and experience of authorship problems. it was a cross sectional survey of 218 faculty members (180 males and 38 females) of various medical universities (dow university of health sciences - karachi, baqai medical university karachi, liaquat university of medical sciences - hyderabad, avicenna medical college lahore, king edward medical university the main outcome measures were awareness and use of icmje criteria for authorship, awareness as to which contributions to research merit authorship, perceptions about gift authorship . the participants were also asked about the problems they faced in deciding authorship and what strategies they wish should be adopted to eliminate gift authorship . majority of the respondents 105 ( 48.2%) out of 218 faculty members who participated in the study were senior registrars and again one hundred twenty eight (58.7%) were from surgery and allied disciplines because the principal investigator was himself a surgeon. ninety six (44.0%) had between one to five publications while 60 (27.5%) had six to ten published papers to their credit (table 1). demographic profile of the respondents (n = 218) one hundred eleven participants (50.9%) said they were aware about the existence of guidelines on criteria for authorship however only twenty two (19.8%) could name this document, only four (1.8%) could correctly state the criteria for authorship suggested by the icmje (table 2). knowledge of icmje criteria of authorship (n = 218) responding as to what they thought of the icmje authorship criteria, 201 (92.7%) agreed with the first criteria as regards substantial contribution to conception and design, acquisition of data and its analysis, interpretation while 186 (85.3%) also agreed with drafting the article and revising it critically and an almost similar number 179 (82.1%) agreed with the final approval of the version to be published. however, only 120 (55.0%) said that all the above three criteria must be met to be eligible for authorship (table 3). views of the participants as regards contributions which merit authorship for scientific publications are given in table 4. attitude about the icmje criteria of authorship(n = 218) faculty view of criteria which alone contribution merits author of scientific publication (n = 218) only 34 (19.7%) of the respondents felt that authorship should be based on contributions while 98 (45.0%) felt it is decided by the main author who also decide the order of authorship (table 5). ninety three (42.7%) stated that they were not included as authors in the study though they deserved to be while sixty three said that they were included as authors though they did not merit. strangely 42 (19.3%) said that they were not aware when they were listed as authors; 52 two said they had been assigned inappropriate co - authorship and the same number had perception of incorrect placing in authorship order (table 6). faculty current trend of co -authorship and order of authorship (n = 218) faculty perception of any problems with authorship (n = 218) fifty percent of respondents in our study had knowledge about the existence of some guidelines regarding criteria for authorship but only19.8% could name it whereas only four out of 218 (1.8%) could state the icmje authorship criteria which is a very dismal picture. hence those participants, who claimed that they were aware of it but had forgotten, could have easily picked it up from there. since they could . in their study fifty out of sixty respondents supported the criteria for authorship though only a few knew about it or used it and only five people could specify all the three criteria and out of them only one knew that all the three criteria s have to be met as against 55% in our study who felt that all three criteria s must be met for authorship. they also reported that gift authorship was quite common which was promoted by pressure to publish, to motivate research team and maintain working relationship. they were of the view that a signed statement which could justify authorship besides contributions by each author could help tackle the issue of gift authorship. their were that there is a gap between the editor s criteria for authorship and researchers practice. they believe that the future criteria for authorship should be agreed by researchers and it should not be imposed by the editors. in a french study pignatell et al. who interviewed 39 investigators who submitted 48 proposals during 1994 - 96, half of the respondents said that they were aware of the authorship criteria and also knew about icmje but most of them disagreed with the obligation to meet all the three criteria justifying co - authorship because they found it too rigid and inapplicable which is similar to the findings in our study. again 59% in the french study had been recipients of gift authorship and they felt that there was a need to have french guidelines for authorship. french researchers had serious reservations regarding third criteria i.e. approval of the final version to be published. few people question ghost or gift authorship and most of them consider it normal which is a serious problem requiring in - depth debate and discussions. they also suggested that guidelines on authorship should be prepared by professionals, by learned societies, representatives of biomedical journals besides public research institutes and national organizations. in a survey from india among teaching faculty, 39% of respondents out of 77 reported conflict on authorship issues like pressure for gift authorship, academic competition, personality differences and intellectual passion besides ownership of data. they also suggested training in ethical concerns in research at undergraduate level. though most authors do understand the authorship issues but as stated by dr. it is not sufficiently considered that men require more often to be reminded than to be informed. in a debate on authorship issues in bmj , scot tim remarked that the present authorship system should continue its slow evolution since it reflects the real power relations in science but richard smith, editor bmj reported that authorship is influenced by power and departmental politics. currie opined that authors saw fraud, misconduct and unfairness to more junior staff and the culprits are most often the senior researchers. those applying for specialists registrars should be asked to choose two of their best publications for inclusion in cv and they should be prepared to discuss one of the two publications they have submitted in the previous year. for consultants merit awards and cme points should be related more to quality and relevance than to number of publications. tramara bates and colleagues in their study reported that as per authors published contributions, number of honorary authors was highest in annals of internal medicine 21.5% followed by bmj 9.5% and jama 0.5%. the number of articles with honorary authors was 60% in annals, 215 in mbj and 4% in jama. out of 6,686 researches only 68% fulfilled the icmje criteria as per author s contributions lists of radiology articles published during 1998 - 2000. these criteria of fulfillment decreased as the number of authors increased, 2,316 researchers (35%) contributed to one or two categories. this study further showed that a total of 2,172 (32.5%) out of 6,686 authors appearing in the bylines did not fulfill the icmje criteria for authorship. in our study over 80% of the respondents opined that designing the study and collection of data were important followed by conceiving the research idea besides literature search and review as regards eligibility for authorship. goodman reported that one third of 84 authors did not meet authorship criteria in an analysis of twelve articles while shapiro after survey of 184 articles with multiple authors from ten medical journals reported that 268 (26%) of 1014 authors had insufficient contributions to research to meet authorship criteria. ana marusic and colleagues did a single blind randomized trial of1462 authors of 232 manuscripts from a general medical journal who answered one of the three different contribution disclosure forms. they asked the respondents to decide in their own words their contribution to the submitted manuscripts. they found that the structure of the contribution disclosure form significantly influenced the number of contributions reported by the authors and their compliance with icmje criteria. they have concluded that editors of journals should not take the existing contribution disclosure and the authorship forms on their face value. more than half of the major papers published in american journal of roentgenology (ajr) had five or more co - authors. the incidence of undeserved co - authors increased from 9% on papers with three authors to 30% in papers with more than six authors which mean that more authors a manuscript has, more are the chances of gift authorship. gift authorship was primarily attributed to those who had some control over the first author because of fear or obligation. moreover a temporary staff member they found was more likely to gift the authorship than a permanent faculty. conception and design, analysis and interpretation and drafting of articles were recognized as the most important of the icmje criteria. final approval and critical revision should be taught as important authorship criteria to the future scientists. vesnailakovac and colleagues in their study which included 919 authors of 201 articles submitted to a general medical journal found that more than two third of the corresponding authors (67.9%) differed in at least one contribution choice between the two disclosure statements made about their own contributions. some others studies report that giving medical students clear guidelines and exposing them to high ethical standards should be a long term solution to the problem of authorship abuses in the field of medical research. a recent study from iran reported that 89% of published bio - medical articles in iran have at least one honorary author. more than 50% of article authors did not meet the authorship criteria according to icmje. about 20% authors confessed they had colleagues omitted from the authors list of the final manuscript. the author of this iranian study concluded that regardless of authorship criteria recommended by icmje and followed by many journals, still there are cases when authorship criteria is not followed. it all shows that we have not yet found an answer which is acceptable to the editors, authors as well as researches as regards authorship and this debate still continues and perhaps will continue for some more time. various suggestions are being put forward to redefine the criteria for authorship but the final word has not yet been spoken. it is also evident from the fact that authorship is an important topic which comes under discussion at various forums. icmje, other bodies of science editors as well as representatives from research institutions and researchers need to come up with authorship guidelines which are acceptable to most of the stakeholders. finding a hundred percent consensus is neither possible nor it should be aimed at since some people will continue to have their own view point on this issue. majority of our participants in the study was junior faculty members i.e. registrars or assistant professors who had just begun their academic career and an overwhelming majority one hundred eighty out of two hundred eighteen participants were male. most of this junior faculty had either not written any or just one to five papers. hence they were either not aware about the existence of any guidelines or familiar with icmje. this was also the main reason that over 98% of them could not correctly state the icmje criteria of authorship. yet another limitation was that in a self - administered questionnaire based survey, participants tend to provide information which may not be 100% correct. medical students should be exposed to the art of medical writing from the beginning and those institutions which have not yet included the subject of medical writing and research methodology it in their curriculum must be advised to do so. similarly regular workshops particularly for junior faculty members, postgraduates on medical writing, guidance on how to plan, conduct study, highlighting the existing guidelines on authorship by various bodies including icmje will help them expose to the scientific publishing world thus improving the quality of their studies as well. majority of our participants in the study was junior faculty members i.e. registrars or assistant professors who had just begun their academic career and an overwhelming majority one hundred eighty out of two hundred eighteen participants were male. most of this junior faculty had either not written any or just one to five papers. hence they were either not aware about the existence of any guidelines or familiar with icmje. this was also the main reason that over 98% of them could not correctly state the icmje criteria of authorship. yet another limitation was that in a self - administered questionnaire based survey, participants tend to provide information which may not be 100% correct. medical students should be exposed to the art of medical writing from the beginning and those institutions which have not yet included the subject of medical writing and research methodology it in their curriculum must be advised to do so. similarly regular workshops particularly for junior faculty members, postgraduates on medical writing, guidance on how to plan, conduct study, highlighting the existing guidelines on authorship by various bodies including icmje will help them expose to the scientific publishing world thus improving the quality of their studies as well. a vast majority of young faculty members are not aware of the existence of authorship criteria by icmje and gift authorship is quite common in pakistan. ethical issues (including plagiarism, informed consent, misconduct, data fabrication and/or falsification, double publication and/or submission, redundancy, etc) have been completely observed by the authors.

abstractthe objective of this study was to assess the knowledge and views of faculty members on criteria for authorship by international committee of medical journal editors (icmje), their current practice of choosing the authors, views on gift authorship and problems they had faced concerning authorship.methodsit was a cross sectional survey from january 2011 to july 2011 among faculty members of various private and public sector medical institutions of pakistan through a self - administered questionnaire. main outcome measures included awareness and use of icmje criteria, which contribution to research merit authorship and their perceptions about gift authorship.two hundred eighteen faculty members (180 males, 38 females) participated in the study. one hundred twenty eight (58.7%) were from surgery and allied disciplines. ninety six percent had published between one to five papers while 60(27.5%) had six to ten papers to their credit. one hundred eleven (50.9%) claimed they were aware about the authorship criteria, only twenty two (19.8%) could name this document. only four (1.8%) could correctly state this. only one hundred twenty (55.0%) said that all three criteria s must be met to be eligible for authorship. ninety three (42.7%) said that they were not included as authors though they deserved it while sixty three said they did not merit but were still included. forty two (19.3%) said that they were not aware when they were listed as authors.a vast majority of young faculty members are not aware of the existence of authorship criteria and gift authorship is quite common.

the reveal registry is a longitudinal registry involving 54 pulmonary hypertension centers in the united states (e - appendix 1). diagnosis was defined as the date of diagnostic right - sided heart catheterization (rhc) occurring at or before the date of enrollment. patients with new diagnoses were defined as those whose diagnostic rhc occurred within 90 days of enrollment. all consecutive patients who, in the opinion of the enrolling investigator, had a clinical diagnosis of pah who group 1 and met the following inclusion criteria were eligible for enrollment: mean pulmonary artery pressure of > 25 mm hg at rest or 30 mm hg with exercise, mean pulmonary capillary wedge pressure or left ventricular end diastolic pressure of 18 mm hg, pvr of 240 dynes / s / cm (divide by 80 for wood units), and 3 months of age. the data in the reveal registry was collected prospectively, but the analyses for this study were performed retrospectively. the database of 3,515 patients was locked on february 4, 2013, for the current analyses. we developed an algorithm (fig 1) to exclude patients with exercise - induced pah, in accordance with the dana point classification criteria, and those with pulmonary capillary wedge pressure > 15 mm hg, who have been shown to differ in many respects from those meeting the traditional hemodynamic definition of pah, and included only patients with ctd - apah. we also excluded those with evidence of significant interstitial lung disease (ild), defined as those with evidence of severe fibrosis on high - resolution ct scan of the chest or we divided the patients with ctd - apah into those with ssc - apah (ssc group) and those with non - ssc - ctd - apah (non - ssc group). strobe diagram of the registry to evaluate early and long - term pah management (reveal) registry patients used in this analysis. we included only patients with ctd - apah who met the strict criteria of world health organization group 1 pulmonary arterial hypertension. ctd - apah = pulmonary arterial hypertension associated with connective tissue disease; hrct = high - resolution ct scan of the chest; ild = interstitial lung disease; non - ssc - ctd = connective tissue disease other than systemic sclerosis; pcwp = pulmonary capillary wedge pressure; ssc = systemic sclerosis; tlc = total lung capacity. baseline characteristics at the time of enrollment were compared between the ssc and non - ssc groups, using the student t or wilcoxon test to compare continuous variables and the or fisher exact test to compare categorical variables. because bnp levels were highly skewed, the variables were log transformed for comparison as continuous variables. cumulative probabilities of survival at 3 years were calculated using the kaplan - meier estimator for both the previously and newly diagnosed populations, and differences between the ssc and non - ssc groups were compared using the log - rank test. cox regression models identified significant predictors of mortality in the ssc and non - ssc populations. all variables identified previously as candidate predictors of mortality in the overall reveal registry population were evaluated in univariate and multivariate models. stepwise selection was used to determine the final model, retaining only variables with p <.05. sas, version 9.1 (sas institute inc) statistical software was used for all analyses. the reveal registry is a longitudinal registry involving 54 pulmonary hypertension centers in the united states (e - appendix 1). diagnosis was defined as the date of diagnostic right - sided heart catheterization (rhc) occurring at or before the date of enrollment. patients with new diagnoses were defined as those whose diagnostic rhc occurred within 90 days of enrollment. all consecutive patients who, in the opinion of the enrolling investigator, had a clinical diagnosis of pah who group 1 and met the following inclusion criteria were eligible for enrollment: mean pulmonary artery pressure of > 25 mm hg at rest or 30 mm hg with exercise, mean pulmonary capillary wedge pressure or left ventricular end diastolic pressure of 18 mm hg, pvr of 240 dynes / s / cm (divide by 80 for wood units), and 3 months of age. the data in the reveal registry was collected prospectively, but the analyses for this study were performed retrospectively. the database of 3,515 patients was locked on february 4, 2013, for the current analyses. we developed an algorithm (fig 1) to exclude patients with exercise - induced pah, in accordance with the dana point classification criteria, and those with pulmonary capillary wedge pressure > 15 mm hg, who have been shown to differ in many respects from those meeting the traditional hemodynamic definition of pah, and included only patients with ctd - apah. we also excluded those with evidence of significant interstitial lung disease (ild), defined as those with evidence of severe fibrosis on high - resolution ct scan of the chest or we divided the patients with ctd - apah into those with ssc - apah (ssc group) and those with non - ssc - ctd - apah (non - ssc group). strobe diagram of the registry to evaluate early and long - term pah management (reveal) registry patients used in this analysis. we included only patients with ctd - apah who met the strict criteria of world health organization group 1 pulmonary arterial hypertension. ctd - apah = pulmonary arterial hypertension associated with connective tissue disease; hrct = high - resolution ct scan of the chest; ild = interstitial lung disease; non - ssc - ctd = connective tissue disease other than systemic sclerosis; pcwp = pulmonary capillary wedge pressure; ssc = systemic sclerosis; tlc = total lung capacity. baseline characteristics at the time of enrollment were compared between the ssc and non - ssc groups, using the student t or wilcoxon test to compare continuous variables and the or fisher exact test to compare categorical variables. because bnp levels were highly skewed, the variables were log transformed for comparison as continuous variables. cumulative probabilities of survival at 3 years were calculated using the kaplan - meier estimator for both the previously and newly diagnosed populations, and differences between the ssc and non - ssc groups were compared using the log - rank test. cox regression models identified significant predictors of mortality in the ssc and non - ssc populations. all variables identified previously as candidate predictors of mortality in the overall reveal registry population were evaluated in univariate and multivariate models. stepwise selection was used to determine the final model, retaining only variables with p <.05. sas, version 9.1 (sas institute inc) statistical software was used for all analyses. of 3,515 patients enrolled in the reveal registry, 815 were identified as having ctd - apah (fig 1). of these, 804 (500 ssc and 304 non - ssc) who did not have significant ild were selected for these analyses. the majority of patients in the non - ssc group had systemic lupus erythematosus - apah or mixed ctd - apah (table 1). patients with ssc were older and had a shorter time between diagnostic rhc and enrollment into the database than did the patients with non - ssc - ctd - apah (table 2). patients with ssc - apah had more severe disease overall, with a higher nyha fc, shorter 6mwd, higher borg dyspnea index, lower dlco, and higher bnp level. patients with ssc - apah were also more likely to have renal insufficiency and pericardial effusions than patients with non - ssc - ctd - apah. although there was a strong trend toward higher mrap in the ssc group, there were no significant differences in hemodynamics or pah - specific therapies at the time of enrollment in the ssc vs non - ssc groups. apah = associated with pulmonary arterial hypertension; ctd = connective tissue disease; non - ssc - ctd = connective tissue disease other than systemic sclerosis; ssc = systemic sclerosis. characteristics, hemodynamics, and cardiac and pulmonary function at enrollment p value calculation uses test for categorical data or fisher exact test for categorical data with small cell counts (5%), and student t test for continuous data. 6mwd = 6-min walk distance; bnp = brain natriuretic peptide; bpm = beats per min; ccb = calcium channel blocker; dlco = diffusion capacity of the lung for carbon monoxide; era = endothelin receptor agonist; fc = functional class; fco = fick cardiac output; mpap = mean pulmonary arterial pressure; mrap = mean right atrial pressure; nyha = new york heart association; pah = pulmonary arterial hypertension; pcwp = pulmonary capillary wedge pressure; pde-5 = phosphodiesterase type-5; pvr = pulmonary vascular resistance; rhc = right - sided heart catheterization. see table 1 legend for expansion of other abbreviations. cardiac output = fco, or, if fco is missing, then cardiac output = thermodilution cardiac output. pvr (wood units) = (mean pulmonary arterial pressure at rest pcwp at rest)/cardiac output, where cardiac output = fco, or, if fco is missing, then cardiac output = thermodilution cardiac output. predicted value based on hankinson et al computation. three - year survival in the ssc group was worse than in the non - ssc group in both the previously and newly diagnosed populations (61.4% 2.7% vs 80.9% 2.7% and 51.2% 4.0% vs 76.4% 4.6%, respectively ; p < three - year survival curves in patients with ssc and non - ssc - ctd - apah . a, three - year survival from enrollment in the newly diagnosed ssc group was 51.2% 4.0% compared with 76.4% 4.6% in the non - ssc - ctd group ( p < .001). b, three - year survival from enrollment in the previously diagnosed ssc group was 61.4% 2.7% compared with 80.9% 2.7% in the non - ssc - ctd group (p < .001). figure 3 shows the univariate analyses of previously identified predictors of mortality from the overall reveal registry cohort in the ssc and non - ssc groups. the following variables were predictive of mortality in both groups: age > 60 years, nyha fc iii or iv status, 6mwd < 165 m, and bnp > 180 pg / ml. unique predictors of mortality in the ssc group, but not the non - ssc group, included male sex, systolic bp (sbp) 110 mm hg, pericardial effusion, dlco 32% predicted, mrap > 20 mm hg within 1 year, pvr > 32 wu, and newly diagnosed status. bnp levels < 50 pg / ml were protective in patients with ssc (hazard ratio = 0.34 ; 95% ci, 0.16 - 0.72 ; p = .005) but not in the non - ssc group (hr = 0.68 ; 95% ci, 0.36 - 1.29 ; p = .24). figure 3 also shows the univariate analyses of additional variables that are relevant to the ctd - apah population. mild to moderate ild was the only feature that increased mortality in patients with non - ssc - ctd - apah but not in patients with ssc - apah (hr = 2.19 ; 95% ci, 1.14 - 4.23 ; p = .02 vs hr = 0.84 ; 95% ci, 0.55 - 1.30 ; p = .44). when compared with ipah, mrap > 20 mm hg within 1 year, pvr > 32 wu, and newly diagnosed status remained unique predictors of death in the ssc - apah group. predictors of mortality for patients with ssc - apah and non - ssc - ctd - apah using univariate cox regression analyses. unique predictors of mortality in the ssc group, but not the non - ssc group, included male sex, sbp 110 mm hg, pericardial effusion, dlco 32% predicted, mrap > 20 mm hg within 1 y, pvr > 32 wu, and newly diagnosed status. bnp levels < 50 pg / ml were protective in patients with ssc, but not in the non - ssc group. mild to moderate ild was the only feature that increased mortality in the non - ssc group but not in patients with ssc. 6mwd = 6-min walk distance; bnp = brain natriuretic peptide; dlco = diffusion capacity of the lung for carbon monoxide; fc = functional class; gfr = glomerular filtration rate; hr = hazard ratio; mrap = mean right atrial pressure; nyha = new york heart association; pvr = pulmonary vascular resistance; sbp = systolic bp; who = world health organization; wu = wood units. , the following variables remained predictive of mortality in both the ssc and non - ssc groups: nyha fc iii or iv status and bnp > 180 pg / ml (table 3). unique predictors of mortality in the ssc group included men > 60 years, sbp 110 mm hg, 6mwd < 165 m, mrap > 20 mm hg within 1 year, and pvr > 32 wu. 6mwd 440 m was protective in the non - ssc group, but not in the ssc group, whereas bnp < 50 pg / ml was protective in the ssc group, but not in the non - ssc group. of 3,515 patients enrolled in the reveal registry, 815 were identified as having ctd - apah (fig 1). of these, 804 (500 ssc and 304 non - ssc) who did not have significant ild were selected for these analyses. the majority of patients in the non - ssc group had systemic lupus erythematosus - apah or mixed ctd - apah (table 1). patients with ssc were older and had a shorter time between diagnostic rhc and enrollment into the database than did the patients with non - ssc - ctd - apah (table 2). patients with ssc - apah had more severe disease overall, with a higher nyha fc, shorter 6mwd, higher borg dyspnea index, lower dlco, and higher bnp level. patients with ssc - apah were also more likely to have renal insufficiency and pericardial effusions than patients with non - ssc - ctd - apah. although there was a strong trend toward higher mrap in the ssc group, there were no significant differences in hemodynamics or pah - specific therapies at the time of enrollment in the ssc vs non - ssc groups. apah = associated with pulmonary arterial hypertension; ctd = connective tissue disease; non - ssc - ctd = connective tissue disease other than systemic sclerosis; ssc = systemic sclerosis. characteristics, hemodynamics, and cardiac and pulmonary function at enrollment p value calculation uses test for categorical data or fisher exact test for categorical data with small cell counts (5%), and student t test for continuous data. 6mwd = 6-min walk distance; bnp = brain natriuretic peptide; bpm = beats per min; ccb = calcium channel blocker; dlco = diffusion capacity of the lung for carbon monoxide; era = endothelin receptor agonist; fc = functional class; fco = fick cardiac output; mpap = mean pulmonary arterial pressure; mrap = mean right atrial pressure; nyha = new york heart association; pah = pulmonary arterial hypertension; pcwp = pulmonary capillary wedge pressure; pde-5 = phosphodiesterase type-5; pvr = pulmonary vascular resistance; rhc = right - sided heart catheterization. see table 1 legend for expansion of other abbreviations. cardiac output = fco, or, if fco is missing, then cardiac output = thermodilution cardiac output. pvr (wood units) = (mean pulmonary arterial pressure at rest pcwp at rest)/cardiac output, where cardiac output = fco, or, if fco is missing, then cardiac output = thermodilution cardiac output. predicted value based on hankinson et al computation. three - year survival in the ssc group was worse than in the non - ssc group in both the previously and newly diagnosed populations (61.4% 2.7% vs 80.9% 2.7% and 51.2% 4.0% vs 76.4% 4.6%, respectively ; p < .001) (fig 2). three - year survival curves in patients with ssc and non - ssc - ctd - apah. a, three - year survival from enrollment in the newly diagnosed ssc group was 51.2% 4.0% compared with 76.4% 4.6% in the non - ssc - ctd group (p < .001). b, three - year survival from enrollment in the previously diagnosed ssc group was 61.4% 2.7% compared with 80.9% 2.7% in the non - ssc - ctd group (p < .001). see figure 1 legend for expansion of abbreviations. figure 3 shows the univariate analyses of previously identified predictors of mortality from the overall reveal registry cohort in the ssc and non - ssc groups. the following variables were predictive of mortality in both groups: age > 60 years, nyha fc iii or iv status, 6mwd < 165 m, and bnp > 180 pg / ml. unique predictors of mortality in the ssc group, but not the non - ssc group, included male sex, systolic bp (sbp) 110 mm hg, pericardial effusion, dlco 32% predicted, mrap > 20 mm hg within 1 year, pvr > 32 wu, and newly diagnosed status. bnp levels < 50 pg / ml were protective in patients with ssc (hazard ratio = 0.34 ; 95% ci, 0.16 - 0.72 ; p = .005) but not in the non - ssc group (hr = 0.68 ; 95% ci, 0.36 - 1.29 ; p = .24). figure 3 also shows the univariate analyses of additional variables that are relevant to the ctd - apah population. mild to moderate ild was the only feature that increased mortality in patients with non - ssc - ctd - apah but not in patients with ssc - apah (hr = 2.19 ; 95% ci, 1.14 - 4.23 ; p = .02 vs hr = 0.84 ; 95% ci, 0.55 - 1.30 ; p = .44). when compared with ipah, mrap > 20 mm hg within 1 year, pvr > 32 wu, and newly diagnosed status remained unique predictors of death in the ssc - apah group. predictors of mortality for patients with ssc - apah and non - ssc - ctd - apah using univariate cox regression analyses. unique predictors of mortality in the ssc group, but not the non - ssc group, included male sex, sbp 110 mm hg, pericardial effusion, dlco 32% predicted, mrap > 20 mm hg within 1 y, pvr > 32 wu, and newly diagnosed status. bnp levels < 50 pg / ml were protective in patients with ssc, but not in the non - ssc group. mild to moderate ild was the only feature that increased mortality in the non - ssc group but not in patients with ssc. 6mwd = 6-min walk distance; bnp = brain natriuretic peptide; dlco = diffusion capacity of the lung for carbon monoxide; fc = functional class; gfr = glomerular filtration rate; hr = hazard ratio; mrap = mean right atrial pressure; nyha = new york heart association; pvr = pulmonary vascular resistance; sbp = systolic bp; who = world health organization; wu = wood units. , the following variables remained predictive of mortality in both the ssc and non - ssc groups: nyha fc iii or iv status and bnp > 180 pg / ml (table 3). unique predictors of mortality in the ssc group included men > 60 years, sbp 110 mm hg, 6mwd < 165 m, mrap > 20 mm hg within 1 year, and pvr > 32 wu. 6mwd 440 m was protective in the non - ssc group, but not in the ssc group, whereas bnp < 50 pg / ml was protective in the ssc group, but not in the non - ssc group. our study provides further evidence that patients with ssc - apah experience higher mortality rates than do patients with other ctd - apah in both incident and prevalent populations. our validate the usefulness of the risk score calculator in patients with ctd - apah, including in patients with ssc - apah. we identified several baseline risk factors that were significantly associated with mortality in the ssc - apah population in comparison with the non - ssc - ctd - apah population, including being an elderly man, having a low sbp, having poor exercise capacity, and having severe hemodynamic indices including elevated mrap and pvr. identifying patients with ssc - apah with high mortality risk based on the presence of these unique predictors of mortality will enable physicians to monitor these patients more closely and escalate therapy when indicated. three - year survival in the newly diagnosed ssc - apah population was 51%, which is similar to survival rates found in other cohorts assessed in the modern treatment era. other studies have found better survival rates (75%-81%) in patients with ssc - apah; these rates are similar to the survival rate of 77% that we and others observed in patients with non - ssc - ctd - apah. this survival discrepancy could be related to early detection algorithms that have been implemented in these ssc - apah cohorts, with the goal to initiate pah - specific therapy when the disease is less severe. survival in patients with non - ssc - ctd - apah appears to be more similar to those with ipah than to those with ssc - apah, despite similar baseline hemodynamics and pah - specific therapies. whether initiating aggressive pah treatment in patients with ssc - apah with a particular high mortality risk may improve outcomes remains an important question to be answered. overall, predictors identified in the multivariate model in ssc - apah were very similar to the core predictors for pah as a whole, including all subtypes. our concur with those of other studies on patients with ssc - apah in that male sex, older age, and fc iii and iv status were significant predictors of death. our confirmed those of a single - center study that identified high pvr as a strong predictor of mortality. unlike these other studies , we did not find that low dlco or glomerular filtration rate were predictive of mortality in the ssc - apah group in multivariate analyses, although they were significant in univariate analyses. lefvre et al identified additional poor prognostic factors in patients with ssc with pulmonary hypertension in a meta - analysis including patients with who groups ii and iii pulmonary hypertension: pericardial effusion, low 6mwd, high mean pulmonary arterial pressure, poor cardiac index, and elevated mrap were poor prognostic factors. although pericardial effusion lost its significance in our multivariate analysis of patients with ssc - apah, poor exercise capacity and elevated mrap remained significant predictors of death. m was protective only in the non - ssc - ctd - apah group in multivariate analyses. a potential explanation for these discrepancies is that patients with ssc can suffer from the presence of contractures and tendon friction rubs that can significantly limit mobility (particularly those with diffuse skin disease) in addition to other factors that limit exercise capacity (such as anemia and joint or muscle inflammation) in patients with other ctds. however, including all variables in the multivariate model without stepwise selection, 6mwd < 165 m was a significant predictor of death in the non - ssc group (hr = 2.03 ; 95% ci, 1.01 - 4.12 ; p = .05), and 6mwd 440 m showed a trend toward a protective effect in the ssc group (hr = 0.62 ; 95% ci, 0.33 - 1.15 ; p = .13). in addition, when we evaluated the effect of 6mwd on mortality risk in the various cutaneous subgroups of ssc, an increase in distance of 100 m was significantly protective in all three groups (p < .001): diffuse hr = 0.53 (95% ci, 0.38 - 0.75); limited 0.59 (95% ci, 0.51 - 0.68); unclassified 0.54 (95% ci, 0.40 - 0.71). in our study, bnp > 180 pg / ml increased the risk of death in both the ssc and non - ssc - apah groups by more than twofold, as has also been shown in patients with ipah. we and others have shown that patients with ssc - apah have markedly elevated bnp and n - terminal - pro - bnp (nt - pro - bnp) levels compared with patients with ipah and patients with non - ssc - ctd - apah. williams et al found in a uk ssc - apah cohort that for every order of magnitude increase in baseline nt - pro - bnp level there was a fourfold increased risk of death (p = .002). in addition, several studies have found that nt - pro - bnp is useful in the screening and early detection of pah in patients with ssc, and this biomarker has been integrated into novel screening algorithms. to our knowledge , our study is the first to show that bnp is an independent predictor of mortality in patients with ctd - apah and ssc - apah, in particular. unfortunately nt - pro - bnp levels were not available in 89% of our ctd - apah cohort, and, therefore, they could not be included in the regression models. to our knowledge, this is the first study to identify low baseline sbp 110 mm hg as an independent predictor of death in patients with ssc - apah. other studies have shown that low sbp, both at peak exercise and upon admission to the hospital for right - sided heart failure, is an independent risk factor for death in pah. a potential pathophysiologic explanation for this finding is that the presence of high right ventricular pressure in a more pronounced effect of low sbp on coronary perfusion. in addition, low sbp may be a sign of low cardiac output, reduced stroke volume, and neurohormonal activation. unless complicated by renal disease, patients with ssc have relatively low baseline bp, and the mean sbp was 119 19 mm hg in the patients with ssc - apah in our study. given that bp can be monitored easily, identification of low baseline sbp as a risk factor in ssc - apah is an important finding. we did not find that mild to moderate ild was predictive of death in patients with ssc - apah. although a significant predictor in the non - ssc - apah group in univariate analysis, it was no longer significant in multivariate analysis. we attempted to exclude patients with substantial ild as defined previously but did not have precise measurements regarding the degree of fibrosis on imaging. the ssc - apah and non - ssc - ctd - apah cohorts are smaller than the overall cohort. thus, differences in significant multivariable predictors may be caused by loss of power as opposed to true differences in predictors for different subtypes. the majority of reveal registry patients, particularly patients who had previous diagnoses, were receiving phosphodiesterase-5 inhibitors, endothelin receptor antagonists, prostacyclins, or a combination. although 86% of the patients with ctd - apah were enrolled at sites that routinely involve a rheumatologist in the diagnosis and care of these patients, misclassification of some patients may have occurred. finally, the analysis only assessed variables available in the reveal registry database. there may be additional factors particular to patients with ctd - apah, such as autoantibody status, that could impact the . in , patients with ssc - apah have higher mortality rates than patients with non - ssc - ctd - apah. our validate the usefulness of the pah risk score in patients with ssc - apah. we have identified unique predictors of mortality in patients with ssc - apah, including being an older man, having a low baseline sbp, having poor exercise capacity, and having an elevated mrap and pvr; these can be used to identify high - risk patients who may benefit from closer monitoring and more aggressive treatment.

: patients with pulmonary arterial hypertension (pah) associated with systemic sclerosis (ssc - apah) experience higher mortality rates than patients with idiopathic disease and those with other connective tissue diseases (ctd - apah). we sought to identify unique predictors of mortality associated with ssc - apah in the ctd - apah population.methods:the registry to evaluate early and long - term pah management (reveal registry) is a multicenter, prospective us - based registry of patients with previously and newly diagnosed (enrollment within 90 days of diagnostic right - sided heart catheterization) pah. cox regression models evaluated all previously identified candidate predictors of mortality in the overall reveal registry population to identify significant predictors of mortality in the ssc - apah (n = 500) vs non - ssc - ctd - apah (n = 304) populations.:three-year survival rates in the previously diagnosed and newly diagnosed ssc - apah group were 61.4% 2.7% and 51.2% 4.0%, respectively, compared with 80.9% 2.7% and 76.4% 4.6%, respectively, in the non - ssc - ctd - apah group (p < .001). in multivariate analyses, men aged > 60 years , systolic bp (sbp) 110 mm hg, 6-min walk distance (6mwd) < 165 m, mean right atrial pressure (mrap) > 20 mm hg within 1 year, and pulmonary vascular resistance (pvr) > 32 wood units remained unique predictors of mortality in the ssc - apah group; 6mwd 440 m was protective in the non - ssc - ctd - apah group, but not the ssc - apah group.:patients with ssc - apah have higher mortality rates than patients with non - ssc - ctd - apah. identifying patients with ssc - apah who are at a particularly high risk of death, including elderly men and patients with low baseline sbp or 6mwd, or markedly elevated mrap or pvr, will enable physicians to identify patients who may benefit from closer monitoring and more aggressive treatment.trial registry: clinicaltrials.gov; no.: nct00370214; url: www.clinicaltrials.gov

cerebral palsy is a nonprogressive central nervous system disorder that in physical impairments and functional limitations that change as the children grow older. among a large number of instruments , for measuring the physical ability of children with cp, the gross motor function classification system (gmfcs) introduced by palisano et al. in 1997 has been widely applied in clinical and research settings. the gmfcs is a five - level classification system that identifies abilities and functional limitations, based on the need of assistive devices of the cerebral palsy child, during self - initiated movements, such as walking and sitting. the system application is quick and easy and it gives a brief description of which level the child resembles based on his / her current gross motor function. the reliability and validity of the gmfcs in differentiating cerebral palsy children with different functional levels have been reported. similarly, the stability of the system over time proved to be very consistent, suggesting that the gmfcs could be used routinely in clinical practice to follow children with cerebral palsy. however, due to the heterogeneous nature of cerebral palsy, some overlap between levels i and ii has been observed and, indeed, anticipated by the authors. gmfcs level i is associated with children with persistent neuromotor abnormalities not as severe as children from level ii. overlap between levels occurred more often when deciding if a child has limitations walking outdoors, going up stairs, jumping, or running. many studies aimed to determine which outcome tools could assist clinicians and researchers to improve the classification levels i and ii of the gmfcs. correct classification at clinical settings has been corroborated by tests such as the gmfm-66, gait velocity, and the weefim mobility. child with cerebral palsy often develops changes in muscle length over time, more common at the hip and knee flexors and at the ankle dorsiflexors. kilgour and colleagues using passive range of motion tests reported that diplegic children levels i and ii of the gmfcs had minimal loss of hip extension compared to a matched control group. since the gmfcs classification is based on functional activities, it would be more interesting to obtain measurements during dynamic activities. in addition, it is expected that muscle shortness is more advanced in diplegic children level ii than level i of the gmfcs. therefore, range of motion comparison between levels i and ii is also relevant. these muscles changes might affect the range and amplitude of the lower limb during dynamic activities such as gait. however, to date, none of the studies tried to discriminate gmfcs levels i and ii according to the angular displacement of the pelvis, hip, knee, and ankle / foot complex during gait. instrumented gait analysis is a complex procedure and a highly costly diagnostic tool; however, the kinematic data obtained provides quantitative information on gait abnormalities that can not be detected during visual observation. raising the knowledge of the gait biomechanical differences between child with cerebral palsy level i and ii of the gmfcs can improve observational gait analysis skills and, at the same time, improve classification accuracy and more coherent physical therapy approaches based on the functional status of children with cerebral palsy. therefore, the research questions for this study were: are there differences in the kinematics gait profiles of the pelvis and lower limb joints during gait between diplegic cerebral palsy children classified as gmfcs levels i and ii? if differences in the kinematics were found, which ones would be the most discriminatory between these groups? a cross - sectional observational study was conducted with diplegic cerebral palsy children classified by a trained physical therapist, as gmfcs levels i and ii. the intra - rater reliability of the physical therapist in assessing the gmfcs levels was excellent (icc = 0.941). all children were community ambulates from outpatient clinics and, together with their parents or guardians, were invited to participate in the study. the temporal and spatial gait parameters and kinematics of the pelvis, hip, knee, and ankle / foot joints were collected on one day in a laboratory. the present study received approval from the ethics committee of the universidade federal de minas gerais, process number etic 088/04. prior to participation, all procedures were explained to the child and his / her parent or guardian and an informed written consent was obtained. twenty - two diplegic cerebral palsy children were included in the study, 15 male and 7 females between the ages of 7 and 12, who could ambulate independently without assistive devices for a minimum of 6 meters without resting. patients were excluded if they had other neurological diseases, botulin injections, or history of orthopedic surgery in the past six months. characteristics of the participants, such as age (years), height (m), and body mass index (bmi, kg / m), were obtained in order to describe the anthropometrics of the groups. three - dimensional kinematics of the right lower limb (hip, knee, and ankle / foot joints) and pelvis during the stance phase of the gait cycle were obtained with a six camera motion analysis system (motion capture unit - qualisys medical ab 411 12, gothenburg, sweden). the children walked barefoot over a 6-meter walkway, for an average of 9 (sd = 3.1) trials at their natural speed. reflective markers and clusters of tracking markers were used to determine coordinate systems and motions of the pelvis, thigh, shank, and ankle / foot segments according to recommendations for minimizing soft tissue artifacts. a footswitch synchronized to the motion system was fixed under the children's foot to determine contact to and loss of contact from the walking surface, and consequently, delimiting stance and swing phases during gait cycle. the ing data were processed through the visual 3d motion analysis software (c - motion, inc, rockville, maryland) where the rigid segments corresponding to the pelvis, shank, thigh, and ankle / foot segments were first created. the position of the reflective anatomical markers were used for attributing coordinate systems for each segment and were located at left and right iliac crest, left and right greater trochanter, medial and lateral epicondyle of the femur, medial and lateral malleoli, 1st and 5th head of the metatarsus, and calcaneal tuberosity. one rigid cluster with 4 noncollinear markers was placed at the base of the sacrum and two nonrigid clusters with 3 noncollinear markers were placed at the medial side of the thigh and shank. data were smoothed using a zero - lag fourth - order butterworth low pass filter with a cut - off frequency of 6 hz. three - dimensional angular motion was calculated using the cardan sequence, defined as the orientation coordinate system of one segment in relation to the orientation of the coordinate system of the adjacent segment. the hip, knee, and ankle / foot joint angles were obtained using as reference the pelvis, thigh, and the shank segments, respectively. the sign convention used in defining the clinical rotation angles were as follows: flexion of the hip and knee, anterior tilt of the pelvis, and ankle dorsiflexion, all of which occur about the lateral - medial or x - axis and were positive angles; adduction of the hip and knee, pelvic obliquity (meaning the height of the iliac crest of the stance foot higher in relation to the height of the iliac crest of the opposite foot) and internal rotation of the ankle / foot complex, all of which occur about the anterior - posterior or y - axis and were considered positive angles; internal rotation of the pelvis, hip and knee joints and adduction of the ankle / foot complex occurs about the distal - proximal or z - axis and all were positive angles. gait velocity (m / s), stride length (m), and cycle time (s) for the entire gait cycle, and swing and stance time (s), were also obtained. baseline characteristics of participants are presented as values, means, and standard deviations (sd). the mean difference between the groups with a 95% ci of the subject's characteristics and temporal and spatial gait parameters were also obtained. a principal component analysis (pca) was applied to the stance phase of the gait waveforms to reduce the data and explore the profiles characterizing typical functions between levels i and ii of the gmfcs. in pca, this technique determines a linear combination of the original variables that are used to summarize a new set of variables called principal components, which are uncorrelated and ordered so that the first component retains most of the variation present in the original data. therefore, each principal component represents a specific feature of the waveform data. the criteria to choose the number of principal components was 90% of the total sample variance. for each pc extracted, a score is calculated for each subject that aids in describing the meaning of the variation component according to the characteristics of each group. the higher the score, the more correlated the subject's waveform is with a specific pc. to interpret the components, two waveforms were created based on the mean waveform one standard deviation of the pc scores times the loading vector for each pc. the principal component scores were analyzed using student's t - test with bonferroni correction for difference between groups. the pcs retained after the discriminant analysis were described according to the discriminant function coefficient to determine its relative importance in separating the groups. group gmfcs level i had 11 children with an average age of 9.1 years (sd : 2.3), average height of 1.3 m (sd : 0.1) and bmi of 16.7 kg / m (sd : 0.2). group level ii, also with 11 children, had average age of 9.8 years (sd : 2.1), height of 1.3 m (sd : 0.1), and bmi of 17.2 kg / m (sd : 3.8, table 1). table 2 describes the temporal and spatial parameters between groups with the 95% confidence interval showing no significant difference between groups. principal component analyses were carried out for three - dimensional angular displacement of the pelvis, hip, knee, and ankle / foot complex. the pc scores generated for each subject in each component were tested for differences between groups. the showed that only the scores from the first component (pc1) of the pelvis and hip joint in the frontal plane were statistically different between the groups (table 3, p < .05). no difference was found in the curve profiles of the knee joint and ankle / foot complex. at the pelvis, figure 1(a) shows the angular displacement of the pelvis during the stance phase (normalized to 100%) between level i and ii groups of the gmfcs. the coefficient of pc1 has all positive values (figure 1(b) ); therefore, it captures the magnitude of the pelvic obliquity angle in the frontal plane during the stance. figure 1(c) shows the mean waveform and the high and low curves created based on the mean waveform one standard deviation of the pc1 score times the loading vector for each pc1. the confirm that, on average, diplegic pc children level ii, during the stance phase of the gait, walked with reduced pelvic obliquity in comparison with children from group level i. the average range of pelvic obliquity during the stance phase of the gait cycle for group level i was 6.2 and for level ii was 3.3. figure 1(d) shows the average angular displacement of the hip joint in the frontal plane between groups during the stance phase of the gait cycle. at the hip joint, two pcs were extracted with pc1 explaining 86.1% and pc2 8.5%, a total of 94.6% of variance explained. pc1, with all positive values, measured the magnitude of the adduction angle of the hip (figure 1(e) ). the mean waveform and the high and low waveforms are shown on figure 1(f) and confirm that children in gmfcs level ii presented reduced hip adduction during the stance phase when compared to cerebral palsy children in level i. the average range of hip adduction / abduction during the stance phase of the gait cycle for group level i was 11.3 and for level ii was 8.9. a stepwise discriminant analysis was conducted with the pc1s of the pelvis and hip in the frontal plane. wilk's lambda score was significant (= .217, ( 2, n = 22) = 29.002, p =.000 ) and showed that both pelvic obliquity and hip abduction angle are stronger discriminant variables, with 95.5% cross - validation. the magnitude of the coefficients of the pcs in the standardized canonical discriminant function showed that pelvic obliquity has higher impact on separating the groups (0.711) followed by hip abduction angle (0.654) during the stance phase of gait. to our knowledge, this is the first study that compared the angular displacement of the pelvis and lower limb joints, between diplegic cerebral palsy children classified according to the gmfcs as levels i and ii. the demonstrated that children with diplegia level ii of the gmfcs significantly reduce the amplitude of pelvic obliquity and hip adduction angles during the stance phase of the gait cycle. gait velocity, stride length, stance / swing and cycle time were similar in outcome for both groups. children classified as gmfcs level i of ages between 7 and 12 years, are expected to walk independently inside and outside their homes, to go up and down stairs, and to run and jump. however, their gait velocity, balance, and motor coordination may be reduced compared to a paired child with normal development. difference between children of gmfcs levels i and ii included limitations in walking outdoors and in the community, on uneven surfaces, and in crowded places. children of level ii may hold onto a rail to climb stairs, may require wheeled mobility when traveling long distances, and their ability to perform gross motor skills such as running and jumping are minimal compared to children in level i. studies showing disagreement between levels i and ii have been reported, suggesting difficulty in classifying if a child has functional limitation or can perform gross motor skills. during normal gait strike, the hemipelvis of the stance limb is aligned with the contralateral hemipelvis. at the beginning of loading response and mid - stance, the contralateral hemipelvis drops in the frontal plane, and the stance hemipelvis is higher around 4 to 7. the effect of pelvic drop is to adjust the length of the support lower limb, helped by some degree of knee flexion, avoiding excessive lower vertical displacement of the center of mass. the hip abduction / adduction displacement at this moment is dependent on the pelvis movement over the femur. while hemipelvis elevation favors adduction of the stance limb, hemipelvis drop favors, on the contralateral side, hip abduction movement. besides saving body energy by preventing greater inferior displacement of the center of mass, hip adduction of the stance limb, also shifts the body center of mass towards the center of rotation of the hip being loaded. the would be a decrease in the external adductor moment of force, favoring the mechanical advantage of the hip abductor muscles during stance. the summation of these actions prevents excessive pelvic drop and the trendelenburg gait. as well, when the support limb begins terminal stance and preswing phases, the opposite will occur. the pelvis will begin to drop, favoring hip abduction which is important to assist the release of the foot from the ground, permitting the body to swing forward. therefore, pelvic obliquity and hip adduction are two mechanisms that work synchronously to maintain stability and forward movement of the body. in diplegic children with cerebral palsy, reduced pelvic obliquity could be a of abductor muscles weakness bilaterally and adductor muscles spasticity. another explanation could be that, by reducing pelvic elevation on the supported limb in the stance phase of the cycle, children with cerebral palsy are probably trying to decrease leg length to facilitate the next initial contact of the same foot. however, this strategy would increase inferior displacement of the center of mass cycle, ing in a less efficient gait. at the hip joint, reduced hip adduction in the stance phase increases the internal abductor moment of the support limb. since children with cerebral palsy normally show weakness of the hip abductor muscles, the gait pattern would be unstable, forcing them to increase base of support or to spend less time in the unipodal phase. the decreased hip adduction could also be a response to alterations in pelvic obliquity, once the loaded hemipelvis is lower than normal, challenging the hip mechanics to generate hip adduction. the combination of reduced pelvic obliquity and hip adduction during the stance phase of the gait cycle, reinforce the evidence that children with cerebral palsy gmfcs level ii are more unstable during gait compared to children in level i. in threatening situations, such as walking on uneven or inclined surfaces, the greater instability of children in gmfcs level ii could justify their need for assistive devices. it also justifies their use a rail to walk up and down stairs, since reduced pelvic obliquity would decrease hip abduction that could also affect hip flexion. although the kinematic analysis was not a tool used to assist the development of gmfcs classification system, the findings of the present study support the differences between levels i and ii of the gmfcs, recently revised by the authors. the discriminant model revealed that pelvic obliquity has a higher impact in discriminating children gmfcs level i from those of level ii. clinically, this shows that pelvic obliquity in the frontal plane explains better the mechanical difference between children in level i and ii. the importance of pelvic obliquity during gait was introduced in 1953 by saunders and coworkers as one of the most important gait marker that minimizes the vertical displacement of the center of mass. in 2001 , della croce and coworkers confirmed that pelvic obliquity and single support knee flexion are the second most important gait determinants in reducing center of mass dislocation and consequently improving gait efficiency. the present study demonstrated that gait velocity was similar between cp children in level i and ii. in a multicentre study conducted with 562 children with cerebral palsy, classified in levels i to iii of the gmfcs, determined that gait velocity is a discriminant factor between levels i and ii. similar were reported by oeffinger et al.. in addition, damiano and abel reported that the temporal and spatial parameters were important indicators of the severity level of the cerebral palsy children. the classification in levels i and ii, proposed by palisano et al., was based primarily in the limitation of the child to execute movements that involved velocity and stability, such as walking, jumping, and going up and down stairs. therefore, we would expect a significant difference between levels i and ii in the gait spatial and temporal markers. it is likely that the different instruments used to capture temporal and spatial gait markers and the fact that our sample was composed only of diplegic cerebral palsy children probably justify the different found in the present study. gait alterations in children with cerebral palsy may also occur in other planes of motion. rodda and graham proposed a classification system for diplegic children based on the kinematic and kinetic analyses focused in the sagittal plane. however, the authors agreed that important alterations may also occur in the frontal and transverse planes. for example, excessive internal pelvic rotation during initial contact contributes to a higher range of hip abduction motion observed during gait strike. on the other hand, increased pelvic obliquity may be secondary to a decrease in the hip and knee sagittal motions. the lack of significant findings in the sagittal plane could be related to the nature of the movement. as in the sagittal plane, the range of motion is greater compared to the other planes, the variability is normally smaller when compared to movements with smaller range of motions. one option would be to increase the sample size, in an attempt to identify the subtle variances that could occur in the sagittal plane. nevertheless, in the present study, even with a relatively small sample size the could discriminate the groups, showing that, in fact, a difference between levels i and ii, although subtle, could be detected with the multivariate analysis technique applied. the present study offers new information on angular displacement in all three planes of gait stance of children with cerebral palsy classified as gmfcs levels i and ii. clinical observation of reduced pelvic obliquity and hip adduction during gait is a difficult task. the literature on observational gait analysis has shown that training and experience are important for a more consistent observation of the pelvic and hip movements during gait. therefore, the kinematic idiosyncrasy of each gmfcs level could be added as one important clinical parameter to help the classification process of mild - to - moderate children with diplegia. in addition, prior knowledge of the biomechanical differences between gmfcs levels i and ii may guide further physical therapy strategies, focused on regaining pelvic obliquity and hip adduction range of motion of the support limb. such strategies may promote gait stability of cerebral palsy children level ii, with less energy expenditure and free from assistive devices.

objective. to determine if gait waveform could discriminate children with diplegic cerebral palsy of the gmfcs levels i and ii. patients. twenty - two children with diplegia, 11 classified as level i and 11 as level ii of the gmfcs, aged 7 to 12 years. methods. gait kinematics included angular displacement of the pelvis and lower limb joints during the stance phase. principal components (pcs) analyses followed by discriminant analysis were conducted. . pc1s of the pelvis and hip in the frontal plane differ significantly between groups and captured 80.5% and 86.1% of the variance, respectively. pc1s captured the magnitude of the pelvic obliquity and hip adduction angle during the stance phase. children gmfcs level ii walked with reduced pelvic obliquity and hip adduction angles, and these variables could discriminate the groups with a cross - validation of 95.5%. . reduced pelvic obliquity and hip adduction were observed between children gmfcs level ii compared to level i. these could help the classification process of mild - to - moderate children with diplegia. in addition, it highlights the importance of rehabilitation programs designed to improve pelvic and hip mobility in the frontal plane of diplegic cerebral palsy children level ii of the gmfcs.