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the myelodysplastic syndrome (mds) is a heterogenous group of stem cell disorders usually characterized by progressive refractory cytopenias with possible progression to acute myeloid leukemia. although cutaneous manifestation in mds is not frequent, mds may be associated with a wide spectrum of skin lesions1 ) and unusual rheumatologic manifestations2,3 ). however, concurrent erythema nodosum and serositis has rarely been reported. we describe a case of mds with erythema nodosum and immune - mediated pericardial effusion. a 34-year - old woman was diagnosed with mds french - american - british (fab) subtype refractory anemia in march, 2000. the patient did not receive any special treatment except intermittent transfusion of packed red cell. in may, 2003 the patient presented with fatigue, myalgia, fever as well as multiple edematous, tender, erythematous to brownish subcutaneous nodular lesions on both upper and lower extremities and trunk (figure 1). laboratory finding indicated hemoglobin 5.8 g / dl, white blood cell count 6.810/l, platelet 4.810/l, and normal liver and kidney function test. biopsy of skin lesion revealed lobular panniculitis consistent with erythema nodosum (figure 2). one month later, dyspnea, chest pain and fever developed and the skin lesions were aggravated. the laboratory tests showed hemoglobin 6.0 g / dl, white blood cell count 4.810/l, platelet 8.010/l, uric acid 2.3 mg / dl, ldh 474 u / l (normal 211 - 423), serum ferritin 1831 ng / ml, serum iron / tibc 145/162 g / dl. chest x - ray and chest ct scan showed cardiomegaly with pulmonary edema (figure 3). after the dose of prednisone was increased to 40 mg / day, the pericardial effusion and skin lesions had remarkably improved, suggesting that these newly developed symptoms might be attributed to the immune - mediated complications of mds. the repeated bone marrow biopsy performed one month later revealed no evidence of progression to acute leukemia. however, the dysplastic changes in the myeloid, erythroid and megakaryocytes lineages had progressed compared to the examination in the prior month. while gradually tapering prednisone until july 2004, she was taking 5 mg / day of prednisone without pericardial effusion. mds can be associated with a wide spectrum of skin lesions including neoplastic cell infiltration, sweet's syndrome, pyoderma gangrenosum, erythema elevatum diutinum, vasculitis, and panniculitis1 ). erythema nodosum is the most frequent clinico - pathological variant of panniculitis and manifests as a cutaneous reaction consisting of inflammatory, tender, nodular lesions, usually located symmetrically on the extensor surfaces of the lower extremities5 ). manifestation of the lesions may be associated with a wide variety of conditions including infections, sarcoidosis, rheumatologic diseases, inflammatory bowel diseases, medications, autoimmune disorders, pregnancy and malignancies6 - 8 ). the appearance of skin lesions in mds may herald its progression to acute myeloid leukemia. although in this case the repeated bone marrow biopsy did not show any findings of progression to acute leukemia, the dysplastic changes in all lineages of hematopoietic cells had progressed with the development of skin lesions. it has been reported that autoimmune manifestations in patients with mds range from asymptomatic serological abnormalities to classic connective tissue disorders such as sjgren's syndrome, relapsing polychondritis, systemic lupus erythematosus, rheumatoid arthritis and mixed connective tissue disease2,3,9 ). in particular, abnormal t - cell responses to antigen presentation and abnormal b - cell and t - cell interactions in mds may be important in the pathogenesis of immune dysregulation leading to the development of an autoimmune phenomenon10 - 12 ). thus, abnormalities in t - cell function might be associated with the development of mds13 ). although pericardiocentesis was not performed in our case, the pericardial effusion rapidly regressed after treatment with high doses of systemic corticosteroid, suggesting that this lesion might be a reactive inflammatory change involving immunologic mechanisms. enright et al.9 ) reported that aggressive therapy with immunosuppressive agents in selected patients often controls autoimmune features associated with mds and may lead to hematological responses in some patients. the most commonly used immunosuppressive agents are oral prednisone and high - dose intravenous methylprednisolone. although there may be poor prognosis with the onset of autoimmune disease in patients with mds, patients with a hematologic response to immunosuppressive therapy have prolonged survival compared with patient who do not. in this case, we noted that treatment with prednisone improved not only erythema nodosum and pericardial effusion, which may be related to an autoimmune manifestation, but also thrombo - cytopenia. in summary , mds may be associated with erythema nodosum with skin lesions that may be a of autoimmune dysfunction. the understanding of the pathophysiology of mds may contribute to explain the autoimmune manifestations and skin lesions in mds.

myelodysplastic syndrome (mds) is a heterogenous group of stem cell disorders usually characterized by progressive refractory cytopenias, which could progress to acute myeloid leukemia. mds may be associated with a wide spectrum of skin lesions, including neoplastic cell infiltration, sweet's syndrome, pyoderma gangrenosum, erythema elevatum diutinum, vasculitis, and panniculitis. however, erythema nodosum is rarely associated with mds. unusual rheumatologic manifestations in patients with mds also have been reported, which range from asymptomatic serological abnormalities to classic connective tissue disorders such as sjogren's syndrome, relapsing polychondritis, systemic lupus erythematosus, rheumatoid arthritis and mixed connective tissue disease. however, concurrent erythema nodosum and serositis has rarely been reported. we describe a case of mds with erythema nodosum and immune - mediated pericardial effusion in a 34-year - old woman.

hepatocellular carcinoma (hcc) has become a global health problem, with more than half a million new cases diagnosed worldwide each year.1 hcc is associated with a high mortality rate, and is the leading cause of liver - related death in patients with compensated cirrhosis.2 the incidence of hcc is increasing in several developed countries, including the us. between 1995 and 1998 alone, there was a 25% increase of hcc incidence in the us.3 it has been estimated that the number of cases of hcc will continue to increase by 81% to the year 2020, predominantly due to hepatitis c (hcv) infection.4 nevertheless, the incidence of hcc is decreasing in some developing countries as a of the implementation of universal hepatitis b vaccination.5 older age (> 50 years), male gender, severity of compensated liver cirrhosis at presentation, and sustained activity of liver disease are important predictors of hcc, independent of etiology of cirrhosis.6 males typically have a two- to four - fold higher risk of developing hcc than females. more than 80% of patients with hcc have cirrhosis, which remains the major risk factor. hcv infection, hepatitis b (hbv) infection, and heavy alcohol use remain the most common causes of cirrhosis in western countries. once the diagnosis of hcc is made and confirmed, there are several treatment options depending on the size and number of tumors, underlying liver function, presence or absence of tumor vascular invasion, and the patient s performance status. hepatic resection may be an option for those with hcc in a noncirrhotic liver or those with hcc and cirrhosis but without portal hypertension and with normal bilirubin. portal hypertension is defined as the presence of hepatic venous pressure gradient greater than 10 mmhg, esophageal varices, or splenomegaly with a platelet count less than 100,000/mm. subjects without relevant portal hypertension and normal bilirubin can achieve a 70% five - year survival after hepatic resection, whereas survival is only 50% in those with portal hypertension, and even lower with both adverse factors.7 hepatic resection is associated with a high tumor recurrence rate, ie, up to 70% in five years, either from true recurrence or development of de novo tumors. liver transplantation is the mainstay treatment for those with hcc and cirrhosis. during transplantation, not only the tumor itself but also the cirrhotic liver that provides the for new tumor development will be removed. for those who meet the milan criteria (solitary hcc less than 5 cm in size or up to three tumors with each tumor less than 3 cm in size) , transplantation provides a five - year survival exceeding 70% while maintaining a low recurrence rate of less than 15%.7,8 in spite of being the best option for patients with hcc, transplantation is limited by the current shortage of available donors. to avoid dropout while awaiting transplantation, transplant candidates usually receive locoregional treatments, such as transarterial chemoembolization or percutaneous ablation, especially if the waiting time exceeds six months.9 due to the small risk of tumor seeding in the needle tract associated with percutaneous ablation, transarterial chemoembolization is more favored as a bridging therapy for transplant candidates. for those with small unresectable hcc, percutaneous ablation, such as percutaneous ethanol injection and radiofrequency ablation, offers comparable survival with that of resection.1013 in addition to similar survival compared with surgery, percutaneous ablation therapy is relatively simple, low - cost, repeatable, and safe. compared with percutaneous ethanol injection, radiofrequency ablation has a higher rate of complete tumor necrosis (radiofrequency ablation 90% versus percutaneous ethanol injection 80%), requires fewer treatment sessions (radiofrequency ablation 1.2 versus percutaneous ethanol injection 4.8), and is associated with better local recurrence - free survival compared with percutaneous ethanol injection. nevertheless, the incidence of adverse events is not significantly different between these two treatment modalities. in addition to being used as a bridging therapy before transplantation, transarterial chemoembolization also serves as a palliative measure for those with unresectable hcc and who are not amenable to percutaneous ablation. in a systematic review of randomized, controlled trials involving various treatment modalities in patients with unresectable hcc, arterial embolization improved two - year survival compared with controls (odds ratio 0.53 ; 95% confidence interval , 0.320.89 ; p = 0.017).14 sensitivity analysis showed a significant benefit of transarterial chemoembolization (or 0.42 ; 95% ci 0.200.88) but none with bland embolization alone (or 0.59 ; 95% ci 0.291.20). radioembolization with microspheres carrying yttrium 90 injected via the intrahepatic arterial route has been proposed as an alternative approach to transarterial chemoembolization.15 this method can deliver radiation to the hcc - associated capillary bed. radioembolization requires only single - dose administration, and has been shown to be therapeutically equivalent to transarterial chemoembolization.16 patients with advanced hcc who are not candidates for local approaches are considered for systemic therapy. systemic therapy with cytotoxic agents, such as doxorubicin, provides marginal benefits without improvement in overall survival.17 investigation in hepatocarcinogenesis has led to the discovery of key molecular targets in hcc, such as angiogenesis, epidermal growth factor receptor, and mammalian target of rapamycin. a growing number of molecularly targeted therapies for hcc are currently at different stages of clinical development.18 sorafenib (nexavir, bayer healthcare ag, leverkusen, germany), a multikinase inhibitor, is the only such drug that has been approved in the us and europe for treatment of unresectable hcc. in this review, we provide a critical appraisal of sorafenib in the management of hcc. sorafenib inhibits multiple tyrosine and serine / threonine kinases involved in cell proliferation and angiogenesis. these kinases include raf kinase, vascular endothelial growth factor receptor (vegfr) kinases, platelet - derived growth factor receptor (pdgfr) kinase, c - kit receptor kinase, ret receptor kinase, and fms - like tyrosine kinase 3.19,20 disruption of pdgfr and vegfr signaling pathways inhibits angiogenesis. raf is an essential serine / threonine kinase in the mitogen - activated protein (map) kinase signaling pathway, and is a downstream effector of ras, which is activated in many human malignancies.21 when the raf - map kinase pathway is activated, extracellular signal - regulated kinase (erk) will become phosphorylated. subsequently, phosphorylated erk (perk) can be translocated to the nucleus where it can regulate gene expression by modulating various transcription factors and target genes. preclinical models have demonstrated that hcc exhibits activation of map signaling and overexpression of angiogenesis factors. sorafenib blocks the raf - map kinase and erk pathway, inhibits tumor angiogenesis, and induces tumor cell apoptosis in hcc cell lines.22 pharmacodynamic assay for raf - map kinase inhibition by sorafenib has been investigated in patients peripheral blood lymphocytes collected at different time points in a phase i study.23 lymphocytes were activated with phorbol myristate acetate, and flow cytometric analyses of perk were performed. the phorbol myristate acetate - induced perk in peripheral blood lymphocytes from six patients was almost completely eradicated by sorafenib after 21 days of the maximally tolerated dose. in a phase ii study of sorafenib in advanced hcc, immunohistochemical study in pretreatment tumor biopsies using an antibody against perk was performed in 33 patients (of 137 enrolled patients), and perk staining was more intense in the nuclei of tumor tissues.24 there was a significant difference in time to progression between patients with higher tumor cell perk staining intensity versus those with lower intensity. a similar finding was also noted in a retrospective biomarker analysis of the phase iii sorafenib hcc assessment randomized protocol (sharp) trial.25 the biomarker study from the sharp trial has further revealed that low hepatocyte growth factor and high c - kit serum levels at baseline were associated with improved survival in multivariate analysis. the recommended dose of sorafenib is 400 mg orally twice a day, and should be administered to patients who are fasting or with a moderate - fat meal. a high - fat meal reduces sorafenib s bioavailability by about 30%. the plasma protein binding of sorafenib is about 99% in patients with normal renal and hepatic function.26 sorafenib is primarily metabolized in the liver, predominantly via phase i oxidation by cytochrome p450 (cyp) 3a4, and phase ii conjugation by uridine 5-diphosphoglucuronosyltransferase.27,28 approximately 50% of orally administered sorafenib is recovered as unchanged drug in the feces, due to either biliary excretion or lack of absorption. the mean terminal elimination half - life of sorafenib is approximately 2438 hours, and sorafenib exposures reach steady state within seven days of dosing.29 because sorafenib is mainly metabolized by cyp3a4, its pharmacokinetics may be affected by drugs modulating the function of cyp3a4. concurrent administration of rifampin, a cyp3a4 inducer, has been shown to decrease the area under the plasma concentration versus time curve (auc) of sorafenib by 37%. other cyp3a4 inducers, including carbamazepine, phenobarbital, and dexamethasone, may decrease the auc of sorafenib.30 neomycin decreases the auc of sorafenib by 54%, probably due to eradication of gastrointestinal bacteria and therefore preventing the enterohepatic recycling of sorafenib.26 due to potential drug interaction, patients taking medications such as warfarin, digoxin, and quinidine may require close monitoring during sorafenib treatment.26,31 it has been shown that the auc for sorafenib is 30%45% lower in japanese or chinese than in caucasian populations, and the clinical significance of this finding remains unknown.30,32 the child - pugh scoring system uses clinical and laboratory information to stratify disease severity, surgical risk, and overall prognosis in patients with liver cirrhosis.33 the auc of sorafenib in patients with mild (child - pugh a) and moderate (child - pugh b) hepatic impairment has been shown to be 23%65% lower than in subjects with normal hepatic function, and there are no significant differences in sorafenib pharmacokinetics between patients with child - pugh a and child - pugh b hepatic impairment.24,34 no significant difference in sorafenib pharmacokinetics has been shown between patients with normal renal function and mild - to - moderate renal insufficiency.30,35 the pharmacokinetics of sorafenib has not been studied in patients with severe hepatic impairment (child - pugh c) or severe renal insufficiency (creatinine clearance less than 30 ml / min). in a phase i pharmacokinetic study of sorafenib in patients with hepatic or renal dysfunction, miller et al showed that patients with severe renal or hepatic dysfunction frequently developed intolerance to sorafenib.35 therefore, it is recommended to start at a lower dose in these patient populations, followed by close monitoring and gradual dose escalation. two phase iii randomized studies, ie, sharp and the asia - pacific study, comparing sorafenib with placebo as first - line systemic treatment in patients with advanced hcc, have demonstrated the efficacy of sorafenib.36,37 patient characteristics and outcome measures of these two studies are summarized in tables 1 and 2. the sharp study was designed for the regulatory approval of sorafenib in the us and europe, and was a randomized, double - blinded, placebo - controlled, phase iii study conducted in europe, the us, and australasia. it enrolled 602 patients with advanced hcc, well - preserved liver function, and no prior systemic therapy. patients were randomized in a 1:1 ratio to receive either oral sorafenib (400 mg twice daily) or placebo. the number of patients needed for this study was calculated on the basis of overall survival, with a power of 90% to detect a 40% improvement in overall survival in the sorafenib group. in the sharp study, patients were well balanced with respect to baseline characteristics (table 1). as summarized in table 2, this study demonstrated a significant improvement in median overall survival on sorafenib versus placebo (10.7 versus 7.9 months ; hazard ratio in the sorafenib group 0.69 ; 95% ci 0.550.87 ; p < 0.001). there was significant prolongation of median time to radiologic progression in the sorafenib group (5.5 versus 2.8 months ; p < 0.001). in the sorafenib group, seven patients (2.3%) had a partial response and 71 (71%) had stable disease (according to the response evaluation criteria in solid tumors), whereas in the placebo group, two patients (1%) had a partial response and 204 patients (67%) had stable disease. the disease - control rate, defined as complete / partial response or stable disease by recist for four weeks from the first demonstration of that rating, was significantly higher in the sorafenib group than in the placebo group (43% versus 32% ; p = 0.002). based on the sharp data, sorafenib was approved by the european medicines agency in october 2007 and by the us food and drug administration in november 2007 for the treatment of patients with unresectable hcc. in order to achieve regulatory approval in china, cheng et al conducted a phase iii randomized study comparing sorafenib with placebo in patients with advanced hcc in the asia - pacific region.37 this study had no predefined primary endpoint, and used the same eligibility criteria as the sharp study. two hundred and twenty - six patients in china, south korea, and taiwan were randomized on an intention - to - treat basis in a 2:1 ratio to sorafenib versus placebo. the number of patients needed for this study was calculated with a power of 83.9% to detect a 20% increase in overall survival in the sorafenib group. overall survival, time to radiologic progression, time to symptomatic progression, disease - control rate, and safety were assessed. overall survival was 6.5 months with sorafenib versus 4.2 months in the placebo group, with an hr of 0.68 (p = 0.014). additionally, there were significant differences in time to radiologic progression and disease - control rate between the sorafenib and placebo groups, but not in time to symptomatic progression. the asia - pacific study has confirmed the of the sharp study, and the magnitude of benefits in overall survival and time to radiologic progression derived from sorafenib was almost the same between these two studies, ie, a 31%32% of relative risk reduction in death, and a 42%43% relative benefit of increase in time to progression. however, the absolute benefit of the increase in overall survival and time to radiologic progression derived from sorafenib was smaller in the asia - pacific study than in the sharp study. additionally, the median overall survival and time to radiologic progression were shorter in asia - pacific study. there were more patients with hbv and severe disease, such as macroscopic vascular invasion and/or extrahepatic spread in the asia - pacific study, which might have contributed to the shorter overall survival and time to radiologic progression than were seen in the sharp trial. patients with hbv - related hcc were shown to have poorer overall survival than patients with hcv - related hcc.38 hbv - related hcc was noted in 73% of patients in the asia - pacific study versus 18% in the sharp trial. a subset analysis of 165 patients with hbv - related hcc in the asia - pacific study indicated that sorafenib improved overall survival and time to radiologic progression independent of hbv status, and the safety profile of sorafenib in patients with hbv was comparable with that for the overall study patients.39 in the sharp study, 30% of patients were infected with hcv, and 25% had alcoholic liver disease. in a subset analysis of 178 patients with hcv - related hcc in the sharp trial, overall survival was 14.0 months in 93 patients receiving sorafenib versus 7.9 months in 85 patients receiving placebo. the disease - control rate was higher in the sorafenib group compared with placebo (44% versus 31%). the safety profile of sorafenib treatment in patients with hcv - associated hcc was similar to that in all patients receiving sorafenib in this study.40 the subgroup analysis in patients with alcohol - related hcc in the sharp study demonstrated a similar outcome and safety profile to the overall population.41 taken together, there is a consistent clinical benefit from sorafenib in hcc, irrespective of hbv / hcv status or alcoholic liver disease. combined analysis of the sharp and asia - pacific trials to examine the effects of macroscopic vascular invasion, extrahepatic spread, and eastern cooperative oncology group (ecog) performance status on outcome has shown that sorafenib was effective and safe for the treatment of advanced hcc in patients globally, irrespective of baseline ecog performance status and the presence or absence of macroscopic vascular invasion and/or extrahepatic spread.42 in the sharp study, 38% of patients had macroscopic vascular invasion and 52% had extrahepatic spread versus 35% with macroscopic vascular invasion and 68% with extrahepatic spread in the asia - pacific study (table 1). however, a significant difference in overall survival based on the presence or absence of macroscopic vascular invasion and/or extrahepatic spread was observed in both trials. for patients treated with sorafenib in the sharp study, median overall survival was 14.5 months in patients without macroscopic vascular invasion or extrahepatic spread versus 8.9 months with macroscopic vascular invasion and/or extrahepatic spread. for patients treated with sorafenib in the asia - pacific trial, median overall survival was 14.3 and 5.6 months, respectively. therefore, for advanced hcc patients with macroscopic vascular invasion and/or extrahepatic spread, there is a dire need for treatment options in addition to sorafenib to improve the outcome. as summarized in table 3, the common drug - related adverse events in patients receiving sorafenib were hand - foot skin reaction, diarrhea, alopecia, fatigue, skin rash, hypertension, nausea, and anorexia. hand - foot skin reaction and diarrhea were the two most frequent severe (grade 3 or 4) toxicities in patients receiving sorafenib, at about 5%10%. for patients on sorafenib, 26%31% required dose reduction, and 20%38% of patients terminated treatment due to toxicities. there seemed to be less treatment discontinuation in the asia - pacific study regardless of whether patients were on sorafenib or placebo than in the sharp study. there might be ethnic differences in hand - foot skin reaction, which occurred more frequently in asian patients. hand - foot skin reaction is manifested by formation of thick hyperkeratotic lesions affecting pressure points and flexure areas.43 these lesions usually develop after 24 weeks on sorafenib treatment and are often painful, which affects patient quality of life.44 a mild hand - foot skin reaction can be treated with lanolin - based or urea - based lotions. protective measures, such as wearing gloves and socks, are recommended to provide pain relief and prevent skin breakdown. dose reduction or interruption of sorafenib is frequently needed in patients with moderate to severe hand - foot skin reaction. sorafenib can be resumed at a lower dose, with gradual escalation when hand - foot skin reaction is resolved. adverse cardiovascular toxicities, including myocardial infarction, hypertension, congestive heart failure, and arrhythmia, are frequently associated with antiangiogenic inhibitors, and are regarded as a class effect.45 hypertension is the most frequent event, and most of the time is clinically manageable. the incidence of hypertension was 5% in the sharp study and 19% in the asia - pacific study, with a 2% incidence of grade 3 or 4 hypertension noted in sorafenibtreated patients in each of the studies. the incidence of severe cardiac ischemia or infarction in sorafenib - treated patients in the sharp study was 3% versus 1% in patients taking placebo.36 sorafenib was associated with hypophosphatemia in 35% of sorafenib - treated patients compared with 11% taking placebo in the sharp study. grade 3 or 4 hypophosphatemia, which can lead to muscle weakness, altered mental status, and cardiac events, was seen in 11% of patients taking sorafenib.32 therefore, sorafenib is not recommended in patients with a recent history of cardiac disease. all patients taking sorafenib should be monitored frequently for symptoms of cardiac disease and have blood levels monitored regularly for hypophosphatemia. hypothyroidism is a common side effect of sorafenib, and the incidence can be as high as 18%.46 sorafenib can enhance t4 and t3 metabolism, which is probably caused by increased type 3 deiodination.47 clinical hypothyroidism occurs about one to seven months after the initiation of sorafenib.48 therefore, it is important to monitor thyroid function periodically in patients receiving sorafenib. in the sharp study, elevated lipase occurred in 40% of patients receiving sorafenib versus 37% for patients on placebo. however, grade 3 or 4 lipase elevation was seen in 9% of patients receiving sorafenib.32 there have been cases reported of acute pancreatitis in patients receiving sorafenib treatment for hcc or renal cell carcinoma, and pancreatitis usually improves after stopping sorafenib.4951 there are limited data on the use of sorafenib in patients with child - pugh b or child - pugh c hepatic impairment. yau et al reported a study involving 15 patients with child - pugh b / c cirrhosis receiving sorafenib for hcc. thirteen patients discontinued treatment prematurely due to treatment toxicities.52 in a phase ii study of sorafenib in hcc, including 98 child - pugh a and 38 child - pugh b patients, there was no significant difference in sorafenib pharmacokinetics between patients with child - pugh a and child - pugh b impairment.53 however, child - pugh b patients had a higher rate of hyperbilirubinemia, encephalopathy, and worsening ascites, and child - pugh b patients did worse, with overall survival of 14 weeks versus 41 weeks for patients with child - pugh a. therefore, it is recommended to reduce the starting dose, perhaps by 50%, with vigilant monitoring of liver function, if sorafenib is to be used in patients with child - pugh b liver cirrhosis.35,54 for hcc patients with child - pugh c, because of their severe underlying disease with limited life expectancy of usually less than three months, sorafenib treatment is not recommended.55,56 in both the sharp and asian - pacific studies, the functional assessment of cancer therapy hepatobiliary symptom index 8 (fhsi-8) was used to assess time to symptomatic progression. fhsi-8 is a patient - oriented outcome instrument designed to assess symptoms and quality of life in hepatobiliary cancer patients in general.57 fhsi-8 analyses in both studies failed to identify any significant difference between the sorafenib and placebo groups. the quality of life of these patients might have been affected by other concurrent diseases, such as liver cirrhosis. additionally, fhsi-8 might not have been sensitive enough to determine small but significant differences in quality of life between these two groups. sorafenib is the first agent that has ed in a survival benefit in hcc, and has validated the role of targeted therapy in this disease. its role in the management of advanced unresectable cancer is now well proven in all subpopulations of hcc having adequate hepatic reserve, and has been incorporated in the consensus - based treatment algorithm globally (see figure 1 for a simplified schema).5860 the role of sorafenib in hcc confined to the liver and as part of multimodality hcc treatment is currently under investigation. ongoing studies will incorporate sorafenib with other treatment modalities, including transarterial chemoembolization, cytotoxic chemotherapy, surgery, and transplantation, in hcc patients.61 further studies are needed to define its dosage and role in patients with child - pugh b and child - pugh c liver cirrhosis. identification of a predictive biomarker signature will help to define the ideal patient population for sorafenib treatment in hcc.

sorafenib is an oral multiple kinase inhibitor that blocks raf, vascular endothelial growth factor receptor, and platelet - derived growth factor receptor. it has been approved in the us and europe for the treatment of advanced hepatocellular carcinoma (hcc). sorafenib has demonstrated a 44% increase in survival for advanced hcc patients, compared with best supportive care alone. we have reviewed the pharmacology, pivotal studies, and safety data for this agent. sorafenib is the first systemic drug demonstrating a significant survival benefit, and is the standard of care for patients with advanced hcc for whom no potential curative option is available.

microvascular decompression (mvd) has been known to be an effective surgical modality as the treatment of various hyperactive neurovascular compression syndromes. its high success rate (over 90% long term cure rate) in the treatment of hemifacial spasm (hfs) has made it the treatment of choice5,10 ). although mvd is known to be a relatively safe procedure, it is rarely reported in the literature to accompany severe disabling complications. common complications of mvd for hfs are also brainstem or cerebellar infarct, hematomas, cerebrospinal fluid leak and cranial nerve palsy, commonly involving 7th and 8th nerve with rare lower cranial nerve palsies are reported1,5,11 ). we have experienced unilateral soft palate palsy without the involvement of vocal fold after mvd for hfs. so far, to our knowledge, such complication is very rare, following mvd. here, a 33-year - old female was presented to our out - patient clinic with a history of left hemifacial spasm for 5 years. intra - operatively, bilateral facial electromyography and brain stem auditory evoked potential was monitored. after the dura incision, gentle retraction of the cerebellum was applied using greenburg retractor and we have exposed the lower cranial nerves. dissection of the arachnoid was done from the rostral border of the lower cranial nerves. the arachnoid band was very tough and difficult to dissect, but no obvious injury occurred to the lower cranial nerve during the dissection. the left anterior inferior cerebellar artery was the offending vessel compressing the facial nerve from the caudal side and decompression was successfully completed without any adverse events and with no change in the intraoperative monitoring of brainstem auditory evoked potential. patient recovered well from anesthesia with reduced facial spasm, compared to the pre - operative state. postoperatively, neurologic exam revealed no abnormalities and also patient's swallowing and vocalization function was intact. barium swallowing test was done to evaluate dysphagia, and minimal nasal regurgitation was demonstrated due to unilateral soft palate palsy. on endoscopic exam, performed by an otolaryngologist, the vocal cord was intact in both sides with unilateral left soft palate palsy, which suggested tensor and/or levator veli palatini muscle weakness (fig . 2). under the impression of possible lower cranial nerve palsy caused by postoperative edema of the nerve, steroid (prednisolone) was prescribed and we decided to observe the symptoms. we encouraged her to sleep on her side; then her symptom improved. despite persistent speech rehabilitation we performed cranial nerve mri to evaluate for the possibility of any structural abnormalities, such as delayed hematoma or possible infection. however, mri showed no abnormal structural lesions or abnormal enhancement, which may suggest infection (fig . 3). the patient continued to receive speech rehabilitation with subjective improvement of swallowing and speech. on postoperative 25th day, the patient developed grade ii facial palsy by house - brackmann grade on the left side. patient's facial palsy improved with administration of acyclovir for 5 days, but hoarseness with swallowing difficulty showed little improvement. the patient received prolonged speech and swallowing rehabilitation therapy, and follow up examination was done before discharge showed improvement of nasalization, but persistent hypernasality. endoscopic examination revealed normalized soft palate palsy and no abnormality in the vocal cord mobility (fig . however, patient 's subjective difficulty of speech still persisted and we decided to observe her symptoms for any changes in the future . cranial nerve palsy has been reported as complication of mvd and can cause symptoms that markedly decrease the quality of life . most commonly, the 7th and 8th cranial nerve are involved with rare lower cranial nerve involvement . among the lower cranial nerves, glossopharyngeal nerve and vagus nerve glossopharyngeal nerve contains both motor and sensory branches that supply the tongue and stylopharyngeus muscle of the pharynx . impairment of glossopharyngeal nerve would cause decreased sensation in the pharynx including taste in the posterior one third of the tongue with difficulty in swallowing and dysphonia2 - 4). our patient developed swallowing difficulty with dysphonia, which suggests possible dysfunction of the 9th nerve. vagus nerve also contains both motor and sensory branches that innervate all striated muscles of the laryngopharynx, except the stylopharyngeus and tensor veli palatini. patients with unilateral high vagal lesions would suffer from breathy dysphonia and aspiration because of impaired vocal cord movement. due to the loss of sensation of the laryngopharynx, causing loss of gag reflex, patients might also suffer severe dysphagia related to the poor coordination of the oro- and hypo - pharyngeal phases of swallowing2 - 4 ). unilateral soft palate palsy with dysphonia and dysphagia was observed in our patient, which suggests unilateral vagus nerve dysfunction, but the sparing of the vocal cord suggest the function of the vagus nerve is only partially impaired. although the functions and innervations of the 9th and 10th nerve are known, it is difficult to exactly locate which lower cranial nerve is causing the symptom by physical examination. many symptoms are subtle and overlap with other diseases, making the diagnosis elusive2 ). common injury to the vagus nerve is accompanied by vocal fold palsy, but our patient only presented with soft palate palsy. in addition, partial injury of the vagus nerve with possible involvement of the glossopharyngeal nerve have never been reported as a complication of mvd in prior literature, thus making it difficult to identify the exact cause of such peculiar symptoms. skull base tumors are the most common cause and trauma or surgery above the skull base and from the cerebellopontine angle can also be a cause. rarely, infection and ischemic vascular insult are reported to cause unilateral vagus nerve palsy3,7,8,12 ). it is important to note that in our patient, the hoarsness and dysphagia developed several days after the surgery. generally, lower cranial palsy caused by trauma would occur immediately after the event, which implies the possibility of other factors, such as infection contributing in the delayed onset of lower cranial nerve palsy. in the literature, some have reported herpes simplex virus (hsv) to be a possible cause of unilateral vagus nerve palsy12 ). in addition, varicella zoster virus is also reported to cause lower cranial nerve palsy6 ). although we have failed to show such connection in our patient, history of delayed facial palsy also suggests the possibility of concurrent viral infection. others have also suggested that delayed facial palsy after mvd in patients with hfs may be caused by viral infections. as hsv is widely known to cause bell's palsy, it could likewise cause reversible inflammation of vagus nerve causing such clinical presentations11 ). the history of relapsing symptom, followed by tapering of steroid, suggests additional evidence that inflammatory reaction contributed in the development of such symptom. antiviral agent, such as acyclovir, is reported to be an effective treatment that we have used for facial palsy of the patient. some reported rare anatomical variants of lower cranial nerves with interconnection between the glossopharyngeal nerve and vagus nerve. such interconnection is so thin and very fragile that it makes them prone to shearing stress14 ). though they have not suggested its clinical implication, it is possible that such interconnection holds some significance in the nerve conduction. as such, it may have been overlooked and damaged during the retraction of the surgery, causing lower cranial nerve palsy. the clinical course of unilateral lower cranial nerve palsy is variable depending on the cause. initial extensive laboratory work up with ct and mr imaging is necessary to evaluate the cause. if no particular structural abnormalities are noted, conservative treatment, including acyclovir and steroid administration with speech therapy, seems to be reasonable. if symptoms persist, surgical treatments, such as medialization laryngoplasty and laryngeal reinnervation, can be considered9,13 ). we have experienced a very rare case of unilateral soft palate palsy, following mvd. although the exact cause of this complication had not been identified, we have suggested possible causes for such rare complication. it is important to keep in mind that mvd is performed to improve the quality of life rather than to save life. although it has relatively little complications, rare and debilitating complications must not be overlooked, and caution must be taken to avoid them. mvd is a very effective and relatively safe surgical modality in the treatment of hfs. the lower cranial nerve palsy we are reporting is one of such complication that can markedly decrease the quality of life of a patient and it is important to note that such delayed complication can occur without evident injury to the nerve during the operation. it is important to consider various etiologies as the treatment, and prognosis differs greatly on the cause. additionally, careful surgical technique with minimal retraction with extra care to avoid damage to the lower cranial nerve during exploration may be important to minimize such complication.

microvascular decompression is a very effective and relatively safe surgical modality in the treatment of hemifacial spasm. but rare debilitating complications have been reported such as cranial nerve dysfunctions. we have experienced a very rare case of unilateral soft palate palsy without the involvement of vocal cord following microvascular decompression. a 33-year - old female presented to our out - patient clinic with a history of left hemifacial spasm for 5 years. on postoperative 5th day, patient started to exhibit hoarsness with swallowing difficulty. symptoms persisted despite rehabilitation. various laboratory work up with magnetic resonance image showed no abnormal lesions. two years after surgery patient showed complete recovery of unitaleral soft palate palsy. various etiologies of unilateral soft palate palsy are reviewed as the treatment and prognosis differs greatly on the cause. although rare, it is important to keep in mind that such complication could occur after microvascular decompression.

hesc culture the hesc line h1 (wa01) was kindly provided by dr. saul sharkis from johns hopkins university, under permission from wicell research institute (5, 16), and hues-17 was kindly provided by dr. all hesc experiments were conducted in accordance with the guidelines for research on human embryonic stem cells, jointly issued by the ministry of science and technology and the ministry of health of china , and approved by the ethical committee of shanghai institutes for biological sciences. hescs were maintained on feeders in hesc medium, which contained 80% dulbecco's modified eagle's medium / ham's f-12 medium (f12), 20% knock - out serum replacement, 1 mm l - glutamine, 0.1 mm -mercaptoethanol, 1% nonessential amino acids, and 4 ng / ml human basic fgf. hescs cells were passaged approximately once a week by incubation in 1 mg / ml collagenase iv for 30 min at 37 c. protein factors or sb431542 were added directly to the culture in the continued presence of conditioned medium (cm). recombinant human activin a, recombinant human bmp-4, and human follistatin were purchased from r&d systems inc. rna isolation and real - time reverse transcription - polymerase chain reaction rna was extracted using trizol reagent for total rna isolation according to the manufacturer's instructions (invitrogen). cdna was synthesized using the revertaid first strand cdna synthesis kit (fermentas). real - time pcr was performed using a synergy brand greeni - based pcr master mixture (toyobo). the expression value of each gene was normalized to the amount of glyceraldehyde-3-phosphate dehydrogenase cdna to calculate a relative amount of rna present in each sample. the expression level of each gene in a single sample was arbitrarily defined as 1 unit. the normalized expression values for all control and treated samples were averaged, and an average -fold change was determined. analysis of variance was conducted between the normalized relative expression values for control and treated samples to determine statistical significance. immunostaining the following antibodies were used: anti - ssea4 (developmental studies hybridoma bank), anti - hcg (r&d systems), and anti - hcg (abcam). western blotting cells were lysed with 1 lysis buffer: 20 mm tris (ph 7.5), 150 mm nacl, 1% triton x-100, 1 mm na3vo4, and complete mini - protease inhibitor mixture (roche). total protein membranes were blocked in tris - buffered saline with 0.1% tween and 5% milk. the following antibodies were used: anti - phospho - smad2/3 (cell signaling), anti - smad2/3 (cell signaling), anti - phospho - smad1 (santa cruz), anti - smad1 (santa cruz), anti - oct4 (santa cruz), and -actin (abcam). primary antibodies were incubated overnight and secondary antibodies for 2 h. proteins were detected with chemiluminescent (pierce). h1 cells were cultured in cm with or without sb431542 for 12 days, and the medium was changed every day. the hcg concentration was analyzed using a hcg elisa kit (reci), which specifically reacts with cg-. the concentration of estradiol and progesterone were analyzed with an elisa kit. activin / nodal signaling has been shown to play a key role in the maintenance of undifferentiated human es cells . to further address the function of activin / nodal signaling in the developmental fate of hescs, and to understand the early developmental mechanisms of human embryogenesis, we inhibited activin / nodal signaling in hescs. two hescs lines, h1 and hues-17, were used in this study, and the obtained from these two cells lines were very similar. for this reason, hescs were cultured without murine embryonic fibroblast feeder cells in cm, or cm plus different concentrations of activin / nodal signaling inhibitors, sb431542 or follistatin, for 6 days; gene expression was analyzed by real - time pcr. consistent with previous studies, the conditioned media to the culture system is sufficient for the maintenance of undifferentiated hescs . sb431542 inhibits the function of activin receptor - like kinase receptors 4/5/7 thereby acting as a selective inhibitor of activin / nodal signaling, but not those of bmps (22, 23). follistatin is an inhibitor of activin by directly binding with activin and preventing the assembly of an active activin - receptor complex . when hescs were cultured in cm supplied with sb431542, the expression levels of p - smad2, and known downstream targets of activin / nodal signaling, namely nodal, lefty - a, and lefty - b, were significantly inhibited (figs . sb431542 is a very potent inhibitor of activin / nodal signaling ; in hescs cultured with cm plus 10 m sb431542, the expression of nodal, lefty - a, and lefty - b decreased to less than 0.1% of hescs cultured with cm . we also determined that hescs underwent differentiation when activin / nodal signaling was inhibited, because the treated cells became flattened and enlarged ( fig . 1b) and that pluripotency markers, such as oct4, nanog, and ssea4 were significantly down - regulated (fig . similar were obtained with follistatin as an inhibitor of activin / nodal signaling in hescs as observed with sb431542 ( fig . 1). these demonstrate that and the inhibition of activin / nodal signaling promoted differentiation of hescs. inhibition of activin / nodal signaling in hescs initiates trophoblast differentiation to determine lineage commitment or differentiation due to inhibition of activin / nodal signaling, we analyzed the induction of lineage - specific marker expression. unlike what we observed with a standard differentiation by embryoid bodies formation, we did not observe a significant up - regulation in expression of ectoderm (neurofilament heavy chain), mesoderm (cardiac actin), or endoderm (1-antitrypsin) markers (fig . 2a), indicating that inhibition of activin / nodal signaling under the monolayer culture condition did not initiate differentiation of endoderm, mesoderm, or ectoderm in hescs. however, the trophoblast marker gcm1 was specifically up - regulated (fig . 2b), which suggests that hescs might have differentiated into trophoblasts when activin / nodal signaling was inhibited. the notion of trophoblast differentiation was further supported by the up - regulation of other trophoblast markers, such as cdx2, gata2, msx2, cg-, and cg-. cg- and cg- are subunits of human chorionic gonadotropin (hcg), which is secreted by giant cells of trophoblast - derived placenta. we also analyzed another key regulator of trophoblast differentiation in mice, eomesodermin (eomes). although eomes plays a key role in mouse trophoblast differentiation, it is a downstream target of activin / nodal signaling in mice and xenopus (26, 27). we observed that eomes showed down - regulation when activin / nodal was inhibited. taken together, these data indicate that the inhibition of activin / nodal signaling in trophoblast differentiation in hescs. notably, we also observed a slight up - regulation of neuroectoderm markers, such as nestin, sox1, sox3, and ngn2, when activin / nodal signaling was inhibited, which supports a recent article by smith et al. the h1 he s cells were cultured under a feeder - free condition and treated with sb 431542 for 6 (a and b) or 12 days (c). then cells were harvested for analyses. a, real - time polymerase chain reaction analysis of endoderm (1-at), mesoderm (cact), and ectoderm (nfh) markers. b, real - time polymerase chain reaction analysis of multiple trophoblast markers. sb431542 up - regulates trophoblast marker expression in a dose - dependent manner. c, differentiated cells form syncytial cells after incubation in cm plus 10 mol / liter sb 431542 for 12 days. the expression level of each gene in h1 hescs maintained on murine embryonic fibroblast feeder cells is arbitrarily defined as 1 unit. 1-at, 1-antitrypsin; cact, cardiac actin; nfh, neurofilament heavy chain; dapi, 4,6-diamidino-2-phenylindole. the h1 he s cells were cultured under a feeder - free condition and treated with sb 431542 for 6 (a and b) or 12 days (c) a, real - time polymerase chain reaction analysis of endoderm (1-at), mesoderm (cact), and ectoderm (nfh) markers. b, real - time polymerase chain reaction analysis of multiple trophoblast markers. sb431542 up - regulates trophoblast marker expression in a dose - dependent manner. c, differentiated cells form syncytial cells after incubation in cm plus 10 mol / liter sb 431542 for 12 days. the expression level of each gene in h1 hescs maintained on murine embryonic fibroblast feeder cells is arbitrarily defined as 1 unit. 1-at, 1-antitrypsin; cact, cardiac actin; nfh, neurofilament heavy chain; dapi, 4,6-diamidino-2-phenylindole. we attempted to differentiate hescs that were growing as embryoid bodies or as a monolayer; however, were similar (supplemental fig . the differentiation of hescs as a monolayer produced higher expressions of trophoblast markers and lower expression of other lineage markers, such as sox3 . therefore, the data presented in this paper pertain to monolayer cultures, unless specifically mentioned . to understand the kinetics of trophoblast differentiation, we performed time course experiments and analyzed marker expression by real - time pcr . the expression of pluripotency markers, namely oct4 and nanog, decreased in a time - dependent manner ( fig . cdx2 has been shown to be the key regulator of trophoblast commitment and subsequent self - renewal in mice ; inhibition of activin / nodal signaling in hescs initiated cdx2 expression after 2 days, and expression rose to a peak on day 6 and decreased thereafter (fig . 3c). gcm1 expression was induced by inhibition of activin / nodal signaling on day 4 and continued to increase throughout differentiation (fig . 3c). two additional markers that often associated with bmp activation and trophoblast commitment, gata2 and msx2, were also dramatically up - regulated and reached a peak level at day 10 (fig . furthermore, cg- and cg- expression significantly increased at day 6 and reached a surprisingly high level on day 12 ( fig . 3c), which suggests that eomes might be dispensable in trophoblast differentiation of hescs. the transient expression of cdx2 suggests that its function could be to induce gcm1 and other trophoblast transcriptional factors, and the down - regulation of cdx2 might allow for further trophoblast maturation. although the hesc is the only available model thus far for studying human embryonic development, the human es cell model may not entirely reflect embryonic development in vivo. to explore this the 8-cell stage mouse embryos were cultured with 10 m sb431542 for 3 days. no gross abnormalities were detected at 4.5 days postcoitum; the inner cell mass and trophoblast formed normally (data not shown). these observations are in accordance with previous reports, demonstrating that activin / nodal signaling is involved in the propagation of mouse embryonic stem cells, but is not involved in the regulation of pluripotency (18, 30). hesc - derived trophoblast cells secrete placental hormones prolonged cultures of hescs in cm plus sb431542 were performed (12 days); the cells continued to develop, and numerous differentiated cells contained multiple nuclei (fig . 2c). reported that syncytial cells were present only among individualized bmp4-treated hescs plated at low density, whereas bmp4-treated hesc colonies form only mononuclear cells. in contrast, the present study demonstrated that the sb431542- or follistatin - treated hesc colonies formed syncytial cells (fig . 2c), which suggests that inhibition of activin / nodal signaling is more efficient than bmp4 in inducing syncytial cell formation. it was not attempted to induce hesc differentiation in individual cells. to further confirm trophoblast differentiation from hescs, both cg- and cg- proteins were detected in a large percentage of differentiated hescs after 12 days treatment with sb431542 or follistatin (fig . the percentage of the cg--expressing cells was 74 5% ( n = 3) when activin / nodal signaling was inhibited by 10 m sb431542, and 66 3% (n = 3) when activin / nodal signaling was inhibited by follistatin, respectively. in addition, during hesc differentiation, the placental hormones, hcg (consisting both a and b subunits), estradiol, and progesterone, were secreted in the supernatant in a time- and dose - dependent manner (fig . inhibition of activin / nodal signaling down - regulates fgf and wnt signals, but up - regulates bmp signals fgf signaling has been shown to be important in the maintenance of hesc pluripotency ( 31, 32), and wnt signaling has been shown to stimulate the proliferation of hescs . previously, we have reported that activin / nodal signaling up - regulates fgf and wnt signaling in hescs. 5a ) was significantly repressed by the inhibition of activin / nodal signaling, but p - smad1 and bmp4 expression was significantly up - regulated (fig . 5, b and c). these observations further strengthen our previous hypothesis that activin / nodal signaling plays a key role in the complex signaling network that maintains the hesc phenotype and function . bmp4-induced trophoblast differentiation correlates with inhibition of activin / nodal signaling as reported by xu et al. , we also observed that hescs differentiated into trophoblasts when cultured in cm plus bmp4 (10 - 50 ng / ml), as evidenced by the down - regulation of pluripotency markers, such as oct4 and nanog (fig . 6a), and the up - regulation of cdx2, gcm1, gata2, cg-, and cg- (fig . 6a). at the same time, expression of lefty - a, lefty - b, and nodal was largely inhibited in a dose - dependent manner (fig . taken together, these indicate that bmp4 was sufficient to inhibit activin / nodal signaling and that bmp4-induced trophoblast differentiation in hescs correlates to the inhibition of activin / nodal signaling . figure 3.trophoblast differentiation in a dose- and time - dependent manner . the h1 he s cells were cultured under a feeder - free condition and treated with sb431542 for 12 days . real - time pcr analyses of the downstream targets of activin / nodal signaling ( a), the pluripotent markers (b), and the trophoblast markers (c), during differentiation of h1 cells to trophoblast cells following induction by sb431542. relative expression levels of each gene were analyzed at 0, 2, 4, 6, 8, 10, and 12 days, respectively, after addition of sb431542. the expression level of each gene at day 0 (prior to the addition of sb431542) is arbitrarily defined as 1 unit. the h1 he s cells were cultured under a feeder - free condition and treated with sb431542 for 12 days. real - time pcr analyses of the downstream targets of activin / nodal signaling (a), the pluripotent markers (b), and the trophoblast markers (c), during differentiation of h1 cells to trophoblast cells following induction by sb431542. relative expression levels of each gene were analyzed at 0, 2, 4, 6, 8, 10, and 12 days, respectively, after addition of sb431542. the expression level of each gene at day 0 (prior to the addition of sb431542) is arbitrarily defined as 1 unit. inhibition of activin / nodal signaling is essential for trophoblast differentiation we further investigated whether inhibition of activin / nodal signaling is essential for hesc trophoblast differentiation. hesc differentiation was induced by incubating the cells in cm supplemented with bmp4 and gradients of activin a. showed that activin a restored the expression of lefty - a, lefty - b, and nodal, indicating release of the bmp inhibition effect on activin / nodal signaling (fig . activin a also significantly inhibited cg- and cg- expression, which was induced by bmp4 ( fig . immunostaining methods were utilized to detect cg- and cg- proteins in hescs after 6 days of treatment with 10 ng / ml bmp4, or 10 ng / ml bmp4 plus 100 ng / ml activin a. bmp4 induced the hescs to produce cg- and cg-. however, the number of cg-- and cg--positive cells was reduced dramatically when activin a was added ( fig . , elisa analyses demonstrated that activin a significantly repressed the placental hormones, hcg, estradiol, and progesterone, in a dose - dependent manner ( fig . therefore, we conclude that inhibition of activin / nodal signaling is essential for trophoblast differentiation of hescs . bmp activation is required for the trophoblast differentiation from hescs we showed that inhibition of activin / nodal signaling induced the expression of bmp4 ( fig . it is interesting to know if the bmp4 induced by inhibition of activin / nodal is required for the trophoblast differentiation . we took advantage of a glycosylphosphatidylinositol - ap deficient hesc line, namely ar1-c1. the bmp signaling depends on a co - receptor, dragon. the function of dragon is disrupted due to the lacking of glycosylphosphatidylinositol anchor. therefore, the extracellular bmp can not bind with the receptor well and the bmp signaling is blocked. the trophoblast development induced by bmps in wild type hescs (g - gfp) is blocked in ar1-c1 hescs, evidenced by absence of the expression of trophoblast markers like cdx2, cg - a, cg - b (fig . the deficiency of bmp signaling can be rescued by transfection of dragon, which indicates that the deficiency of trophoblast development is caused by deficiency of bmp signaling, not any other signal, in the ar1-c1 cell . we expected that if activation of bmp signaling by bmps was not required for trophoblast differentiation when activin / nodal signaling is repressed, the ar1-c1 cells would differentiate into trophoblast when activin / nodal signaling is repressed . if activation of bmp signaling by bmps is required, the ar1-c1 cells would not differentiate into trophoblast when activin / nodal signaling is repressed . when the ar1-c1 cells were treated with sb431542 to inhibit activin / nodal signaling, no evidence of trophoblast differentiation was observed ( fig . therefore, our data indicated that both inhibition of activin / nodal and activation of bmp signaling were required for trophoblast differentiation from hescs . the first cell lineage segregation in human embryonic development takes place at the blastocyst stage, when the trophoblast segregates from the inner cell mass . due to ethical and practical reasons, it has been difficult to determine the key signals in this event . we, along with others, have previously shown that activin / nodal signaling maintains pluripotency of hescs (16 - 18, 36). in the present study , it is demonstrated that hescs develop into trophoblasts, when activin / nodal signaling is inhibited (fig . 2). based on these observations, we propose that the segregation of the trophoblast from the inner cell mass is controlled by activin / nodal signaling. in the human morula, the cells that receive active activin / nodal signals form the inner cell mass; other cells that do not receive sufficient activin / nodal signals develop into the trophoblast. this suggests that activin / nodal signaling regulates the first differentiation event of human embryonic development. xu et al. showed that bmp4 is able to initiate trophoblast differentiation. to further address the mechanisms that control cell lineage segregation at the human blastocyst stage, the relation of bmp signal to inhibition of activin / nodal signaling was investigated. showed that the effect of bmp4 correlates to inhibition of activin / nodal signaling. in addition, inhibition of activin / nodal signaling induced trophoblast differentiation (figs . 2 and 3), whereas activin / nodal signaling inhibited trophoblast differentiation ing from bmp4 signals (fig we conclude that inhibition of activin / nodal signaling is essential for trophoblast differentiation of hescs . our data also showed that when activin / nodal was repressed, bmp4 was induced . this raised the possibility that bmp4 induced by activin / nodal repression promotes trophoblast differentiation . we used the bmp co - receptor, dragon, deficient hescs to investigate if the bmp4 induced by activin / nodal repression is required for trophoblast differentiation. we found that trophoblast differentiation was still blocked when activin / nodal was repressed. our data suggested that bmp signaling is still required for trophoblast development even when activin / nodal is repressed. the observation should not be simply interpreted that they are upstream and downstream. because trophoblast induction of bmp also depends on the inhibition of activin / nodal. activin / nodal inhibition induces the expression of bmp and activates bmp signaling; bmp signaling further inhibits activin / nodal. both inhibition of activin / nodal and activation of the bmp signal our observation reveals that a novel mechanism in which a critical interaction of two related but antagonizing signals by activin / nodal and bmp regulates the fate determination of hescs in culture, and possibly also true for human embryo in vivo. a and b, immunofluorescence for cg- and cg-. h1 cells were treated with cm, or cm plus 10 mol / liter sb431542 or 300 ng / ml follistatin for 12 days. c, immunoassays of placental hormones. conditioned culture medium from h1 cells cultured in cm; cm + 1 sb; cm + 10 sb; cm + 30 fs; or cm + 300 fs were collected at the indicated times and subjected to immunoassays for hcg, estradiol (e2), and progesterone (prog). cm + 1sb, cm plus 1 mol / liter sb431542; cm + 10sb, cm plus 10 mol / liter sb431542; cm + 3fs, cm plus 3 ng / ml follistatin; cm + 30fs, cm plus 30 ng / ml follistatin; cm + 300fs, cm plus 300 ng / ml follistatin. hesc - derived trophoblast cells secrete placental hormones. a and b, immunofluorescence for cg- and cg-. h1 cells were treated with cm, or cm plus 10 mol / liter sb431542 or 300 ng / ml follistatin for 12 days. c, immunoassays of placental hormones. conditioned culture medium from h1 cells cultured in cm; cm + 1 sb; cm + 10 sb; cm + 30 fs; or cm + 300 fs were collected at the indicated times and subjected to immunoassays for hcg, estradiol (e2), and progesterone (prog). cm + 1sb, cm plus 1 mol / liter sb431542; cm + 10sb, cm plus 10 mol / liter sb431542; cm + 3fs, cm plus 3 ng / ml follistatin; cm + 30fs, cm plus 30 ng / ml follistatin; cm + 300fs, cm plus 300 ng / ml follistatin. figure 5.inhibition of activin / nodal signaling down - regulates fgf and wnt signals, but up - regulates bmp signals. the h1 he s cells were cultured under a feeder - free condition and treated with sb431542 for 6 days. then cells were harvested for real - time polymerase chain reaction analysis of ligands of the fgf (a), wnt (a), and bmp (b) signaling pathways and western analysis of oct4, smad2, p - smad2, smad1, and p - smad1 (c). inhibition of activin / nodal signaling down - regulates fgf and wnt signals, but up - regulates bmp signals. the h1 he s cells were cultured under a feeder - free condition and treated with sb431542 for 6 days. then cells were harvested for real - time polymerase chain reaction analysis of ligands of the fgf (a), wnt (a), and bmp (b) signaling pathways and western analysis of oct4, smad2, p - smad2, smad1, and p - smad1 (c). figure 6.bmp4-induced trophoblast differentiation correlates with and depends on inhibition of activin / nodal signaling. real - time polymerase chain reaction analysis of pluripotent markers, trophoblast markers, and downstream targets of activin / nodal signaling were performed. a, bmp4 promoted the trophoblast differentiation of hescs; b, the trophoblast induction effect of bmp4 correlated with the inhibition of activin / nodal signal; c, activin a restores expression of lefty - a, lefty - b, and nodal; d, real - time pcr analysis of the expression of cg- and cg-; e, the h1 he s cells were cultured feeder free with cm plus 10 ng / ml bmp4 and 100 ng / ml activin a, immunofluorescence of cg- and cg- indicates that activin a inhibit the effect of bmp4; f, immunoassays of the placental hormones, hcg, estradiol (e2), and progesterone (prog). cell culture supernatant of hescs cultured on mef, in cm, cm + 10b, cm + 10b + 1a, cm + 10b + 10a, or cm + 10b + 100a were collected at the indicated times and subjected to immunoassays for human chorionic gonadotropin, estradiol, and progesterone. abbreviations: cm+10b, cm plus 10 ng / ml bmp4; cm+10b+1a, cm plus 10 ng / ml bmp4 and 1 ng / ml activin a; cm+10b+10a, cm plus 10 ng / ml bmp4 and 10 ng / ml activin a; cm+10b+100a, cm plus 10 ng / ml bmp4 and 100 ng / ml activin a; acta, activin a. bmp4-induced trophoblast differentiation correlates with and depends on inhibition of activin / nodal signaling. the h1 he s cells were cultured feeder free under the labeled conditions. real - time polymerase chain reaction analysis of pluripotent markers, trophoblast markers, and downstream targets of activin / nodal signaling were performed. a, bmp4 promoted the trophoblast differentiation of hescs; b, the trophoblast induction effect of bmp4 correlated with the inhibition of activin / nodal signal; c, activin a restores expression of lefty - a, lefty - b, and nodal; d, real - time pcr analysis of the expression of cg- and cg-; e, the h1 he s cells were cultured feeder free with cm plus 10 ng / ml bmp4 and 100 ng / ml activin a, immunofluorescence of cg- and cg- indicates that activin a inhibit the effect of bmp4; f, immunoassays of the placental hormones, hcg, estradiol (e2), and progesterone (prog). cell culture supernatant of hescs cultured on mef, in cm, cm + 10b, cm + 10b + 1a, cm + 10b + 10a, or cm + 10b + 100a were collected at the indicated times and subjected to immunoassays for human chorionic gonadotropin, estradiol, and progesterone. abbreviations: cm+10b, cm plus 10 ng / ml bmp4; cm+10b+1a, cm plus 10 ng / ml bmp4 and 1 ng / ml activin a; cm+10b+10a, cm plus 10 ng / ml bmp4 and 10 ng / ml activin a; cm+10b+100a, cm plus 10 ng / ml bmp4 and 100 ng / ml activin a; acta, activin a. figure 7.activation of bmp4 is required for trophoblast differentiation. the parental (g - gfp) and ar1-c1 he s cells were cultured under a feeder - free condition and treated with bmp4 (50 ng / ml) or sb431542 (10 m) for 10 days. b, immunofluorescent staining for trophoectoderm markers troma - i (red) from the differentiated g - gfp and ar1-c1 cells. the parental (g - gfp) and ar1-c1 he s cells were cultured under a feeder - free condition and treated with bmp4 (50 ng / ml) or sb431542 (10 m) for 10 days. b, immunofluorescent staining for trophoectoderm markers troma - i (red) from the differentiated g - gfp and ar1-c1 cells. in contrast, reported that inhibition of activin / nodal signaling promotes specification of human embryonic stem cells into neuroectoderm. we did observe a very slight up - regulation of neuroectoderm markers (supplemental fig . s1); however, we also observed a dramatic up - regulation of trophoblast markers (figs . 2 and 3 and supplemental fig . did not analyze trophoblast marker expression, it is likely they overlooked the dramatic differentiation of trophoblast in their experiments, which led to improper . little is known about normal human development during the early post - implantation period. although the mouse is the typical model for experimental mammalian embryology, early structures, including the placenta, extraembryonic membranes, and the egg cylinder, all differ substantially from the corresponding human structure. our display that although the most key transcriptional factors exhibit similar expression between hescs and mouse escs, some genes, such as eomes, are completely different. eomes has been reported to be essential for trophoblast development in mice ; however, when activin / nodal signaling is inhibited, causing hescs to differentiate into trophoblasts, the expression of eomes is down - regulated. this suggests that eomes are not essential for human trophoblast differentiation, which might imply that there are substantial differences between mouse and human early development. human and mouse es cells are both blastocyst - derived; however, they are not equivalent. the mechanisms that human and mouse es cells use to maintain stemness differ greatly (16 - 18, 30 - 32, 38 - 40), as well as their developmental potential, especially the capacity to form cells of the trophoblast lineage (8, 13, 41 - 43). hescs and the mouse epiblast stem cell use the same signaling pathways to maintain pluripotency, hescs can differentiate into all embryonic germ layers, as well as trophoblasts (5, 8, 13, 41). in contrast, mouse es cells are capable of reconstituting all cell types of the body, but do not routinely exhibit a capacity for trophoblast cell differentiation (42, 43, 45). these differences highlight the fact that hescs are a unique and irreplaceable model for studying early human developmental events. human es cells will be particularly valuable for studying development and function of tissues that differ between mice and humans. hescs give rise to early human cell types that were previously almost unobtainable, which is a major advantage; however, ethical considerations, as well as the practicalities, will make it extremely difficult to validate in vitro with in vivo significance. we demonstrate that combinatorial signals of activin / nodal and bmp regulate lineage segregation of early human embryo stem cells in vitro; however, a direct role for activin / nodal signaling in early human embryonic lineage segregation has not been demonstrated in vivo. expression profiles, attained by analysis of est counts at the ncbi database, shows that activin a, follistatin, and bmps are all expressed in the human ovary and/or uterus, which implies their function during early development. the challenge for the future will be to determine whether activin / nodal and bmp signals play a role in early lineage segregation of human embryo in vivo, and to establish the key transcriptional factor pathways in human embryo trophoblast differentiation using hescs as a model.

cell fate commitment of pre - implantation blastocysts, to either the inner cell mass or trophoblast, is the first step in cell lineage segregation of the developing human embryo. however, the intercellular signals that control fate determination of these cells remain obscure. human embryonic stem cells (hescs) provide a unique model for studying human early embryonic development. we have previously shown that activin / nodal signaling contributes to maintaining pluripotency of hescs, which are derivatives of the inner cell mass. here we further demonstrate that the inhibition of activin / nodal signaling in the loss of hesc pluripotency and trophoblast differentiation, similar to bmp4-induced trophoblast differentiation from hescs. we also show that the trophoblast induction effect of bmp4 correlates with and depends on the inhibition of activin / nodal signaling. however, the activation of bmp signaling is still required for trophoblast differentiation when activin / nodal signaling is inhibited. these data reveal that the early lineage segregation of hescs is determined by the combinatorial signals of activin / nodal and bmp.

dental resin composites have borne witness to remarkable refinements in recent years, especially due to filler size reduction, which has enhanced the polishability and wear resistance of these materials. an important factor driving the achievements made in advanced resin composite properties has been nanotechnology. however, despite the ongoing improvements in resin composites, a concern remains about their chemical and enzymatic degradation in the oral environment. apart from the degradation caused by saliva, by foodstuffs and by beverages, bleaching procedures can potentially cause softening and increased surface roughness to resin composites, depending on the resin composite and on the bleaching agent used. contradictory data has been reported regarding the effect of bleaching agents on microhardness and surface roughness of resin composites. although the authors of some investigations have noticed a reduction in surface microhardness of resin composites after exposure to bleaching agents, authors of other papers have described no change or increase in surface microhardness of composites after bleaching. in regard to surface roughness, the application of bleaching agents to resin composites has been reported, in previous studies, as not changing or increasing surface roughness. bleached resin composites have been observed to stain more easily than unbleached counterparts, probably as a of softening and increased surface roughness. although the literature is still conflicting regarding the susceptibility of bleached and unbleached composites to staining, an important issue is that staining solutions can also soften resin composites. in fact, red wine and coffee, commonly used as staining solutions in the literature, have been proven to decrease microhardness of resin composites. therefore, both bleaching agents and beverages with staining capacity can decrease the microhardness of resin composites, including nanostrucutured materials. considering the softening effect produced by staining solutions, it is important to evaluate whether resin composites that had been previously in contact with bleaching agents would be affected by further softening and darkening as a of being immersed in different staining solutions. this study aimed at elucidating this issue by evaluating the effect of staining solutions on microhardness and on the shade changes of a nanofilled composite resin previously in contact with bleaching agents. table 1 states the main materials used, the staining solutions and their compositions, ph values, manufacturers, batch numbers and manufacturers directions. nanofilled composite resin, bleaching agents and their composition, manufacturers, batch numbers, directions and ph values a total of 135 disk - shaped specimens were prepared with a nanofilled resin composite (filtek supreme xt - 3 m espe, st . paul, mn, usa), which was inserted with a metal spatula (minelli no . 1 - golgran, so paulo, sp, brazil) in circular acrylic molds (10 mm diameter 2 mm thick). a mylar strip (dentart - polidental, so paulo, brazil) was placed on top of and pressed flat with a microscope slide plus a 500 g weight for 30 s. the composite was photocured with a led unit (radii - cal sdi - melbourne, victoria, australia - 1,200 mw / cm) for 20 s. following preparation , specimens were stored in relative humidity at 37c for 24 h. the finishing and polishing steps were performed with al2o3 abrasive disks (sof - lex pop on - 3 m espe, st paul, mn, usa) of decreasing coarseness. the specimens were then stored at 37c for another 24 h. the specimens were randomly divided into three groups (n = 45) to be subjected to one of the bleaching agents. an individual acetate tray was fabricated in a vacuum - forming machine (p7/bio - art equip odontolgicos ltda . , so carlos, sp, brazil) for each specimen, which would be then exposed to 10% or 16% carbamide peroxide (cp) bleaching agents. these bleaching agents were applied for 4 h / day, for 14 days, simulating the at - home bleaching technique. in regard to the group exposed to the 35% hydrogen peroxide (hp) agent (hp whiteness hp 35% fgm produtos odontolgicos, joinville, sc, brazil), the product was applied with a disposable applicator (microbrush - vigodent, rio de janeiro, rj, brazil) for 45 min (three sequential 15-min applications) once a week for three consecutive weeks. when the bleaching time was completed, the specimens were rinsed thoroughly with distilled water and stored in distilled water at 37c. when the bleaching procedures were completed, specimens of each group were allocated into three subgroups (n = 15) to be immersed daily in 200 ml of either coffee (melitta, so paulo, brazil) or red wine (concha y toro, santiago, chile), as described in table 1. specimens were immersed in staining solutions or distilled water for 3 h / day, at room temperature, over 40 days. after every 3-h period of immersion time, the specimens were rinsed thoroughly with distilled water and stored in distilled water at 37c. color shade was measured using a spectrophotometer (vita easyshade - vita zahnfabrik, bad sckingen, germany) and the data were expressed based on the vita shade guide and then converted into scores, as can be seen in table 2. color shade was recorded on three different occasions: baseline, after bleaching and after staining. vita shade guide scores three knoop microhardness indents were carried out on the top surface of each specimen, using a hvs-1000 microhardness tester (panambra, so paulo, sp, brazil), under a 50-g load, applied for 15 s. measurements were performed at the baseline, following bleaching and after staining procedures. microhardness data were analyzed using an analysis of variance (anova) for split - plot design and tukey's test, whereas the color shade recordings were submitted to kruskal - wallis and dunn's tests. statistical procedures were performed with sas 6.11 (sas institute inc ., cary, nc, usa). a total of 135 disk - shaped specimens were prepared with a nanofilled resin composite (filtek supreme xt - 3 m espe, st . paul, mn, usa), which was inserted with a metal spatula (minelli no . 1 - golgran, so paulo, sp, brazil) in circular acrylic molds (10 mm diameter 2 mm thick). a mylar strip (dentart - polidental, so paulo, brazil) was placed on top of and pressed flat with a microscope slide plus a 500 g weight for 30 s. the composite was photocured with a led unit (radii - cal sdi - melbourne, victoria, australia - 1,200 mw / cm) for 20 s. following preparation, specimens were stored in relative humidity at 37c for 24 h. the finishing and polishing steps were performed with al2o3 abrasive disks (sof - lex pop on - 3 m espe, st paul, mn, usa) of decreasing coarseness. the specimens were randomly divided into three groups (n = 45) to be subjected to one of the bleaching agents. an individual acetate tray was fabricated in a vacuum - forming machine (p7/bio - art equip odontolgicos ltda . , so carlos, sp, brazil) for each specimen, which would be then exposed to 10% or 16% carbamide peroxide (cp) bleaching agents. these bleaching agents were applied for 4 h / day, for 14 days, simulating the at - home bleaching technique. in regard to the group exposed to the 35% hydrogen peroxide (hp) agent (hp whiteness hp 35% fgm produtos odontolgicos, joinville, sc, brazil), the product was applied with a disposable applicator (microbrush - vigodent, rio de janeiro, rj, brazil) for 45 min (three sequential 15-min applications) once a week for three consecutive weeks. when the bleaching time was completed, the specimens were rinsed thoroughly with distilled water and stored in distilled water at 37c. when the bleaching procedures were completed, specimens of each group were allocated into three subgroups (n = 15) to be immersed daily in 200 ml of either coffee (melitta, so paulo, brazil) or red wine (concha y toro, santiago, chile), as described in table 1. specimens were immersed in staining solutions or distilled water for 3 h / day, at room temperature, over 40 days. after every 3-h period of immersion time, the specimens were rinsed thoroughly with distilled water and stored in distilled water at 37c. color shade was measured using a spectrophotometer (vita easyshade - vita zahnfabrik, bad sckingen, germany) and the data were expressed based on the vita shade guide and then converted into scores, as can be seen in table 2. color shade was recorded on three different occasions: baseline, after bleaching and after staining. three knoop microhardness indents were carried out on the top surface of each specimen, using a hvs-1000 microhardness tester (panambra, so paulo, sp, brazil), under a 50-g load, applied for 15 s. measurements were performed at the baseline, following bleaching and after staining procedures. microhardness data were analyzed using an analysis of variance (anova) for split - plot design and tukey's test, whereas the color shade recordings were submitted to kruskal - wallis and dunn's tests. statistical procedures were performed with sas 6.11 (sas institute inc ., cary, nc, usa). means and standard deviations (sd) of microhardness values and color shade recordings of the nanofilled resin composite at baseline, after bleaching and after staining, are shown in tables 3 and 4, respectively. mean microhardness values (sd) of nanofilled resin composite at the baseline, after bleaching and after staining medians and color shade ranges of nanofilled resin composite at the baseline, after bleaching and after staining in regard to the microhardness data, anova for split - plot design showed no significant interaction between the bleaching agent and the staining solution (p = 0.4384). bleaching agents significantly influenced the microhardness values of the nanofilled resin composite (p = 0.0003). lower microhardness values were noticed when the composite had been in contact with 35% hp (p < 0.0001). no difference was noticed between the microhardness values produced by the 10% and 16% cp bleaching agent. overall, there was a significant reduction in the microhardness values from the baseline to the post - staining condition (p < 0.0001). the microhardness values of the nanofilled resin composite that had been previously exposed to the bleaching agents were lower when red wine or coffee were used as staining solutions, when compared to distilled water. in terms of color change, as examined spectrophotometrically, kruskal wallis and dunn tests demonstrated that the nanofilled resin composite was lighter after bleaching with the 35% hp agent (p < 0.0500). regardless of the bleaching agent, no difference was observed for the color shade recorded for the nanofilled resin composite as a of being immersed either in red wine or coffee, both of which produced a darker color than distilled water. the of the present study showed that the bleaching agents significantly influenced nanofilled resin composite microhardness, which was reduced when the resin composite was exposed to the 35% hp bleaching agent. the adverse effects of this agent at high concentrations (such as 35% or 38%) have been demonstrated in previous in vitro studies. it has been speculated that hydrogen - peroxide - based bleaching agents may have high oxidation and reduction capability, thus generating free radicals. these radicals have been thought to degrade the resinous matrix and disrupt the filler / matrix interface, effects which cause increasing water sorption and filler detachment and which, in turn, augment surface roughness. in addition, the oxidative capacity of peroxides may cleave the composite polymer - chains. in this context, the effects caused by 10% cp and cp 16% agents on the surface microhardness of the nanofilled resin composite were significantly lower than those produced by the 35% hp. while some studies have shown significant reduction in knoop microhardness after at - home bleaching, others have demonstrated the opposite. differences in protocols of daily use and in bleaching agents may explain such contradictory findings. have reported a significant decrease in nanofilled composite after a 6 h / day bleaching period with 10% cp (as opposed to the 4-h period used in the current study). the authors here cited hypothesized that the bleaching agent had sufficient time to diffuse into the high molecular weight organic matrix of the nanofilled composite. it is worth noting, however, that these investigations were conducted in in vitro conditions, in which specimens are commonly stored in distilled water. an in situ study demonstrated that bleaching with 15% cp had no effect on nanofilled composite microhardness. the specimens immersed in distilled water (control) showed decreased microhardness, which probably occurred because of previous exposure to bleaching agents. this finding may be attributed to the composition of the nanofilled resin composite, the inherent characteristics of the staining solutions and the storage protocol adopted. in this study, red wine and coffee produced lower microhardness values in the resin composite than distilled water. no difference was found between the microhardness values obtained as a of using red wine or coffee. although the red wine had a lower ph (4.2) than the coffee (ph 5.5) and an alcoholic strength of 12.5% overall factors (lower ph and average alcohol by volume) which seem to affect the resin matrix crosslink, the filler / matrix interface and the filler itself the coffee was used at a temperature above 37c, a condition that may have accelerated the resin composite degradation process. color change was evaluated with the vita easyshade spectrophotometer, which allows tooth shade to be determined based on the vita classical scale. the data from the this study shows that no significant difference existed between the color of the specimens at the baseline and in post - bleaching conditions, except when 35% hp was used, which made the nanofilled composite lighter. this may be attributed to a higher proportion of hydrogen and the more acidic ph of the 35% bleaching gel, as compared to the other home - use gels tested in this study. in fact, color changes of composites have not been clinically detectable after the application of 10% cp agents. the assessment of color stability of resin composites following contact with different immersion media has shown that composites are not inert in the oral environment. the of color evaluation showed that, after immersion in coffee and red wine, specimens which had been previously exposed to bleaching agents were significantly darker than those exposed to distilled water. it has been reported that color changes may occur in different restorative materials after bleaching followed by immersion in staining solutions, including red wine and coffee. apart from the pigments contained in red wine and coffee, the alcoholic content of the red wine (12.5% by volume) and the coffee temperature used in this study may have softened the organic matrix of the composite and allowed dye absorption. in this study, the effect on nanofilled resin caused by bleaching followed by staining was evident, i.e. microhardness decreased after bleaching and the samples became lighter when subjected to 35% hp. resin composite previously in contact with bleaching agents showed softening and darkening as a of being stained in red wine and coffee.

objectives: the present study aimed to investigate the effect of staining solutions on microhardness and shade changes of a nanofilled resin composite, which had been previously in contact with bleaching agents.materials and methods: a total of 135 disk - shaped specimens (10 mm 2 mm) were fabricated with a nanofilled resin (filtek supreme) and photocured with a light emission diode (led) unit and then allocated into three groups to be bleached with 10% or 16% carbamide peroxide (cp) bleaching agents or a 35% hydrogen peroxide (hp) product. following bleaching, specimens within each group were subdivided into three groups to be immersed in coffee, red wine or distilled water. microhardness and color were monitored at baseline, after bleaching and after staining.:analysis of variance for split - plot design showed lower microhardness values when the composite had been in contact with hp (p < 0.0001). the specimens immersed in red wine and coffee provided lower microhardness values than those immersed in distilled water, regardless of the bleaching agent to which the composites were previously exposed. kruskal wallis and dunn tests demonstrated that the composite was lighter after bleaching with a 35% hp agent (p < 0.0500).: the composite was darker as a of being immersed either in red wine or coffee, regardless of the bleaching agent.

prostate cancer (pca) is the most frequently diagnosed malignancy and the second leading cause of cancer - specific deaths in men in most industrialized western countries. recently, the incidence of this disease has significantly increased in asian countries, including korea, owing to the reported widespread use of prostate - specific antigen (psa) testing and downward stage migration. nevertheless, recent reports have shown that up to 15% of contemporary patients still harbor lymph node invasion (lni) at extended pelvic lymph node dissection (plnd) in pca. many studies have reported preoperative nomograms for predicting lni after radical prostatectomy (rp) and plnd, and these predictive models were reconstructed by using an extended plnd procedure. although recent studies have demonstrated a significantly higher rate of lni in patients undergoing extended plnd relative to patients undergoing only limited plnd, it is a common policy to spare plnd or to perform limited plnd in patients with a low risk of lni because of the widely applied early detection of pca and screening programs and the ing stage migration. therefore, it became necessary to find a preoperative tool for predicting lni before surgery. recently, genome - wide association studies have identified several genetic variants that are associated with risks for pca and have provided additional predictive gain to prostate - related prognosis. these genetic data have been applied to actual clinical situations and have been validated as genetic biomarkers in pca. thus, in the present study, by using 242,186 single - nucleotide polymorphisms (snps) in an exome array from 341 korean patients with pca who underwent rp and plnd, we investigated significant genetic variants for lni and showed how additional predictive gain was achieved by reconstructing a clinicogenetic model. after we obtained institutional review board approval (irb number, b-1312/232 - 302), we enrolled 1,002 patients with pca from november 2003 to july 2013. blood specimens were prospectively collected from all of the patients, and genomic dna was extracted by use of purelink genomic dna maxi kits (invitrogen, carlsbad, ca, usa). genomic dna was diluted to 1 g / ml and stored at -20. we included patients who underwent rp and plnd, and most of the patients had undergone a limited extent of plnd. we excluded patients who had undergone neoadjuvant hormone or radiation therapy, prostate biopsies at another institution, or prostate biopsies with < 12 cores taken. we also excluded patients who had a postoperative follow - up of less than 1 year. accordingly, 341 patients were enrolled with complete records of clinical data including data on lni and biochemical recurrence (bcr). transrectal ultrasound - guided multicore biopsies were obtained from all male patients by use of a biopsy gun with an automatic firing mechanism. bcr was defined as two consecutive psa increases of 0.2 ng / ml or higher within at least 2 months after rp. study samples were processed on the humanexome beadchip 12v1 - 1 system (illumina inc ., san diego, ca, usa), which included 242,901 markers focused on protein - altering variants. details concerning snp content and selection strategies can be found at the exome array design webpage (http://genome.sph.umich.edu/wiki/exome _ chip_design). after additional visual inspection of snps with call rates of < 0.99 and snps with minor allele frequencies of < 0.002, 242,186 of 242,901 attempted markers (99.71%) were successfully genotyped with call rates > 95% (average call rate, 99.98%). in total, 1,001 of 1,002 individuals (99.9%) were successfully genotyped (call rate > 98%). for the 242,186 snps that passed quality control, genotype concordance among one individual per pair of six known twin pairs and six unknown duplicates were excluded. we performed principal components analysis twice, once excluding hapmap samples to identify population outliers and a second time including hapmap samples to interpret outliers. to avoid artifacts due to family relatedness, we computed the principal components by using snp loadings estimated from a subset of 7,304 notclose relatives. we defined close relatives as an estimated genome - wide identical - by - descent proportion of alleles shared of > 0.10. we estimated identical - by - descent sharing by using plink's " -genome " option38 and performed principal components analysis by using smartpca37 on a linkage - disequilibrium - pruned set of 22,464 autosomal snps. these were obtained by removing large - scale, high - linkage disequilibrium (ld) regions, snps with a minimum allele frequency < 0.01, or snps with a hardy - weinberg equilibrium p - value<10 and by performing ld pruning by using the plink option " --indep - pairwise 50 5 0.2. " after inspecting the first 10 principal components, we identified 12 population outliers, 9 of which had self - reported non - finnish ancestry. snp genotype frequencies were examined for hardy - weinberg equilibrium by using the chi - square statistic and all were found to be consistent (p>0.05). the data were analyzed by using an unconditional logistic regression to calculate an odds ratio (or) as an estimate of the relative risk of lni associated with snp genotypes. to determine the association between the genotype and haplotype distributions, a logistic analysis was performed with control for age (continuous value) as a covariate to eliminate or reduce any confounding factors that could influence the findings. lewontin's d' (|d'|) and the ld coefficient r were examined to measure ld between all pairs of biallelic loci. the haplotypes were inferred from the successfully genotyped snps by using the phase algorithm ver. 2.0, and sas 9.1 (sas institute inc ., the effective number of independent marker loci was calculated by using snpspd software ( http://www.genepi.qimr.edu.au/general/dalen/snpspd/). a total of 341 patients were stratified into two groups on the basis of lni. comparing patients with and without lni, we assessed the differences among the clinicopathological profiles of patients by using the chi - square test, fisher exact test, and the mann - whitney test. the multivariate analyses on models adjusted for age, initial psa, biopsy gleason scores, clinical stage, percentage of positive core, and maximal tumor length in a core were performed to identify an independent predictor of lni. the predictive accuracy for this multivariate model was assessed by the receiver operating characteristics - derived area under the curve (auc) analysis. another multivariate model was created by using the additional genetic information derived from the exome array, and predictive accuracy was assessed by using the same method. the two aucs were subsequently compared by using the mantel - haenszel test. the cox proportional hazard model for predicting bcr analyses was conducted by adjusting the clinicopathologic parameters, such as pathologic gleason scores, extracapsular extension, seminal vesicle invasion, positive surgical margin, and lni, with or without genetic factors from the exome array. bcr - free survival was measured from the day of surgery to the date of the last follow - up period or until the date of bcr. the predictive accuracy for predicting bcr from each model was compared by use of similar methods. the spss ver. 15.0 (spss inc ., a two - tailed p<0.05 was considered to be statistically significant for all of the analyses . after we obtained institutional review board approval ( irb number, b-1312/232 - 302), we enrolled 1,002 patients with pca from november 2003 to july 2013. blood specimens were prospectively collected from all of the patients, and genomic dna was extracted by use of purelink genomic dna maxi kits (invitrogen, carlsbad, ca, usa). genomic dna was diluted to 1 g / ml and stored at -20. we included patients who underwent rp and plnd, and most of the patients had undergone a limited extent of plnd. we excluded patients who had undergone neoadjuvant hormone or radiation therapy, prostate biopsies at another institution, or prostate biopsies with < 12 cores taken. we also excluded patients who had a postoperative follow - up of less than 1 year. accordingly, 341 patients were enrolled with complete records of clinical data including data on lni and biochemical recurrence (bcr). transrectal ultrasound - guided multicore biopsies were obtained from all male patients by use of a biopsy gun with an automatic firing mechanism. bcr was defined as two consecutive psa increases of 0.2 ng / ml or higher within at least 2 months after rp. study samples were processed on the humanexome beadchip 12v1 - 1 system (illumina inc ., san diego, ca, usa), which included 242,901 markers focused on protein - altering variants. details concerning snp content and selection strategies can be found at the exome array design webpage (http://genome.sph.umich.edu/wiki/exome _ chip_design). after additional visual inspection of snps with call rates of < 0.99 and snps with minor allele frequencies of < 0.002, 242,186 of 242,901 attempted markers (99.71%) were successfully genotyped with call rates > 95% (average call rate, 99.98%). in total, 1,001 of 1,002 individuals (99.9%) were successfully genotyped (call rate > 98%). for the 242,186 snps that passed quality control, genotype concordance among the 104 blind duplicate sample pairs was 99.998%. one individual per pair of six known twin pairs and six unknown duplicates were excluded. we performed principal components analysis twice, once excluding hapmap samples to identify population outliers and a second time including hapmap samples to interpret outliers. to avoid artifacts due to family relatedness, we computed the principal components by using snp loadings estimated from a subset of 7,304 notclose relatives. we defined close relatives as an estimated genome - wide identical - by - descent proportion of alleles shared of > 0.10. we estimated identical - by - descent sharing by using plink's " -genome " option38 and performed principal components analysis by using smartpca37 on a linkage - disequilibrium - pruned set of 22,464 autosomal snps. these were obtained by removing large - scale, high - linkage disequilibrium (ld) regions, snps with a minimum allele frequency < 0.01, or snps with a hardy - weinberg equilibrium p - value<10 and by performing ld pruning by using the plink option " --indep - pairwise 50 5 0.2. " after inspecting the first 10 principal components, we identified 12 population outliers, 9 of which had self - reported non - finnish ancestry. snp genotype frequencies were examined for hardy - weinberg equilibrium by using the chi - square statistic and all were found to be consistent (p>0.05). the data were analyzed by using an unconditional logistic regression to calculate an odds ratio (or) as an estimate of the relative risk of lni associated with snp genotypes. to determine the association between the genotype and haplotype distributions, a logistic analysis was performed with control for age (continuous value) as a covariate to eliminate or reduce any confounding factors that could influence the findings. lewontin's d' (|d'|) and the ld coefficient r were examined to measure ld between all pairs of biallelic loci. the haplotypes were inferred from the successfully genotyped snps by using the phase algorithm ver. 2.0, and sas 9.1 (sas institute inc ., the effective number of independent marker loci was calculated by using snpspd software ( http://www.genepi.qimr.edu.au/general/dalen/snpspd/). a total of 341 patients were stratified into two groups on the basis of lni. comparing patients with and without lni, we assessed the differences among the clinicopathological profiles of patients by using the chi - square test, fisher exact test, and the mann - whitney test. the multivariate analyses on models adjusted for age, initial psa, biopsy gleason scores, clinical stage, percentage of positive core, and maximal tumor length in a core were performed to identify an independent predictor of lni. the predictive accuracy for this multivariate model was assessed by the receiver operating characteristics - derived area under the curve (auc) analysis. another multivariate model was created by using the additional genetic information derived from the exome array, and predictive accuracy was assessed by using the same method. the cox proportional hazard model for predicting bcr analyses was conducted by adjusting the clinicopathologic parameters, such as pathologic gleason scores, extracapsular extension, seminal vesicle invasion, positive surgical margin, and lni, with or without genetic factors from the exome array. bcr - free survival was measured from the day of surgery to the date of the last follow - up period or until the date of bcr. the predictive accuracy for predicting bcr from each model was compared by use of similar methods. the spss ver. 15.0 (spss inc ., a two - tailed p<0.05 was considered to be statistically significant for all of the analyses . among 341 patients with pca, 24 patients ( 7.04%) had lni after rp and plnd (table 1). patients with lni displayed significantly higher biopsy gleason scores, percentage of positive core, and maximal tumor length in a core before rp. the rates of extracapsular extension, seminal vesicle invasion, positive surgical margin and bcr were higher in the lni group after rp. there was no significant difference in the rate of lni according to surgical method: open rp, 5.3% (7/131); robotic rp, 8.1% (17/210). genotype frequencies in patients with and without lni were analyzed by using a logistic regression model (fig . the of genotyping 242,186 snps by use of exome arrays showed that five snps ( rs75444444, rs8055236, rs2301277, rs9300039, and rs6908581) were significantly associated with lni in men who underwent rp (table 2). these significant snps were all positively correlated with lni; ors and significance levels are shown in table 2; however, the aforementioned snps were not significant after multiple testing with false - discovery rate. the initial serum psa levels, biopsy gleason scores, clinical stage, percentage of positive cores, and maximal tumor length in a core were significant factors for lni in the univariate analysis. the multivariate models incorporated these variables and age, including and excluding the snp variants, as shown in table 3. the biopsy gleason scores and maximal tumor length in a core were significant factors for predicting lni in a multivariate logistic model; the predictive accuracy for this model was 80.7% (95% confidence interval , 0.762 - 0.847). by adding the genetic variant factors, the clinicogenetic multivariate model showed that predictive accuracy was 93.2% (95% ci, 0.900 - 0.956). thus, the accuracy was increased within a significant level (difference between area, 0.124 ; 95% ci, 0.0357 - 0.213 ; p=0.006) (fig . the clinicopathological multivariate models showed that pathologic gleason scores, seminal vesicle invasion, positive surgical margin, and lni were significant factors for predicting bcr . selected snp - related lni was also a significant factor for bcr in the multivariate cox proportional models ( hazard ratio, 2.244 ; 95% ci, 1.030 - 4.888 ; p=0.042). the auc increased from 71.8% in the model without genetic factors to 72.3% in the model with genetic factors (p=0.346). in the present study, we investigated potential genetic biomarkers for predicting lni among patients with pca who underwent rp and plnd. the logistic regression analysis suggested that five snps were significantly associated with the risk for lni. moreover, we applied the information derived from the genetic studies to an actual clinical model based on previously established clinical factors and found an additional predictive gain for predicting lni. the first nomogram introduced by briganti et al. showed 76% accuracy by adjusting for psa, clinical stage, and biopsy gleason scores. in another nomogram that included percentage of positive cores as a covariate, afterwards, many studies that contained external and internal validation of these nomograms showed a higher accuracy level. recent updated nomograms predicting lni showed 87.6% accuracy in 588 patients with clinically localized pca who underwent rp and extended plnd. showed externally validated updated nomograms among 1,282 pca men and showed 82.9% accuracy. in our setting, including biopsy parameters, the multivariate model for predicting lni was 80.7%; however, with the addition of the genetic parameters to the multivariate model, accuracy was significantly increased to 93.2%. although recently published nomograms related to pca prognosis need to be improved by including new variables, models based on plasma or tissue quantitative biomarkers have recently been proposed. however, although associations between snps and pca have been reported, whether including any of these snps or a combination might improve the classic nomograms has not been explored. huang et al. showed that among 320 patients with pca after rp, 3 of 20 snps were significantly associated with bcr (p<0.02) after adjustment for clinicopathological factors such as age, preoperative psa, pathologic stage, and surgical margin. likewise, we performed a broader investigation by analyzing 242,186 snps among men who underwent rp by use of a custom humanexome beadchip and applied our genetic information from the exome array to the clinical model. as a because the action of snps is generally understood to be a function of multiple variant combinations and not a single action, it was important to find many potential genetic variant outcomes. the relationship between the five snps selected from our series and pca has not yet been investigated. rs75444444, which is located in chromosome 19 (muc16), is known as the largest glycoprotein of the mucin family. it is normally expressed in the epithelial lining of various tissues and is elevated in some organ malignancies. the high rs8055236 (ier3) expression revealed in the prostate and bladder was described as being regulated by p53 and involved in the proapoptotic action by the tumor suppressor. rs2301277 (pbx2) works as a transcriptional activator for valosin - containing protein expression in breast cancer and is known to be an important factor for cell survival. however, an accurate mechanism of action of these snps in pca has not been published; it is necessary to confirm the genetic variants and lymph node metastasis in pca through molecular work. actually, our selected snp, which is related to lni, had significance to pca - related bcr in the multivariate analysis; these markers or the combination of these markers had a likelihood of being a potential biomarker for pca. although the ideal candidate for plnd still needs to be defined, general agreement has been reached that if plnd is contemplated, it should be extended to reach an adequate nodal staging accuracy. consequently, if an lni prediction model is developed, it must be based on an extended plnd series. conversely, applying predictive tools developed on patients treated with limited plnd might significantly underestimate the true risk of lni. however, lni was detected even in low - risk patients with pca in whom plnd is not indicated in the current era; therefore, we need the available nomogram in this real clinical setting. our predictive model had a higher accuracy rate above 90% and consisted of preoperative parameters with genetic information obtained from serum. therefore, by adding a recently published nomogram obtained from an extended plnd setting, the external validation of our selected biomarker exhibited an excellent model for predicting lni. owing to the limited extent of plnd, the small number of positive lymph nodes was a limitation. the sample size was another limitation; however, all men included in this study were of a homogeneous racial population. pca diagnosed in asian, american, and european men may have innate differences associated with racial and environmental factors. because pca is a hormone - dependent disease, various investigators have suggested that racial variations in the serum levels of testosterone and derivatives may contribute to differences in pca risks and prognoses among different races. differences in the hormonal milieu and a lack of psa screening have been speculated to play a role in the generally more aggressive profile of pca diagnosed in contemporary korean men compared with their western counterparts. therefore, our data from an asian population may differ from the data for a western population. another limitation was that our data should be validated in other independent cohorts for persistent significance. despite these limitations, a potential predictive marker should be judged on the basis of its capacity to improve the preexisting optimized predictive model rather than simply on the basis of its status as an independent variable. we showed that five snps were significant predictors of lni. furthermore, the addition of genetic information from exome arrays effectively enhanced the predictive accuracy of our multivariate model, which incorporated various clinical factors. these need to be validated in future studies but may lead to the development of an accurate model that predicts lni in the preoperative setting.

purposegenetic variations among prostate cancer (pca) patients who underwent radical prostatectomy (rp) and pelvic lymph node dissection were evaluated to predict lymph node invasion (lni). exome arrays were used to develop a clinicogenetic model that combined clinical data related to pca and individual genetic variations.materials and methodswe genotyped 242,186 single - nucleotide polymorphisms (snps) by using a custom humanexome beadchip v1.0 (illumina inc .) from the blood dna of 341 patients with pca. the genetic data were analyzed to calculate an odds ratio as an estimate of the relative risk of lni. we compared the accuracies of the multivariate logistic model incorporating clinical factors between the included and excluded selected snps. the cox proportional hazard models with or without genetic factors for predicting biochemical recurrence (bcr) were analyzed.the genetic analysis indicated that five snps (rs75444444, rs8055236, rs2301277, rs9300039, and rs6908581) were significant for predicting lni in patients with pca. when a multivariate model incorporating clinical factors was devised to predict lni, the predictive accuracy of the multivariate model was 80.7%. by adding genetic factors in the aforementioned multivariate model, the predictive accuracy increased to 93.2% (p=0.006). these genetic variations were significant factors for predicting bcr after adjustment for other variables and after adding the predictive gain to bcr.based on the of the exome array, the selected snps were predictors for lni. the addition of individualized genetic information effectively enhanced the predictive accuracy of lni and bcr among patients with pca who underwent rp.

breast mri accuracy for assessing residual disease is good and surpasses other diagnostic means. diagnostic accuracy of mri seems to be affected by treatment regimen and breast cancer subtype. neoadjuvant chemotherapy (nac) is defined as the administration of chemotherapy to treat invasive breast cancer before local treatment (i.e. surgery). although in breast cancer patients nac was primarily used for treatment of locally advanced disease stages, its use has been extended to the treatment of early stage breast cancers in order to enable breast - conserving therapy for patients who would otherwise undergo mastectomy. in a large meta - analysis by mauri et al., no significant differences in survival or overall disease progression was observed between patients receiving adjuvant or neoadjuvant chemotherapy. some physicians prefer nac instead of the adjuvant chemotherapy because of the ability to assess tumour response in vivo. in this situation, reliable assessment of pathological tumour response to nac is vital in order to select the most appropriate surgical plan. an imaging modality that could assess tumour response to nac would be beneficial, providing it could detect any residual disease present. in addition, pathological complete response (pcr, i.e. the absence of any residual invasive tumour cells) on nac has shown to be a prognostic factor for overall better survival, disease - free survival and recurrence - free survival. in the future many examinations have been proposed to evaluate residual disease and/or complete response to therapy (such as clinical examination, mammography and ultrasound), but their accuracy was only of modest degree. parallel to these findings, (contrast - enhanced) magnetic resonance imaging (mri) of the breast proved to be superior to mammography and ultrasound with respect to assessing tumour extent, presence of additional foci (i.e. multifocality and/or multicentricity) and the presence of contralateral breast tumours. therefore, mri might be a promising imaging tool to assess therapy response in the nac setting and for assessing pcr. several reviews have been recently published that assessed the ability of breast mri to predict pcr in patients receiving nac. however, pcr is only achieved by a minority of patients and as a consequence, the role of breast mri in neoadjuvant chemotherapy was not investigated for a substantial number of patients that still have some degree of residual disease after treatment. in this systematic review, we aimed to address the role of breast mri in assessing both residual disease extent and pcr after nac in breast cancer patients. our study purpose was to assess the role of magnetic resonance imaging (mri) in the evaluation in all patients receiving neoadjuvant chemotherapy for invasive breast cancer. for this systematic review, embase, the cochrane library, medline and citations as provided by pubmed were searched until 1 july 2012, using the search terms breast, breast neoplasms, breast neoplasm, breast cancer, breast carcinoma and breast lesion combined with the search terms magnetic resonance imaging, mri, mr mammography and neoadjuvant therapy, neoadjuvant chemotherapy, neoadjuvant systemic therapy, neoadjuvant, chemotherapy, primary therapy and initial therapy. only original articles were considered for inclusion (i.e. no reviews, brief communications or letters to the editor). studies found through these search terms were assessed for potential eligibility by reading the abstracts first and then applying inclusion and exclusion criteria. included were only those in which breast mri was performed at baseline and prior to surgery (but after completion of neoadjuvant chemotherapy). in addition, the ability of mri to assess pcr was one of our study aims. rates of pcr to nac may vary, depending on the treatment regime used: 615 % in antracycline - based therapies, up to 30 % when adding taxanes. therefore, in order to have some reliable information on the ability of mri to assess residual disease and also pcr, eligible studies should have a sufficiently large study population. to be eligible for this review , we decided that a study should consist of at least 25 patients (in the final analysis) with newly diagnosed, histologically proven breast cancer undergoing neoadjuvant chemotherapy who were imaged using clinical mri scanners (i.e. minimum 1.5 t). studies were not excluded if other imaging modalities were performed parallel to mri in order to evaluate treatment response. after this initial assessment included: first author, year of publication, study design (retrospective or prospective), blinding procedures, population size, mean patient age and range, magnetic field strength, contrast agent / dose used, breast cancer stage at inclusion, tumour histology, breast cancer subtypes, chemotherapy regimen, imaging response assessment and histopathological response assessment. while scoring the extraction forms in consensus, some studies were excluded if the study outcome proved not to contain information on residual disease evaluation by mri. all reported p - values 0.05 were considered statistically significant. the large heterogeneity observed in the included studies precluded us from pooling data (see also discussion section), which is why we chose to use descriptive statistics in this review. since this was a systematic review, no approval from our institutional review board was necessary. in the primary literature search, 3,119 potential studies were identified, of which 515 were double in various searches, leaving 2,604 studies after the primary search. after reading the abstracts, 2,444 were excluded from further evaluation, leaving 160 studies to be analysed using the inclusion and exclusion criteria. in these studies, two additional studies were identified by manually searching the references in the manuscripts. in this analysis , another 98 studies did not comply with our eligibility criteria and were subsequently excluded, leaving 64 studies to be reviewed using the extraction form and consensus reading. this led to the exclusion of another 29 studies, because they did not address the topic of residual disease assessment with mri after neoadjuvant chemotherapy. therefore, a total of 35 studies were eligible for this systematic review. 1detailed overview of study selection detailed overview of study selection the majority of studies were prospective in design. a total of 2,359 patients were included in the studies (mean 65.5 patients per study, range 30216). three studies were performed on a 3-t mri scanner, four on both 1.5- and 3-t scanners, and the remaining studies on 1.5-t mri scanners. in all studies, a commercially available gadolinium - based contrast agent was used for breast mri at regular clinical administration doses. interestingly, there was a remarkable heterogeneity in breast cancer stages and subtypes, neoadjuvant chemotherapy regimens, and methods used for assessing response in both imaging and histopathological analyses (tables 1 and 2). this heterogeneity precluded us from further pooling of data in a meta-analysis.table 1overview of included studiesauthorsyearstudy designpopulation sizeage in years (range)breast cancer stage of included patientsbreast cancer typesbreast cancer subtypesabraham et al.1996prospective3950iia, iib, iiia, iiibidc, ilc, mixed ductal and lobular carcinomaer, presserman et al.2001prospective3346iiia, iiib, iiic, to large for breast conserving therapyidc, ilc, inflammatoryer, prrieber et al.2002prospective5851not reportedidc, ilc, dcis, mucinous carcinoma, ductolobular carcinomanot reportedpartridge et al.2002prospective5247not reportednot reportednot reportedcheung et al.2003prospective3345not reportedidc, ilc, mucinous carcinomanot reporteddenis et al.2004prospective4048labcnot reportednot reportedwarren et al.2004retrospective6746not reportednot reporteder, prmartincich et al.2004prospective3049ii, iii, inoperable locally - advanced breast canceridc, ilcer, prschott et al.2005prospective4348iib, iiia, t1n0/1, t2n0/1idc, ilc, mixed ductal and lobular carcinoma, anaplastic carcinomaeryeh et al.2005prospective3145iib, iiia, iiib, iiicidc, ilc, mixed ductal and lobular carcinoma, invasvive carcinoma n.o.s.not reportedbelli et al.2006prospective4554iia, iib, iiia, iiib, idc, ilc, mucinous carcinoma, tubular carcinomaer, prsegara et al.2007prospective6850clinical stage i, ii, and iiiidc, ilc, mixed ductal and lobular carcinoma; sometimes dcis or lcis present in lesionser, pr, her2kim et al.2007prospective5042iib, iiia, iiib, iiicidc, ilcer, prchen et al.2007prospective5150ii, iii, ividc, ilcher2bhattacharyya et al.2008prospective3242>4 cm, large tumours in small breasts, node involvementnot reportednot reportedmoon et al.2009prospective19546 (not reported)not reportednot reporteder, pr, her2wright et al.2010prospective4847iib, iiia, iiibidc, ilc, invasive micropapillary carcinomaer, pr, her2woodhams et al.2010prospective69not reportednot reportedidc, ilc, dcis, lcis, mucinous carcinoma, othernot reportedpark et al.2010retrospective5344iia, iib, iiia, iiibidc, mucinous carcinoma, mixed ductal and lobular carcinomaer, pr, her2de los santos et al.2010retrospective8150not reportedidc, ilc, mixed ductal and lobular carcinoma, carcinoma n.o.s.er, pr, her2straver et al.2010retrospective20846>3 cm and/or n+idc, ilc, carcinoma n.o.s.er, pr, her2nakahara et al.2010prospective8648not reportedidc, papillotubular carcinoma, solid - tubular carcinoma, scirrhous carcinomaer, her2wang et al.2010prospective4348ii, iiiidcnot reporteddongfeng et al.2011prospective6055 (not reported)i, iia, iib, iiia, iiib, iiicidc, mucinous carcinomaer, her2fangberget et al.2011prospective3151not reportedidc, ilcer, pr, her2guarneri et al.2011retrospective5948iia, iib, iiia, iiib, iiicidc, ilc, other n.o.s.er, pr, her2loo et al.2011prospective11846>3 cmidc, ilc, adenocarcinoma n.o.s.er, her2shin et al.2011prospective4343labc / inflammatory breast cancer and at least n1, or unsuitable for bct, or skin / chest wall involvementidc, micropapillary carcinomaer, her2lyou et al.2011retrospective5744not reportedidc, ilcnot reportedchen et al.2011prospective5049not reportedidc, ilc, invasive cancer with squamous differentiationer, pr, her2, ki-67kim et al.2012prospective5549iia, iib, iiia, iiib, iiic, ividc, ilc, mucinous carcinomanot reportedkuzucan et al.2012retrospective5446not reportedidc, ilc, mixed ductal carcinoma with lobular featureser, pr, her2, ki-67takeda et al.2012prospective3751not reportednot reportednot reportedshin et al.2012retrospective9046iia, iib, iiia, iiib, iiicidc, ilc, metaplastic carcinoma, micropapillary carcinomaer, pr, her2hylton et al.2012prospective21648t3 tumour of at least 3 cmidc, ilc, mixed ductolobular carcinoma, mucinous carcinomaer, pr, her2park et al.2012retrospective3444tumour size > 2idc, mucinous carcinoma, mixed ductolobular carcinomaher2overview of included studies regarding year of publication, study design, size of study population, age of population, breast cancer stages of patients included, breast cancer types observed in the respective studies and their subtypeslabc locally advanced breast cancer, bct breast - conserving therapy, idc invasive ductal carcinoma, ilc invasive lobular carcinoma, dcis ductal carcinoma in situ, n.o.s. not otherwise specified, er estrogen receptor, pr progesterone receptor, and her human epidermal growth factor receptortable 2overview of included studiesauthorchemotherapy regimenresponse assessmentfield strength (tesla)pathologic assessmentabraham et al.45 cycles of doxorubicinwho1.54 categories: no residual disease; disease in a single quadrant only; multiquadrant small residual disease; multiquadrant extensive diseaseesserman et al.4 cycles of doxorubicin / cyclophosphamidewho1.5correlation with tumour size measurementsrieber et al.35 cycles of anthracyclines and epirubicine / paclitaxel, or anthracyclines and cyclophosphamideno who or recist1.5correlation with tumour size measurementspartridge et al.4 cycles of doxorubicin and cyclophosphamide followed by up to 12 cycles of paclitaxelno who or recist1.5correlation with tumour size measurementscheung et al.3 cycles of paclitaxel and epirubicinrecist1.5correlation with tumour size measurementsdenis et al.5-fluorouracil / epirubicine, or 6 cycles of docetaxel or 8 cycles of docetaxel / epirubicinerecist1.5correlation with tumour size measurementswarren et al.6 cycles of doxorubicin / cyclophosphamide, or 6 cycles of doxorubicin / docetaxel, or 4 cycles epirubicin then 4 cycles cyclophosphamide, or 4 cycles epirubicin, or docetaxel / herceptinno who or recist1.5uicc response criteria / nhsbsp guidelinesmartincich et al.4 cycles of doxorubicin bolus, followed by paclitaxel (n = 29). in one case, doxorubicin was omitted because of low baseline left ventricular ejection fractionwho1.55 grades: some alteration in individual cells but no reduction in overall number of tumour cells; mild loss of invasive tumour cells but still high cellularity; estimated > 90 % loss of tumour cells; only small clusters of disease remaing, or only in situ component, or only tumour stroma remaining; pathologic complete responseschott et al.4 cycles of doxorubicin / docetaxelno who or recist1.5definitions of response: pcr: no viable invasive cancer or dcis.. partial response and progressive disease: not definedyeh et al.4 cycles of doxorubicin, then 9 cycles of paclitaxel (or vice versa)recist1.5equal to pathology if longest tumour diameter was within 30 % of pathology tumour size. not equal if tumour diameter was less than 70 % of pathology tumour size (underestimation) or more than 130 % of pathology tumour size (overestimation)belli et al.3 cycles of cyclophosphamide, methotrexate, and 5-fluorouracil alternated with adriamycine, or 6 cycles of epirubicin and taxolewho1.5correlation with tumour size measurementssegara et al.er/pr/her2 negative: 4 cycles cisplatin. her2 positive: either trastuzumab / vinorelbine or trastuzumab / carboplatin / docetaxelrecist1.55 grades: some alteration in individual cells but no reduction in overall number of tumour cells; mild loss of invasive tumour cells but still high cellularity; estimated > 90 % loss of tumour cells; only small clusters of disease remaing, or only in situ component, or only tumour stroma remaining; pathologic complete responsekim et al.3 cycles of doxorubicine / docetaxelwho1.5equal to pathology if tumour size was within 50 % of pathology tumour size. not equal if tumour size was less than 50 % of pathology tumour size (underestimation) or more than 150 % of pathology tumour size (overestimation)chen et al.2 cycles of doxorubicin and cyclophosphamide, then either 2 cycles of additional cyclophosphamide or taxane - based regimen (paclitaxel or nab - paclitaxel combined with carboplatin . + also received trastuzumab, her2- also received bevacizumabrecist1.53 categories : no residual malignancy or cancer cells; no residual invasive cancer, but dcis present; residual invasive cancer. categories 1 and 2 are considered pcrbhattacharyya et al.6 cycles of doxorubicin or epirubicin and cyclophosphamiderecist1.5correlation with tumour size measurementsmoon et al.taxane/anthracycline regimen, or anthracycline regimen, or trastuzumabno who or recist1.5correlation with tumour size measurementswright et al.epirubicin/taxotere (n = 30); doxorubicin / cyclophosphamid (n = 17); cyclophosphamide / taxotere / herceptin (n = 1)no who or recist1.55 grades: some alteration in individual cells but no reduction in overall number of tumour cells; mild loss of invasive tumour cells but still high cellularity; estimated > 90 % loss of tumour cells; only small clusters of disease remaing, or only in situ component, or only tumour stroma remaining; pathologic complete responsewoodhams et al.4 cycles of anthracycline / cyclophosphamide, followed by 4 cycles of paclitaxelnot applicable1.54 categories: no residual disease; disease in single quadrant only; multiple residual diseases including eic limited to one quadrant; multiple residual disease including eic in 2 or more quadrantspark et al.3 cycles of docetaxel / doxorubicinrecist1.5correlation with tumour size measurementsde los santos et al.4 cycles of doxorubicin, then 4 cycles of paclitaxel, then 4 cycles of cyclophosphamiderecist1.5assessment for pcrstraver et al.her2-: 6 cycles of doxorubicin / cyclophosphamide, 6 cycles of doxorubicin / docetaxel, or 6 cycles of capecitabine / docetaxel, or the combination of 3 cycles of doxorubicin / cyclophosphamide plus 3 cycles of capecitabine / docetaxel. +: doxorubicin / cyclophosphamide and after 2005 paclitaxel, trastuzumab, carboplatinno who or recist1.5/3assessment for pcrnakahara et al.46 cycles of epirubicin / cyclophosphamide, with or without doxifluridine. in 17 cases continuation with 46 cycles docetaxel or 12 cycles paclitaxel. in 15 cases 6 cycles of docetaxel / epirubicin / cyclophosphamide. in her2 +, also taxanes (n = 7) or taxanes / trastuzumab (n = 4)recist1.5japanese breast cancer society classification systemwang et al.taxol/carboplatin n.o.s.no who or recist1.5correlation with tumour size measurementsdongfeng et al.4 cycles of paclitaxel / pirarubicinrecist3correlation with tumour size measurementsfangberget et al.letrozol therapy (n = 2); or 4 cycles 5-fluorouracil / epirubicin / cyclophosphamide, followed by 2 additional cycles (n = 10), or switch to taxans (n = 10) or taxans / trastuzumab (n = 8)recist1.5correlation with tumour size measurementsguarneri et al.48 cycles of either epirubicin / paclitaxel or paclitaxel, followed by 5-fluorouacil / epirubicin / cyclophosphamide. some her2 + patients also received trastuzumab (n = 25)no who or recist1.5correlation with tumour size measurementsloo et al.different regimes: majority of her2-recieved 6 cycles of cyclosphosphamide / doxorubicin, some her2 - 6 cycles of capecitibine / docetaxel, or doxorubicin / docetaxel; if no response, switch to 3 cycles of cyclophosphamide / doxorubicin, followed by 3 cycles of capecitibine / docetaxel; her2 + recieved 8 weeks of paclitaxel / trastuzumab / carboplatin, followed by either 2 8 weeks paclitaxel / trastuzumab / carboplatin, or 4 cycles of trastuzumab / fluorouacil / cyclophosphamidetwo approaches: breast response index, and dichotomously (presence or absence of residual disease). pcr and near - pcr were considered on category (pcr)1.5/3world health organisation grading of tumoursshin et al.8 cycles of adriamycin / cyclophosphamide / docetaxel, or 8 cycles of capecitabine / vinorelbine / docetaxelrecist1.55 grades: some alteration in individual cells but no reduction in overall number of tumour cells; mild loss of invasive tumour cells but still high cellularity; estimated > 90 % loss of tumour cells; only small clusters of disease remaining, or only in situ component, or only tumour stroma remaining; pathologic complete responselyou et al.36 cycles of taxane / anthracycline, or 34 cycles of anthracycline - based regimen, or 6 cycles of trastuzumab / taxane regimenrecist1.5correlation with tumour size measurementschen et al.2 cycles of doxorubicin, followed by cyclophosphamide biweekly, followed by 12 weeks of taxane based regimen; or only taxane - based regimennot reported33 grades: no residual cancer cells; no residual invasive cancer cells but ductal carcinoma in situ; residual invasive cancer. pcr was defined as being category 1 and 2kim et al.taxane/anthracycline based regimen, orrecist1.5/35 grades: some alteration in individual cells but no reduction in overall number of tumour cells; mild loss of invasive tumour cells but still high cellularity; estimated > 90 % loss of tumour cells; only small clusters of disease remaining, or only in situ component, or only tumour stroma remaining; pathologic complete responsekuzucan et al.combination of doxorubicin and cyclophosphamide and taxane based regimen; or only cyclophosphamide; or only taxane - based regimennot reported1.5/3pathologic complete response defined as the absence of invasive cancertakeda et al.docetaxel and cyclophosphamide for 36 cyclesno who or recist3pathologic complete response defined as the absence of invasive cancershin et al.4 cycles of doxorubicin / cyclophosphamide; or 4 cycles of cyclophosphamide followed by 4 cycles of docetaxel; or doxorubicin / docetaxel; or 5-fluorouracil / epirubicin / cyclophosphamide; or 6 cycles of trastuzumab / paclitaxelrecist1.5pcr classified into two categories: no residual disease, or absence of invasive cancer, but dcis presenthylton et al.anthracycline-cyclophosphamide regimen alone or followed by a taxanerecist1.5residual cancer burden and pathologic complete response; pcr defined as the absence of invasive cancerpark et al.3 cycles of doxorubicin / docetaxel; or (if her2-positive) 6 cycles of paclitaxel / gemcitabine / trastuzumabno who or recist1.55 grades: some alteration in individual cells but no reduction in overall number of tumour cells; mild loss of invasive tumour cells but still high cellularity; estimated > 90 % loss of tumour cells; only small clusters of disease remaining, or only in situ component, or only tumour stroma remaining; pathologic complete response pathologic complete response defined as the absence of invasive cancer. pathologic complete response defined as the absence of invasive cancer pcr classified into two categories: no residual disease, or absence of invasive cancer, but dcis present. residual cancer burden and pathologic complete response; pcr defined as the absence of invasive canceroverview of included studies regarding chemotherapy regimen(s) that were used in the study, method of response evaluation, field strength of the mri scanner used, method of pathologic response assessmentn.o.s. not otherwise specified, er estrogen receptor, pr progesterone receptor, her human epidermal growth factor receptor, recist response evaluation criteria in solid tumours, who world health organisation, pcr pathologic complete response, dcis ductal carcinoma in situ, eic extensive intraductal component, uicc union for international cancer control and nhsbsp national health service breast screening programme overview of included studies overview of included studies regarding year of publication, study design, size of study population, age of population, breast cancer stages of patients included, breast cancer types observed in the respective studies and their subtypes labc locally advanced breast cancer, bct breast - conserving therapy, idc invasive ductal carcinoma, ilc invasive lobular carcinoma, dcis ductal carcinoma in situ, n.o.s. not otherwise specified, er estrogen receptor, pr progesterone receptor, and her human epidermal growth factor receptor overview of included studies overview of included studies regarding chemotherapy regimen(s) that were used in the study, method of response evaluation, field strength of the mri scanner used, method of pathologic response assessment n.o.s. not otherwise specified, er estrogen receptor, pr progesterone receptor, her human epidermal growth factor receptor, recist response evaluation criteria in solid tumours, who world health organisation, pcr pathologic complete response, dcis ductal carcinoma in situ, eic extensive intraductal component, uicc union for international cancer control and nhsbsp national health service breast screening programme seventeen studies calculated correlation coefficients for the comparison of mri tumour measurements compared with histopathological . correlation coefficients varied from poor to excellent, but the median value was 0.698 (range 0.210.982, table 3). nonetheless, two studies reported non - significant correlation coefficients. in the relatively small (n = 59) retrospective study by guarneri et al., the correlation coefficients were similar for mri (0.53) and ultrasound (0.66) when compared to histopathology, and both were non - significant. in the study of 86 women by nakahara , the correlation coefficient of all included patients was remarkably low (0.21), but rose to a strikingly high and statistically significant value of 0.92 when only triple - negative breast cancer types were analysed. a weak correlation coefficient (0.30) was also presented by chen et al. however, when four cases with a size discrepancy larger than 5 cm were excluded (all her2-negative tumours presenting as non - mass - like enhancement), the correlation coefficient increased remarkably to 0.76 (p < 0.001). furthermore, both overestimation and underestimation were observed by multiple studies.table 3correlation coefficients of mri and histopathological tumour measurementsauthorcorrelation coefficientp - valuepartridge et al.0.89<0.001cheung et al.0.982<0.001martincich et al.0.72<0.001segara et al.0.749<0.0001kim et al.0.645<0.001moon et al.0.584nawright et al.0.49napark et al.0.667nanakahara et al.0.21nswang et al.0.866<0.01dongfeng et al.0.698<0.001fangberget et al.0.87<0.001guarneri et al.0.53nsshin et al.0.97nachen et al.0.300.03kim et al.0.619<0.0001shin et al.0.781nana not available, ns not significant2011 paper, 2012 papertable 4diagnostic accuracies of mri for predicting pathologic complete responseauthorsensitivity (%) specificity (%) ppv (% npv ( %) accuracy (%) prevalencerieber et al.42897371720.21schott et al.25975094890.10bhattacharyya et al.80895696na0.13moon et al.3896nanana0.15de los santos et al.92507280na0.40fangberget et al.3896nanana0.37park et al.1007047100770.21shin et al.7770nanana0.3310091nanana5370nananappv positive predictive value, npv negative predictive value, na not available, tcho total choline - containing compoundsthe are presented for dynamic, contrast - enhanced mri, with the exception of: diffusion - weighted imaging and mr spectroscopy parameters correlation coefficients of mri and histopathological tumour measurements na not available, ns not significant 2011 paper, 2012 paper diagnostic accuracies of mri for predicting pathologic complete response ppv positive predictive value, npv negative predictive value, na not available, tcho total choline - containing compounds the are presented for dynamic, contrast - enhanced mri, with the exception of: diffusion - weighted imaging and mr spectroscopy parameters although correlation coefficients are useful tools to describe mri s ability to assess response to nac, it could mask the truth, since the same trend in all studies could in excellent correlation between histopathological and mri measurements, yet the actual estimation of pcr might not be accurate. bhattacharyya et al. reported an overestimation of > 10 mm in 4 of 32 cases. belli et al. described a mean overestimation and underestimation of 2.1 and 2.0 mm, respectively. these numbers were 20.2 and 13.8 mm in the study by denis et al.. partridge et al. found the smallest deviation with an overestimation of mri measurements of only 0.9 mm. found a mean size difference of 1.6 and 6.0 mm, respectively. with respect to pcr prediction with mri, sensitivity was considered to be the proportion of patients with pcr that were correctly classified with mri as complete responders. specificity was considered to be the proportion of patients with non - pcr correctly classified by mri as non - responders. to illustrate, a sensitivity of 62 % in these studies meant that in 62 out of 100 patients, mri was able to correctly identify patients with pcr (i.e. mri did not show any residual enhancement). eight studies calculated the diagnostic accuracies for mri in predicting pcr (table 4). two studies reported diagnostic accuracies for diffusion - weighted mr imaging, and both reported a sensitivity of 100 %. only one study evaluated the potential of mr spectroscopy parameters (total choline - containing compounds, tcho) and reported a sensitivity of 53 % and a specificity of 70 %. the remaining studies used dynamic, contrast - enhanced mr imaging. median (and range) sensitivity and specificity were 42 % (2592 %) and 89 % (5097 %), respectively. if reported, median (and range) ppv and npv were 64 % (5073 %) and 87 % (7196 %), respectively. interestingly, only three studies compared diagnostic accuracy of mri and ultrasound for assessing residual disease. in these studies, ultrasound was less accurate than mri. mri was also more accurate than mammography in assessing residual disease, but only one study performed this comparison. finally, mri was more accurate than physical examination for assessing residual disease, which was examined by three studies. in this systematic review, we aimed to analyse the available data on mri accuracy for assessing residual disease and pcr after neoadjuvant chemotherapy in breast cancer patients. many studies compared the measured tumour diameter or volumes on mri with pathological as the gold standard. correlation coefficients for these comparisons were good to excellent, but both overestimation and underestimation of the mri size measurements were frequently observed. although these correlation coefficients were good, it does not necessarily mean that agreement between these measurements is good. the majority of studies did not investigate the agreement between these measurements, for instance by using bland - altman plots. contrast - enhanced breast mri is superior to other imaging modalities to assess breast tumour extent and the presence of multicentricity or multifocality. however, overestimation of tumour extent is a well - known phenomenon in (preoperative) breast mri and was also observed in the nac setting. confounding factors in overestimating tumour size might be: reactive inflammation caused by tumour response and healing, surrounding sclerosis and necrosis, multiple scattered lesions and presence of accompanying ductal carcinoma in situ. in theory, overestimation of tumour size by mri could in an altered surgical treatment plan for the individual patient, with the risk of achieving wider resection margins (with poorer cosmetic ) or performing unnecessary mastectomy (where breast - conserving therapy would have been possible). in contrast to overestimation of tumour size by mri, underestimation was also observed in the nac setting. causes might be antivascular effects of docetaxel (ing in less tumour enhancement), lack of inflammatory response surrounding the tumour in docetaxel patients, more extensive ductal carcinoma in situ components and partial volume effects in very small foci of residual disease. underestimation of residual disease could lead to positive resection margins with viable residual tumour cells, necessitating re - surgery. in addition, positive resection margins are associated with an increased long - term risk of disease recurrence in patients who have undergone breast - conserving therapy. straver et al. attempted to create an mri - based model that could help in surgical decision making. in this study, mri underestimated tumour size > 20 mm in 17 % of the patients, in 13 % leading to an incorrect decision to perform breast - conserving surgery. from their study, they concluded that baseline tumour size, tumour size reduction and cancer subtype should be taken into account in the optimal selection of patients eligible for breast - conserving therapy. if reported, median ppv and npv were 64 % and 87 %, respectively. in a meta - analysis of 25 studies, yuan et al. showed that pooled weighted estimates of sensitivity and specificity of mri for demonstrating pcr were 63 % (range 5670 %) and 91 % (range 8992 %). these findings were concordant with the observations made by wu et al., who performed a meta - analysis of 34 studies. they concluded that the sensitivity and specificity of contrast - enhanced breast mri to predict pcr were 68 % and 91 %, respectively. the variation in these findings, especially in specificity, might be explained by the differences in included studies, since these recent publications additionally included studies with smaller populations and lower mri field strengths. since pcr is only achieved in up to 30 % of patients, we think that a minimum number of ten patients is too low to accurately use pcr as a study outcome. therefore, we chose a minimum number of 25 patients to be included in our final analysis. despite the differences in these reviews and our current study, the diagnostic accuracy of breast mri to predict pcr seems to have a high specificity and npv versus only moderate sensitivity and ppv. nevertheless, the varying in the separate studies (and their range) of the recent reviews show that mri's accuracy for assessing pcr is still under debate and that it is too early to use it as a decision - making tool in studies that investigate other treatment strategies after pcr besides surgery. although the number of studies was small, contrast - enhanced mri outperformed physical examination, ultrasound and mammography in accurately assessing residual disease. in physical examination, this is most likely explained by fibrosis surrounding the tumour bed as a of the therapy. this fibrotic tissue remains hard, and as such it could lead to misinterpretation of residual disease. these fibrotic changes can also be observed in mammography and ultrasound and can not be easily discerned from residual tumour tissue, but can be excluded by mri since this fibrotic tissue does not show any enhancement after contrast administration. in addition, the diagnostic accuracy of mammography is strongly dependent on breast density, being lower in breasts with extremely dense fibroglandular tissue. if performed in the right period of the menstrual cycle (day 314 in premenopausal women), the accuracy of breast mri was less influenced by breast density. almost all papers used contrast - enhanced breast mri for the evaluation of residual disease and pcr after nac. some studies additionally investigated the ability of diffusion - weighted imaging (dwi) for assessing pcr after nac. in dwi , mri is used to assess the brownian motions of water molecules within a certain tissue of interest. in the cell - rich environment of tumours, this in increased signal intensity on so - called diffusion - weigted images, with corresponding low values for the apparent - diffusion coefficient or adc. woodhams et al. demonstrated that the sensitivity, specificity and accuracy of dwi for assessing pcr was higher than contrast - enhanced breast mri, but the differences observed were not significant with p - values of 0.31, 0.08 and 0.06, respectively. in the study by park et al. , dwi was compared to positron emission computed tomography (pet - ct) and showed a slightly higher auc for predicting pcr when compared to pet - ct, although this difference was not statistically significant in their population of 34 patients, of which 7 achieved pcr after therapy. , three different mri techniques were compared: dynamic contrast - enhanced mri, dwi and mr spectroscopy. they concluded that the change in adc after treatment was the most accurate predictor of pcr. with an auc of 0.96, they found that the optimal cutoff value for percentage adc change was 40.7 %, yielding a sensitivity of 100 % and a specificity of 91 %. first, publication bias is a study limitation that merits attention in each systematic review. small studies with less favorable tend to be published less frequently or not at all. with this potential bias in mind , one should realise that the current positive findings of mri accuracy after nac might be overestimated. second, the lack of study uniformity prevented us from performing a meta - analysis. therefore, we chose to perform a systematic review of the selected studies and provide a descriptive presentation of the observed findings instead of performing a meta - analysis that uses statistical models to adjust for this heterogeneity to some extent. variations in study aim, chemotherapy regimens, response assessment criteria in both imaging and pathological analysis, patient populations and breast cancer subtypes precluded us from drawing more definitive . for example, chen et al. showed in their study that mri can predict pcr accurately in her2-positive patients, but a high false - negative rate was observed in her2-negative patients, especially when they received anti - angiogenic drugs. loo et al. showed in their study of 118 patients that response monitoring after nac is effective in triple - negative or her2-positive breast cancer subtypes, but is inaccurate in er - positive / her2-negative subtypes. in their 2011 publication, chen et al. demonstrated that mr imaging accuracy was higher for her2-positive cancer types than for her2-negative tumours (88 % versus 82 %). in the same study , they showed that the average size discrepancy in cases with ki-67 staining of < 10 % was greater than in cases with ki-67 staining of > 40 %. denis et al. showed that mri frequently underestimated residual tumour size in taxane - containing treatments, most likely because of the antivascular effects of these drugs, ing in less enhancement on contrast - enhanced mri. although no significant differences between who and recist criteria in imaging response assessment were observed in other cancer types, multiple response criteria were used in the selected studies. similarly, there are no widely accepted response assessment criteria for pathology. the most important issue in this assessment is the extent of residual dcis. whether or not dcis is included in the analysis might partly explain the differences observed in mri over- and underestimation. from a clinical point of view, it would be most interesting to assess mri accuracy if dcis were included in the definition of pcr, since dcis should also be excised during surgery and identification of dcis extension by mri remains challenging. only four studies had a population size > 100 subjects and most of the studies were single - centre studies. the statistical noise will be smaller if the sample sizes are increased in (future) multicentre studies in order to assess the true accuracy of mri in the nac setting with greater confidence. in summary, breast mri accuracy for assessing residual disease after neoadjuvant chemotherapy is good, but multiple factors, such as cancer subtype and treatment regimen, can influence mri accuracy and should be considered in clinical decision making. clinical decision making based on mri should therefore be made prudently with these limitations in mind. regardless of the many potential confounders described in this review, we feel that assessment of nac response with mri is promising and ready for more multicentre studies that are able to address these shortcomings.

objectivesthis systematic review aimed to assess the role of magnetic resonance imaging (mri) in evaluating residual disease extent and the ability to detect pathologic complete response (pcr) after neoadjuvant chemotherapy for invasive breast cancer.methodspubmed, the cochrane library, medline, and embase databases were searched for relevant studies published until 1 july 2012. after primary selection, two reviewers independently assessed the content of each eligible study using a standardised extraction form and pre - defined inclusion and exclusion criteria.a total of 35 eligible studies were selected. correlation coefficients of residual tumour size assessed by mri and pathology were good, with a median value of 0.698. reported sensitivity, specificity, positive predictive value and negative predictive value for predicting pcr with mri ranged from 25 to 100 %, 5097 %, 4773 % and 71100 %, respectively. both overestimation and underestimation were observed. mri proved more accurate in determining residual disease than physical examination, mammography and ultrasound. diagnostic accuracy of mri after neoadjuvant chemotherapy could be influenced by treatment regimen and breast cancer subtype.breast mri accuracy for assessing residual disease after neoadjuvant chemotherapy is good and surpasses other diagnostic means. however, both overestimation and underestimation of residual disease extent could be observed.main messages breast mri accuracy for assessing residual disease is good and surpasses other diagnostic means. correlation coefficients of residual tumour size assessed by mri and pathology were considered good. however, both overestimation and underestimation of residual disease were observed. diagnostic accuracy of mri seems to be affected by treatment regimen and breast cancer subtype.

seborrheic dermatitis is a very common papulosquamous dermatoses, affecting over 5% of the general population with male predilections. this disease is characterized by mild to severe erythema, often accompanied by oily thick scales and mild inflammations. it typically affects areas containing sebaceous glands, particularly the face, ears, scalp, and upper part of the trunk. since seborrheic dermatitis commonly has a chronic and relapsing course with no permanent cure, various types of treatment have been introduced. the mainstays of treating facial seborrheic dermatitis rely on topical corticosteroids, antifungals, or a combination of both. although topical steroids rapidly reduce the severity of the disease by anti - inflammatory action, side effects associated with prolonged application and early relapse after discontinuation of treatment limit its wide usage. topical antifungal agents are also frequently used in order to decrease the colonization of lipophylic yeasts, malassezia (pityrosporum ovale), but they show a relatively slow onset of the anti - inflammatory effect compared to topical steroids. pimecrolimus cream 1% is a new topical macrolactam immunomodulator widely used for the treatment of atopic dermatitis via inhibition of t - cell activation and proinflammatory cytokine production. the cream has also shown to be effective in the treatment of other inflammatory skin disorders. crutchfield suggested that pimecrolimus cream 1% could be used as an effective treatment for facial seborrheic dermatitis. rigopoulos et al. conducted a randomized open - label clinical trial with pimecrolimus cream 1% and betamethasone 17-valerate 0.1% cream, and reported that pimecrolimus may be an excellent alternative modality for treating seborrheic dermatitis. we herein add our experience of rapid and sustained therapeutic response of pimecrolimus cream 1% in the treatment of facial seborrheic dermatitis. this study was a 4-week, open - label, clinical trial to investigate the efficacy of pimecrolimus cream 1% for the treatment of facial seborrheic dermatitis. it was conducted after the notice of purpose and obtaining signed informed consent from all subjects. a total of 20 patients with a clinical diagnosis of facial seborrheic dermatitis were enrolled. they composed of 16 males and 4 females with a mean age of 47.9 yr (range, 22 - 79) and a mean duration of 3.8 yr (range, 1 - 13) of dermatitis. patients who had taken topical or systemic treatment of corticosteroids, retinoids, or immune - modulating agents within 28 days prior to the study were excluded. patients with any significant medical condition that could compromise immune responsiveness (lymphoma, acquired immunodefiuency syndrome, other immunodeficiency disorders, or a history of malignant diseases) were excluded. exclusion criteria also included pregnancy, lactating women, and patients with other facial dermatoses. enrolled patients were instructed to stop using systemic and topical antimicrobials and antifungal agents including tar, zinc pyrithione, or selenium sulfate for 14 days before starting the treatment, but the uas of non - medicated shampoo and facial cleansers were allowed the use of the study. moreover, patients were informed to apply pimecrolimus cream 1% (norvatis, basel, switzerland) to affected facial areas only in thin layers, twice daily for 4 consecutive weeks. if complete resolution of seborrheic dermatitis occurred early in the study, patients were instructed to use pimecrolimus cream for an additional week. the evaluation of patients were performed at baseline and at week 1, 2, and 4 for the severity of seborrheic dermatitis. four different sites (forehead / eyebrows, nasolabial folds, perioral areas / chin, and posterior aspect of the ears) were clinically assessed for erythema, scaling, and pruritus using a 4-point scale: 0, absent; 1, mild; 2, moderate; and 3, severe. scores of each clinical symptom and sign at the baseline were compared to those at subsequent visits to assess the efficacy of pimecrolimus cream. in addition, patients themselves and physicians clinically performed the global assessments of the severity of seborrheic dermatitis at each visit as: completely clear (no evidence of seborrheic dermatitis); almost clear (> 90% of clearance); marked improvement (75 - 90% of clearance); moderate improvement (50 - 75% of clearance); slight improvement (25 - 50% of clearance); no change compared with baseline; or worse compared with baseline. local adverse reactions such as burning sensation, tingling, pain or soreness, or signs of viral or bacterial infections were recorded throughout the study as none, mild, moderate, or severe. a follow - up survey for recurrence of any erythema, scalings, or pruritus after the discontinuation of pimecrolimus was done by telephone. to assess the efficacy of pimecrolimus, data from scales of erythema, scaling, and pruritus were statistically analyzed by means of a repeated - measures analysis of variance (anova) model. the study patients had commonly been managed with topical corticosteroids, whereas only 1 patient had had a history of using a topical antifungal agent before visiting our clinic. the 4-week duration of clinical study with pimecrolimus cream 1% was completed in 16 patients, whereas 4 patients discontinued treatment before the completion of the study by the reason of early resolution with their symptoms and signs. at the baseline, all patients had erythema, with a mean grade of 5.15; 19 (95%) patients had scaling, with a mean grade of 3.70; and 17 (85%) patients had pruritus, with a mean grade of 4.12. 1 shows the significant decrements in the mean severity scores of each clinical feature during the 4-week application of pimecrolimus cream 1%, and the most prominent reduction of the disease severity was observed at the end of the first week. at week 1, the mean score of scaling improved most rapidly at 73.0% compared to the mean baseline score. this improvement continued over the following week at 85.1%, and the overall scaling score showed a mean reduction of 91.9% at week 4 (p<0.001). similar decrements of mean scores were also observed with erythema (p<0.001) and pruritus (p<0.001) at reductions of 53.4%, and 68.7% at week 1, 69.9% and 88.6% at week 2, and 87.4% and 91.5% at week 4 compared to the mean baseline scores, respectively. these changes were statistically significant (p<0.001) and clinically well correlated with photographs (fig . 2, 3). global improvement of severity assessed by investigators and patients at the week 1 and 4 of treatment are shown in table 1. similar to the symptoms and signs, the patients' subjective response to pimecrolimus was mostly significant at the end of the first week. investigators noticed above the moderate improvement of severity in all patients after the first week of treatment. at week 4, 3 patients (15%) reported marked improvement, 6 (30%) almost clear, and 11 (55%) complete clear of the dermatitis. from physician's point of view, 12 subjects (60%) including 4 prematurely discontinued patients experienced a complete clearance of clinical signs and symptoms within 4 weeks of pimecrolimus treatment. a total of 9 patients (45%) reported mild (1 patient) to moderate (8 patients) burning or tingling sensations lasting for approximately 20 - 30 min and mostly during the first 2 or 3 days of treatment. these local adverse events did not require any further management nor did they lead to premature discontinuation of the therapy. after the completion of the study, 12 patients were available for follow - up telephone surveys 4 to 8 weeks later. three patients had continued to apply pimecrolimus cream 1% once daily and did not notice any relapse of symptoms or signs of the disease for 8 weeks. nine patients not using any medications were aware of recurrence of the disease, mostly between 3 to 8 weeks after the discontinuation of pimecrolimus. five out of 9 patients remained free of the disease for at least 4 weeks after the discontinuation of pimecrolimus, whereas 4 patients experienced relapse within 4 weeks (table 2). recent studies proposed that pimecrolimus cream 1% is an effective and safe modality in controlling the symptoms and signs of seborrheic dermatitis (table 3). in the study by rigopoulos et al. to determine the efficacy of pimecrolimus in comparison with potent topical corticosteroid in the treatment of seborrheic dermatitis, the complete resolution of symptoms was rapidly achieved within 9 days in both the pimecrolimus - treated and the topical corticosteroid - treated group. although both agents were highly effective in the treatment of seborrheic dermatitis, the relapses were more common, rapid, and severe in the corticosteroid - treated group. treated 19 patients of seborrheic dermatitis with pimecrolimus, and clearance was observed after 1 to 3 weeks of treatment. in addition, patients who were previously unresponsive to topical corticosteroid and antifungals also showed clearance of seborrheic dermatitis with pimecrolimus treatment. these indicate that pimecrolimus cream 1% may be an effective nonsteroidal alternative for the treatment of seborrheic dermatitis. similar were reported in the use of topical tacrolimus for seborrheic dermatitis. in our study, consistent with those previous reports, the mean severity scores of erythema, scaling, and pruritus showed statistically significant decrements after a 4-week application of pimecrolimus cream 1% relative to the baseline. the evident therapeutic responses mostly appeared within the first week of treatment and continued to improve until the end of the study. improvements in global assessment of disease severity determined by patients and investigators also showed excellent during the first week of the application, and they experienced continuous improvements throughout the duration of the study. according to the follow - up survey, improved states were maintained for at least 4 weeks in 5 out of 9 patients who did not use any medications after the completion of the study. previous studies have indicated that seborrheic dermatitis might develop as a of inflammatory immune reactions to the lipophilic yeast malassezia. humoral and cell - mediated immune response to malassezia has also been advocated. improvement of the disease by antifungal medications supports the possible role of malassezia. pimecrolimus selectively inhibits t cell activation via inhibition of the calcineurin - dependent nuclear transcription factor of activated t cells. it prevents the release of cytokines and proinflammatory mediators involved in the inflammatory response. accordingly, the beneficial effect of pimecrolimus on the course of seborrheic dermatitis in our study is thought to be due to its anti - inflammatory and immunomodulatory properties. however, the possibility of direct antifungal activities of pimecrolimus can not be excluded, since a recent report documented that tacrolimus, another topical immunomodulator, has antifungal activities against some of the malassezia species. the safety of pimecrolimus cream has been well documented, and in contrast to topical corticosteroids it does not induce skin atrophy or other corticosteroid - related adverse events, especially on the face. burning sensations and tingling are well - known local side effects of pimecrolimus, and 9 of 20 (45%) patients experienced these events in our study. however, most reactions were minimal and transient, beginning on day 1 and resolving within 3 days of treatment. no pain, soreness, or signs of viral or bacterial infections were detected. in , our findings further support the previous reports that pimecrolimus cream 1% can be an effective and well - tolerated modality in the treatment of facial seborrheic dermatitis. however, since the most of previous encouraging reports had been studied in small numbers and in short - term, further clinical troals involving controlled, long - term follow - up protocols with a large scale of patients are warranted to assess the precise efficacy and preventive action of pimecrolimus in the management of seborrheic dermatitis.

pimecrolimus cream 1% has shown to be effective in patients with a variety of inflammatory cutaneous disorders. and it might be a useful modality in the treatment of seborrheic dermatitis. this prospective study was aimed at assessing the efficacy and tolerability of pimecrolimus cream 1% in the treatment of facial seborrheic dermatitis. twenty patients were instructed to apply pimecrolimus cream 1% for 4 consecutive weeks. assessment of the disease severity was performed at baseline and at week 1, 2, and 4. clinical assessments of erythema, scaling, and pruritus were measured using a 4-point scale. global assessments of the disease severity by patients and investigators were performed at each visit. mean clinical scores of erythema, scaling, and pruritus significantly improved by 87.4%, 91.9%, and 91.5% respectively at week 4 (p<0.001). improvements in the global assessment of disease severity determined by patients and investigators also showed excellent . no specific adverse events other than transient burning and tingling sensations were noted. the relapse of facial seborrheic dermatitis was mostly observed between 3 to 8 weeks after the discontinuation of pimecrolimus. we suggest that the topical application of pimecrolimus cream 1% can be an effective and safe alternative for treatment of facial seborrheic dermatitis.

hernias through the foramen of winslow are extremely rare, accounting for 0.1% of all abdominal hernias we present a case of a patient with strangulated ileum herniated through the foramen of winslow. recent literature review was undertaken on pubmed as a search platform using the keywords foramen of winslow and hernia. an emergency computed tomography scan revealed a loop of ileum in the lesser sac. at emergency laparotomy, a herniated loop of ileum that had become strangulated at its entry to the lesser sac via the foramen of winslow was confirmed. the loop of ileum was reduced but was nonviable, which had to be resected with a primary anastomosis. herniation through the foramen of winslow is a difficult diagnosis and must not be missed. early cross - sectional imaging and surgical intervention are advised in order to reduce morbidity. hernias through the foramen of winslow are extremely rare, accounting for 0.1% of all abdominal hernias1 and 8% of all internal hernias. two - thirds of cases reported were herniation of ileum followed by a mobile cecum or ascending colon.2,3 seldomly, the gallbladder or the omentum has herniated through. as the symptoms are so nonspecific, delayed clinical diagnosis is often observed, ing in bowel strangulation and high mortality rate between 36% and 49%.4 nonviable bowel is often identified only at laparotomy. we present a case of a patient with strangulated ileum herniated through the foramen of winslow. a 66-year - old man, with no known past medical or surgical history, presented acutely with a few hours history of severe colicky epigastric pain and vomiting. he had similar milder and short - lived episodes of pain in the past, but he had put these episodes down to muscular pain, for which he had never sought medical attention. there was no report of alleviation of pain on truncal flexion. on admission, examination was unremarkable except for mild tenderness of the epigastrium, but the patient was in extreme pain. his blood profile showed a mild neutrophilia of 12.4 10/l and a raised c - reactive protein of 130 mg / l. blood lactate and base excess of an arterial gas sample were also within normal limits. an emergency computed tomography scan of the abdomen revealed distended loops of small bowel showing reduced enhancement, which were located within the lesser sac. both afferent and efferent limbs were visualized in the space between the liver hilum and inferior vena cava, which was widened. the appearance was in keeping with an internal hernia ing in a closed - loop obstruction (figs . 1 and 2). this revealed a loop of mid - ileum herniating through the foramen of winslow, and the herniated loop of ileum had become strangulated. the reported demographic for bowel herniation through the foramen of winslow is usually men with a manual occupation, aged between 61 and 69 years. some have suggested that cholecystectomy might be a risk factor.5 other postulated risk factors6 include abnormally long bowel mesentery, abnormally enlarged foramen of winslow, and a defect in the gastrohepatic ligament. herniation through the foramen of winslow is rare because the normal peritoneal orifice is kept closed by normal intra - abdominal pressure ( fig. erskine7 has also postulated the failure of the right colon to retroperitonealize along with changes in the intra - abdominal pressure as a contributing factor. the presence of jaundice has been described due to direct compression of the hepatic pedicle.5 the obstruction in our patient was very proximal, rendering minimal nasogastric drainage. our patient had no previous abdominal surgery or trauma, and the cross - sectional imaging suggested small bowel ischemia, and therefore laparotomy was mandated. there are many more common causes of epigastric pain; however, these conditions can be excluded quickly. unfortunately, many cases of this condition have been identified at autopsy. some have reported that gas - containing intestinal loops are high in abdomen and medial - posterior to stomach associated with small bowel obstruction. cross - sectional imaging is considered the diagnostic modality of choice,8 largely as a consequence of clinical diagnostic uncertainty.9 classical signs include mesenteric vessels stretching anterior to the inferior vena cava and posterior to the portal vein associated with bowel obstruction in the lesser sac. there were no obvious risk factors in this gentleman, except that he is an average manual worker. this condition is rare, difficult to diagnose, and does not always have the obvious risk factors. a wide kocher s maneuver or opening the gastrohepatic ligaments may be required. due to diagnostic uncertainty even with high - resolution computed tomography, open surgery is usually performed. however, this has led some experienced surgeons to investigate initially with laparoscopy. successful laparoscopic management for the foramen of winslow herniation has now been described.10 the debate continues as to whether the foramen of winslow ought to be closed in order to prevent recurrence. to date, there has not been a report of recurrence probably due to adhesions obliterating the foramen and tethering the remainder of the small bowel. experts have also warned of the potential significant negative consequences of closing the defect: portal vein thrombosis or obstructive jaundice. internal herniation through the foramen of winslow is a difficult clinical diagnosis and must not be missed.

introductionhernias through the foramen of winslow are extremely rare, accounting for 0.1% of all abdominal hernias. delayed diagnosis is often observed, ing in bowel strangulation and high mortality.methodwe present a case of a patient with strangulated ileum herniated through the foramen of winslow. recent literature review was undertaken on pubmed as a search platform using the keywords foramen of winslow and hernia.case summarya 66-year - old man presented acutely with severe epigastric pain and vomiting. an emergency computed tomography scan revealed a loop of ileum in the lesser sac. at emergency laparotomy, a herniated loop of ileum that had become strangulated at its entry to the lesser sac via the foramen of winslow was confirmed. the loop of ileum was reduced but was nonviable, which had to be resected with a primary anastomosis. the patient s postoperative recovery was uneventful.herniation through the foramen of winslow is a difficult diagnosis and must not be missed. early cross - sectional imaging and surgical intervention are advised in order to reduce morbidity.

intractable nausea and vomiting is a commonly encountered problem in any general medicine or gastroenterology service. several causes from infection to chronic medical conditions, such as diabetic gastroparesis and gastroparesis due to multiple causes, can be listed. neuromyelitis optica (nmo), also known as devic's disease, is a rare autoimmune disorder in which a patient's immune system attacks their own nervous system, especially the optic nerves and the spinal cord, leading to loss of vision and spinal cord dysfunction. the likely target of the autoimmune attack, at least in some patients with nmo, has been identified as a protein of the nervous system astrocytes called aquaporin 4. typically, nmo is diagnosed in patients presenting with a rapid onset of blindness in one or both eyes, followed within days or weeks by varying degrees of paralysis in the arms and legs. in most cases, however, the interval between optic neuritis and transverse myelitis is significantly longer, in many cases can occur after some years of quiescence. after the initial attack, nmo follows a largely unpredictable course. besides muscle weakness and reduced sensation, bladder and bowel incontinence the disease can also occur as clusters of attacks months or years apart, followed by partial recovery during periods of remission. another form of nmo, in which an individual only has a single, severe attack extending over a month or two, is most likely a distinct disease that affects men and women with equal frequency. the onset of nmo varies from childhood to adulthood, with two peaks, one in childhood and the other in adults (mostly in their 40s). for a while, nmo was considered to be a severe variant of multiple sclerosis (ms) because both can cause attacks of optic neuritis and myelitis. recent discoveries, however, suggest it is a separate disease. nmo is different from ms in the severity of its attacks and its tendency to solely strike the optic nerves and spinal cord at the beginning of the disease. symptoms outside of the optic nerves and spinal cord are rare, although certain symptoms, including uncontrollable vomiting and hiccups, are now recognized as relatively specific symptoms of nmo that are due to brainstem involvement, specifically the area postrema. we present our encounter with a 38-year - old female with no past medical history who presented to the emergency department with complaints of intractable nausea, vomiting, hiccups and abdominal pain. around thanksgiving 2012, she had had bronchitis and sinusitis. she had been treated with tylenol cold and cough, a z - pak, inhalers and tessalon perles. this had progressively worsened into intractable nausea and vomiting about 4 days following thanksgiving and lasted till presentation, which was about 2 weeks after onset. complete blood count revealed hemoglobin of 10.1 g / dl, a hematocrit of 31.2%, a platelet count of 225 103/l and white blood cell count of 6.9 10/l. serum electrolytes were as follows: na 136 mmol / l, k 3.6 mmol / l, cl 105 mmol / l, hco3 23 mmol / l, bun 10 mg / dl and creatinine 1.3 mg / dl. she had a right upper quadrant ultrasound that showed some stones within the gallbladder, but no findings suggestive of acute cholecystitis. gastroenterology was consulted and she had an egd that showed mild esophagitis for which she was placed on a proton pump inhibitor. serial daily labs including complete blood count and basic metabolic profile did not reveal any abnormalities. while in the hospital, she was noticed to have a positive babinski's test on daily physical examination, which was a new finding that had been absent on presentation. at this point she had an mri without contrast showing moderate diffuse atrophy, most particularly in the occipital area (she denied any prior head trauma or problems at birth) and very few scattered white matter hyperintensities (she has a history of tobacco abuse). she went on to have a brain mri with and without contrast with showing small foci of linear abnormal t2 signal with questionable associated vague enhancement at the posterior aspect of the brainstem, in the region that anatomically corresponds to the area postrema (fig . 1, fig this finding was suspicious for a variant presentation of nmo with intractable nausea / vomiting . following this finding, she was started on 1 g of iv solu - medrol daily . the day after her first dose of solu - medrol, she was symptom - free and asked for a diet, which she tolerated well . however, the patient started feeling better on solu - medrol and deferred the procedure, saying she would consider it if her symptoms recurred . after 5 days on iv solu - medrol and still symptom - free, she was transitioned to oral steroids and discharged with a taper . she was asked to follow up with the neurology clinic and her primary care doctor . other lab work done included tsh, rpr, heavy metals, uds and serum ace, which all came back negative or within normal range . at the clinic, she was started on rituximab and has remained symptom - free till the writing of this article . intractable nausea and vomiting is a commonly encountered problem in any general medicine or gastroenterology service . several causes from infection to chronic medical conditions, such as diabetic gastroparesis and gastroparesis due to multiple causes, can be listed . nmo, also known as devic 's disease, is a rare autoimmune disorder in which a patient 's immune system affects the optic nerves and spinal cord, leading to loss of vision and spinal cord dysfunction . spinal cord dysfunction is mainly manifested as weakness, paralysis, numbness and possibly bowel and bladder incontinence . due to these symptoms, nmo can often be confused with ms . as rare a disease as nmo is, it is even rarer for intractable nausea and vomiting to be the only initial presenting symptoms, and this is sparsely mentioned in reviewed literature . nmo is caused by the development of autoantibodies ( nmo - igg) against the protein aquaporin 4 found in the cell membranes of the processes of astrocytes that surround and protect the blood brain barrier. like in ms, demyelination of the optic nerve and spinal cord is also known to occur in nmo, but the exact mechanism is still to be described. however, astrocyte destruction is thought to be more severe than demyelination and as such probably plays a bigger role in the pathogenesis of the disease. they proposed that at least two out of three supporting criteria in addition to the above - listed absolute criteria are needed to make a diagnosis of nmo. the three supporting criteria are: a contiguous spinal cord lesion on mri extending over three or more segments. initial brain mri not meeting the usual diagnostic criteria for ms. seropositivity for nmo - igg. conducted a case - control study on the sensitivity and specificity of nmo - igg in the diagnosis of the disease and concluded that sensitivity and specificity were 73% (95% ci 6086) and 91% (95% ci 79100) for nmo. of 14 seropositive cases identified incidentally, 12 had nmo or a high - risk syndrome for the disease. different variants of the disease with unique presenting features have been described. in a case series, apiwattanakul et al. reported on 12 aquaporin 4 antibody - positive patients (12% of seropositive mayo clinic patients identified since 2005) whose initial presenting symptom of nmo was intractable vomiting. vomiting lasted a median of 4 weeks (range 2 days to 80 weeks). optic neuritis or transverse myelitis developed after vomiting onset in 11 patients. at last evaluation (median 48 months after vomiting onset), 7 patients fulfilled the diagnostic criteria for nmo. from their study they suggested that the aquaporin 4-rich area postrema might be a first point of attack in nmo. the disease has recently been associated with several comorbid conditions including collagen vascular diseases, autoantibody syndromes, infections with varicella zoster virus, epstein - barr virus and hiv, and exposure to clioquinol and anti - tuberculosis drugs. a high level of suspicion on the part of the clinician is necessary for early diagnosis, especially in cases where the only initial presenting symptoms are nausea and vomiting. it is also pertinent to note that nmo - igg - negative disease has been described as well, making diagnosis in these circumstances quite challenging. in terms of treatment, intravenous glucocorticoid (typically 1 g of iv solu - medrol daily over 5 days) is the recommended treatment during acute attacks. azathioprine, mycophenolate mofetil or rituximab is used to achieve immunosuppression on a chronic state, thereby preventing relapse. patients will typically require this for at least 5 years (especially if they are nmo - igg - seropositive) even if they have had just one attack. it is our hope that our experience and this article highlight a less often considered cause of intractable nausea and vomiting. if this had been considered earlier, this patient could have achieved a quicker turnaround, with improved health outcomes and cost.

neuromyelitis optica, also known as devic's disease, is a rare autoimmune disorder in which a patient's immune system affects the optic nerves and the spinal cord, leading to loss of vision and spinal cord dysfunction. we present our experience with a 38-year - old female who presented to our facility with complaints of intractable nausea and vomiting. after extensive evaluation, she was found to have neuromyelitis optica. her symptoms completely resolved following institution of appropriate therapy. she made a significant recovery and has since been placed on chronic immunosuppressive therapy. through this article we hope to bring attention to a significant cause of intractable nausea and vomiting that may often be forgotten in general medicine or gastroenterology services.

service - learning as a teaching methodology has roots in the experiential education movement of the 1950 s and 1960 s. experiential education was based on the concept that the quality of student learning was measured by the meaning students placed on their experience. developing knowledge through action, specifically through actions that benefitted course - based, credit - bearing educational experience in which students (a) participate in an organized service activity that meets identified community needs and (b) reflect on the service activity in such a way as to gain further understanding of course content, a broader appreciation of the discipline, and an enhanced sense of civic responsibility . according to the national service - learning clearinghouse, almost 30% of the 6.7 million students in public and private 4 year institutions of higher education report participating in a course where service is part of the curriculum. service - learning in the medical and allied health professions has been a critical component of the curriculum with projects aimed at providing a service to the community and hands - on experience for students. bentley and ellison described a service - learning project designed to teach nursing students key elements of delivering community services to pregnant teens. nursing students (n = 20) partnered with early head start to address the needs of pregnant teenagers and help them transition from pregnancy to parenthood. specific course objectives were outlined in advance to give students, faculty and community partners clear guidelines for evaluation. make an appointment with the adolescent once per week for six weeks to implement nursing interventions identified from the supporting data following the assessments. to attend the adolescent s delivery if she happens to deliver during this semester. bentley and ellison found that students were able to understand the at - risk population at a deeper level, overcoming preconceived ideas of pregnant teens. in addition, the faculty found that student s commitment to community service following the class increased. health education programs have also included service - learning projects in their curricula as a means of enhancing the preparation of health educators. geiger and werner described a service - learning assignment completed by undergraduate health and elementary education students (n = 27). the assignment was designed to reflect the competencies outlined in the national commission of health education credentialing. students engaged in learning by attending lectures on relevant topics, assisting health teachers with instructional planning, and presenting information to elementary school students. student evaluations reflected a positive experience and an ability to overcome preconceived barriers relating to teaching in an inner - city school environment. cooperating teacher evaluations provided valuable feedback to both faculty and students relating to future course preparations. freewheelers designed to increase the mobility of wheel - chair bound nursing home clients (n = 15) through a stretching and strength training program facilitated by kinesiology students (n = 22) enrolled in a motor development laboratory class. clearly articulated learning objectives relating to motor development education as well as community service needs were provided. in addition, students were challenged to consider and reflect upon their stereotypes around aging adults. each nursing home resident was assigned to three students from the class with one hour sessions scheduled three times a week on monday, wednesday and friday for eight weeks of the 15-week semester. the students reported greater understanding of motor development over the life span and a deeper appreciation for older adults. in this section, a definition of service - learning was proposed, with examples provided of service - learning projects from nursing, health education and kinesiology. while the spectrum of service - learning projects is broad, all contain some structure of objectives, accountability, and partnering. some service - learning projects take place over the course of a 15-week semester, while other projects are event - based with students completing assignments in preparation for a single event. in this section, a brief description of three service - learning projects conducted by faculty and students from westfield state college will be provided. the original homeschooler s field day was a collaborative effort with students enrolled in an analysis of teaching physical education class, sport promotions class and a community - based homeschooler s programming organization. the now annual event has grown to include faculty and students from the biology department, as well as students enrolled in a kinesiology laboratory class. homeschooler s field day was a collaborative event organized and implemented by students and faculty for homeschooler s in the new england area to engage in fun, motor - skilled activities. in this one day event homeschooled children (n = 162) ages 513 pre - registered and participated in an organized field day scheduled from 11:00 a.m.2:00 p.m. homeschooler s divided into younger (ages 58) and older (ages 913) groups progress through a series of eight 1520 minute stations. kinesiology students developed stations relating to biomechanical principles that could be taught and practiced in a 15-minute station. sport promotions students developed objectives and implemented strategies relating to event promotion, logistics and risk management. movement science majors not enrolled in the classes responsible for the field day were invited to participate as volunteers. parents were invited to join in any activities, adding to the complexity of the teaching for the students in charge of each station. homeschooler s, parents, students and faculty reported positive experiences, with all participants providing valuable feedback for future events. although not one of the original objectives, student stereotypes and attitudes about homeschooler s and the structure of education were challenged. in addition, students reported greater appreciation for the level of planning and preparation required for a successful event. hope for hollie provides an example of a service - learning project that not only engaged students and the larger community, but made a meaningful and lasting difference in the world. hope for hollie was a sports promotions class service - learning project that was undertaken to raise funds for a student who was recovering from meningitis. the students in the sports promotion class were offered an opportunity to apply the principles of sport event management by envisioning, initiating, planning, organizing, implementing and evaluating a real event. the upper - level class (n = 18) consisted of movement science, business and communications seniors. because of student internship and work commitments, the event a run / walk was planned for a friday in april, during the scheduled class from 12:30 p.m. participation objectives included the registration of 100 participants and awareness objectives relating to risks associated with meningitis were also developed. because of the interdisciplinary nature of the class, students were able to draw on their strengths to achieve a successful outcome. movement science majors assisted with risk management, developed pre - walk exercise programs for participants, and contacted exercise - related sponsors. communications majors assisted with the development of a logo, news releases and attractive flyers / promotions. students reported learning about sport promotions, but also learning life lessons about the goodness of humankind. the day of the walk was one of my best days all year, actually of my life. i had no idea how good it would feel to be able to give hollie the check. this was the most rewarding class i have ever taken, not only academically but also on a personal level... we bonded with our classmates on a level that usually never happens in a class, we were able to work closely and learn from students from different majors and we were able to take what we learned in the classroom and put it to use in the real world. elementary school students (n = 84) in the pioneer valley chinese immersion charter school (pvcics) begin their kindergarten and first grade education with 75% of their day taught in mandarin chinese. drawing on the successful experiences from the homeschooler s field day and hope for hollie events, two faculty from the movement science department at westfield state college initiated, organized and implemented a service - learning event to be taught primarily in chinese. balance across boundaries was the collaborative effort of students (n = 30) enrolled in movement science classes in kinesiology, rehabilitation for special populations, supervising sport and fitness programs and a physical science class for teachers preparing to teach physics. a series of four stations were developed to teach balance using simplified chinese characters for numbers and animals. for example, one station, entitled, balance bingo had the simplified chinese characters for numbers 15 marked in large squares with the characters chinese symbol for an animal at the heading. the elementary school students who landed on a specific square called out the name of the animal and number in chinese the college students repeated in chinese and performed a balance activity for the elementary school student to copy. while the pronunciation of the elementary school students was superior to that of the college students, the ability to communicate movement in another language both challenged and simplified the event. several students reported an inability to understand the elementary school students, but an ability to overcome the language barrier by performing a movement. likewise, students reported admiration for the efforts of the kindergarten students to communicate in chinese. while not an objective of the program, students reported a positive shift in attitude toward chinese - speaking individuals. elementary school students expressed pride in their ability to communicate in their immersion language, and their ability to learn new skills relating to balance. some projects have involved only one class, while others have involved several different classes and disciplines. service - learning provides a methodology and structure that embraces interdisciplinary, intergenerational and international experiences. the original homeschooler s field day was a collaborative effort with students enrolled in an analysis of teaching physical education class, sport promotions class and a community - based homeschooler s programming organization. the now annual event has grown to include faculty and students from the biology department, as well as students enrolled in a kinesiology laboratory class. homeschooler s field day was a collaborative event organized and implemented by students and faculty for homeschooler s in the new england area to engage in fun, motor - skilled activities. in this one day event homeschooled children (n = 162) ages 513 pre - registered and participated in an organized field day scheduled from 11:00 a.m.2:00 p.m. homeschooler s divided into younger (ages 58) and older (ages 913) groups progress through a series of eight 1520 minute stations. learning objectives for students were developed relevant to their course. kinesiology students developed stations relating to biomechanical principles that could be taught and practiced in a 15-minute station. sport promotions students developed objectives and implemented strategies relating to event promotion, logistics and risk management. movement science majors not enrolled in the classes responsible for the field day were invited to participate as volunteers. parents were invited to join in any activities, adding to the complexity of the teaching for the students in charge of each station. homeschooler s, parents, students and faculty reported positive experiences, with all participants providing valuable feedback for future events. although not one of the original objectives, student stereotypes and attitudes about homeschooler s and the structure of education were challenged. in addition, students reported greater appreciation for the level of planning and preparation required for a successful event. hope for hollie provides an example of a service - learning project that not only engaged students and the larger community, but made a meaningful and lasting difference in the world. hope for hollie was a sports promotions class service - learning project that was undertaken to raise funds for a student who was recovering from meningitis. the students in the sports promotion class were offered an opportunity to apply the principles of sport event management by envisioning, initiating, planning, organizing, implementing and evaluating a real event. the upper - level class (n = 18) consisted of movement science, business and communications seniors. because of student internship and work commitments, the event a run / walk was planned for a friday in april, during the scheduled class from 12:30 p.m. participation objectives included the registration of 100 participants and awareness objectives relating to risks associated with meningitis were also developed. because of the interdisciplinary nature of the class, students were able to draw on their strengths to achieve a successful outcome. movement science majors assisted with risk management, developed pre - walk exercise programs for participants, and contacted exercise - related sponsors. communications majors assisted with the development of a logo, news releases and attractive flyers / promotions. students reported learning about sport promotions, but also learning life lessons about the goodness of humankind. the day of the walk was one of my best days all year, actually of my life. i had no idea how good it would feel to be able to give hollie the check. this was the most rewarding class i have ever taken, not only academically but also on a personal level... we bonded with our classmates on a level that usually never happens in a class, we were able to work closely and learn from students from different majors and we were able to take what we learned in the classroom and put it to use in the real world. elementary school students (n = 84) in the pioneer valley chinese immersion charter school (pvcics) begin their kindergarten and first grade education with 75% of their day taught in mandarin chinese. drawing on the successful experiences from the homeschooler s field day and hope for hollie events, two faculty from the movement science department at westfield state college initiated, organized and implemented a service - learning event to be taught primarily in chinese. balance across boundaries was the collaborative effort of students (n = 30) enrolled in movement science classes in kinesiology, rehabilitation for special populations, supervising sport and fitness programs and a physical science class for teachers preparing to teach physics. a series of four stations were developed to teach balance using simplified chinese characters for numbers and animals. for example, one station, entitled, balance bingo had the simplified chinese characters for numbers 15 marked in large squares with the characters chinese symbol for an animal at the heading. the elementary school students who landed on a specific square called out the name of the animal and number in chinese. the college students repeated in chinese and performed a balance activity for the elementary school student to copy. while the pronunciation of the elementary school students was superior to that of the college students, the ability to communicate movement in another language both challenged and simplified the event. several students reported an inability to understand the elementary school students, but an ability to overcome the language barrier by performing a movement. likewise, students reported admiration for the efforts of the kindergarten students to communicate in chinese. while not an objective of the program, students reported a positive shift in attitude toward chinese - speaking individuals. elementary school students expressed pride in their ability to communicate in their immersion language, and their ability to learn new skills relating to balance. some projects have involved only one class, while others have involved several different classes and disciplines. service - learning provides a methodology and structure that embraces interdisciplinary, intergenerational and international experiences. one of the primary purposes of service - learning education is to expose students to diverse individuals and cultures. preparing students to live in a global community enables them to transition from academia to the workplace more smoothly. traditionally, this exposure to different cultures has taken place in the context of study abroad programs. community service programs designed to assist an under - served population have long been a part of campus ministry and study abroad programming. while worthy and useful, many such programs could be defined more as volunteer than service - learning. however, structured, service - learning projects that provide students an opportunity to learn about different cultures have been developed throughout the world. parsi and list described the powerful experiences reported by medical students involved in a service - learning project in nairobi, kenya. the authors recommended designing service - learning programs that promote cultural sensitivity, individual and community empowerment and provide education about the historical and global forces that shape communities. riner and becklenberg described a partnership with a nicaraguan community and the indiana university school of nursing that provided a rich opportunity for nursing students to learn about working in a multi - cultural, interdisciplinary team of healthcare professionals. ngai described a service - learning project implemented at the chinese university of hong kong in which students provided services to needy individuals within the context of a general education class. while service - learning projects are often designed to assist an at - risk population, many provide students an opportunity to move beyond the confines of a classroom or case study to real people in real situations. as exercise science professionals , we have ample opportunities to develop cultural sensitivity and provide diverse experiences for our students to gain exposure to the global community. in this paper, service - learning has been defined and several examples of service - learning have been provided. a challenge of developing effective service - learning projects is the logistics and ongoing evaluation. many colleges and universities have an office dedicated to service - learning education. working in unison with a service - learning coordinator many study - abroad programs provide service - learning opportunities for students. however, not many such programs are discipline specific. educating and collaborating with study abroad coordinators to educate, envision and organize exercise science related service - learning programs may increase the likelihood of students having discipline specific opportunities. the number of international students enrolled at colleges and universities in the united states has grown exponentially. international student organization leaders can make connections for students to participate in collaborative service - learning projects. for example, international students who are active in sports and physical activities not often played in the united states can partner with exercise science students for a series of laboratory activities. the international students will have an opportunity to teach a skill, take pride in their sport, and forge new relationships. exercise science students can analyze the skills necessary to be successful in the sport, learn more about the origins and history of the culture and sport, or develop specific training programs for athletes involved in the sport. in addition to on - campus opportunities, locally - based, culturally diverse communities provide another opportunity to engage students in service - learning projects focused on physical activity and exercise science. language need not be a barrier to program development. some examples of simple, service - learning projects that can be conducted with minimal language skills are: a student - directed, 8-week walking program with individuals from a local international community at a designated time and place; a 4-week playground project in which students engage caregivers and children from a local international community in motor skill activities; a stretching and/or strength program designed to increase mobility of a given population, perhaps from a local ethnic church. while examples of effective service - learning projects abound, it is critical that objectives relating to increasing cultural sensitivity and exposing students to diverse cultures be articulated in advance. as advocates for healthy lifestyles and ambassadors of the exercise is medicine campaign, we have a unique opportunity to envision, create and provide service - learning programming that challenges students to be agents of change. service - learning education provides a vehicle for applying theory to practice and engaging students in programs that develop reflective, global citizens prepared to face the relevant issues and challenges of the future. a challenge of developing effective service - learning projects is the logistics and ongoing evaluation. many study - abroad programs provide service - learning opportunities for students. however, not many such programs are discipline specific. educating and collaborating with study abroad coordinators to educate, envision and organize exercise science related service - learning programs may increase the likelihood of students having discipline specific opportunities. the number of international students enrolled at colleges and universities in the united states has grown exponentially. international student organization leaders can make connections for students to participate in collaborative service - learning projects. for example, international students who are active in sports and physical activities not often played in the united states can partner with exercise science students for a series of laboratory activities. the international students will have an opportunity to teach a skill, take pride in their sport, and forge new relationships. exercise science students can analyze the skills necessary to be successful in the sport, learn more about the origins and history of the culture and sport, or develop specific training programs for athletes involved in the sport. in addition to on - campus opportunities, locally - based, culturally diverse communities provide another opportunity to engage students in service - learning projects focused on physical activity and exercise science. language need not be a barrier to program development. some examples of simple, service - learning projects that can be conducted with minimal language skills are: a student - directed, 8-week walking program with individuals from a local international community at a designated time and place; a 4-week playground project in which students engage caregivers and children from a local international community in motor skill activities; a stretching and/or strength program designed to increase mobility of a given population, perhaps from a local ethnic church. while examples of effective service - learning projects abound, it is critical that objectives relating to increasing cultural sensitivity and exposing students to diverse cultures be articulated in advance. as advocates for healthy lifestyles and ambassadors of the exercise is medicine campaign, we have a unique opportunity to envision, create and provide service - learning programming that challenges students to be agents of change. service - learning education provides a vehicle for applying theory to practice and engaging students in programs that develop reflective, global citizens prepared to face the relevant issues and challenges of the future.

service - learning provides students, faculty and community partners an opportunity to go beyond classroom and laboratory instruction to the application of theoretical principles, and the development of civic responsibility by forging meaningful relationships with community partners. service - learning also enables faculty to broaden the cultural sensitivity and international exposure of students by engaging students in directed, purposeful projects aimed at supporting the goals of both the college and the international partners. we have a unique opportunity in exercise science to make lasting changes and improve global conditions relating to student learning and the physical inactivity crisis. the purpose of this article is to define service - learning, describe different types of service - learning projects, and discuss service - learning opportunities in international settings.

phenylketonuria is an autosomal recessive metabolic disease which may cause brain insult in the developing brain, consequently, leading to progressive neurodevelopmental delay. in this genetic metabolic disorder, the hepatic enzyme, this enzyme is necessary to break down amino acid phenylalanine to tyrosine. in deficiency of this enzyme, phenylketonuria is classified into classic phenylketonuria (pku) which indicates phenylalanine (phe) levels higher than 1200 mol / l and mild pku in which phe levels are between 600 mol / l and 1200 mol / l. in mild hyperphenylalaninemia, phe level is higher than normal limits, but below 600 mol. in early infants with phenylketonuria, the serum phenylalanine level is in normal limits at birth, but begins to rise within the first few hours of life. excessive phenylalanine is generally believed to be responsible for the brain insult leading to progressive mental retardation and seizure disorder. without treatment, cognitive delay becomes evident within 6 months of age and is progressive. although the principal biochemical defect is obvious in inborn errors of metabolism such as pku and other similar disorders, the exact neurodysfunction defect is not clearly definite. clinical manifestations in untreated infants with pku include growth failure, microcephaly, seizures, dermatitis, and intellectual defect. remarkable clinical findings are light colored skin, hair and eyes, eczematous rash and behavioral disorders. different studies reported 25% generalized or partial seizures in phenylketonuria. long eeg recording can detect abnormal epileptic discharge with or without clinical seizure or convulsion. eeg activity is categorized into normal and abnormal activity which is classified into type 1 and type 2 of continuous and hyperactive rapid tracing and in highly abnormal activity, inactive, paroxysmal and low voltage plus theta activity. in this study , we evaluated the association between eeg abnormality and developmental delay as well as behavioral disorders in patients diagnosed as phenylketonuria. in this case - control study, 105 pku patients (50 male, 55 female ; mean age, 8.5 6.2 years) who had been referred to the neurometabolic clinic of mofid children hospital, tehran, iran from 2010 to 2011 were evaluated. these patients were diagnosed as pku patients through newborn screening tests or later during follow - up evaluation for neurodevelopmental delay at the hospital. the diagnosis of classic pku was made on the basis of serum phe level higher than 6 mg / dl in untreated infants, (we used mg / dl for measuring serum phe level .) , the patients were matched according to sex, age, and the serum phenylalanine level. then all the patients were referred to the eeg laboratory for eeg recording (after sleep deprivation for 10 to 12 hours). pediatric neurologists took the history of seizures and all the patients who had recent seizures were excluded and only patients who were seizure - free for at least six months before the study were enrolled. based on eeg recording, the patients were divided into two groups: the case group in which the patients had an abnormal eeg and the control group which consisted of those with normal eegs. we evaluated the developmental score of the two groups with the asq questionnaire (age and stage questionnaire). after estimation of developmental score in cases and controls, we detected the developmental quotient (dq) of patients using the below equation: dq= developmental ageactual age 100. we also used the test of child symptom inventory-4 (csi-4) for evaluation of behavioral disorders in children. this behavior rating scale screens for dsm - iv emotional and behavioral disorders in children between 5 and 14 years of age. we evaluated generalized anxiety disorder, attention deficit, hyperactivity, attention deficit hyperactivity disorder, oppositional defiant disorder, compulsive disorder, obsession, and autistic behavioral disorder in two groups. we considered an eeg with normal activity and mild epileptic discharge as mild abnormal eeg. eegs with an abnormal activity and definitely abnormal by showing paroxysmal epileptic discharge were considered as moderately abnormal. severely abnormal eegs were eegs with continuous spikes and wave discharges or the hypsarrhythmia pattern. finally, the data were compared between the patients (cases and controls) and anova and chi - square test were applied for statistical analysis. we evaluated 105 patients (50 boys, 55 girls) with the diagnosis of pku. the mean age of the patients was 8.5 6.2 years. of these pku patients, 52.3% (55 patients) had seizure (more than 6 months before the study) and 47.7% (50 patients) were clinically seizure - free. the case group consisted of 70 patients (66.6%) with an abnormal eeg and the controls were 35 patients (33.4%) with a normal eeg. in the case group, 37 patients (52.8%) had a mild abnormal eeg, 10 patients (14.2%) had moderate abnormal eeg and 23 patients (32.8%) had severe abnormality in their eeg (as we considered in our evaluation and mentioned before). in the case group, phe levels were 14 mg / dl (mean, 21.4 9.1 mg / dl) at diagnosis, 9 mg / dl (mean, 10.3 1.6 mg / dl) after 6 months of diagnosis, and 4 mg / dl (mean, 7.31 4.1 mg / dl) at the time of our evaluation. the serum phe levels in controls were 12 mg / dl (mean, 17.3 6.1 mg / dl) at diagnosis and from 6 mg / dl (mean, 8.4 2.1 mg / dl) after six months of treatment to 4 mg / dl (mean, 7.39 3.6 mg / dl) at the time of our study. there was no significant difference between mean phenylalanine levels in the abnormal and normal eeg groups at the time of diagnosis, after six months, and at our evaluation. our group was matched according to serum phe level (p > 0.05) (table 1). in the case group (patients with an abnormal eeg), 47% (33 patients out of 70 patients) had no clinical seizure. there was no significant difference between age in the two groups regarding dq levels (above and below 80) (p = 0.2) and no significant difference in dq levels between genders (p = 0.7). comparison between head circumference (hc) and the dq level showed a significant difference between hc and dq level in pku patients (p = 0.04) (table 2). distribution of dq levels in the abnormal and normal eeg groups (cases and controls) revealed a significant difference; an abnormal eeg was associated with a higher percentage of low dq levels (90% versus 57.1%) (p = 0.001) (table 3). patients in the case group (abnormal eeg) according to severity of abnormality in eeg were categorized into three classes. distribution of high and low dq levels in these three subgroups showed a significant difference (p = 0.001) (table 4). in other words, a severe abnormal eeg was associated with a higher percentage of low dq levels. in the behavioral - emotional scale evaluation, the frequency of attention deficit was 85.7% in the case group and 40% in the controls. 85.7% of the cases showed attention deficit hyperactivity disorders in comparison with 40% in the controls. oppositional defiant disorders were reported 84.2% in the case group and 34.2% in the controls. autistic behavior was detected in 57.1% of the cases and 28.5% of the controls. totally, the frequency of behavioral disorder was 85.7% in cases and 42.8% in control patients. there was a significant relationship between behavioral disorder and eeg abnormality in our study (p < 0.001) (table 5). in neurometabolic diseases, neurodevelopmental delay, seizures and behavioral disturbances are the prominent presentations. according to our knowledge , different previous studies revealed the relationship between phe level and eeg abnormality and also between phe level and developmental delay, but there are limited studies about the relationship between eeg abnormality and developmental delay in pku patients. studied the effects of treatment of phenylalanine level (special diet) on the eeg of six pku patients. computerized eeg analysis showed activity appearance in the low frequency band (25 cycle / second), dominant rhythm frequency changes and synchronous degree changes between previous identified frequencies. this study described the association between phenylalanine level and eeg changes, when phe level increased, the eeg changes intensified and in contrast when the phe level decreased the eeg changes reversed, indicating that phe or its metabolites lead to eeg abnormalities. in our study, the case group consisted of 70 patients (66.6%) with abnormal eegs. in our evaluation, we could not find any correlations between phe level and eeg changes because we matched phe level in the two groups. between patients with abnormal eegs (the cases) there was a significant relationship between eeg findings and phe level (p < 0.001). according to p. t. gross et al. review, in eeg recording of pku patients, about 45% of the patients had an abnormal eeg and nearly 30% had a normal eeg in the beginning which became abnormal later. study showed 25% of the patients had seizure, but more than 50% had an abnormal electroencephalogram, which means some pku patients had an abnormal eeg without any clinical seizure. in our study, 44% (28 patients) of the patients with abnormal eegs (out of 63 patients in the case group) had no clinical seizure. karimzadeh and tabarestani reviewing study reported negative effects of this epileptic form discharge on the choice reaction time, verbal and nonverbal communication and behavioral disorder. our study revealed 85.7% of the patients in the case group and 42.8% of the children in the control group had a pattern of behavioral disorders. these behavioral disorders were in accordance with their eeg abnormalities and behavioral disorders in phenylketonuria patients regardless of seizure and phe level. in smith et study, the rutter behavior questionnaire was used for evaluation of common abnormal behaviors in 544 patients with phenylketonuria (8-year - old children). children had routine screening testing in the early months of screening program; anxiety, hyperactivity and less responsibility were detected. all patients received treatment before 4 months. they described that these abnormal behaviors may be the of both psychologic stress and neurologic impairment. rolle - daya et al. also studied the eeg findings of 90 patients with classic pku in one clinic. 73% of these patients who were diagnosed and were treated early had a normal eeg and 23% had mild abnormal activity. in patients diagnosed after 6 months of age, 31% had normal eegs, 24% had abnormal activity, and 45% had abnormal paroxysmal discharges. thirty - four children with phenylketonuria with early treatment were studied by pietz et al. they determined the prognostic value of age on the special diet in the first three months of life and iq score and eeg were investigated. higher iq scores were detected in patients with strict dietary control in comparison with lose control. generalized slowing and paroxysmal activity (with or without spikes) were seen more frequently compared to the controls. eeg abnormality was increased independent of iq development and eeg abnormality showed no relationship with the onset age or the quality of dietary treatment. in our present study, we found a significant difference between dq levels in the abnormal eeg group. 74.7% of the patients with abnormal eeg had a low dq level; therefore, we had a significant relationship between abnormal eeg and the low dq levels (table 3). in addition, we found a significant correlation between severity of abnormal eegs and the level of dq in pku patients (table 4). according to our study, (in pku patients matched in phe levels) , there was a significant relationship between the abnormal eegs and the low dq level as well as the abnormal eegs and behavioral disorders. based on our findings, the prevalence of seizure was less than eeg abnormality in pku patients. there was a significant relevance between eeg abnormality and behavioral disorders and developmental score in phenylketonuria regardless of seizure or phenylalanine level. the authors believe that physicians should pay attention to these paroxysmal epileptic discharges in pku patients more than before and treatment of these eeg abnormalities may affect developmental scores or may lead to correction of some behavioral disorders in these patients.

. brain defect leading to developmental delay is one of the clinical manifestations of phenylketonuria. the aim of this study was to evaluate the association between eeg abnormality and developmental delay / behavioral disorders in phenylketonuria. patients and methods. 105 phenylketonuria patients, who were diagnosed through newborn screening tests or during follow - up evaluation, were enrolled. patients who were seizure - free for at least six months before the study were included. the developmental score were evaluated by the asq questionnaire (age - stage questionnaire) and the test of child symptom inventory-4 (csi-4), respectively. . 55 patients had a history of seizure more than 6 months before the study. seventy had abnormal eeg (cases) and 35 had normal eeg (controls). there was no significant difference between mean phenylalanine levels in the abnormal and normal eeg groups at the time of diagnosis, after six months and at our evaluation. distribution of dq level in the abnormal and normal eeg groups revealed a significant difference. an abnormal eeg was associated with a higher percentage of low dq levels. . paroxysmal epileptic discharges in pku patients are important. treatment of these eeg abnormalities may affect developmental scores or may lead to correction of some behavioral disorders in patients.

leishmaniasis is a vector borne disease, affecting 98 countries and territories around the world which is transmitted by the bite of the female sandfly phlebotomine (who, 2010). this disease causes a considerable morbidity and mortality in tropical and sub - tropical countries. leishmaniasis presents with four major clinical forms: visceral, cutaneous, diffuse cutaneous and mucocutaneous leishmaniasis. 90% of the cases occur in seven countries including iran, afghanistan, peru, saudi arabia, syria, algeria, and brazil (desjeux, 2004). it has been estimated that each year 1 - 1.5 million new cases of cl and 500,000 cases of visceral leishmaniasis occur. overall, the prevalence is 12 million and the population at risk is 350 million (desjeux, 2004). the epidemiology of cl has been significantly changed, mainly because of ecological factors, population displacements and individual risk factors (desjeux, 2001). vector and reservoir control methods are difficult due to the diverse ecology of many species of sandfly vectors and reservoirs. the standard drugs, pentavalent antimonials, have been used for decades with severe side - effects. moreover, prolonged use and resistant forms have been frequently reported (hadigi et al ., 2006). in iran two epidemiological forms of cl are present: zoonotic cl (zcl) caused by l. major and anthroponotic cl (acl) caused by leishmania tropica. the latter species are prevalent in kerman province (sherifi et al ., 2012 and nadim et al ., 1995) and have developed resistance to antimonials (hadigi et al ., 2006 ; pour et al ., 2010). in the present study , we characterized the ultrastructure alteration of different cell organelles in glucantime unresponsive l. tropica isolates compared with standard strains or sensitive isolates. the evaluation of resistant isolates is important because it permits the development of diagnostic tools for early recognition of unresponsiveness, thus avoiding inefficient and often toxic chemotherapy. to the best of our knowledge , there is no previous study that has been done on electron microscopic changes in unresponsive and sensitive isolates of l. tropica in clinical settings. this study is experimental and was done in the city of bam during 2008 - 2012 in kerman province, south - eastern iran. bam is a well - known focus for acl, where vaccine trials of single and multiple doses of l. major against l. tropica were conducted (noazi et al ., 2008, 2009). a devastating earthquake had struck bam in 2003 destroying nearly 90% of medical and social infrastructures. ever since, the mean annual incidence of acl has significantly increased in this region (sharifi et al ., 2011). the samples were taken from 66 patients who were referred to the cl treatment center in bam. a questionnaire was completed for each individual, recording their demographic characteristics and cl status including age, sex, area of residence, number, type and location of cl lesions, and history of the disease. consent of the patient was obtained and all patients received proper medication free of charge. the scraping smears provided from the edge of active lesions with sterile blades were fixed with methanol, stained by giemsa, and examined under a compound light microscope for amastigote form simultaneously. skin scrapings were inoculated for mass production of promastigotes into nnn biphasic medium and incubated at 241c for 7 days. promastigotes were then sub - cultured in rpmi 1640 medium (gibco) containing 15% of heat - inactivated fetal calf serum (gibco) with streptomycin (200 mg / ml), and penicillin (200 units / ml) (gibco), and incubated at 241c. the cultures were checked every 7 days for promastigote growth for up to 28 days (sharifi et al ., 1999). diagnosis was confirmed by smear and culture media, while identification of species was carried out by nested - pcr, as it was previously performed elsewhere (unpublished data) based on national guidelines. resistant case was referred to those cl - infected patients who did not heal after receiving two full courses of treatment by glucantime. on the other hand, a sensitive case was an infected individual who often responded to a full course of glucantime. the l. tropica standard strain (mhom / ir/02/mash2) was provided by the center for research and training in skin diseases and leprosy, tehran university of medical sciences, tehran, iran. to prepare the specimens for transmission electron imaging, promastigotes were fixated with 3% cold glutaraldehyde (merck chemical co.) for at least half an hour. the samples were centrifuged and resuspended in 0.1 m sodium cacodylate buffer (ph 7.2). then, it was again centrifuged, the supernatant was discarded, and resuspended in 3% agar 50c. the promastigotes were further fixed in 1% osmium tetroxide for 2 hours, washed with buffer, and dehydrated through series of ethanol concentrations. using an ultramicrotome, the blocks were cut into 1 m sections, then stained by toluidine blue and were examined under a light microscope to confirm the preparation success (oryan and mehrabani, 2007). the rest of the electron microscopic sample preparation and imaging were done at janelia research campus, howard hughes medical institute (hhmi). the imaging at hhmi was performed with a scanning transmission electron microscope (stem) detector. this detector was developed utilizing a carl zeiss, ultra 55 zeiss schottky field emission sem system, ing a low voltage (2030 kv, which is a few times smaller than the voltage used in conventional tem) high throughput stem (lvstem). the detector is based on a scintillator and photo - multiplier tube technology and the detector's optics geometry gets the highest signal to noise ratio in an image (bolorizadeh, 2009). furthermore, the column design of the sem, having no cross over point and having a capability of upgrading to high current modes (tens of na), made it an ideal candidate for this study. electron microscopic studies prior to imaging were prepared with 40 nm pioloform substrates on 0.5 mm tem slot grids. then, 5 nm carbon was thermally evaporated onto the top surface (the side facing of primary electron beam) of the tem grids, to eliminate any charging during the electron beam imaging. to make the grids more hydrophilic, the grids were glow discharged (using nitrogen dc plasma) prior to laying down the sections on the grid slots. immediately after the grid substrate preparation, the resin blocks were cut into 40 nm thick sections using an ultramicrotome and laid on the prepared tem slot grids. these sections were post - stained with 1% osmium tetroxide at room temperature for 30 minutes. all the images in this paper were taken with a 30 kev, 16 na electron beam at 10 mhz bandwidth (0.1 sec per pixel) without any image noise reduction and alteration. our ultrastructural findings in unresponsive promastigote revealed that the cytoplasmic mitochondrion was mostly circular. the cytoplasmic microtubules were seen in the middle of the promastigote. the nucleus was bigger than normal and located close to the posterior end of the kinetoplast. the kinetoplast, which is enlarged in figure 1c, was not degenerated as in the sensitive strain the plasma membrane was smooth with normal thickness. (b) nucleus became swollen, nuclear chromatin fragmented and dispensed instead of rimming around nuclear membrane. n, nucleus; k, kinetoplast; v, vesicle; m, mitochondrion; f, flagellum; mi, microtubule; pm, plasma membrane; v, vacuole; fp, flagellar pocket. horizontal bars are 3 m while the vertical bar is 1m the ultrastructural finding in sensitive promastigote revealed the cytoplasmic mitochondrion that was swollen, elongated and located at a short distance from the plasma membrane of the promastigote. the nucleus became elongated and the nuclear chromatin was fragmented and dispensed instead of rimming around the nuclear membrane. its location was at a further distance from the kinetoplast at the posterior end of the organism as compared with the standard or unresponsive isolates as compared with the standard or unresponsive isolates. ultrathin sections of sensitive strain of promastigote forms of leishmania tropica revealed different ultrastructural alteration induced by treatment with meglumine antimoniate (glucantime). (a) nucleus became elongated; nuclear chromatin became fragmented and dispensed instead of rimming around nuclear membrane, mitochondrial swelling, loss of its matrix and cristae. bars are 3 m electron microscopy of standard sample showing: (a) few vesicles scattered throughout the cytoplasm. (b) normal mitochondria, (c) mitochondrion with normal matrix and cristae the mitochondrion - kinetoplast's complex loss, its matrix and cristae, condensation of dna core and disintegration into a complex of thin filaments. meglumine treatment induced intense vesicles which could be seen around and under the nucleus, mostly at the posterior end of the promastigote. intense vacuolization of promastigote was observed in sensitive patients. the vacuoles which were not seen in the standard stain were visible close to the kinetoplast and flagellum and at the posterior end of the promastigote. the ultrastructural findings of standard sample revealed few vesicles which were located throughout the cytoplasm and also at the posterior end of the promastigote stage. the nucleus had a normal size and was located at a short distance posterior to the kinetoplast. the cytoplasmic mitochondria were circular and located throughout the promastigote. the cytoplasmic microtubules were located at a short distance from the cytoplasmic membrane. the discussed for unresponsive, sensitive, and standard promastigotes are given in table 1. this study is experimental and was done in the city of bam during 2008 - 2012 in kerman province, south - eastern iran. bam is a well - known focus for acl, where vaccine trials of single and multiple doses of l. major against l. tropica were conducted (noazi et al ., 2008, 2009). a devastating earthquake had struck bam in 2003 destroying nearly 90% of medical and social infrastructures. ever since, the mean annual incidence of acl has significantly increased in this region (sharifi et al ., 2011). the samples were taken from 66 patients who were referred to the cl treatment center in bam. a questionnaire was completed for each individual, recording their demographic characteristics and cl status including age, sex, area of residence, number, type and location of cl lesions, and history of the disease. consent of the patient was obtained and all patients received proper medication free of charge. the scraping smears provided from the edge of active lesions with sterile blades were fixed with methanol, stained by giemsa, and examined under a compound light microscope for amastigote form simultaneously. skin scrapings were inoculated for mass production of promastigotes into nnn biphasic medium and incubated at 241c for 7 days. promastigotes were then sub - cultured in rpmi 1640 medium (gibco) containing 15% of heat - inactivated fetal calf serum (gibco) with streptomycin (200 mg / ml), and penicillin (200 units / ml) (gibco), and incubated at 241c. the cultures were checked every 7 days for promastigote growth for up to 28 days (sharifi et al ., 1999). diagnosis was confirmed by smear and culture media, while identification of species was carried out by nested - pcr, as it was previously performed elsewhere (unpublished data) based on national guidelines. resistant case was referred to those cl - infected patients who did not heal after receiving two full courses of treatment by glucantime. on the other hand, a sensitive case was an infected individual who often responded to a full course of glucantime. the l. tropica standard strain (mhom / ir/02/mash2) was provided by the center for research and training in skin diseases and leprosy, tehran university of medical sciences, tehran, iran. to prepare the specimens for transmission electron imaging, promastigotes were fixated with 3% cold glutaraldehyde (merck chemical co.) for at least half an hour. the samples were centrifuged and resuspended in 0.1 m sodium cacodylate buffer (ph 7.2). then, it was again centrifuged, the supernatant was discarded, and resuspended in 3% agar 50c. the promastigotes were further fixed in 1% osmium tetroxide for 2 hours, washed with buffer, and dehydrated through series of ethanol concentrations. using an ultramicrotome, the blocks were cut into 1 m sections, then stained by toluidine blue and were examined under a light microscope to confirm the preparation success (oryan and mehrabani, 2007). the rest of the electron microscopic sample preparation and imaging were done at janelia research campus, howard hughes medical institute (hhmi). the imaging at hhmi was performed with a scanning transmission electron microscope (stem) detector. this detector was developed utilizing a carl zeiss, ultra 55 zeiss schottky field emission sem system, ing a low voltage (2030 kv, which is a few times smaller than the voltage used in conventional tem) high throughput stem (lvstem). the detector is based on a scintillator and photo - multiplier tube technology and the detector's optics geometry gets the highest signal to noise ratio in an image (bolorizadeh, 2009). furthermore, the column design of the sem, having no cross over point and having a capability of upgrading to high current modes (tens of na), made it an ideal candidate for this study. electron microscopic studies prior to imaging were prepared with 40 nm pioloform substrates on 0.5 mm tem slot grids. then, 5 nm carbon was thermally evaporated onto the top surface (the side facing of primary electron beam) of the tem grids, to eliminate any charging during the electron beam imaging. to make the grids more hydrophilic, the grids were glow discharged (using nitrogen dc plasma) prior to laying down the sections on the grid slots. immediately after the grid substrate preparation, the resin blocks were cut into 40 nm thick sections using an ultramicrotome and laid on the prepared tem slot grids. these sections were post - stained with 1% osmium tetroxide at room temperature for 30 minutes. all the images in this paper were taken with a 30 kev, 16 na electron beam at 10 mhz bandwidth (0.1 sec per pixel) without any image noise reduction and alteration. this study is experimental and was done in the city of bam during 2008 - 2012 in kerman province, south - eastern iran. bam is a well - known focus for acl, where vaccine trials of single and multiple doses of l. major against l. tropica were conducted (noazi et al ., 2008, 2009). a devastating earthquake had struck bam in 2003 destroying nearly 90% of medical and social infrastructures. ever since, the mean annual incidence of acl has significantly increased in this region (sharifi et al ., 2011). the samples were taken from 66 patients who were referred to the cl treatment center in bam. a questionnaire was completed for each individual, recording their demographic characteristics and cl status including age, sex, area of residence, number, type and location of cl lesions, and history of the disease. consent of the patient was obtained and all patients received proper medication free of charge. the scraping smears provided from the edge of active lesions with sterile blades were fixed with methanol, stained by giemsa, and examined under a compound light microscope for amastigote form simultaneously. skin scrapings were inoculated for mass production of promastigotes into nnn biphasic medium and incubated at 241c for 7 days. promastigotes were then sub - cultured in rpmi 1640 medium (gibco) containing 15% of heat - inactivated fetal calf serum (gibco) with streptomycin (200 mg / ml), and penicillin (200 units / ml) (gibco), and incubated at 241c. the cultures were checked every 7 days for promastigote growth for up to 28 days (sharifi et al ., 1999). diagnosis was confirmed by smear and culture media, while identification of species was carried out by nested - pcr, as it was previously performed elsewhere (unpublished data) based on national guidelines. resistant case was referred to those cl - infected patients who did not heal after receiving two full courses of treatment by glucantime. on the other hand, a sensitive case was an infected individual who often responded to a full course of glucantime. the l. tropica standard strain (mhom / ir/02/mash2) was provided by the center for research and training in skin diseases and leprosy, tehran university of medical sciences, tehran, iran. to prepare the specimens for transmission electron imaging, promastigotes were fixated with 3% cold glutaraldehyde (merck chemical co.) for at least half an hour. the samples were centrifuged and resuspended in 0.1 m sodium cacodylate buffer (ph 7.2). then, it was again centrifuged, the supernatant was discarded, and resuspended in 3% agar 50c. the promastigotes were further fixed in 1% osmium tetroxide for 2 hours, washed with buffer, and dehydrated through series of ethanol concentrations. the samples were embedded in agar-100 resin and baked at 65c for 24 hours. using an ultramicrotome, the blocks were cut into 1 m sections, then stained by toluidine blue and were examined under a light microscope to confirm the preparation success (oryan and mehrabani, 2007). the rest of the electron microscopic sample preparation and imaging were done at janelia research campus, howard hughes medical institute (hhmi). the imaging at hhmi was performed with a scanning transmission electron microscope (stem) detector. this detector was developed utilizing a carl zeiss, ultra 55 zeiss schottky field emission sem system, ing a low voltage (2030 kv, which is a few times smaller than the voltage used in conventional tem) high throughput stem (lvstem). the detector is based on a scintillator and photo - multiplier tube technology and the detector's optics geometry gets the highest signal to noise ratio in an image (bolorizadeh, 2009). furthermore, the column design of the sem, having no cross over point and having a capability of upgrading to high current modes (tens of na), made it an ideal candidate for this study. electron microscopic studies prior to imaging were prepared with 40 nm pioloform substrates on 0.5 mm tem slot grids. then, 5 nm carbon was thermally evaporated onto the top surface (the side facing of primary electron beam) of the tem grids, to eliminate any charging during the electron beam imaging. to make the grids more hydrophilic, the grids were glow discharged (using nitrogen dc plasma) prior to laying down the sections on the grid slots. immediately after the grid substrate preparation, the resin blocks were cut into 40 nm thick sections using an ultramicrotome and laid on the prepared tem slot grids. these sections were post - stained with 1% osmium tetroxide at room temperature for 30 minutes. all the images in this paper were taken with a 30 kev, 16 na electron beam at 10 mhz bandwidth (0.1 sec per pixel) without any image noise reduction and alteration. our ultrastructural findings in unresponsive promastigote revealed that the cytoplasmic mitochondrion was mostly circular. the cytoplasmic microtubules were seen in the middle of the promastigote. the nucleus was bigger than normal and located close to the posterior end of the kinetoplast. the kinetoplast, which is enlarged in figure 1c, was not degenerated as in the sensitive strain the plasma membrane was smooth with normal thickness. (b) nucleus became swollen, nuclear chromatin fragmented and dispensed instead of rimming around nuclear membrane. n, nucleus; k, kinetoplast; v, vesicle; m, mitochondrion; f, flagellum; mi, microtubule; pm, plasma membrane; v, vacuole; fp, flagellar pocket. horizontal bars are 3 m while the vertical bar is 1m the ultrastructural finding in sensitive promastigote revealed the cytoplasmic mitochondrion that was swollen, elongated and located at a short distance from the plasma membrane of the promastigote. the nucleus became elongated and the nuclear chromatin was fragmented and dispensed instead of rimming around the nuclear membrane. its location was at a further distance from the kinetoplast at the posterior end of the organism as compared with the standard or unresponsive isolates as compared with the standard or unresponsive isolates. ultrathin sections of sensitive strain of promastigote forms of leishmania tropica revealed different ultrastructural alteration induced by treatment with meglumine antimoniate (glucantime). (a) nucleus became elongated; nuclear chromatin became fragmented and dispensed instead of rimming around nuclear membrane, mitochondrial swelling, loss of its matrix and cristae. bars are 3 m electron microscopy of standard sample showing: (a) few vesicles scattered throughout the cytoplasm. (b) normal mitochondria, (c) mitochondrion with normal matrix and cristae the mitochondrion - kinetoplast's complex loss, its matrix and cristae, condensation of dna core and disintegration into a complex of thin filaments. meglumine treatment induced intense vesicles which could be seen around and under the nucleus, mostly at the posterior end of the promastigote. intense vacuolization of promastigote was observed in sensitive patients. the vacuoles which were not seen in the standard stain were visible close to the kinetoplast and flagellum and at the posterior end of the promastigote. the ultrastructural findings of standard sample revealed few vesicles which were located throughout the cytoplasm and also at the posterior end of the promastigote stage. the nucleus had a normal size and was located at a short distance posterior to the kinetoplast. the cytoplasmic mitochondria were circular and located throughout the promastigote. the cytoplasmic microtubules were located at a short distance from the cytoplasmic membrane. the discussed for unresponsive, sensitive, and standard promastigotes are given in table 1. this electron microscopic study was done to evaluate the effect of drugs on l. tropica parasite due to the development of drug resistance. we demonstrated the effect of glucantime on various l. tropica promastigote organelles to provide a better understanding of drug - resistance. the indicated that meglumine acted mainly on the mitochondrial physiology of this species which was distributed in branches close to the promastigote membrane and in the region rich in dna called kinetoplast (de souza, 2009). our ultrastructural observation of the mitochondria and kinetoplast showed disintegration into a complex of thin filaments, condensation of dna core, loss of its matrix and cristae. similar findings were observed for l. amazonesis with different drugs (rodrigues et al ., 2005, 2007, mitochondria are essential organelles in energy metabolism, oxidative phosphorylation, and respiration . they also have a vital role in the survival of the organism and are an exceptionally chemotherapeutic target ( job et al ., 2012). another deleterious effect was also observed on the microtubules, they disappeared in sensitive promastigote. the microtubules play important roles in intracellular transport, morphogenesis, and maintenance of cellular shape, and are involved in mitosis (bell, 1998). the extensive increase in vacuole size and number in sensitive strains indicated the cessation of the cell cycle which is consistent with previous findings by de macedo and his colleagues. other ultrastructural alteration was structural changes in the nucleus including nuclear chromatin which was fragmented and dispensed instead of rimming around the nuclear membrane and became elongated. in a study that was done by de macedo and his colleagues, they observed many changes in mitochondria, nucleus, vesicles and mitochondrion - kinetoplast complexes in l. amazonensis treated with amiodarone drugs. a study by rodrigues and colleagues showed the effect of drugs on the organelles of l. amazonensis as alteration of mitochondrion such as swelling, loss of cristae and matrix content. they also observed a large vacuole close to the flagellar pocket and small ones inside it. we also observed intense vacuolization of promastigotes, marked swelling of cytoplasmic mitochondrion and its elongation. all of these alterations in sensitive promastigotes are characteristics of cell death: necrosis and apoptosis. similar findings were observed by de macedo and his colleagues who showed the effect of amio drugs on the organelles of l. amazonensis. it seems that resistant forms get more amastigote like structure and more atrophic but viable organelles close to each other for better hemostasis of the parasites. kinetoplasts are the most important damageable organelles. with respect to ultrastructural findings in l. tropica, so far, no study has been done on l. tropica isolates unresponsive to glucantime. to the best of our knowledge , this study is the only investigation performed on ultrastructure of unresponsive and sensitive isolates. the significance and relevance of these changes might help understand drug resistance patterns and help localize the best target site for inactivating the organism. further, ultrastructural studies from skin biopsies of different forms of cl are needed to evaluate in vivo changes of amastigotes. kinetoplasts are the most important damageable organellesmicrotubules play important roles in surveillance of leishmania. kinetoplasts are the most important damageable organelles microtubules play important roles in surveillance of leishmania.

: the control of leishmaniasis faces serious challenges because of resistance to the first - line antimonial drugs. we aimed to evaluate the differences in organelle changes of cultivated promastigotes obtained from skin lesions of sensitive and unresponsive isolates to meglumine antimoniate (glucantime) by electron microscopy.material and methods: this study was done in bam city, southeastern iran, in which the incidence of disease has sharply increased since the earthquake in 2003. the samples were taken from 66 patients who were referred to the cutaneous leishmaniasis (cl) treatment center in bam. a questionnaire was completed for each individual, recording their demographic characteristics and cl status. the scraping smears provided from the edge of active lesions with sterile blades were fixed with methanol, stained by giemsa, and examined under a compound light microscope for amastigote form simultaneously. to prepare the specimens for transmission electron imaging , promastigotes were centrifuged and resuspened.:transmission electron microscopic study of the cultivated promastigotes revealed that there were alterations in the organelles and structures of sensitive isolates compared with unresponsive and standard ones. organelles and structures such as mitochondria, kinetoplast, microtubules, cytoplasmic vacuoles, plasma membrane and vesicles were studied. the alterations such as disintegration of kinetoplast into thin filaments and condensation of kinetoplast dna core, changes in size, number and location of vesicles and microtubules were observed. we noted intense cytoplasmic vacuolization, and considerable swelling of mitochondria.:the significance and relevance of these changes might help understand drug resistance patterns and help localize the best target site for inactivating the organism.

industrial growth, technological advancements, and higher living standards in today's society contribute to the ever increasing generation of solid wastes. industrial park is one of the most important manufacturing bases and an indicator of development in each country. however, each manufacturing process usually generates some amount of nonconsumable waste causing adverse effects on environment. geographical information system (gis) technologies are effectively used in the process of site selection, which is a spatial problem. despite the consensus of all countries to achieve the goal of zero waste in industries, the accomplishment of this goal is predicted to be difficult and generation of various wastes arising from manufacturing processes is currently inevitable. a focus on sources of solid waste with respect to management is justified by the fact that waste characteristics and composition differ according to source. the appearance of such problems caused some countries to move their waste to developing countries in order to be saved from the probable risks of the hazardous waste. uncontrolled expansion of industries and ignoring environmental principles in industrial development and overuse have caused much environmental problems. a number of countries have ratified state and national legislation for managing and controlling hazardous waste whose main purpose is to minimize the potential risks of industrial hazardous waste for humans and the environment. in iran, despite the excess growth of industries, urbanization, and industrial centralization around major cities particularly tehran, no fundamental effort has been yet made in the field of industrial waste disposal, and subsequently, there are few rules for controlling and disposal of such materials. evidence shows that solid, semisolid, and liquid wastes of the factories are disposed regardless of health issues and effluent of these industries enters the absorbing wells or spread in surrounding land. solid waste is piled up around the factories for a period, and then a part of the waste is incinerated outdoor and some part is transferred out of the factories along with household waste by municipal general service. large scale land use transportation from agricultural to industrial has made tehran a city for a large center for industries centralization and their peripheral activities. because of the importance of industrial hazardous waste minimization in prevention of pollutant emissions in the environment, it is emphasized by current laws in many countries. moreover, establishment and development of minimization programs have been adopted more than ever through legislation of laws and more restrictions in controlling hazardous waste and prevention of leaving waste in the environment. the application of waste minimization techniques does not necessarily mean the use of complicated technologies or investments on expensive machines. many methods require a simple change in the process of production or in transferring the materials. generally, hazardous waste minimization methods can be used for any production process and the mutual factor in all techniques is the reduction of production costs. in this study, basic and technical information required for the waste management were collected and analyzed due to the increasing growth of industries and the need for developing efficient methods in proportion to the conditions of the studied region. in fact, the main objective of this study is to examine the quantity of industrial solid waste regarding the production line of cb province, iran. the population of this study included all operational industries in 19 industrial zones of cb province, iran, which were studied for 8 months from march to october, 2009. data were collected partly through questionnaires and partly through field investigation and literature review on the subject on matter, which was carried out using various books and articles. industrial zones of the province were identified in order to specify the production resources. in this regard, detailed information on the industrial zones was taken from the iran small industries and industrial parks organization, in cb province. metal industries with 95 units and electricity and electronics industries with 10 units had maximum and minimum numbers of units in the province, respectively. the first stage of the study including separation and classification of the industrial units was done based on their activities and locations according to the available statistics in the province. the questionnaire contained type of industrial group, the number of personnel, the kind of product and raw materials, the amount and type of generated waste, waste physical state, frequency of generation, method of storage and maintenance, recycling methods, final disposal, and the responsible organizations for waste collection in order to identify industrial waste in cb province. various questionnaires presented by different organizations such as recycle organization, environmental protection organization, and ministry of industries were analyzed and finally a questionnaire containing two forms was prepared. according to the investigations, 19 industrial parks with the total area of 2356.10 hectares were under the coverage of industrial parks organization. among these 19 industrial parks, shahrekord, lordegan, saman, faradnbeh, junghan, farokhshahr, ben, and dastgerd industrial parks had 154, 22, 16, 1, 2, 3, 4, and 3 units, respectively. therefore, shahrekord with 154 units (or 49.8%) and faradnbeh with 1 unit (or 0.3 percent) had the most and the least units, respectively. a number of 309 questionnaires were completed by the industrial units. generally, these industries performed various activities, so the amount of industrial waste was influenced by the kind of activities. in this regard, these industries were divided according to the classification of ministry of industries. food, metal, chemical, nonmetallic minerals, cellulose, textile, and electrical and electronics industries comprised 21, 30.4, 22, 12.6, 7.1, 3.8, and 2.9 percent of the evaluated industries in this study. the workers of these 14 industrial parks were 6033 people. based on table 1, the total industrial waste was 1246 tons per month, and semihousehold waste per capita (for food consumption of the workers) was 350 gram per day. table 2 shows that the amount of wastes generated in cb province was approximately 32 ton per month. according to table 3, the total food waste generated in studied industries was an average of 65516 kg per month which depended on the number of workers. herbal waste was 384 tons a month of which 99.4% and 0.6% were generated in food and chemical industries, respectively. herbal waste of food industries encompassed wheat, straw, bran, fruit, vegetable, and dried fruit waste. herbal waste of chemical industries was related to yarn and fabric dyeing industries. in these industries, flour and doughy waste was generated in industries like macaroni manufacturing, noodle making, and industrial bread. the amount of flour and doughy waste was 22.3 tons per month in the province which was totally generated in food industries. sweets, chocolate, and candy waste was 6.5 tons per month which was totally generated and recycled in food industries. meat and bone waste encompassed chicken and fish packaging waste and sausage production waste which was totally generated in food industries. according to investigations, wood waste was generated in metal, chemical, cellulose, and electricity and electronics industries in a form of wooden pallets which entered the factories along with purchase of tools like the engines of various machines and variety of metal ingots. the amount of wood waste was 17 tons per month, of which the maximum amount of 55.2 percent was related to metal industries. the amounts of plastic waste generated in food, metal, nonmetallic minerals, cellulose, textiles, and electrical and electronics industries were 40.8, 1.9, 0.8, 0.3, 0.3, and 2.2 percent, respectively. these types of waste were recycled in chemical industries; however, no action has been yet done for recycling these wastes by other industries. the amount of glass waste in the entire study was 8.2 tons per month of which 91.8, 6, and 2.2 percent were generated in nonmetallic minerals, cellulose, and food industries, respectively. the amount of paper and cardboard waste was 84.5 tons per month of which 83.1 percent was generated in cellulose industries and 12.2 percent was generated in food and other industries. ferrous metals waste included iron chips, iron scraps, and snipping which was 160 tons per month of which 94.8, 6, and 0.3 percent were generated in metal and cellulose industries, respectively. nonferrous metal waste included aluminum, copper, and cast iron of which the maximum amount was related to electricity and electronics industries for the use of aluminum and copper in wire and cable industry. the amount of nonferrous metal waste in studied samples was 5.7 tons per month of which 52.2, 31.4, 8.7, 1.2, and 3.5 percent were generated in electricity and electronics, metal, cellulose, nonmetal, and food industries, respectively. gunny waste consisting of sugar and flour sacks generated 4003 kg per month of which 81.9, 15.6, and 2.5 percent were generated from food, chemical, and metal industries. it should be noted that 100 percent of these wastes were reused and exploited in the same industries. trash waste included concrete, mosaics, ceramics, and stone body waste which were totally generated in nonmetal industries. the amount of generated pvc waste in the studied zones was 2.5 tons per month of which chemical, and electricity and electronics industries comprised 2300 kg (91.6 percent) and 70 kg (2.8 percent), respectively. the amount of generated pet waste was 18 tons (93.7 percent) per month of which 200 kg (1.1 percent) and 935 kg (5.2 percent) were generated in metal and food industries, respectively. the amount of polystyrene waste was 5.8 tons per month which was totally generated in chemical industries. the amount of polypropylene waste was 6.2 tons per month of which 96.8 percent and 3.2 percent were generated in chemical and textile industries, respectively. soil waste consisting of wheat soil and bentonite clay was used for vinegar treatment in industrial vinegar - making and stabilization of edible oils. furthermore, burnt sand waste (co2 silica sand co2 waste) is contained in soil waste. the amount of soil waste generated in the province was approximately 130 tons per month of which 96.6% and 3.4% were generated in food and metal industries, respectively. as noted above, burnt sand waste was classified in this category and was generated in cast iron foundry industry. the amount of yarn and fabric waste in all studied zones was about 7.4 tons a month. these wastes included yarn and fabrics, wool, fiber, and polymer of which 93.9 percent and 6.1 percent were generated in textile and chemical industries. the amount of chemical and special waste in all studied zones was about 21 tons per month of which 3, 41, 36.9, 16.6, 2, and 4 percent were generated in metal, chemical, nonmetal, cellulose, textile, and electricity and electronics industries. the initial chemical waste produced from industries which produced detergents and disinfectants comprised 1 percent of the raw chemical materials. these wastes consisted of sulfuric acid, diethanolamine, coconut fatty acid, betaine, essence, resin, sulfate, salt, and so forth. the amount of these wastes was 1775 kg per month which comprised 8.1 percent of the total chemical waste. the amount of color waste was 581 kg per month which comprised 2.6 percent of the total special waste. it should be noted that this type of waste was found in metal, chemical, cellulose, and textile industries. the amount of adhesives waste was 172 kg per month which was related to chemical and cellulose industries and was most generated in card board industry. a study on the industries of industrial parks in other region of iran showed that 250 kg of waste containing degradable materials and scrap iron was transferred out of the city and incinerated outdoor by municipal service. overestablishment of industries and conversion of agricultural land to industrial land in tehran, iran, have changed the city to a large center for industries centralization and their peripheral activities. in this study, the maximum amount of generated sludge waste was 8 tons per month which was related to nonmetal industries that was generated in a mosaic - building industrial unit in boroujen industrial zone. the tar waste generated in the province was 220 kg per month which was related to the waterproof membrane industries. the zinc oxide waste was 35 kg per month which comprised 0.1 percent of total special waste in the studied industrial parks. moreover, zinc clay waste was generated by a zinc ingot production cooperative in shahrekord. it was 600 kg per month which comprised 2.7 percent of total chemical and special waste. an ideal waste disposal site is that one which is located fairly close to the source of the waste, has easy transportation access, is not located in a low - lying area or floodplain, and is underlain by geologically stable, strong, and competent rock material. the site selection depends on several factors like land use, environmental, hydrology, socioeconomic, and so forth. according to the literature , the following criteria can be applied for site selection: site must be close to at least a street with a buffer of 30 m;site must not be too far from a transfer station;site must be 3 km from residential areas, with the exception of areas with barriers;there should be a minimum distance of 100 m between site and roads;site must be on a suitable soil;site should be constructed in areas which do not have an important economic or ecological value.such kinds of criteria could be combined in a hierarchal structure shown in figure 1. site must be close to at least a street with a buffer of 30 m; site must not be too far from a transfer station; site must be 3 km from residential areas, with the exception of areas with barriers; there should be a minimum distance of 100 m between site and roads; site must be on a suitable soil; site should be constructed in areas which do not have an important economic or ecological value. the combination of gis and multicriteria decision models (mcdm) can be useful in this regard. such approach provides flexible methods for exploring relationships among geographic data and assisting experts from diverse fields in pooling their knowledge to solve complex problems. a list of potential sites which generally satisfy the minimum requirements can be identified for the purpose of effective sanitary landfill - type waste disposal. among these areas, the integration of gis in multicriteria decision analysis (mcda) is a powerful tool in solving disposal site selection problem, because it provides efficient spatial data manipulation and presentation. one of the most important issues in industrial waste management is its special waste which distinguishes the industrial waste from the general waste. the waste of this category includes chemical raw materials, color, adhesives, tar, zinc oxide, zinc clay, and sludge. the total generated waste in the studied industrial zones was 1246 tons per month, of which 48.2, 14.9, 6.7, 22, 0.9, 0.6, and 6.5 percent were generated in food, metal, chemical, nonmetallic minerals, textile, electrical and electronics, and cellulose industries, respectively. the achieved in this study are considered the first step toward proper industrial waste management especially in recycling and disposal of waste. according to the of this study, environmental general department recommended to use rewards and punishments for reduction of hazardous waste. coordination with financial institutions for giving loans with lower interests in order to take environmental actions or imposing large fines for each kilogram of generated hazardous waste gives instances of reward and punishment. collection, delivery, and disposal of hazardous waste should be done by private section and the officials of environmental general department supervise the performance of the private section. costs of monitoring water, soil, and weather analyses in hazardous waste disposal site should be adopted from pollutant industries and the environmental report on the landfill should be submitted to environmental department every three months. regarding fragile ecosystem of cb province, layout and extension of industrial parks in future finally, an approach to the integration of gis and mcdm was introduced as a powerful tool in solving disposal site selection problem considering several criteria.

the aim of this study is the comprehensive planning for integrated management of solid waste at the industrial parks. the share of each industrial group including food, metal, chemical, non - metallic minerals, textile, electrical and electronical, and cellulose industries were 48.2, 14.9, 6.7, 22, 0.9, 0.6, and 6.5 percent, respectively. the showed that nearly half of total industrial waste produced from the range of biological materials are biodegradable and discharging them without observing environmental regulations leads to short - term pollution and nuisance in the acceptor environment. also some parts of case study waste were recyclable which is considerable from viewpoint of economical and environmental pollution. long - term impacts will appear due to improper site selection of disposal from the spatial standpoint. in this way , an approach for site selection using several socioeconomic, physical, and environmental criteria based on multicriteria decision making model (mcdm) is introduced. health risks and environment pollution such as soil and surface water may be done. it is essential to revise the studied industries layout, particularly those units which produce special waste which should be more cautious. also stricter enforcement is required as an effective step in reducing the harmful impacts of it.

radiotherapy (rt) in malignancies can be broadly classified into radical and palliative with regards to the intention of treatment. radical rt primarily is delivered with an intention to cure, and associated toxicities can be anticipated and are acceptable as an expected side effect. however, when cure seems unlikely, the intention of rt becomes palliative, wherein the goal is to achieve durable symptom relief at the shortest expense of time and resources, and while inflicting the least possible toxicity. given that primum non nocere (first, do no harm) is the principle behind palliative rt, the treatment program is designed to cause the least possible adverse impact upon the patient in all possible aspects. while radical rt programs can often last for around half - a - dozen weeks, often with superimposed administration of concurrent chemotherapy, palliative rt most often consists of an overall lesser dose of radiation, delivered in large fraction sizes (hypofractionated) and alone without chemotherapy. further, palliative rt is less expensive, since it involves simple planning and treatment without the need for elaborate conformal plans. overall, palliative rt utilizes sub - therapeutic doses since the goal is not to achieve a complete eradication of tumor. as an example, while head and neck squamous cell carcinomas (hnscc) often require > 70 gy, delivered concurrently with chemotherapy to achieve a reasonable tumor control probability (tcp), palliation can be achieved at much lesser doses in the order of 3045 gy delivered mostly in hypofractionated regimens (utilization of more than 2 gy per fraction). in very rare instances, it is not impossible to come across patients who have achieved excellent survival after treatment with palliative intent, in spite of overall rt doses being well below the doses required for expectations of a good tcp. thus, recognizing the existence of such outcomes in the clinic, we intended to analyze the frequency of such occurrences of excellent outcomes after palliative radiation doses. this study is a retrospective analysis of hospital records of the regional cancer centre, shimla, india. patients treated with palliative rt between 1 january 2001 and 31 december 2006 were included, provided their hospital records fulfilled the inclusion criteria. eligibility criteria for inclusion into analysis we purposefully excluded malignancies of the breast and prostate, given that these malignancies often have a long natural history. we also excluded malignancies of the central nervous system given the poor survival with or without rt. we also excluded salivary gland adenoid cystic malignancies, given that these malignancies too have a very long natural history wherein patients survive for years even after cannon - ball pulmonary metastases. we excluded hematological and lympho - reticular malignancies since these patients often follow - up elsewhere at departments specializing in hematological oncology. we excluded patients who, after initiation of palliative rt, later on received treatment with radical intent, either with the use of additional doses of radiation, chemotherapy, or surgery. overall, 963 patients were eligible for analysis , of whom 23 patients (2.4%) were alive for at least 5 years since the initiation of palliative rt. of these patients who had survived at least 5 years since palliative rt, 73.9% (17 of 23 patients) were free of disease without any clinical or radiological evidence of residual or recurrent malignancy. site - specific distribution of patients eligible for analysis interestingly, in spite of palliative rt being delivered after patients were considered unlikely to benefit from a full - course radical therapy, 1.6%, 2.6%, 5.5%, and 3.5% of patients with lung cancer, gynecological cancer, head / neck cancer, and esophageal cancer, respectively, were alive for more than 5 years of follow - up. less than one - fourth of patients surviving for 5 years after treatment had any evidence of residual or recurrence. the individual characteristics of patients who survived at least for 5 years since the initiation of palliative rt with regards to the diagnosis, age at diagnosis, sex, reason for choosing palliative approach, dose and fractionation of palliative rt, and current status have been presented in tables 35. characteristics of patients of lung cancer surviving 5 years after palliative rt characteristics of patients of gynecological cancer surviving 5 years after palliative rt characteristics of patients of head and neck, and esophageal cancer surviving 5 years after palliative rt palliative rt is based upon the principles of maximizing symptom relief with minimal consumption of time and resources, and causing the least possible concern to the patient with regards to span of treatment and toxicities afforded. lower total time as well as lower total dose is the hallmark of palliative rt. as an example, control of symptoms arising from superior vena caval obstruction syndrome with lung carcinoma is effective even with 20 gy delivered over 5 days, with 5 fractions of 4 gy each. however, a radical rt regime for a patient of lung cancer could take anywhere between 5 and 6 weeks. similarly, a patient of bulky neck nodal disease, which is threatening to rupture, may effectively be palliated with doses as less as 30 gy delivered over 10 fractions of 3 gy each. however, a radical regime consists of at least 70 gy delivered over 35 fractions over 67 weeks, often with concurrent chemotherapy. the toxicities suffered from the higher total dose and the cost incurred by the protracted radical rt regimen can be justifiable, given the chance of cure. however, in the case of palliative rt, since there is an assumption of a low or absent probability of cure, the patient is offered a short course of radiation, which delivers higher dose per fraction, which can be expected to palliate the local symptoms, and at the same time not adding to agony by means of treatment - related toxicity. often, a percentage of patients may not enjoy the expected survival outcomes even after a fully radical course of chemo - radiotherapy. conversely, the opposite, that is, expectations of cure or long - term survival can often be kindled (in the patient and in the clinician) when a patient initiated on palliative rt demonstrates very rapid tumor shrinkage. however, more often than not, patients succumb either due to disseminated disease, local recurrence with aggressive repopulation, or a combination of distant and local disease progression. a detailed literature search done on literature indexes (pubmed, pubmed central, scopus, hinari, index copernicus, google scholar, embase, cinahl, and doaj) with the terms such as long term survival after palliative radiotherapy, cure after palliative therapy, and tumour control sub - therapeutic radiation dose did not yield any study, either prospective or retrospective, regarding long - term survival after sub - therapeutic / palliative radiation doses. we performed a retrospective analysis utilizing hospital records of patients who were treated with palliative rt from 1 january 2001 to 31 december 2006. we chose december 2006 as the cutoff so as to ensure that every patient included into the analysis had the opportunity to complete at least 5 years of follow - up. though our original intention was to perform a kaplan meier analysis for survival of all eligible patients who had undergone palliative rt, we found it not feasible given the frequent migration of patients to other centers for treatment and a high attrition rate owing to the inherent logistic difficulties arising from the mountainous terrain of this region. undeniably, many patients who died after having received symptom control could indeed have succumbed to intercurrent illnesses such as infectious diseases and other non - oncological causes concerning the cardiovascular and pulmonary systems. nevertheless, we observed that 2.4% of patients had indeed survived for at least 5 years in spite of the treatment intent not having focused on the goal of prolonged survival. though the percentage value (2.4%) of patients surviving 5 years after palliative rt may seem trivial, the absolute numbers are significant given the large sample size. it is very likely that these very patients could not have survived if in case they were transitioned to supportive care instead of palliative rt. this also is striking, given that often patients do not enjoy survival in spite of a full course of radical chemo - radiotherapy, whereas some patients (as illustrated in our analysis) very rarely may indeed benefit excellent survival outcomes even with sub - curative doses of palliative radiation. this could be in part due to various biological factors, such as immunological clearance, induction of apoptotic signals by radiation, or possibly a lack of aggressive clonogens capable of accelerated repopulation. while some cancers such as small cell lung cancer (sclc) and poorly differentiated cervical carcinomas are more radiosensitive compared to other malignancies, it must also be acknowledged that tumors of the same diagnosis may have different biological behaviors in different patients. further, a prospective multivariate analysis of various potential factors contributing to exquisite radiosensitivity even with palliative doses can confirm or refute the role of parameters such as, but not limited to, histology, grade of differentiation, immunological indicators including circulating cytokines, anti - tumor antibodies, and growth factor receptors such as epidermal growth factor receptor 1 (egfr1), her-2-neu, and vascular endothelial growth factor receptor (vegfr). to conclude, we remind the reader that rt in a palliative role has excellent efficacy with regards to the control of pain and bleeding, and also with the relief of obstructive symptoms such as dyspnea and dysphagia. in addition, as our study suggests, there could be a benefit with regards to prolongation of survival. thus, even seemingly unfit patients must be assessed for the potential utility of palliative rt before the decision is made to transition to terminal end - of - life supportive care. the take - home message for oncologists and palliative care professionals would be that patients not given definitive therapy, but having responded exceptionally well to palliative treatment (e.g. a patient having achieved near - complete response after a course of palliative rt or a patient with markedly improved performance status after completion of palliative therapy, etc .) must be closely followed up and considered for further therapy, such as the use of additional chemotherapy for sclc, or the use of additional rt and chemotherapy for patients of cervical and head / neck cancers. summing it up, the noble principle of primum non nocere, which translates to first, do not harm should not be interpreted in a way as if it were encouraging therapeutic nihilism, wherein the clinician's pessimism with regards to the available therapeutic modalities could diminish a patient's chance of obtaining benefit from therapy.

context: palliative radiotherapy aims at symptom alleviation and improvement of quality of life. it may be effective in conferring a reasonable quantum of local control, as well as possibly prolonging survival on the short term. however, there can be rare instances where long - term survival, or even cure, from palliative radiotherapy, which mostly uses sub - therapeutic doses.aim:to categorize and characterize the patients with long - term survival and/or cure after palliative radiotherapy.materials and methods: this study is a retrospective analysis of hospital records of patients treated with palliative radiotherapy from 2001 to 2006 at the regional cancer centre, shimla.:of the analyzed 963 patients who received palliative radiotherapy, 2.4% (n = 23) survived at least 5 years, with a large majority of these surviving patients (73.9%, n = 17) being free of disease.:in addition to providing valuable symptom relief, palliative radiotherapy utilizing sub - therapeutic doses may, in a small proportion of patients, bestow long - term survival, and possibly cure. rationally, such a favorable, but rare outcome can not be expected with supportive care alone.

an 82-year - male presented with severe pain in the low back and both buttocks that was exacerbated by lumbar motion for 3 days. the intensity of the pain was 9/10 on a visual analogue scale (vas). the pain was relieved when he rested in bed, and aggravated by weight loading or motion. the physical examination revealed a reduction in all back movements and tenderness over the l4 spinous process. the patient had straight - leg - raising restriction of 70 in his right leg and 75 in his left leg. the laboratory values were normal, including the erythrocyte sedimentation rate and c - reactive protein. plain radiographs showed hyperostosis in the lumbar spine (fig . 1) and magnetic resonance imaging (mri) revealed a schmorl's node of l4 with adjacent marrow edema (fig . the spinal canal was narrow at the l3 - 4 and l4 - 5 levels . we performed nerve blocks on the l4 ramus communicans nerve . with the patient prone on the operating table, a 22-gauge spinal needle ( spinocan, b.brown, germany) was directed underneath the pedicle to rest slightly anterior to the superior aspect of the neural foramen. the needle was advanced until it was positioned at the inferior third aspect of the vertebral body under lateral fluoroscopic view. with the needle in the proper position, a mixture of 0.5 ml of 2% mepivacaine and 1.5 ml of contrast medium was injected to confirm the absence of intravascular, somatic nerve root, or epidural placement (fig . , a mixture of 2 ml of 1% mepivacaine and 10 mg of triamcinolone was injected on each side . his pain improved dramatically immediately after the nerve block ( vas score 2/10); there were no serious procedure - related complications. at the 1-month follow - up schmorl first described cartilaginous nodal herniation of the disc into an adjacent vertebral body in 1927. the majority of authors report that typical schmorl's nodes are usually asymptomatic. however, an acute or inflamed schmorl's node may be symptomatic and clinically significant. schmorl's nodes are most commonly located at the middle third of the inferior endplate, near the thoracolumbar junction. schmorl's nodes occur when the cartilaginous endplate of the vertebral body has been disrupted by an intrinsic abnormality of the endplate itself such as indentations left by the regression of the chorda dorsalis, ossification gaps, vascular channels, scheuermann's disease or by alterations in the subchondral bone itself such as osteomalacia, hyperparathyroidism, paget's disease, infection, neoplasm, trauma or mechanical overuse, scheuermann's disease, and osteoporosis. such weakening of the endplate is not a necessary prerequisite for extrusion and is thought to be present as an underlying cause only in a small percentage of schmorl's node cases. most schmorl's nodes form after axial - loading trauma in the preferential extrusion of nuclear material through the vertebral endplate, rather than through an intact, normal annulus fibrosus. schmorl's nodes are commonly seen on radiographs or at autopsy. in most cases, it is a radiographic or autopsy diagnosis. the demonstration of disc prolapse by radiography is usually possible only after the bony reaction of the vertebral body has developed an osseous sclerotic bone casting. mri has contributed to rapid improvements in the fundamental understanding of the bone marrow and its anatomy and physiology. in all symptomatic cases, the vertebral body marrow surrounding the schmorl's node had low signal intensity on t1-weighted images and high signal intensity on t2-weighted images. in our case, the vertebral body marrow surrounding the schmorl's node was seen as low signal intensity on t1-weighted images and high signal intensity on t2-weighted images, so we made an early diagnosis of a symptomatic schmorl's node. the principal branches of the lumbar sympathetic trunks are the rami communicantes to the lumbar ventral rami. white rami communicantes are distributed to the l1 and l2 ventral rami, and grey rami communicantes are distributed to every lumbar ventral ramus. the number of rami communicantes to each lumbar nerve varies from one to three, and exceptionally may be as high as five. in general, the rami communicantes reach the ventral rami by passing through the tunnels deep to the psoas muscle that lie along the concave lateral surfaces of the lumbar vertebral bodies. these tunnels direct them to the lower borders of the transverse processes where the rami communicantes join the ventral rami just outside the intervertebral foramina. the source of the nerve endings in the lumbar discs and vertebral bodies, are two extensive microscopic plexuses of nerve that accompany the anterior and posterior longitudinal ligaments. the anterior plexus bridges the two lumbar sympathetic trunks and covers the anterior longitudinal ligament. it is formed by branches of the sympathetic trunks and branches from the proximal ends of the grey rami communicantes. the posterior plexus is derived from the sinuvertebral nerves and accompanies the posterior longitudinal ligament. the anterior and posterior plexuses are connected around the lateral aspects of the vertebral bodies and discs by way of a lateral plexus that is formed by branches of the grey rami communicantes. the anterior and posterior plexuses supply superficial branches that innervate the periosteum of the vertebral bodies, and long penetrating branches that enter the intervertebral discs and vertebral bodies. through these branches this type of nerve block was initiated from the basis of the observation of the course of gray ramus communicans, containing unmyelinated postganglionic fibers which rejoin dorsal and ventral rami, distributing around vertebral body wall and coursing into anterior disc. it is known that gray ramus communicans nerve provides the greatest source of disc innervations and vertebral column. with respect to complications, chandler et al. reported potential risks, including infection, bowel puncture, intravascular injection, somatic nerve root trauma, intrathecal or epidural injection, kidney puncture with a far lateral approach and pneumothorax in thoracic approach. mri of patients with symptomatic schmorl's nodes has demonstrated inflammation and edema in the vertebral body, localized to the area around the schmorl's node. inflammatory change in the vertebral body marrow induced by intraosseous fracture and some biological reaction to the intraspongious disc materials might cause pain. after the fracture has healed and the inflammation subsided, the schmorl's node would be asymptomatic in analogy with an old vertebral compression fracture. symptomatic schmorl's nodes should be treated similarly to vertebral compression fracture, and conservative treatment is the first choice. if conservative treatment fails, surgery with anterior interbody fusion might be indicated. in our case , the schmorl's node was treated similarly to a vertebral compression fracture, and we successfully reduced the pain after blocking the ramus communicans nerve. the pain fell to a vas score of 2, and the patient was discharged 1 week after the block. most schmorl's nodes are asymptomatic, although acute or inflammatory schmorl's nodes are painful and clinically significant. some patients suffer from disabling pain due to schmorl's nodes, despite conservative treatment. a rami communicans nerve block is one treatment modality for patients with back pain due to a schmorl's node.

histologically, schmorl's nodes are defined as the loss of nuclear material through the cartilage plate, growth plate, and end plate into the vertebral body. most schmorl's nodes are asymptomatic, although there are some reports of symptomatic schmorl's nodes, which should be treated similarly to vertebral compression fractures, with conservative treatment as the first choice. we report the case that we reduced the pain by blocking the ramus communicans nerve in a patient with schmorl's node.

hepatocellular carcinoma (hcc) accounts for more than a million new cases each year worldwide and is the third leading cause of cancer - related death worldwide. its incidence in the united states has shown a dramatic rise over the past few decades and is expected to increase in the coming years. liver transplantation (lt) is in many cases the ideal therapy for hcc, providing not only oncologic resection but also replacement of a diseased organ. an important study by mazzaferro and colleagues demonstrated that if lt was limited to those with early hcc, long - term post - lt survival was excellent. based on this study and others, lt has become the standard of care for those hcc that satisfy the eponymous milan criteria (a single tumor under 5 cm, or 3 tumors each under 3 cm, without evidence of metastatic disease / vascular invasion) on radiological imaging. however, despite advances in imaging techniques, almost 20% of hccs are understaged on pre - lt radiological studies and are found on explant to exceed milan criteria, an established risk factor for post - lt hcc recurrence. specific predictors of post - lt recurrence are tumor size, multifocality, and vascular invasion. the efficacy of liver transplantation for hcc is therefore limited by tumor recurrence and the lack of effective preventive strategies for those at high recurrence risk. sorafenib is a multiple tyrosine kinase inhibitor, which inhibits tumor angiogenesis by inhibition of the vascular endothelial growth factor (vegf) and platelet derived growth factor (pdgf) signaling pathway and is the only fda - approved systemic chemotherapeutic agent for the treatment of advanced hcc at this time. we therefore undertook this pilot study to examine the preemptive use of sorafenib in the prevention of hcc recurrence post - lt in those with high - risk explant characteristics. we sought to assess the tolerability and safety of sorafenib in this population and examined rates of hcc recurrence and post - lt survival. all explants were sectioned at 5 mm intervals and examined by an experienced hepatopathologist. patients were offered entry if their explant examination demonstrated viable tumor exceeding milan criteria, specifically any of the following. 3 or more viable tumors (if 3 tumors, at least one over 3 cm diameter), one viable tumor greater than 5 cm in diameter, or evidence of micro- or macrovascular invasion. exclusion criteria were as follows: pathologic features of mixed hepatocellular cancer with cholangiocarcinoma, contraindication to sorafenib, absence of informed consent, and use of sirolimus as part of the immunosuppressant regimen. patients were started on sorafenib 200 mg by mouth once daily within 1224 weeks of lt, and the dose was titrated upwards to the target dose of 400 mg twice daily. patients underwent abdominal mri and ct scan of the chest every three months or earlier if clinically indicated, for hcc surveillance. the 7 patients who satisfied entry criteria were maintained on standard immunosuppression, and sirolimus use was prohibited. to identify a comparison group, we reviewed explant pathology of all patients transplanted for hcc from 2003 (initiation of the liver transplant program at our institution) onwards. we identified 12 patients who exceeded milan criteria on explant examination and who did not receive sorafenib preemptively after lt. of these, 9 patients had undergone lt prior to protocol initiation, and 3 patients declined treatment with sorafenib when offered entry into this protocol. there were no significant changes in the immunosuppression protocols or the pretransplant treatment of hcc between the two groups. secondary end points included rate of hcc recurrence, time to recurrence, and mortality. demographic characteristics, clinical variables, and transplant outcomes of patients included in the study were compared using means for continuous variables and frequencies for categorical variables. differences in characteristics between the adjuvant therapy group and historical comparison group were compared using the fisher's exact test and t - test. posttransplant survival and hcc recurrence between the two groups were compared using kaplan - meier (k - m) curves. the majority of patients in both groups were male, and hepatitis c virus (hcv) represented the primary underlying etiology of liver disease. all patients underwent either contrast - enhanced magnetic resonance imaging (mri) or computerized tomographic (ct) scan prior to lt. , even though no patients were noted to have > 3 nodules on pre - lt imaging, 50% and 42.8%, respectively, in the historical and the treated group were found to have multifocal disease on explant examination. vascular invasion was noted on microscopic examination of the explants (i.e., microvascular invasion) in 33% and 28% of patients in each group. the mean alpha - fetoprotein (afp) level at lt was not significantly different between groups. all patients received locoregional therapy (lrt) prior to lt, except 1 patient in the treatment group, with an equivalent number of lrt sessions between groups. five patients with recurrent hcc in the historical comparison group were treated with sorafenib after recurrence was documented. there were no significant differences between the two groups in terms of the number of tumors, presence of microvascular invasion, mean / maximum tumor diameter, tumor differentiation, and percentage of necrosis. the most frequent side effects of sorafenib were skin rash and diarrhea (58.3% of treated patients experiencing either or both adverse effects). twenty - two percent of patients experienced hand - foot syndrome. nausea and fatigue were also noted as side effects of sorafenib. there were no differences in the adverse effect profile of patients treated thereafter, patients were started at low doses of 200 mg daily and increased to full doses (400 mg twice daily) as tolerated. the mean follow - up for treated patients was longer than that for those in the historical group but this difference did not reach statistical significance (1125 versus 840 days, p value = 0.18). the overall rate of hcc recurrence was lower in the adjuvant therapy group compared to historical controls, (29% versus 75%, p = 0.07, table 3). disease - free 1-year survival for sorafenib and the comparison group was 100% and 66%, respectively; overall 1-year survival was 100% and 92%, respectively. two of 7 sorafenib treated patients suffered hcc recurrence (at 14 months and 52 months), one patient is deceased; the other is alive 5 months following documented recurrence. of the comparison group, 9 out of 12 patients the median time to recurrence among those treated with adjuvant therapy was longer but not statistically significant (1053 days versus 620 days, p = 0.08). the lung represented the most common site of recurrence (6 patients), followed by the liver (3 patients); 2 patients had multiple sites of recurrence. survival rates at study were higher for the treatment group (85.7%) compared to the historical arm (33.3%) one patient is alive at the current time 4 months from date of documented recurrence. the mortality incidence for the two groups it can be seen that the historical comparison group appears to have a higher mortality (75%) compared to the adjuvant therapy group (14%). however, because of the small sample size and the large number of censored observation, the sample size was effectively only 9 patients. this number is too small to use the tests of homogeneity over strata that require larger sample sizes in order to be meaningful. lt offers excellent survival outcomes in patients with hcc who satisfy well - established selection criteria. these criteria are however based on pre - lt imaging studies, which have been shown to underestimate tumor stage in approximately 20% of patients. patients with explant characteristics of tumor multifocality and vascular invasion are at high risk of recurrence. we have previously shown that, in those who exceed milan criteria on explant, the hazard ratio for hcc recurrence increased to 3.14 (p value 0.03) compared to those whose hccs on explant satisfied milan criteria. as a consequence, hcc recurrence after lt occurs at a significant rate, ranging from 3.5 to 26%, with median rates of 13%. this represents an important limitation to the efficacy of lt for malignant liver disease and may not be an appropriate use of an increasingly scarce resource. adjuvant chemotherapy with 5-fluorouracil / carboplatin, cisplatin / adriamycin, or doxorubicin has been studied, but published reports document conflicting efficacy rates and an unfavorable toxicity profile. the use of conventional chemotherapeutic regimes appears to be constrained by toxicity and difficulty of administration. the recent development of molecularly targeted therapies with acceptable side effect profiles and oral availability may provide an opportunity to reevaluate our approach to adjuvant treatments. sorafenib is the first of these agents to have been fda approved for the treatment of advanced hcc, and we believe it offers an opportunity for use in the prevention of hcc recurrence in high - risk individuals following lt. saab et al. described 8 patients treated with adjuvant sorafenib therapy and reported recurrence rates of 12.5%, as compared to 50% in a control population. as in our population, sorafenib appeared safe. a study from china consisted of a somewhat ill - defined population, but also appeared to confirm overall enhanced survival rates in patients offered sorafenib. our study comprises a homogenous group of patients, carefully monitored for recurrence with a longer period of follow - up than previously reported. as a , we are able to report on delayed recurrence (52 months) in one of the treated patients. while it is not possible to base mechanistic insights on such a limited sample, our experience raises tantalizing questions about the mechanism of action of sorafenib suggesting that it may render tumor cells studies that have examined the use of sorafenib prior to liver transplantation have shown mixed , with a suggestion of increased biliary complications and acute cellular rejection following lt. several studies have examined sorafenib as treatment for recurrent hcc following lt. other therapies have included surgery, systemic chemotherapy, as well as mammalian target of rapamycin (m - tor) inhibitors. overall have been disappointing, suggesting that recurrent hcc following lt is invariably a lethal and unresponsive disease. our small pilot study was primarily designed to assess the safety and tolerability of sorafenib. we found that sorafenib was well tolerated in the majority of patients, with increased tolerability if the dose was gradually titrated upward instead of started at a high dose. the main adverse event was a skin rash, which usually responded well to topical therapy and dose adjustment. there were no observed interactions with immunosuppressant regimens and no episodes of acute cellular rejection. however, there was a striking reduction in the rate of recurrence among treated, as compared to a historical comparison group, matched by explant features. all but one patient with hcc recurrence have succumbed to their disease, and hence the overall mortality rate in the historical control group was much higher (75%) compared to treated patients (14%). both preemptively treated patients who developed recurrence did so at a much later time point than those who were not treated, raising the possibility that sorafenib may delay the time to recurrence. however, the 5 treated patients without recurrence have now been followed for a mean time of 1125 days and there was no statistically significant difference in follow - up times. our study suffers from the limitation of being a single - center experience with a small number of patients. the comparison group was historical and therefore could not be controlled for many variables. in summary, we have demonstrated that the use of sorafenib in patients transplanted for high - risk hcc is a safe, feasible, and well - tolerated intervention. in our small sample, sorafenib use was significantly associated with a lower risk of hcc recurrence and improved survival. larger, prospective controlled trials will be required and have in fact been planned, to further examine the preemptive use of sorafenib and similar agents in this population, so that we may expand the armamentarium of therapies that are available to this challenging group of patients.

the efficacy of liver transplantation (lt) for hepatocellular (hcc) is limited by tumor recurrence rates of 1015%. we undertook this pilot study to examine the use of sorafenib as adjuvant therapy in high - risk lt recipients. methods. we prospectively enrolled patients transplanted for hcc into a treatment protocol utilizing sorafenib if their explant examination showed evidence of viable tumor exceeding milan criteria. we utilized as historical controls patients transplanted previously, whose explant tumor characteristics exceeded milan criteria, but who were not preemptively treated with sorafenib. wilcoxon two - sample test and fisher's exact test were used to compare survival and recurrence rates between the two groups. . seven patients were treated with sorafenib and compared to 12 historical controls. two of 7 treated patients suffered from hcc recurrence. of the comparison group, 9 experienced hcc recurrence and all succumbed to disease. dose reduction improved tolerance of drug. the overall rate of hcc recurrence was decreased in the adjuvant therapy group compared to historical controls (29% versus 75%, p = 0.07). disease free 1-year survival for the treated versus untreated group was 100% versus 66%, respectively. . adjuvant use of sorafenib is safe and decreases risk of hcc recurrence in high - risk lt recipients.

hepatitis c virus (hcv) is the major causative pathogen of chronic liver diseases with a risk of progression to cirrhosis and hepatocellular carcinoma. currently, ribavirin is one of the major agents used in combination therapy with interferon for chronic hepatitis c. during this treatment, however, patients sometimes suffer ribavirin - induced hemolytic anemia as an adverse event, necessitating discontinuation of the therapy or reduction of the dosage of anti - viral agents 1, 2. the mechanism of the hemolysis induced by ribavirin has not been clearly determined, but oxidative stress and subsequent membrane fragility of erythrocytes have been suggested to play an important role 3,4. after administration, ribavirin is converted to its phosphorylated form within the cell using adenosine triphosphate (atp). however, phosphorylated ribavirin is not converted back to the dephosphorylated form because erythrocytes lack the enzymes necessary for dephosphorylation. thus, the level of atp in cells is reduced as a of ribavirin phosphorylation 4, 5. the metabolic system in erythrocytes consists of glycolysis and the pentose phosphate cycle, for which atp is essential. the pentose phosphate cycle begins with glucose-6-phosphate (g6p), which is the first intermediate metabolite produced by glycolysis, and thus it interacts with glycolysis closely. it has been hypothesized that deficiency of atp causes impairment of the glycolytic system, thus triggering an energy crisis within erythrocytes, and inducing oxidative stress, leading to membrane fragility through impairment of the pentose phosphate cycle. in the present study, to investigate changes in the intermediate metabolites of glycolysis and the pentose phosphate cycle in erythrocytes, we carried out the metabolomics analysis of erythrocytes from patients with chronic hepatitis c, who had received antiviral therapy with pegylated interferon plus ribavirin. it was anticipated that this approach would help to clarify the mechanism responsible for ribavirin - induced hemolysis. eight patients with chronic hepatitis c (4 men and 4 women, aged 54.111.6 yr, meanstandard deviation) were treated with pegylated interferon plus ribavirin in accordance with the japanese standard guideline for treatment of chronic hepatitis c 6. erythrocytes were collected from the patients before and one week after the start of treatment, separated from whole blood by centrifugation, and resuspended in normal saline solution at a concentration of 40010/l. for disruption of the erythrocyte membranes, the levels of intermediate metabolites in erythrocytes were measured by capillary electrophoresis - time - of - flight mass spectrometry (ce - tofms) as reported previously 7 - 9. informed consent was obtained from all patients, and the study was approved by the institutional ethics committee. wilcoxon matched - pairs signed rank test was used for statistical analysis, and differences at p < 0.05 were considered to be significant. hemolytic anemia induced by ribavirin is one of the serious adverse events associated with therapy for chronic hepatitis c 1, 2. the erythrocyte count observed in this study showed a significant reduction at 2 weeks after the start of ribavirin administration (452.043.910/l vs. 421.752.410/l : pretreatment vs. 2 weeks after treatment, meanstandard deviation, p=0.028). the drop in the erythrocyte count progressed and became 388.158.010/l at 4 weeks after the start of ribavirin administration. although the mechanism of this hemolysis has not been precisely clarified, a reduction of atp due to ribavirin phosphorylation has been suggested 10. a recent study showed that ribavirin - induced anemia is primarily due to the effect of the drug on guanosine triphosphate, consequently leading to a reduction of atp in erythrocytes 11. to clarify this mechanism, we focused on the levels of intermediate metabolites of glycolysis and the pentose phosphate cycle in erythrocytes. the changes in the levels of intermediate metabolites belonging to the glycolytic system and the pentose phosphate cycle in erythrocytes between samples collected before and after ribavirin administration are shown in table 1. as ribavirin was taken up by erythrocytes, atp was consumed for its phosphorylation, and as erythrocytes lack the enzymes necessary for ribavirin dephosphorylation, atp deficiency progressed and eventually inhibited the glycolytic system. here we found that the levels of the intermediate metabolites of glycolysis in erythrocytes, including g6p, fructose-6-phosphate, 2, 3-diphosphoglyceric acid and 3-phosphoglyceric acid, were significantly lower one week after ribavirin administration than before the start of treatment. g6p is the key metabolite that is central to both glycolysis and the pentose phosphate cycle in erythrocytes. g6p is metabolized to 6-phosphogluconic acid, d - ribose-5-phosphate and d - sedoheptulose-7-phosphate to supply nicotinamide adenine dinucleotide phosphate (nadph) for the pentose phosphate cycle. the levels of these metabolites in patients' erythrocytes were all reduced after ribavirin administration, among which that of d - ribose-5-phosphate became significantly lower. this indicated that the decrease in the level of g6p influenced the activity of the pentose phosphate cycle and caused a decrease of nadph. nadph has an important role in the reduction of glutathione (gsh) in concert with glutathione reductase. thus it was suggested that the decrease of nadph ing from impairment of the pentose phosphate cycle led to a decrease in the concentration of reduced gsh, consequently increasing the degree of oxidative stress in erythrocytes. in fact, a previous study has shown that ribavirin treatment causes a significant increase of malondialdehyde and methemoglobin in erythrocytes, both being typical products of oxidative damage 4. taken together, the data suggest that two major events in erythrocytes - an energy crisis due to atp deficiency in the glycolytic system and consequent oxidative stress due to impairment of the pentose phosphate cycle - are closely related to the occurrence of hemolysis associated with ribavirin. the single nucleotide polymorphisms (snps) of inosine triphosphatase (itpa) gene are shown to be strongly associated with ribavirin - induced hemolytic anemia in chronic hepatitis c 12. further studies are needed to investigate a relation between snps of itpa gene and levels of intermediate metabolites for glycolytic system and pentose phosphate cycle in a larger cohort of patients. in the treatment of chronic hepatitis c, adequate doses of ribavirin are closely associated with viral clearance, and prevention of both viral breakthrough and emergence of resistant variants 2. for achieving the maximum effect of antiviral therapy, the of this study suggest that some form of medication strategy for both the energy crisis and oxidative stress caused by ribavirin may be important for prevention of erythrocyte hemolysis.

ribavirin is one of the major agents used in combination therapy with interferon for chronic hepatitis c, but is often associated with hemolytic anemia as a serious adverse event. employing metabolome analysis , we demonstrated that the concentrations of intermediate metabolites produced by glycolysis and the pentose phosphate cycle in patients' erythrocytes were significantly decreased after administration of ribavirin. our findings suggest that hemolysis associated with ribavirin is triggered by an energy crisis and consequent oxidative stress, thus having implications for the prevention of such hemolysis.

opposite to the term fertility, there is another important aspect called infertility, which is defined as inability to conceive for at least 1 year after regular and unprotected sexual intercourse. the causes for infertility can be generally categorized as male factor, female factor, combination, and unexplained or idiopathic factor. the world health organization (who) reported mean infertility rate as 1215% globally, with a prevalence of 24.9% in iran in 2004. an imbalance in any of these systems leads to infertility and predisposes the couples to mental and psychological problems such as anxiety, depression, and mental pressure, which, at times, in social problems such as domestic violence. in various studies , it has been reported that the stressful experience of infertility is accompanied with a vast spectrum of psychological problems including impulsive behaviors and spread of anger, the feeling of helplessness and rejection, as well as marital problems. meanwhile, couples negative self - concept and low self - esteem drive them to have unreal and early expectations. on couples failure to achieve their unreal expectations (e.g., wishing to have a child) misbehavior is among the important issues associated with public health, which is not only observed in all geographical areas and religions and racial groups, but also in all levels of education, occupation, and economic and social classes. several studies have reported some factors associated with infertility that can predispose women to misbehavior and increase the rate of violence by twofold. as infertility and its effects may vary in difference cultures, the prevalence of violence has been reported differently in various studies. reported the rate of domestic violence incidence among infertile women as 33.6% in turkey, and this rate was reported as 61.8% in iran. meanwhile, leung et al. reported the incidence of violence to be only 1.8% in hong kong. there are controversial in the context of the effect of interventional factors such as infertility on the incidence of marital reactions. most of the conducted studies investigated physical violence, although non - physical violence can lead to major complications and due to lack of studies in this subject. therefore, the present study aimed at determining the association between infertility factors and non - physical partner abuse in infertile couples referring to infertility centers in isfahan in 2013. this is a descriptive cross - sectional study conducted on 262 infertile couples (131 male and 131 female) who referred to the infertility centers of isfahan and met the inclusion criteria. inclusion criteria were primary infertility, infertility with a diagnosed cause, and absence of any diagnosed mental and psychological diseases. data collection tool comprised a demographic characteristics questionnaire, and non - physical abuse of partner scale (npaps) as well as partner abuse scale: non - physical (pasnp), both of which include 25 items that were separately completed and measured for each subject. npaps (25-item tool) is one of the care scales in which the concept of the abuser from the level of the abuse delivered to his / her partner is measured. it has been designed to measure the level or severity of non - physical abuse delivered upon the partner, based on the abuser's self report. pasnp is also a 25-item tool to measure the degree or severity of non - physical behavioral abuse delivered to the clients by their spouse or partner. these two questionnaires, designed by james dublio and garner to measure non - physical abuse, enjoy an excellent internal consistency with = 0.90, and were adopted for the first time in iran. the present research was approved in isfahan university of medical sciences and its related ethics committee. for questionnaire localization, reliability and validity of this questionnaire were established by face and content validity with the help of the academic members in nursing and midwifery school and school of educational sciences of isfahan university. its validity and reliability were confirmed with cronbach alpha = 0.84. after obtaining an informed consent from the subjects, both questionnaires are scored based on a likert's seven - point scale as follows: never (score 1), seldom (score 2), sometimes (score 3), usually (score 4), often (score 5), frequently (score 6), and always (score 7), with a calculated mean out of 100. the collected data were analyzed by descriptive statistics (mean, sd, min, max) and interventional statistics (independent t - test) in spss 16 with a significance level of p < 0.05. the are presented in tables 13. mean of npaps and pasnp in infertile couples the relationship between infertility factors, and npaps and pasnp in women the relationship between infertility factors, and npaps and pasnp in men female and male subjects mean ages were 27.5 and 31.9 years, respectively. about 90.8% of the men and 26.9% of the women were working. the highest education level of the men and women was high school diploma (43.5% and 46.6%, respectively). the most common cause of infertility (31.3%) was male factor, and the least (17.5%) was combination factor. as shown in table 1, the mean scores of npaps were 23.1 10.3 in men and 21.3 10.6 in women out of 100. the mean scores of pasnp were 13.8 1.9 in men and 20.3 13.4 in women out of 100. independent t - test showed no significant difference in the mean scores of npaps between men and women (p = 0.165), although the mean scores of pasnp showed a significant difference between men and women (p < 0.001). the highest scores of npaps and pasnp were 23.3 and 26.5 in female factor, while the lowest were 17 and 16.5 in idiopathic factor among infertile women. one - way analysis of variance (anova) showed no significant association between infertility factor and score of npaps, while it showed a significant association between pasnp and female factor of infertility (p < 0.01). the showed that when the infertility factor was female factor or combination factor, npaps had the highest mean score of 20.2 among infertile men. meanwhile, one - way anova showed no significant association between infertility factor and the score of npaps (p = 0.85). when the female factors was a cause of infertility the highest score of pasnp (16.7) in men was observed. meanwhile, one - way anova showed no significant association between the factor of infertility and npaps in men (p = 0.15). the of the present study showed that the most common cause of infertility was male factor, which is consistent with the studies of el kissi et al. and chiaffarino et al. meanwhile, volgsten et al., in sweden, reported female factor, idiopathic factor, and male factor as the causes for infertility respectively. li et al., in a study conducted in china, reported idiopathic factor as the most common cause for infertility (43.9%), while female factor (20.8%), male factor (20.3%), and combined factor (15%) were ranked after that, respectively. direkavand moghadam et al., in thier meta - analysis study, reported an equal rate for male and female factors of infertility. with regard to the aforementioned studies, it is found that the prevalence of infertility has been reported differently in various studies. it should be noted that in references, the male factor was reported to be 2540%, the female factor 40 - 55%, the combined factor 10%, and the idiopathic factor was reported as 10%. nowadays, there is an ascending trend for the male factor of infertility due to an increase in industrial occupations and more involvement of men in industries, as well as their contact with environmental and industrial pollutants. isfahan infertility center (from which most of the subjects were selected) acts as a referral infertility center where couples with a diagnosed infertility factor are referred from other cities to receive professional treatment and undergo fertility assistive techniques. this can be the reason for the high prevalence of male factor infertility found in the present study. there is no doubt that having a child plays a pivotal role in couples happiness in their marital life. when they are deprived from having their biological offspring in their common life, they not only miss the meaning in their life but also develop conflicts in their interpersonal communication, which predisposes them to mental and psychological problems such as anxiety, depression, and mental pressure, consequently leading to domestic violence. the of the present study showed that npaps was lowered in women, compared to men, although the mean scores of npaps showed no significant difference between men and women. pasnp was also found to be more among women, compared to men, but its mean score showed a significant difference between men and women. several studies showed that infertility and the attitude toward that puts the couples in an emotional crisis, and meantime, couples negative self - concept and low self - esteem drive them toward inappropriate behaviors such as violence to relieve their discomfort. other studies on domestic violence investigated the npaps just from the viewpoint of one of the couples. meanwhile, in the present study, in addition to npaps, the adopted questionnaire investigated pasnp from the viewpoint of both the couples. akyuz et al., in a study on infertile women, showed a significant association between the score of violence and infertility factor in such a way that the level of violence was higher in infertility with female factor. one study has reported the rate of domestic violence as 61.8% among infertile women, although all the participants were women with female factor infertility. as the of the present study showed no significant difference in the factor of infertility and npaps, and mean npaps was not high in women, it can be reasoned that as women with female factor infertility felt somehow defective, they manifested a higher pasnp, although they did not really undergo a higher behavioral abuse. they argue that infertile women are more predisposed to depression, anxiety, stress, and stressful events that can make a potential for behavioral abuse. one of the major goals of marriage in women is fertility, and infertility, in fact, ruins this value. therefore, women accept to undergo difficult fertility techniques to preserve their condition, which may predispose them to many mental diseases, depression, and other psychological disorders. on the other hand, infertile women face other threats such as a divorce or their spouse's remarriage, which notably affect their pasnp. the showed that infertility can act as a factor to make the couples closer to each other and to have a mutual cooperation in infertility treatment, and generally improves their marital relationship, as their npaps and pasnp were found lower in this group of couples in the present study, compared to other studies. both couples should be simultaneously considered in fertility counseling sessions, as well as in medical and clinical treatments given by infertility centers and the associated professionals.

: infertility predisposes the couples to mental and psychological problems such as anxiety, depression, anger, and partner abuse. this study aimed to investigate the association between infertility factors and the non - physical abuse between infertile spouses.materials and methods: this is a descriptive cross - sectional study conducted on 262 infertile couples (131 female and 131 male), selected through convenient sampling, who referred to infertility centers in isfahan. data were collected by partner abuse scale: non - physical (pasnp), designed to measure the non - physical abuse experienced in relationship with partner and non - physical abuse of partner scale (npaps), designed to measure the non - physical abuse delivered upon the partner. all data were analyzed through spss version 16.:mean scores of npaps were 23.1% and 21.3% in men and women, respectively. mean scores of pasnp were 13.8% and 20.3% among men and women, respectively. there was a significant difference in the mean scores of perceived non - physical partner abuse between men and women (p < 0.001). there was also a significant difference in the mean scores of perceived non - physical partner abuse and factor of infertility (p < 0.01).: perceived non - physical abuse and delivered non - physical abuse upon the partner were low among infertile couples. women had a higher perception of abuse when the cause of infertility was female factor, compared to men. however, special attention should be paid to infertile couples. marital counseling, besides infertility counseling, should be conducted for these couples.

growth disorder in children is one of the important health problems in the world, especially in developing countries. regardless of the underlying disease, as the main cause of growth disorders, assessment and correction of nutritional status of these children are very important. given the fundamental importance of this issue and ascending tendency to use complementary medicine in the world, this article discusses the traditional iranian philosopher s views on the role of nutrition in child development. this study reviews textbooks of traditional medicine, particularly in the field of pediatric medicine with a focus on canon of medicine of avicenna. temperament is the physiological concept of the human body in traditional medicine and has an important role in health, diagnosis, and treatment of disease. generally, children are born with warm and wet temperament that provides the best condition for growth. however, the personal temperament of each child determines growth, the need for a variety of food groups, and even physical activity. special lifestyle orders are discussed in detail according to these differentiations and nutritional management is the most important factor considered. in spite of advances in classical medicine in the prevention and treatment of many diseases, there are still a lot of therapeutic challenges in many health problems. temperamental approach to the human body in traditional medicine provides a different perspective on the medicine. reflecting on temperamental view in child development may lead to a better understanding of the diagnosis and treatment of diseases. on the other hand, further research studies based on the reform of nutrition with respect to temperament should be considered as a new strategy in the management of developmental disorders.

: growth and development are the basic science in pediatric medicine. growth disorder in children is one of the important health problems in the world, especially in developing countries. regardless of the underlying disease, as the main cause of growth disorders, assessment and correction of nutritional status of these children are very important. given the fundamental importance of this issue and ascending tendency to use complementary medicine in the world, this article discusses the traditional iranian philosopher s views on the role of nutrition in child development.methods:this study reviews textbooks of traditional medicine, particularly in the field of pediatric medicine with a focus on canon of medicine of avicenna.:temperament is the physiological concept of the human body in traditional medicine and has an important role in health, diagnosis, and treatment of disease. generally, children are born with warm and wet temperament that provides the best condition for growth. however, the personal temperament of each child determines growth, the need for a variety of food groups, and even physical activity. different appetite and food preferences in children show temperamental variation. therefore, children need special management regarding special temperament. in iranian traditional medicine, special lifestyle orders are discussed in detail according to these differentiations and nutritional management is the most important factor considered.:in spite of advances in classical medicine in the prevention and treatment of many diseases, there are still a lot of therapeutic challenges in many health problems. temperamental approach to the human body in traditional medicine provides a different perspective on the medicine. reflecting on temperamental view in child development may lead to a better understanding of the diagnosis and treatment of diseases. on the other hand, further research studies based on the reform of nutrition with respect to temperament should be considered as a new strategy in the management of developmental disorders.

rubinstein - taybi syndrome is a congenital malformation disorder characterized by short stature, mental retardation, microcephaly, broad great toe and thumb, and various facial abnormalities (hypoplastic maxilla, prominent beaked nose, low - set ears, antimongoloid palpebral fissures).1 first described in 1963 by rubinstein and taybi, this condition occurs in approximately one in 100,000 newborns. most patients with rubinstein - taybi syndrome have a mutation of the creb binding protein gene which codes for a regulatory protein in fetal development.2 most cases are isolated, but parental autosomal dominant transmission can be seen.3 various ocular abnormalities linked with this syndrome (84%) include glaucoma, down - slanting palpebral fissures, epicanthus, strabismus, cataract, refractive error, colobomata, highly arched eyebrows, nasolacrimal duct obstruction, and anterior segment dysgenesis.1,4 van genderen et al found a high frequency of electroretinographic abnormalities in 14 of 18 patients (78%), eight with decreased cone responses and six with decreased cone and rod responses. similarly, 18 of 23 patients examined (78%) showed mild to severe fundus abnormalities including absent macular reflexes, increased reddening of the foveal area, unusual distribution of pigmentation, lacquer cracks, and chorioretinal coloboma.1 while there are numerous publications on rubinstein - taybi syndrome, the literature on related ocular disturbances is limited. to our knowledge, there are no reports of fluorescein angiography evaluation of rubinstein - taybi syndrome. we report fluorescein angiography findings of vascular attenuation, prolonged arteriovenous transition time, delayed venous filling, small vessel leakage, and peripheral avascularity in a child with rubinstein - taybi syndrome. a 6-year - old male, born at 34 weeks gestation with rubinstein - taybi syndrome due to creb binding protein gene mutation underwent fluorescein angiography to characterize better his retinal vasculature attenuation. the patient had characteristic clinical features of rubinstein - taybi syndrome, including microcephaly, down - slanting palpebral fissures, broad thumbs and first toes (figure 1), bicuspid aortic valve, and an ocular history of high myopia 6.00 diopters, exotropia, epiblepharon, and congenital glaucoma status post trabeculotomy ou and baerveldt glaucoma implant os. anterior segment examination showed clear corneas ou, clear lens od, small nasal cataract os, superonasal tube os, and posterior synechiae os. fundus examination showed sharp discs with significant glaucomatous cupping ou and optociliary shunt vessel os. choroidal vasculature was prominent due to diffuse retinal atrophy with scattered focal retinal pigment epithelial changes ou (figure 2). fluorescein angiography showed retinal vascular attenuation, prolonged arteriovenous transition time with delayed venous filling od (figure 3), and late small vessel leakage nasally os (figure 4). a total of 360 degrees of peripheral avascularity was also evident ou (figure 5). vital signs, including heart rate, blood pressure, and oxygen saturations were within normal limits throughout the examination. the present case has many of the ocular findings commonly reported in rubinstein - taybi syndrome including down - slanting palpebral fissures, glaucoma, high myopia, strabismus, and retinal dystrophy.5,6 the fluorescein angiography findings of rubinstein - taybi syndrome identified in this case include vascular attenuation, prolonged arteriovenous transition time with delayed venous filling, small vessel leakage, and peripheral avascularity. prolonged transit time has been identified in glaucoma, but the marked delay in this case is unexpected because intraocular pressures were controlled.7 prolonged transit time can also occur in chorioretinal inflammatory disorders.8 an inflammatory process may have contributed to the prolonged transit time in this case. the late small vessel leakage showed that retinal vascular inflammation was present, and recent baerveldt glaucoma implant surgery may have been a contributing factor. peripheral avascularity, common in retinopathy of prematurity, familial exudative vitreoretinopathy, and incontinentia pigmenti, has not been described previously in rubinstein - taybi syndrome. the degree of peripheral avascularity in the present case is not typical for a birth history of 34 weeks gestational age. mutation of the creb binding protein responsible for growth retardation and other skeletal and cardiac abnormalities in rubinstein - taybi syndrome may also affect vascularization of the retinal periphery. further investigation with fluorescein angiography is warranted to characterize better the retinal vascular abnormalities of rubinstein - taybi syndrome.

the purpose of this report is to describe the fluorescein angiography findings in a case of rubinstein - taybi syndrome. fundus photography and fluorescein angiography were performed on a 6-year - old male with rubinstein - taybi syndrome due to creb binding protein gene mutation. fundus photography showed glaucomatous cupping and diffusely attenuated retinal vasculature. choroidal vasculature was prominent due to diffuse retinal atrophy with scattered focal retinal pigment epithelial changes. fluorescein angiography showed retinal vascular attenuation, prolonged arteriovenous transit time with delayed venous filling, late small vessel leakage, and 360 degrees of peripheral avascularity. peripheral retinal avascularity and retinal vascular inflammation evidenced by late small vessel leakage can be demonstrated by fluorescein angiography in the retinal dystrophy of rubinstein - taybi syndrome.

differential migration in males and females has been demonstrated in various bird species (cristol et al . it has been extensively studied, in relation to spring migration, that when selection favours earlier arrivals of a given sex ( usually males) at the breeding grounds, due to competition for high - quality territories and mates (kokko 1999). much less attention has been paid to differential migration in the autumn, although passage to the wintering grounds may also be time - constrained in a different manner for the two sexes. we investigated the autumn passage of molecularly sexed male and female aquatic warblers acrocephalus paludicola, with regard to their age and body condition. the aquatic warbler is a globally threatened passerine; its population size and range have decreased by 90 % over the last century (flade and lachmann 2008). during autumn, the birds migrate to a tropical sub - saharan african location (buchanan et al . 2011 ; flade et al . 2011 ; foucher et al . 2013 ; salewski et al . 2013) through western (de by 1990 ; aquatic warbler conservation team 1999) and possibly southern europe (salewski et al . 2013). earlier studies of the aquatic warbler's autumn migration based on small samples did not examine sex differences (de by 1990 ; atienza et al . 2001 ; julliard et al . 2006 ; migulez et al . 2009 ; neto et al . 2010 ; le nev et al . 2011). however, in view of the breeding biology of the species only the female provides care for the offspring; the male is free of parental duties after the mating period (dyrcz et al . one might also expect that the two - peak pattern of autumn migration ( neto et al . this study was carried out at donges on the loire estuary ( 47 18 ' 17.19 " n, 2 2 ' 9.51 " w), one of the most important sites in france for capturing the aquatic warbler (le nev et al . 2011). we performed the fieldwork in 2011, covering the entire autumn migration period of adult aquatic warblers and almost the entire migration period for young birds in this area (18 july15 september ; le nev et al . we captured the birds in 25 mist - nets placed in the reedbed habitat, opened just before local sunrise for circa 6 h ( weather permitting). we used tape - luring for the whole capturing period (played from about 1 h before local sunrise until the nets were closed). we measured the right wing with a ruler (0.5 mm) to provide a surrogate measure of body size and body mass using an electronic scale (0.1 g, my weigh pointscale 5.0). we determined the fat score according to the 4-score scale proposed by the french ringing centre, crbpo - musum paris. we took 15 body feathers from each bird for molecular sexing, including the primer pair p2 and p8 (according to the method of griffiths et al . we extracted dna from the proximal tip of feathers using the sherlock ax kit for biological tracks ( a&a biotechnology, gdynia, poland). we sampled 176 of 179 (98 %) birds captured during the whole study period, sexing all of them successfully. the median capture dates for different sex and age groups ranged from 11 to 20 august (fig . 1). in adults, the timing of migration differed between sexes (mann whitney u test, z29,13 = 3.73, p < 0.001), with the median date for males being 8 days earlier than for females (fig . 1). immature males and females migrated at a similar time (z99,35 = 1.30, p = 0.19), with only a 1-day difference between median dates (fig . 1). adult males migrated earlier than immature males (9-day difference in the median date ; z29,99 = 5.55, p < 0.001 ; fig . 1), whereas the timing of adult and immature females was similar (only a 2-day difference in the median date ; z13,35 = 1.02, p = 0.31 ; fig . 1timing of autumn migration in aquatic warblers grouped by ages and sex at a stopover site in the loire estuary ( donges, france). numbers in brackets denote the relevant sample size timing of autumn migration in aquatic warblers grouped by ages and sex at a stopover site in the loire estuary (donges, france). numbers in brackets denote the relevant sample size body mass was not affected by body size (ancova, wing length f1,170 = 1.72, p = 0.19) or by sex (f1,170 = 0.64, p = 0.43). only age was found to affect body mass, with adults being on average 6 % heavier than immature birds (f1,170 = 14.85, p < 0.001 ; fig . 2). the age by sex interaction was not significant (f1,170 = 0.04, p = 0.84).fig. 2size - adjusted body mass (mean and 95 % confidence intervals) in aquatic warblers of both sex and age categories during autumn migration at the loire estuary stopover site. numbers above the bars denote the relevant sample size size - adjusted body mass (mean and 95 % confidence intervals) in aquatic warblers of both sex and age categories during autumn migration at the loire estuary stopover site. numbers above the bars denote the relevant sample size adult males had similar fat scores to adult females (median fat score for both sexes = 2 ; z29,13 = 0.15, p = 0.88); immature males and immature females also had similar fat scores (median fat score for both sexes = 2 ; z99,35 = 0.94, p = 0.34). a comparison of the fat scores across age groups revealed no differences in males (z29,99 = 0.20, p = 0.84) or females (z13,35 = 0.59, p = 0.55). the sex ratio differed significantly from parity in both adults (chi - squared test, 1 = 4.13, p = 0.04) and immature birds (1 = 20.91, p < 0.001). the numbers of captured adult and immature males were 2.2 and 2.8 times higher, respectively, than the corresponding female age groups. we found sex differences in the timing of autumn migration in adult aquatic warblers, with males migrating significantly earlier than adult females. however, immature birds of both sexes migrated at a similar time. this pattern of migration, in which non - parenting adults migrate first, followed by parenting adults and young is common in ducks and shorebirds (newton 2011). the existence of similar patterns in different taxonomic groups with similar breeding biology emphasizes the link between breeding system and migration behaviour. although the studied site may be considered representative (le nev et al . 2011), we can not exclude the possibility that the differential migration we observed might be specific to this particular site and season. the males may have stopped predominantly in the study site (perhaps due to tape - luring, see below), whilst the females may have stopped preferentially at another site. it is also possible that the 2011 season was an unusual breeding year with a lot of second brood attempts, this would account for the late passage of adult females and immature birds. a further examination of the autumn migration with respect to sex differences in timing, perhaps across several stopover sites, is desirable. the frequency distribution of the capture of adult aquatic warblers of both sexes was more concentrated in time than that of immature birds, indicating differential migration of the age groups. the most likely explanation for the pattern observed is the difference in previous migration experience (e.g., ellegren 1990). tegetmeyer and colleagues found some intra - specific variation in timing of moulting, which could be related to the age (or sex) groups. however, currently, there are no published data on age- or sex - based winter segregation. however, in line with reports on the aquatic warbler from other stopover sites, and on other acrocephalus species (julliard et al . 2006 ; jakubas and wojczulanis - jakubas 2010), we found a tendency towards higher body masses, but not fat scores in adults compared to immature birds. this might be explained by more efficient foraging in experienced or higher social status adults. the discrepancy between the for body mass and fat scores might be accounted for by the low resolution of the fat score system used and/or a nonlinear relationship between fat mass and fat score (hedenstrm et al . however, sex ratios in the population at nestling stage as well as in wintering areas seem to be balanced ( giessing 2002 ; dyrcz et al . 2004 ; vogel 2009). the most likely explanation for the biased sex ratio we observed is therefore the tape - luring used in the present study. this procedure caused a sixfold increase in the capture rate of the aquatic warbler (julliard et al . it has also been shown that in some species ( to date, these do not include the aquatic warbler), tape - luring may efficiently attract males, but not females (e.g. lecoq and catry 2003). however, we can not entirely rule out the possibility that the sex ratio observed during migration reflects a sex difference in migration routes. females may have more frequently used an alternative, more direct migration route (e.g. via southern europe, salewski et al . 2013 ; but note that only males were investigated in this study), as they have less time after breeding for migration than males. these uncertainties indicate clearly that further studies investigating the effects of sex on the migration behaviour of aquatic warblers are needed.

relatively little attention has been paid to sex differences in the migration of birds in autumn. we studied the autumn migration strategy of molecularly sexed males and females in the globally threatened aquatic warbler acrocephalus paludicola. we captured 176 birds at a stopover site in the loire estuary at donges, france. the median date for the passage of adults was 8 days earlier in males than females, although the timing of migration in first - year males and females was similar. this indicates that males, who are without parental duties, can start their migration earlier than females and first - year birds. adults were significantly heavier than immature birds but did not have higher fat scores. in both age categories, more males (two to three times more) were captured. however, various factors (including tape - luring) can affect observed sex ratio.

it is estimated that 382 million people all over the world had diabetes mellitus in 2013. chronic loss of retinal neurons occurs due to increased frequency of apoptosis and activation of glial cells. vulnerability to neurons exists before any sign of vascular damage. retinal barrier, vasoregression, and impairment of neurovascular interaction. homocysteine is a by - product of transmethylation reactions and is detoxified by methionine synthetase, which depends on vitamin b12 and folate as coenzymes for proper function. elevated total plasma levels of homocysteine has been found to be an independent risk factor for retinal vascular occlusive disease. homocysteine has been found to be involved in a complex and dynamic way in vascular injury and repair, thus contributing to the development of diabetic microangiophathy. therefore, strategies for controlling the level of homocysteine by supplementation with folic acid or vitamin b12 may be potential treatment strategies to ameliorate neurodegeneration. the present study was conducted to evaluate the status of serum levels of vitamin b12, folic acid, and homocysteine in diabetic retinopathy and the correlation with retinal nerve fiber layer (rnfl) thinning on spectral domain optical coherence tomography (sd - oct). institutional review board approval was obtained, and the study was performed in accordance with the tenets of the declaration of helsinki. sample size was calculated to be 80 according to the standard sample size calculation formula. the study was conducted in adherence to the arvo guidelines regarding the ethical use of human subjects in research. in this tertiary care center based cross - sectional study, 60 consecutive cases of type 2 diabetes mellitus and 20 healthy controls (presenting for refraction) aged between 40 and 65 years were included after informed voluntary consent was obtained. the 60 cases of type 2 diabetes mellitus were divided into three groups: patients with diabetes without retinopathy (n = 20), patients with non - proliferative diabetic retinopathy with macular edema (npdr ; n = 20), and patients with proliferative diabetic retinopathy with macular edema (pdr ; n = 20), based on the early treatment diabetic retinopathy study (etdrs) classification. level of diabetic retinopathy was graded according to the eye with the more severe form of disease. the duration of illness was defined as the duration from the time of the diagnosis of diabetes mellitus given to the participant until the time of the examination. exclusion criteria included ocular or systemic diseases affecting the retinal vasculature (hypertension), nervous system (alzheimer disease, peripheral neuropathy, glaucoma, age - related macular degeneration, end stage renal disease), previous intravitreal injection(s), ophthalmic surgical or laser interventions, cases on medications, vitamin, or antioxidant supplements, and cases with signal strength 5 or below on the optical coherence tomography examination. the examination consisted of an explanation of the study, measurement of the blood pressure, refraction and assessment of the logmar best - corrected visual acuity (logmar is expressed as the decadic logarithm of the minimum angle of resolution with 20/20 line equivalent to logmar 0.00 and the 20/200 line to logmar 1.0), and slit - lamp biomicroscopy of the anterior segment. further, goldmann applanation tonometry, gonioscopy, and computerized static perimetry using the full threshold 242 program of the humphrey perimeter (humphrey field analyzer, zeiss / humphrey systems, dublin, ca) were performed. fundus examination was performed with slit - lamp biomicroscopy with a 90-diopter lens and indirect ophthalmoscopy. fundus photography was performed in all cases using a zeiss fundus camera ff 450 plus with pixel width of 0.0054 and image size 25881958. subsequently, all the study subjects were evaluated using sd - oct (cirrus high definition oct ; carl zeiss meditec inc ., every study subject underwent rnfl thickness analysis using the optic disc cube 200200 feature ( figure 1). the average retinal nerve fiber layer thickness was noted. retinal nerve fiber thickness analysis using optic disc cube 200x200 feature depicting thickness map, thickness deviation, tsnit (temporal, superior, nasal, inferior, temporal) normative data and extracted tomograph of both eyes. the quadrant map shows thinning in the superior quadrant of both eyes and thinning in inferior quadrant of left eye. blood samples were collected from the study subjects in the morning after an overnight fasting interval of 68 h. the samples were drawn by vein puncture using a 5 ml metal - free plastic syringe fitted with a 24-gauge stainless steel needle (nirlife ; nirma limited, sachana, india) under contamination - controlled conditions, and the blood samples were collected in a 4 ml vacutainer (vaku-8 ; hindustan syringes and medical devices limited ; faridabad, india) that contained heparin as an anticoagulant. the volume of the samples ranged from 4 ml to 5 ml. for separation of plasma, the citrated blood was centrifuged at 200 g for 10 min, and the supernatant platelet - rich plasma was aspirated out into another plastic tube. fasting and post - prandial blood glucose levels was estimated using the glucose oxidase method. glycated hemoglobin was measured using the bio - rad d-10 (bio - rad laboratories, inc ., hercules, ca) that employs the high - power liquid chromatography cation exchange chromatography method. the serum folic acid assay was performed using elecsys folate iii (roche diagnostics ; indianapolis, in) assay method that employs a competitive test principle using natural folate binding protein (fbp) specific for folate. the serum vitamin b12 assay was performed using the elecsys vitamin b12 (roche diagnostics) assay method that employs a competitive test principle using the intrinsic factor specific for vitamin b12. assay of homocysteine was performed using the human homocysteine enzyme - linked immunosorbent assay (elisa) kit (mybiosource, inc ., san diego, ca). the reagents in the kit were prepared following the standard protocol provided with the kit. human homocysteine standard samples were added to corresponding wells (100 l for each well), and the 0 nmol / ml well was filled with standard diluent. serial dilutions of the homocysteine standard were performed using 50, 25, 12.5, 6.25, 3.12, 1.56, and 0.78 nmol / ml concentrations to the standard curve following the manufacturer s instructions and run in parallel. the serum sample was diluted ten times with the sample dilution for the best detection range of the elisa kit, and further of the serum levels of homocysteine multiplied by 10. standard sample diluent (300 l) was added to each of the seven microtiter wells. diluent (300 l) was pipetted out from one tube to another tube sequentially. reaction wells were sealed with adhesive tape and incubated at 37 c for 90 min. biotinylated human homocysteine antibody liquid was prepared 30 min in advance. biotinylated human homocysteine antibody liquid was added to each well (100 l for each). reaction wells were sealed and incubated at 37 c for 60 min. enzyme - conjugate liquid was prepared 30 min in advance. enzyme - conjugate liquid was added to each well except the blank wells (100 l for each). color reagent liquid (100 l) was added to individual wells (also to the blank well) and incubated at 37 c. when the color of the high concentration of the standard curve became darker and the color gradient appeared, the hatching was stopped. color reagent c (100 l) was added to individual wells (also to the blank well) and mixed well. the intensity of the color was read over the elisa plate reader (synergy ht ; biotek, winooski, vt) at 450 nm. the calibration curve of the standard homocysteine was plotted against the homocysteine on the y - axis and the concentration on absorbance on the x - axis. the standard curve was drawn using professional curve software (curveexpert 1.3 ; hyams development ; madison, al) to analyze and compute the . the continuous variables of the study groups were compared with one - factor anova (anova). distribution of variables such as visual acuity, homocysteine, and vitamin b12 was skewed. therefore, log and antilog conversions were made to analyze these variables. for pair - wise comparison between the groups, the newman the discrete (categorical) variables were compared with a chi - square test. the logmar vision score of two groups (npdr and pdr) was compared with an independent student t test. all analyses were performed using statistica 6.0 software package (dell, inc ., round rock, tx). assay of homocysteine was performed using the human homocysteine enzyme - linked immunosorbent assay (elisa) kit (mybiosource, inc ., san diego, ca). the reagents in the kit were prepared following the standard protocol provided with the kit. human homocysteine standard samples were added to corresponding wells (100 l for each well), and the 0 nmol / ml well was filled with standard diluent. serial dilutions of the homocysteine standard were performed using 50, 25, 12.5, 6.25, 3.12, 1.56, and 0.78 nmol / ml concentrations to the standard curve following the manufacturer s instructions and run in parallel. the serum sample was diluted ten times with the sample dilution for the best detection range of the elisa kit, and further of the serum levels of homocysteine multiplied by 10. standard sample diluent (300 l) was added to each of the seven microtiter wells. diluent (300 l) was pipetted out from one tube to another tube sequentially. reaction wells were sealed with adhesive tape and incubated at 37 c for 90 min. biotinylated human homocysteine antibody liquid was prepared 30 min in advance. biotinylated human homocysteine antibody liquid was added to each well (100 l for each). reaction wells were sealed and incubated at 37 c for 60 min. enzyme - conjugate liquid was prepared 30 min in advance. enzyme - conjugate liquid was added to each well except the blank wells (100 l for each). color reagent liquid (100 l) was added to individual wells (also to the blank well) and incubated at 37 c. when the color of the high concentration of the standard curve became darker and the color gradient appeared, the hatching was stopped. color reagent c (100 l) was added to individual wells (also to the blank well) and mixed well. the intensity of the color was read over the elisa plate reader (synergy ht ; biotek, winooski, vt) at 450 nm. the calibration curve of the standard homocysteine was plotted against the homocysteine on the y - axis and the concentration on absorbance on the x - axis. the standard curve was drawn using professional curve software (curveexpert 1.3 ; hyams development ; madison, al) to analyze and compute the . the continuous variables of the study groups were compared with one - factor anova (anova). the kolmogorov distribution of variables such as visual acuity, homocysteine, and vitamin b12 was skewed. therefore, log and antilog conversions were made to analyze these variables. for pair - wise comparison between the groups, the newman the discrete (categorical) variables were compared with a chi - square test. the logmar vision score of two groups (npdr and pdr) was compared with an independent student t test. all analyses were performed using statistica 6.0 software package (dell, inc ., round rock, tx). table 1 summarizes the mean age, duration of type 2 diabetes mellitus, visual acuity (logmar), fasting and post - prandial blood glucose levels, serum levels of homocysteine, vitamin b12, and folic acid, average rnfl thickness, and central sub - field thickness in the study groups. no statistically significant difference in the mean age of the study groups was observed (f = 1.404, p = 0.248). the chi - square test revealed similar sex proportions among all four groups (male / female : 12/8 versus 12/8 versus 13/7 versus 10/10, 2 = 1.152 ; p = 0.334). a statistically significant difference in the mean duration of diabetes mellitus of the study groups was observed (f = 25.95, p<0.001). the level of significance was accepted at p<0.05. * a compared to control group, b compared to nodr group, c compared to npdr group. the mean glycated hemoglobin in the control, nodr, npdr, and pdr groups was 6.211.02% (33 to 56 mmol / mol), 6.390.41% (42 to 51 mmol / mol), 8.352.53% (40 to 95 mmol / mol), and 7.951.69% (45 to 82 mmol / mol), respectively. the difference in the glycated hemoglobin levels was statistically significant between the study groups (f = 8.95 ; p<0.001). pearson correlation analysis revealed a statistically significant correlation between glycated hemoglobin and homocysteine (r = 0.231 ; p=0.039), vitamin b12 (r = 0.363 ; p = 0.001), folic acid (r = 0.358 ; p = 0.001), and average rnfl thickness (r = 0.357 ; p = 0.001). the mean blood pressure (systolic/ diastolic) levels in the control, no dr, npdr, and pdr groups were 114/74, 126/78, 134/82, and 144/86 mmhg, respectively. in the comparison of visual acuity , anova revealed a statistically significant difference in visual acuity in each group (f = 47.192, p<0.0001). the anova showed that the difference in the serum levels of homocysteine was statistically significant between the study groups (f = 53.79 ; p<0.001 ; figure 2). the anova also showed that the difference in average rnfl thickness and central sub - field thickness was statistically significant between the study groups (p<0.001 and p<0.001, respectively). the serum levels of folic acid and vitamin b12 were observed to be within the normal reference range (220 ng / ml and 200900 pg / ml, respectively). box and whisker plot graphically representing the descriptive statistics for serum levels of homocysteine among the study groups. the horizontal line within the box indicates the median, boundaries of the box indicate the 25th- and 75th -percentile, and the whiskers indicate the highest and lowest values. an increase in serum level of homocysteine with increase in severity of diabetic retinopathy pearson correlation analysis revealed a statistically significant negative correlation between homocysteine with average rnfl thickness (r = 0.315 ; p = 0.004 ; figure 3 ; table 2) and a positive correlation with central sub - field thickness (r = 0.240 ; p = 0.032). scatter plot showing a negative correlation between the serum levels of homocysteine and the average retinal nerve fiber layer thickness observed on pearson correlation analysis. this study was conducted to determine the correlation between the serum levels of homocysteine, vitamin b12, and folic acid and alterations in rnfl thickness in cases of diabetes mellitus. a previous study by corra et al. found an association between a risk factor such as the duration of diabetes mellitus and the severity of retinopathy. in the present study, a statistically significant difference existed among the study groups in the comparison of the duration of diabetes. it was concluded that increased duration of diabetes mellitus is associated with the increased severity of diabetic retinopathy. in this study, visual acuity was found to decrease with the increase in the severity of retinopathy. homocysteine is a by - product of transmethylation reactions and is detoxified by methionine synthetase, which depends on vitamin b12 and folate as coenzymes for proper function. the role of homocysteine has been induced in several microvascular complications of diabetes mellitus, including diabetic retinopathy. found significantly raised fasting plasma levels of homocysteine in patients with diabetes mellitus with microalbuminuria or proliferative retinopathy that were not attributable to confounders, such as age, sex, smoking, or dissimilar plasma folate and vitamin b12 concentrations. several other studies also reported a statistically significant increase in the total plasma level of homocysteine and a statistically significant decrease in the serum levels of vitamin b12 and folic acid in diabetic retinopathy. our study demonstrated that the difference in the serum level of homocysteine was statistically significant between the study groups. although the mean serum levels of folic acid and vitamin b12 among the cases were found to be within the normal reference range, the values were on the lower side of normal. the mean serum levels of folic acid and vitamin b12 in the controls were found to be on the higher side of the normal reference range. it can be concluded that increased serum levels of homocysteine are associated with increased severity of diabetic retinopathy. the mechanism of action of homocysteine leading to retinal neurodegeneration has been studied in several animal models. several studies suggested that homocysteine induces apoptosis in retinal ganglion cells with the expression of bax (bcl-2-associated x protein), a proapoptotic protein, as it was found in increased levels in diabetic retinas. in addition, increased levels of terminal nick - end labeling positive cells that correlated with increased levels of caspases (the enzymes involved in apoptosis) in the neural retinas of diabetic rats have been found. ganapathy et al. concluded that homocysteine - induced ganglion cell loss involves the dysregulation of mitochondrial dynamics, in vivo and in vitro. disruption of glutamate homeostasis in the diabetic retina initiates the development of diabetic retinopathy. the major cause of neuronal cell death following glutamate - induced activation of n - methyl d - aspartate (nmda) receptors is generation of free radicals that induce apoptosis. therefore, strategies for decreasing the level of extracellular glutamate or inhibiting the activation of nmda receptors may decrease neurotoxicity and cell death. in vitro studies of retinal ganglion cells and in vivo studies in the brain , flash electroretinography, contrast sensitivity, color vision, and short - wavelength automated perimetry have demonstrated functional deficits in the neuronal component of the diabetic retina. our study for the first time provides clinical evidence of a correlation between increased serum levels of homocysteine and rnfl damage in an in vivo human diabetic retina. the difference in the average rnfl thickness levels was statistically significant between the study groups. increased severity of retinopathy was associated with decreased average rnfl thickness that can be attributed to increased levels of homocysteine. the severity of diabetic retinopathy has been reported to correlate with macular thickness parameters on sd - oct. mean macular thickness, retinal thickness, foveal thickness, and central macular thickness have been shown to correlate with the severity of diabetic retinopathy. a recent study we conducted demonstrated that central sub - field thickness is associated with the severity of diabetic retinopathy. of the present study are also in concordance with the findings of this previous research. the novelty of our study lies in the demonstration of a correlation between increased serum levels of homocysteine and in vivo retinal nerve fiber layer thinning in the human diabetic retina. a correlation between increased serum levels of homocysteine and increased severity of retinopathy was also found. the novelty of our study lies in the demonstration of a correlation between increased serum levels of homocysteine and in vivo retinal nerve fiber layer thinning in the human diabetic retina. a correlation between increased serum levels of homocysteine and increased severity of retinopathy was also found.

purposeto study the correlation between serum levels of vitamin b12, folic acid, and homocysteine and the severity of diabetic retinopathy and the correlation with retinal nerve fiber layer (rnfl) thinning on spectral domain optical coherence tomography (sd - oct).methodsin a tertiary care center based prospective cross - sectional study, 60 consecutive cases and 20 healthy controls in the age group of 4065 years were included. the eyes of the cases were divided into three groups according to early treatment diabetic retinopathy study (etdrs) classification: diabetes mellitus without retinopathy (n = 20), non - proliferative diabetic retinopathy with macular edema (n = 20), and proliferative diabetic retinopathy with macular edema (n = 20). the serum levels of vitamin b12 and folic acid were measured using a standard protocol. the serum homocysteine assay was performed using an enzyme - linked immunosorbent assay (elisa) kit. average rnfl thickness was measured using sd - oct. statistical analysis was used to assess the correlations between the study variables.increased severity of diabetic retinopathy was found to correlate with an increase in the serum levels of homocysteine (f = 53.79 ; p<0.001). the mean serum levels of vitamin b12 and folic acid were found to be within the normal reference range. a positive correlation was found between retinal nerve fiber layer thinning and serum levels of homocysteine (p<0.001).this study, for the first time, demonstrated a correlation between increased homocysteine with a decrease in rnfl thickness and increased severity of diabetic retinopathy.

, it was estimated that in the usa there were nearly 210,000 new cases of invasive breast cancer and more than 40,000 deaths. axillary node status is a major prognostic factor in early - stage disease, and this information is important for treatment. axillary involvement is found in 10%30% of patients with t1 (< 2 cm) tumours. this rate reaches 45% for small t2 tumours (2.13 cm) and 55%70% for larger tumours routine axillary lymphadenectomy adds the risk of lymphedema, sensory disturbances, and chronic pain. sentinel lymph node biopsy is a minimal invasive method of checking the potential nodal involvement. it is based on the assumption of an orderly progression of lymph node invasion by metastatic cells from tumour site. thus, the nodal basin is free of malignancy if the sentinel lymph node is not involved. patients with metastasis to a sentinel node would undergo either immediate or delayed completion lymph node dissection. randomized trials demonstrated a marked reduction of complications associated with the sentinel lymph node biopsy when compared with axillary lymph node dissection. in the almanac trial, more than 1,000 patients were randomized to undergo either axillary lymphadenectomy or sentinel node biopsy. lymphedema was present in 13% of the axillary lymphadenectomy group and in 5% of the sentinel node group 12 months after surgery. in 2005, guidelines from the american society of clinical oncology stressed that a multidisciplinary team should aim at a sentinel node identification rate of 85% with a false - negative rate of 5% or less in order to abandon axillary dissection. false - negative rate is the proportion of axillary node dissection, positive cases with a negative sentinel node at biopsy. false - negative cases may from massive involvement of the real sentinel node, a circumstance that interferes with the uptake of both radiocolloid and dye and lymph flow that goes to a node other than the true sentinel node. a meta - analysis of 69 trials with a total of 8,059 patients in whom sentinel node biopsy was followed by axillary dissection showed substantial variability in the performance of the technique throughout different centers. however, recent from large multi - institutional trials showed that all have achieved excellent identification rates, ranging from 93% to 97%, but that none achieved a false - negative rate lower than 5%. the lowest false - negative rates were obtained in the 2 studies in which preoperative lymphoscintigraphy and dual mapping during surgery were required. however, if only blue sentinel nodes are considered, the false - negative rate was 9.1%. clinical indications for this approach have been changing through the years and there is still a debate on some of them. many centers use sentinel node biopsy only in patients with a unifocal tumour smaller than 3 cm, whereas others have extended the application to patients with large t2 or t3 (> 5 cm) tumours, multifocal / multicentric carcinomas, or to patients who have received neoadjuvant chemotherapy. currently, the sentinel node biopsy procedure is recognized as the standard treatment for stages i and ii. in these stages , this approach has a positive node rate similar to those observed after lymphadenectomy, a significant decrease in morbidity and similar nodal relapse rates at 5 years. no significant differences on disease - free survival, overall survival, and local control of disease were seen in case of negative sentinel node. (i) pregnancy is no contraindication for sentinel node biopsy, but only for blue dye, and it has been demonstrated that the dose to the foetus from this procedure is negligible. (ii) the evidence regarding the safety of sentinel node biopsy is mainly based on studies including t1 and small t2 tumours only. however, in patients with larger tumours (t3-t4), the false negative rate has been similar and no increased axillary recurrence has been reported. (iii) multifocal breast cancer is defined as separate foci of ductal carcinoma more than 2 cm apart within the same quadrant, while multicentric breast cancer indicates the presence of separate independent foci of carcinoma in different quadrants. the prevalence of axillary metastases and false negative is higher in multifocal or multicentric tumours. however, the reported axillary recurrence rates are acceptable also in patients with multifocal or multicentric tumours. however, invasion is missed in up to 40% of patients in the preoperative diagnosis. therefore, sentinel node biopsy is recommended in patients undergoing mastectomy. in patients with breast conservation , sentinel lymph node biopsy can be performed as a second operation if invasion is detected in the surgical specimen. (v) palpable axillary nodes may be tumour negative in up to 40% of the patients. preoperative axillary ultrasound with fine needle aspiration cytology or core needle biopsy from the suspicious nodes is a widely accepted policy. in many units, sentinel node biopsy is performed also in patients with palpable nodes if negative in the preoperative diagnosis. (vi) internal mammary sentinel node detection rate is significantly affected by the depth of radiopharmaceutical injection. it is generally recognized that mapping of inner mammary chain requires deep injection (peritumoural or intratumoural) of radiotracer. with this approach, internal mammary chain sentinel nodes have been detected in about 30% of patients with breast cancer, of which about 60%90% could be harvested during surgery and 11%27% of them will have metastases. however, the significance of internal mammary sentinel node biopsy is under debate. there is evidence that mapping it leads to stage migration and modification of treatment planning with respect to radiotherapy and systemic therapy, but more evidence is necessary to support that it will improve the outcome of treatment and survival. a second sentinel node biopsy may be performed in patients with a local recurrence after breast conservation and negative axillary sentinel node biopsy. sentinel node biopsy can be performed in patients undergoing breast surgery due to a local recurrence after breast conservation in dcis. furthermore, plastic surgery with breast augmentation or reduction does not contraindicate the procedure. in prior excisional biopsy the lymph drainage is probably changed in patients who have undergone previous breast surgery (oncologic and nononcologic). extra - axillary drainage is identified more frequently in reoperative sentinel node biopsy than in former sentinel node biopsy. however, there are evidences that sentinel node biopsy performed in the area of previous breast biopsy do not affect the accuracy of the procedure. (vii) before neoadjuvant chemotherapy, sentinel node biopsy gives a more precise axillary staging, with more information about the nodal spread. but it may delay the beginning of the therapy, and two surgeries can be necessary. after neoadjuvant chemotherapy, the sentinel node biopsy may lead to an underestimation of the initial stage. on the other hand, pathologic complete response in the axilla can be achieved in up to 40% of the patients. available data show that there are no significant differences in the success rate of sentinel node biopsy according to clinical tumour size or clinical nodal status, and that the false - negative rate is not affected by tumour response to chemotherapy. despite this, the current controversy in this scenario lies on the question of axillary lymph node dissection after a positive sentinel node biopsy, mainly stimulated by the recent publication of the acosog - z0011 data. the sentinel node procedure uses a radiotracer, a blue dye, or both to find the node. these colloids allow sentinel node visualization with a gamma camera before surgery and intraoperative detection with a hand - held gamma probe. controversies exist with regard to the selection of agents, the size of the particles of the radiotracer, the optimal route for injection, time to scintigraphy and intraoperative detection, and whether or not extra - axillary lymph nodes should be considered as well. mariani et al. suggested that tc - labeled colloids with most of the particles in the 100 to 200 nm size range would be ideal for sentinel node biopsy in breast cancer. tc - labeled colloids of human serum albumin are often used in europe, tc - sulfur colloid is used in the united states (sometimes after filtration through a 0.1 or 0.2 mm membrane), and tc - antimony trisulfide in australia. there is no established difference between a 1-day protocol (same - day imaging and surgery) and a 2-day protocol. after an experience of almost 20 years, it is generally assumed that both deep and superficial injection approaches are valid techniques and may be complementary. in most early sentinel node studies, the tracer was injected around the tumour and such peritumoral injection was considered the gold standard against which all other mapping techniques were tested. many investigators have reported good using injection into the breast skin over the tumour or using a periareolar, subareolar, or even intratumoural injection. one clearly established advantage of deep injections (peritumoral / intratumoural) is its ability to also reveal extra - axillary drainage. on the other hand, superficial injection techniques (subdermal / areolar) provide a faster lymphatic drainage, yield more radioactive counts at the axillary sentinel nodes, and are independent of the palpable or nonpalpable nature of the tumour. hence, the tracer is not always transported to the same axillary node, regardless the injection site. however, if the goal is axillary staging only, a superficial tracer injection is preferable due to better visualization of axillary sentinel nodes. if the aim is to stage also the extra - axillary nodal basins, peri- or intratumoural injection should be applied. lymphoscintigraphy has been an essential component for the preoperative sentinel node identification in breast cancer. lymphoscintigraphy has the potential to both improve accuracy and reduce morbidity relative to gamma probe alone by providing the surgeon with a roadmap of lymphatic drainage and the location of sentinel nodes. to identify all sentinel nodes and avoid confusion with a stasis in a lymphatic vessel, with planar scintigraphy, combining 2 views may help prevent some sentinel nodes from being missed (figure 1). the functional information from spect can be combined with the morphological information from ct by applying both techniques in one session. the ing spect / ct fused images depict sentinel nodes in an anatomical landscape providing a helpful roadmap for surgeons. in recent years, spect / ct has been used in breast cancer patients with unusual or complex drainage. spect / ct can also detect hot nodes missed by planar imaging because of shine - through from the injection site or in overweight patients (figure 2). differentiating a true sentinel node from a secondary echelon node also, lymphatics of a tumor site can drain simultaneously to more than one sentinel node. lymphoscintigraphy is able to identify sentinel nodes in a majority of cases by acquiring early and delayed planar images. in current protocols, spect / ct is performed following delayed planar images (mostly 24 hours after tracer administration). however, it is necessary to specify the criteria for sentinel node identification on preoperative images. major criteria to identify lymph nodes as sentinel nodes are the visualization of lymphatic ducts, the time of appearance, the lymph node basin, and the intensity of lymph node uptake. following these criteria visualized radioactive lymph nodes may be classified as followsdefinitively sentinel nodes: this category concerns all lymph nodes draining from the site of the primary tumour through an own lymphatic vessel, or a single radioactive lymph node in a lymph node basin. highly probable sentinel nodes: this category includes lymph nodes appearing between the injection site and a first draining node, or nodes with increasing uptake appearing in other lymph node stationsless probable sentinel nodes: all higher echelon nodes may be included in this category. definitively sentinel nodes: this category concerns all lymph nodes draining from the site of the primary tumour through an own lymphatic vessel, or a single radioactive lymph node in a lymph node basin. highly probable sentinel nodes: this category includes lymph nodes appearing between the injection site and a first draining node, or nodes with increasing uptake appearing in other lymph node stations less probable sentinel nodes: all higher echelon nodes may be included in this category. the use of these categories to characterize radioactive lymph nodes is also helpful for clinical decision making. lymph nodes of the first two categories (definitively sentinel node or highly probable sentinel node) are the nodes recognized by the nuclear physician and that must be removed at the operation room by the surgeon. less probable sentinel nodes may sometimes be removed depending on the degree of remaining radioactivity measured by the gamma probe during the control of the surgical field. isosulfan blue is of greater use in the united states, and patent blue v in europe. data from nsabp b-32 and acosog - z0010 with isosulfan, and from almanac with patent blue v, showed that the overall risk of allergic reaction is close to 1% for both dyes, with an approximately 0.1% risk of severe reactions (grade iii). despite a risk of allergic reactions to blue dye, most teams favour the combinative mapping procedure. in one multicentric study, the false - negative rate was 17.7% if only 1 node was resected, 10% if 2, 6.9% if 3, 5.5% if 4, and 1% if 5 or more. these should not imply removal of multiple nodes for an optimal sentinel node procedure. careful palpation by the surgeon of the operative field is also required to identify any suggestive large, hard nonblue and nonradioactive nodes. during the last decade, intraoperative imaging devices have become available for clinical practice and can be used during surgery as they provide information that can be combined with data obtained with conventional gamma probes. however, since nonimaging probes are still the standard equipment for detection of radiolabeled tissue in the operating room, the role of intraoperative imaging is generally limited and constitutes an additional aid to the surgeon. intraoperative imaging with portable gamma cameras provides real - time imaging with a global overview of all radioactive hot spots in the whole surgical field. its position can be adjusted to also show sentinel nodes near the injection area, which can easily be missed when using the non - imaging probe. some authors have tried to clarify the added value of portable gamma camera in clinical practice. in fact, there is no consensus on the real need for an intraoperative imaging device to help detection of the sentinel lymph node. the usefulness of the portable gamma cameras in breast cancer patients lies on when no conventional gamma camera is available or in particular cases with extra - axillary drainage (intramammary and internal mammary chain nodes) or when the sentinel lymph node is located very close to the injection site. although the majority of these cases can be solved with the presurgical information provided by spect / ct, real - time images acquired with a portable gamma camera can be an alternative to hybrid imaging. on the other hand, the preoperative anatomical information obtained by spect / ct appears to lead to a more optimal use of portable devices for sentinel localization in the operation room. using an intraoperative imaging device implies the possibility to better planning the procedure and to monitor the lymphatic basin before and after removal of the hot nodes, so to verify completeness of lymph node excision. after excision of each lymph node, a new image is acquired and compared with the situation before excision. if focal radioactivity remains at the same location, it is concluded that another possible sentinel lymph node is still in place. thanks to novel technological possibilities, combining a spatial localization system and two tracking targets to be fixed on a conventional, hand - held gamma probe in new 3d visualization of the traditional acoustic signal of the gamma probe. in this regard, the most recent development is the system so - called free - hand spect, in which a continuous positioning system installed in the operating room is based on a fix - pointing device, on the patient's body and, respectively, on the hand - held gamma counting probe, thus permitting a virtual reconstruction in a 3d environment. the surgeon can easily check location and depth of the foci of radioactivity accumulation to be resected, and this 3d information may be further used for precise localization and targeting of the radioactive sentinel lymph node(s) and of tumour tissue. the device can ensure permanent assistance and transparent documentation of soft tissue removal during the intervention. on the other hand, the possibility of combining the current radiopharmaceuticals with other agents opens new fields to explore. in this regard , a radiolabeled nanocolloid agent has been combined with icg, a fluorescent agent, for sentinel node detection. in contrast to the use of a single - fluorescent agent, this bimodal tracer may allow the surgeons to integrate the standard approach based on radioguided detection with a portable gamma camera with a new optical modality based on fluorescent signal detection. this approach is being successfully applied in several malignancies and to localize sentinel nodes outside the axilla in breast cancer (figure 3). for all these new intraoperative modalities, the preoperative anatomical spect / ct acquisition remains essential and is the starting point for surgical planning. before sending for histological examination, any lymph node removed should be rechecked by the probe to demonstrate that they are radioactive. histopathological assessment of the sentinel lymph node is the golden standard procedure for the subsequent management of the conservative surgery in breast cancer patients. the sentinel nodes are assessed intraoperatively using imprint cytology, frozen sectioning, or both, and more thoroughly after the operation. the sensitivity of the intraoperative diagnosis in variable and many units do not adopt it at all. some molecular methods have been used previously for sentinel node diagnosis but have shown a lack of reproducibility, a longer time for the intraoperative assessment, and an inability to study the whole lymph node. a new molecular method has been developed recently, based on an one - step nucleic acid amplification (osna) method. this procedure is in the phase of validation in many centres, although there are others that routinely apply this method. in summary, after two decades of sentinel lymph node biopsy use in breast cancer, this technique is the current standard of care for locoregional staging. however, some concerns remain as there is no only one standardized technique and many controversies are still unsolved. however, with the recent technologic and histologic developments, a more accurate and refinate technique can be achieved by virtually identifying-100% of sentinel nodes and reduce the false negative rate.

axillary node status is a major prognostic factor in early - stage disease. traditional staging needs levels i and ii axillary lymph node dissection. axillary involvement is found in 10%30% of patients with t1 (< 2 cm) tumours. sentinel lymph node biopsy is a minimal invasive method of checking the potential nodal involvement. it is based on the assumption of an orderly progression of lymph node invasion by metastatic cells from tumour site. thus, when sentinel node is free of metastases the remaining nodes are free, too (with a false negative rate lesser than 5%). moreover, randomized trials demonstrated a marked reduction of complications associated with the sentinel lymph node biopsy when compared with axillary lymph node dissection. currently, the sentinel node biopsy procedure is recognized as the standard treatment for stages i and ii. in these stages , this approach has a positive node rate similar to those observed after lymphadenectomy, a significant decrease in morbidity and similar nodal relapse rates at 5 years. in this review, the indications and contraindications of the sentinel node biopsy are summarized and the methodological aspects discussed. finally, the new technologic and histologic developments allow to develop a more accurate and refinate technique that can achieve virtually the identification of 100% of sentinel nodes and reduce the false negative rate.

nonalcoholic fatty liver disease (nafld) has become a global health problem in both adults and children, the prevalence of which has doubled during last 20 years in tune with the increasing incidence of obesity and insulin resistance. improper regulation of lipogenesis and lipid oxidation is a prime factor in the genesis of fatty liver. lipogenesis encompasses the processes of fatty acid synthesis and subsequent formation of triglyceride (tg). sterol regulatory elemental binding protein-1c (srebp-1c) belongs to a family of transcription factors that are sensitive to cellular availability of cholesterol and promotes fatty acid synthesis and lipid deposition. srebp-1c regulates lipogenesis by increasing the expression of genes involved in fatty acid biosynthesis such as acetyl coa carboxylase, fatty acid synthase, and stearoyl coa desaturase. srebp-1c has been implicated in the development of human metabolic disorders like obesity, type 2 diabetes, dyslipidemia, atherosclerosis, lipodystrophy, and metabolic syndrome. lds, long considered as inert substances, are now recognized for their dynamic role in lipid metabolism. they are structurally similar to circulating lipoproteins, consisting of a core of esterified lipids (tg, cholesterol ester, retinol esters, or ether lipids, depending on the cell type) covered by a phospholipid monolayer, free cholesterol, and a coat of specific proteins. the ld proteins include the founding member perilipin (plin1), adipophilin (plin2, also known as adipocyte differentiation - related protein ( adrp) ), tail - interacting protein of 47 kilo daltons (tip47, plin3), s3 - 12 (plin4), and myocardial lipid droplet protein (plin5). these are collectively known as the pat family of proteins named after perilipin, adipophilin, and tip47. another protein termed fat - specific protein 27 (fsp27), although not a member of pat family proteins, is recognized to be a structural protein present in ld and is specifically involved in diet - induced fatty liver. ld proteins are involved in lipid metabolism and transport, intracellular trafficking, signaling, and cytoskeletal organization. ld proteins also regulate lipolysis in adipose tissue by modulating the access of hormone sensitive lipase (hsl) to lipid surface. the ld proteins also serve to regulate lipolysis of tg rich droplets in response to insulin and adrenergic stimulation. peroxisome proliferator - activated receptor- (ppar-) is a nuclear receptor protein and is the first member of ppar family cloned in 1990. among the three isoforms of ppar family-, , and , ppar- is predominantly present in liver and is involved in the activation of hepatic lipid catabolism by targeting genes involved in cellular fatty acid uptake and transport, mitochondrial and peroxisomal fatty acid uptake, and -oxidation. among the various endogenous ligands for ppar-, long - chain fatty acids are the most studied. thus ppar- serves as a lipid sensor and directs lipid metabolism towards oxidation when upregulated by agonists. grape seed proanthocyanidins (gsp) is a complex mixture of oligomeric compounds possessing high antioxidant activity with preventive effects on some forms of cancer and oxidative injury. proanthocyanidin is richly present in red wine, and its content in red wine is reported to be 117.18 96.06 mg the first clue to its lipid - lowering effect came from the french paradox, which refers to the lower mortality rates of cardiovascular disease despite high intake of saturated fats due to red wine consumption. in fact studies have shown that gsp can lower postprandial plasma tgs and apolipoproteins levels in healthy rats. the insulin - sensitizing agent metformin (met) is used as a treatment strategy in nafld patients due to the role of insulin resistance in the pathogenesis of nafld. since nafld is a multifactorial disease, combination therapy is recommended to address the individual components like hyperlipidemia and insulin resistance. we hypothesize that gsp may help to increase the oxidation of lipids by reducing the lipogenic pathway and lipid storage while met may improve the hepatic insulin sensitivity. hence, in this study, we compared the effects of gsp and met individually and in combination against diet - induced nafld. proanthocyanidin - rich extract from grape seed (gsp, gravinol - super) was kindly provided by kikkoman co. (chiba, japan). the grape seed extract contained 89% proanthocyanidin, 6% monomers, and 5% other materials. the normal rat feed was obtained from sai enterprise, chennai, india, which contained 60% starch, 22.08% protein, and 4.38% fat. this commercial diet provided 382.61 cal/100 g. the high - fat / fructose diet (hffd) prepared in our laboratory contained 45% fructose, 20% fat, (10% beef tallow, 10% groundnut oil), and 22.5% casein and provided 471.25 cal/100 g. this study was conducted in strict accordance with the guidelines of the committee for the purpose of control and supervision on experiments on animals (cpcsea). all procedures were approved and adhered to the guidelines of the institutional animal ethical committee (iaec). six - week - old male albino wistar rats (n = 6) were purchased from rajah muthiah medical college and hospital (rmmc and h, annamalai university) and left for acclimatization in the department animal house for a week. obesity was induced in rats by feeding hffd. therapeutic intervention with gsp or met or combination of both was initiated after 30 days of hffd feeding. gsp (100 mg / kg b.w / day in water) and met (50 mg / kg b.w / day in water) were administered orally once a day to the respective groups from the 31st day till the 45th day. for combination treatment, gsp and met were administered at an interval of 4 hours. at the end of the experiment, the rats were fasted for 18 h. after decapitation, blood samples were collected in tubes containing edta and centrifuged to obtain plasma. oral glucose tolerance test (ogtt) was carried out on the 44th day after an overnight fast (12 hours) as described elsewhere. for this, animals were given glucose (2 g / kg b.w, oral) after collecting fasting blood samples. additional blood samples were drawn after one hour and two hours by sinoocular puncture in heparinized test tubes and centrifuged at 3000 g for 10 minutes to separate plasma. plasma insulin was assayed using an enzyme - linked immunosorbent assay kit (accubind, monobind chemicals, ltd ., total lipids, extracted with chloroform - methanol mixture ( 2 : 1 ( v / v) ) from liver, were evaporated to dryness and used for the estimation. estimation of cholesterol, tg, and ffas in plasma and liver was carried out following procedures described earlier. high density lipoprotein cholesterol (hdl - c) was determined following the kit procedure (agappe diagnostics) and low density lipoprotein cholesterol (ldl - c) and very low density lipoprotein cholesterol (vldl - c) were calculated using the following equations: vldl (mg / dl) = tg/5; ldl (mg / dl) = total cholesterol(hdl - c + vldl - c). total rna was isolated from rat liver using the trizol reagent (invitrogen, carlsbad, ca, usa). rna concentration was assessed in a biophotometer (eppendorf, hamburg, germany) and reverse - transcribed using m - mulv - reverse transcriptase (fermentas) and oligo dt primers (invitrogen). cdna was quantified using spectrophotometer (eppendorf) and amplified using the maxima sybr green qpcr master mix (fermentas, pittsburgh, usa). amplification of target genes detailed in table 1 was performed using realplex mastercycler (eppendorf) using gapdh as endogenous control. the amount of target gene, normalized to gapdh and relative to a calibrator, was determined by the arithmetic formula 2 by the comparative ct method. histologic analyses of liver were performed after liver tissue samples were fixed at room temperature in 4% formaldehyde and embedded in paraffin. five - micrometer sections were mounted on glass slides, deparaffinized in xylol, and stained for hematoxylin and eosin to evaluate steatosis and inflammation. localization of lipids was performed by oil red o staining of frozen - liver sections, followed by counterstaining with hematoxylin for visualization of the nuclei. the statistical significance of differences between groups was determined by one - way anova followed by the duncan's multiple comparison tests. ogtt (figure 1(a) ) show a rise in glucose levels in hffd - fed rats by 2.8-, 2.9-, and 2.3-fold in plasma samples collected at 0, 60, and 120 min, respectively, after an oral glucose challenge compared to con. combination of gsp and met showed a normal response and an additive improvement towards glucose load. isi0,120 (figure 1(b) ), a measure of insulin sensitivity, assessed using the ogtt values, was significantly decreased in hffd - fed rats as compared to con rats. the values of isi0,120 were 2.13- and 2.45-fold, respectively, in gsp and met group compared to hffd. combination of gsp and met showed additive improvement in isi values (by 2.97-fold) compared to hffd. plasma and hepatic cholesterol, tg, and ffa levels are given in figures 2(a)2(c) and figures 3(a)3(c), respectively. hffd - fed rats showed increased cholesterol (2-fold in plasma and 2.3-fold in liver), tg (3-fold in plasma and liver), and ffa (2.6-fold in plasma and 2.5-fold in liver) compared to con rats. these were reduced effectively and significantly after treatment with gsp in plasma (by 29%, 49%, and 45% of cholesterol, tg and ffa, resp .) and liver (by 46%, 45%, and 45% of cholesterol, tg and ffa, resp .) compared to hffd - fed rats. met was not much effective compared to gsp although it reduced the lipids levels in the plasma (by 8%, 10%, and 10% in cholesterol, tg and ffa, resp .) and liver (by 25%, 29%, and 15% of cholesterol, tg and ffa, resp .) compared to hffd group. combination of met with gsp in hffd - fed rats showed marked improvement in plasma (by 27%, 44% and 41% of cholesterol, tg and ffa, resp .) and liver (by 40%, 38%, and 42% of cholesterol, tg and ffa, resp .) compared to hffd group of rats. plasma lipoproteins, namely, hdl - c, ldl - c, and vldl - c were analyzed, and the values are presented in figures 4(a)4(c). hffd - fed rats showed 55% reduction in hdl - c and 5.1- and 3-fold increase in ldl - c and vldl - c values, respectively, compared to control rats. compared to that of hffd - fed rats, 0.9- fold increase in hdl - c and 52% and 47% decrease in the ldl - c and vldl - c were observed during gsp treatment. met showed 37% improvement in hdl - c and 16% and 8% decrease in ldl - c and vldl - c, respectively, whereas the combination of met with gsp showed similar effects to that of gsp alone compared to hffd - fed rats (99% increase in hdl - c and 49% and 44% decrease in ldl - c, and vldl - c resp .). mrna expression of the lipogenic transcription factor, srebp-1c (figure 5(a) ), and the rate limiting enzyme in cholesterol biosynthesis and hmg coa reductase (figure 5(b) ), was found to be 6.4- and 4.5-fold, respectively, in liver of hffd - fed rats compared to con rats. this increase was attenuated significantly by gsp and met supplementation. with respect to con, the expression levels of srebp1c and hmg coa reductase were 3.7- and 3.2- fold in gsp and 5- and 3.8-fold in met, respectively. combination of met and gsp showed similar effects as that of gsp in mrna expression of srebp-1c and hmg coa reductase (see figures 5a and 5b in supplementary material available online at http://dx.doi.org/10.1155/2013/153897). mrna expression of the fat sensor and ppar- was found to be markedly lower (by 68%) in liver of hffd - fed rats compared to con (figure 6). + gsp, the expression was improved by 56% compared to hffd indicating improved fatty acid oxidation despite continuous hffd feeding. mrna expression of ppar- in met administered rats was improved by 37% compared to hffd which is less than that induced by gsp alone. in met and gsp treatment group, ppar- expression increased by 54% with respect to hffd group (see figure 6 in supplementary material available online at http://dx.doi.org/10.1155/2013/153897). increase in the mrna expression of genes specific to ld proteins like perilipin, adipophilin, tip47, and fsp27 by 3.5-, 7.8-, 4.1- and 6.4-folds, respectively, was observed during hffd feeding compared to con. however, treatment groups showed reduced expression of ld proteins, compared to hffd groups. the expression of perilipin, adipophilin, tip47, and fsp27 was 1.4-, 3.5-, 1.9- and 2.5-folds in hffd + gsp, and 2.5-, 5.5-, 3.5- and 4.9-folds in hffd + met with respect to con. in gsp + met group, the expression of perilipin, adipophilin, tip47, and fsp27 was 1.5-, 3.7-, 1.9- and 2.5-folds respectively, with respect to con (figure 7) (see figures 7a, 7b, 7c and 7d in supplementary material available online at http://dx.doi.org/10.1155/2013/153897). the extent of inflammation and steatosis was examined histologically in liver sections using hematoxylin and eosin staining. the representative photographs show (figure 8) severe micro- and macrovesicular fatty changes with dense perivascular inflammatory infiltration in tissues from hffd - fed rats (figure 8(b) ). upon gsp administration to hffd - fed rats, the inflammatory infiltration is reduced to a minimum with mild steatosis (figure 8(c) ). hffd + met group of rats show moderate amount of inflammatory infiltration with microvesicular degeneration of hepatocytes (figure 8(d) ). hffd + gsp + met group of rats show hepatocytes with marked reduction of inflammation and steatosis (figure 8(e) ). liver architecture of gsp - treated control rats (figure 8(f) ) appears normal as that of control rat liver (figure 8(a) ). figure 9 shows accumulation of lds in liver sections examined by oil red o staining. sections from liver tissue of hffd - fed rats show severe steatosis and ballooning degeneration of hepatocytes (figure 9(b) ). hffd + met showed moderate steatosis (figure 9(d) ), whereas hffd + gsp showed minimal amount of steatosis (figure 9(c) ). hffd + gsp + met showed hepatocytes with occasional areas of steatosis (figure 9(e) ). con (figure 9(a) ) and con + gsp (figure 9(f) ) show liver sections with normal lipid distribution. the present study examined whether the supplementation of gsp, met, or both could exert protective effects against hffd - induced obesity and nafld in wistar rats. the expression profiles of genes related to lipogenesis and fatty acid oxidation in liver were examined. the two, administration of gsp showed more favorable effect on hepatic steatosis in hffd - induced obese rats as compared to met.. altered plasma lipid profile and lipoprotein abnormalities like decreased hdl - c and increased ldl - c, and vldl - c were observed in hffd - fed rats. accumulation of tg, a chief matrix of the lipid droplets in hepatocytes, is accelerated under conditions such as insulin resistance or hypertriglyceridemia, if left without appropriate treatment. accumulation of intracellular lds in nonadipose tissues is recognized as a strong prognostic factor for the development of insulin resistance in obesity. a recent study has shown that reducing adrp and tip47 in the liver via antisense oligonucleotide treatment attenuated steatosis and improved insulin sensitivity and glucose metabolism in c57bl/6j mice fed with high - fat diet. knockdown of fsp27 in the ob / ob mouse liver partially improved the fatty liver suggesting that fsp27 plays a vital role in the development of liver steatosis. given the importance of pat family proteins in nafld, we investigated their status in hffd - fed rats, and in the treatment groups. in hffd rats we observed that gsp reduces the levels of the ld - specific proteins. also, considering the reduced tg content in liver and plasma and liver histology using oil red o, it is clear that gsp has the ability to reduce tg storage and ld formation. histological examination of liver using hematoxylin and eosin and oil red o staining showed increased accumulation of inflammatory cells and lds, with macrovesicular fatty changes and hepatocyte ballooning in liver of hffd group of rats. gsp administration to nafld rats reduced steatosis to microvesicular fatty changes and inflammatory cell infiltration whereas met showed reduced inflammation. however, in met + gsp group, the expected combined positive effects were not observed, the reason for which is yet to be explored. srebp-1c controls hepatic de novo lipogenesis (dnl) primarily by regulation of expression of genes involved in dnl and lipid homeostasis. therefore, this transcription factor is a favored candidate in experiments that investigate the role of hypolipidemic agents in the prevention of fatty liver disease. similarly, hmg coa reductase is an enzyme of much importance in lipogenesis since it catalyzes the rate - limiting step in cholesterol biosynthesis. increase in this enzyme indicates increased production of cholesterol. the observed increase in mrna expression of srebp-1c and hmg coa reductase in hffd - fed rat liver ppar- mediates -oxidation of fatty acids, and agonists of ppar- possess the property of reducing lipid dystrophy and obesity. ppar- activates some of the key enzymes of -oxidation and -oxidation of fatty acids in liver. multitude of studies have postulated that, during diet - induced obesity, fatty acid oxidation is left incomplete accompanied by mitochondrial lipid overload and dysfunction. gsp treatment reduced the levels of ffa in plasma and liver and also improved the expression of ppar-. the lipid regulatory mechanism of gsp on lipid metabolism is observed to be at the genetic level. additionally, gsp may improve cellular fatty acid uptake and mitochondrial function which needs to be studied in future. in , we have shown that gsp protects against hepatic steatosis in obesity by suppressing lipogenesis and promoting -oxidation in liver. gsp reduces ld formation by reducing hepatic tg content, regulates lipid biosynthesis, and promotes -oxidation through its effects on srebp-1c, ld proteins, and ppar- mrna expression. since nafld and insulin resistance are closely associated, we linked and studied the action of gsp along with met, an insulin sensitizer, on lipid abnormalities. although met showed significant restoration of lipid levels, comparative point out that gsp is more effective. histopathological examination provides good evidence for the lipid - lowering property of gsp whereas met does not show a strong action against lipid abnormalities. combination of met with gsp showed effective improvement in insulin sensitivity and better reduction in hyperlipidemia compared to met alone. the effects of met and gsp on the parameter studied are not additive during combination treatment, the reason for which is unclear at this stage. combination of drugs with different mechanisms of action has always been encouraged by clinicians in order to obtain the best . further studies are warranted to attest their putative positive effects of gsp and met on other components of nafld and to see if nafld can be dealt using the combination of these two drugs.

nonalcoholic fatty liver disease (nafld), a premorbid condition, lacks proper management owing to multitude of abnormalities. in this study, we compared the effects of a potent antioxidant, grape seed proanthocyanidins (gsp), and an insulin sensitizer, metformin (met), in high - fat - fructose - diet- (hffd-) induced albino wistar rat model of nafld. either gsp (100 mg / kg b.w) or met (50 mg / kg b.w) or both were administered as therapeutic options. hffd - fed rats showed abnormal plasma lipid profile, inflammation, and steatosis of the liver when examined by biochemical and histology techniques. increased lipid storage, lipogenesis, and reduced lipolysis were evident from mrna expression studies of hepatic lipid droplets (ld) proteins, sterol regulatory element binding 1c (srebp 1c), and peroxisome proliferator activated receptor- (ppar-). gsp administration to hffd - fed rats caused 69% reduction in hepatic tg levels, whereas met caused only 23%. the combination treatment reduced tg levels by 63%. gsp reduced the mrna expression of srebp1c and ld proteins and increased that of ppar- more effectively compared to met in hffd - induced hyperlipidemic rats. combination of met and gsp improved the metabolism of lipids effectively, but the effect was not additive in restoring lipid levels.

while the first reports of mammalian adult neurogenesis in the 1960s were met with little fanfare, the revival of the field in the 1990s incited both excitement and skepticism; until that time, it was accepted that after birth, the brain could only lose and not gain neurons. however, with the advent of new techniques to label dividing cells and distinguish neuronal from glial populations, evidence for adult neurogenesis accumulated throughout the 1990s and 2000s. it is now well established that the birth of new neurons in the adult brain occurs in two regions: the subventricular zone of the lateral ventricles and the hippocampus. increasingly sophisticated questions have since been asked, refining our image of how these newborn neurons are regulated and how they function. in this review , we will cover several themes that have emerged in the last few years in the study of adult hippocampal neurogenesis. these themes share a common goal of defining the unique contributions of adult - born cells, albeit at different levels of study: learning and memory, physiology, hippocampal anatomy, and comparative biology. both positive and negative alterations in neurogenesis affect discrimination between memories of recalled events and similar present events, also referred to as behavioral pattern separation. (b) young adult - born hippocampal neurons have unique physiology and circuitry that may contribute to pattern separation. although links are largely speculative, current hypotheses have attempted to link pattern separation to the period of hyperexcitability in newborn neurons. (c) the position of newborn cells within the dentate gyrus may alter their fate and function. differences in proliferation, maturation, and function of newborn neurons have been identified along the longitudinal axis of the dentate gyrus. (d) the function of new neurons in humans may be unique when compared with other mammals. differences in maturation rates, newborn neuron proportion, and anatomy between humans and animal models are now being recognized and studied. current inquiries into the functional significance of adult - born neurons have heavily emphasized the concept of pattern separation. pattern separation, a term appropriated from computational models of neural networks, has been loosely used to describe the process by which similar, but not identical, patterns of inputs are classified as distinct. at the behavioral level, these inputs reflect both external sensory input and internally generated representations of a previously experienced environment or event. by manipulating the rate of adult neurogenesis, several groups have shown that newborn neurons are critical for making fine discriminations between neighboring spatial locations or highly similar environments in tests of working memory and long - term memory. ablating adult neurogenesis via irradiation impairs performance on a touchscreen task in which mice must discriminate between two adjacent stimuli presented simultaneously in close proximity. the same irradiation treatment interfered with the recall of nearby arm locations after a brief delay (< 30 s) on a radial arm maze task. in contrast, an increase in rates of neurogenesis via running improves the performance on the touchscreen task. improving the survival of newborn neurons via a genetic strategy similarly enhances the ability to discriminate between two similar contexts during contextual fear conditioning, a distinction that arises over the course of several days of training. notably, the contribution of adult neurogenesis in each of these cases emerges only on more difficult discriminations. when difficulty is reduced by either increasing the spatial separation or making the contextual conditioning environments more distinct, both impaired and enhanced neurogenesis groups perform comparably to controls. these data support the hypothesis that the advantage provided by adult - born neurons grows as the overlap between two input patterns increases. newborn neurons that are 3 - 4 weeks of age are particularly distinct in function from mature granule cells (see below). optogenetic silencing of newborn cells at 4 weeks of age, but not at 2 or 8 weeks, impairs the retrieval of both previously learned spatial location on the morris water maze and of a previously learned fear - conditioned context. from these studies, it remains unclear whether these 4-week - old cells contribute solely to the retrieval of an existing memory or whether they also play a role in encoding. genetic suppression of mature granule cell activity, while leaving younger granule cells less than 4 weeks of age intact, also improves contextual fear discrimination of two similar contexts. conversely, this young cell - dominated dentate gyrus impairs performance on a water maze task requiring the opposite process pattern completion when navigation must be completed using only a subset of the cues present during training. this study suggests that a dentate gyrus network dominated by young granule cells is biased towards interpreting similar but not identical inputs as distinct, whereas older granule cells are biased towards interpreting similar inputs as equivalent. however, the behaviorally optimal amount of pattern separation versus completion will depend on the relevance of particular stimuli to the demands of the task and motivation of the individual. it is possible that a deficit in pattern separation does not automatically imply an enhancement of pattern completion but rather a global deficit in the detailed recall of a particular context. the physiological properties of newborn neurons and their effects on dentate gyrus circuitry provide some explanation for their time - limited role in pattern separation as described above. newborn neurons have a developmental time window during which they exhibit unique intrinsic and circuit - level properties. physiology and novel circuit tracing studies in mice have revealed details in newborn neuron development. days after birth, newly generated cells respond to ambient -aminobutyric acid (gaba) with tonic activation due to a high concentration of intracellular chloride that leads to depolarization. in 1 - 2 weeks after 2 - 3 weeks, they start expressing glutamatergic receptors, and subsequently, the direction of the chloride gradient switches such that gabaergic input hyperpolarizes newborn neurons. at around 1 month, new neurons receive synaptic glutamatergic input from the entorhinal cortex, similar to mature cells. however, at this time point, new neurons have a lower density of gaba inputs and inhibitory post - synaptic currents (ipscs) compared with mature granule neurons. once fully mature (about 6 weeks after birth), newborn neurons are essentially indistinguishable physiologically from developmentally born granule neurons. hence, at 4 weeks after birth, new neurons receive input from the same sources as mature granule cells but, because of their unique properties, they are hyperexcitable and show broader tuning than surrounding mature granule cells; young neurons are more likely to respond to two separate inputs than mature granule cells. the effect of this developmental time course is a period during which excitation dominates over inhibition. by contrast, inputs to fully mature granule neurons are biased towards inhibition, as they fire very infrequently. adult neurogenesis therefore serves to make the principal cells of the dentate gyrus physiologically heterogeneous and distinct subpopulations are constantly shifting: as one newborn neuron subpopulation matures, another develops to replace it. the physiological differences between mature granule cells and 4-week - old newborn neurons suggest at least two mechanisms for how neurogenesis may affect pattern separation and completion. one mechanism involves interpreting the activity of individual granule cells directly in response to two different inputs as being reflective of overall dentate gyrus activity. in this case, mature granule neurons, which require high input specificity and maintain a high activation threshold, potentially serve as good pattern separators. the hyperexcitability and broad tuning properties of newborn neurons would conversely suggest that they could serve as pattern integrators. however, this hypothesis is inconsistent with the behavioral evidence discussed above, in which increases in the newborn neuron population improve pattern separation. an alternative mechanism is to consider how local dentate gyrus interneurons may influence overall dentate gyrus activity. both young and mature granule cells stimulate local inhibitory interneurons either directly or indirectly via mossy cells in the hilus. those interneurons in turn also send efferents to granule cells but with a higher density of inhibitory inputs to mature as compared with young neurons (as described above). thus, by dampening the activity of mature granule cells, newborn cells increase their influence on the network. by this proposed mechanism, increased numbers of hyperexcitable neurons paradoxically make the dentate gyrus sparser, supporting a role for newborn neurons in enhancing pattern separation function. while the evidence for a role for newborn neurons in pattern separation is accumulating, we are also beginning to appreciate that this function depends on the location of new cells within the dentate gyrus. the dentate gyrus can be anatomically distinguished along the dorsal - ventral (septo - temporal) axis, and it is becoming increasingly recognized that newborn neurons arising from the dentate gyrus may be functionally distinguished along this axis. differences in the generation and maturation of adult - born neurons appear along the dorsal - ventral axis. new neurons in the dorsal dentate gyrus may mature significantly faster than in the ventral dentate gyrus because of higher levels of basal local network activity in the former. these distinctions might arise from the region - specific variation in cortical and subcortical connections to the hippocampus. a newborn neuron's maturation time and therefore its unique properties is linked with its position within the dentate gyrus. an additional distinction of newborn neuron function may be related to the idea that the dorsal hippocampus is involved in spatial learning and memory, whereas the ventral hippocampus has been linked with emotional behavior. with these associations , it has been proposed that a cognitive - specific task or a stimulus with emotional valence would selectively affect neurogenesis in the dorsal and ventral hippocampus, respectively when adult rats learned a spatial water maze task, enhanced neurogenesis was observed in the dorsal, but not ventral, hippocampus, as predicted. in probing the activation of granule cells after water maze learning, c - fos expression was higher in the dorsal than ventral hippocampus when considering the overall granule cell population, but among the adult - born subpopulation alone, c - fos expression was higher in the ventral hippocampus. acute stress or administration of corticosterone in adult rats was predicted to alter neurogenesis in the ventral hippocampus, when, in fact, neurogenesis increases were observed selectively in the dorsal hippocampus. therefore, although there is evidence for cognition primarily involving the dorsal hippocampus and emotional behavior utilizing mainly the ventral hippocampus, the links with neurogenesis require further refinement. collectively, these studies highlight the idea that newborn neurons can be born at the same time yet mature differently, and/or subserve unique functions depending on their position along the long axis of the dentate gyrus. the nature and consequence of having variability within a newborn neuron population along the dorsal - ventral axis are usually not reported and warrant re - evaluation of previous studies and further attention in future studies. while we have known for 15 years that adult neurogenesis takes place in the adult human, most of our understanding of mammalian adult neurogenesis comes from studies of rodents. therefore, it has been tempting to view our knowledge of rodent adult neurogenesis from an anthropocentric viewpoint. however, recent adult neurogenesis studies in primates and humans are reshaping ideas of how new neurons may function within us. as suggested from evaluating non - human primates, the developmental timeline of newborn neurons in humans may be much longer than previously estimated. two - thirds of brdu cells continued to express the immature neuron marker doublecortin (dcx) at 6 months in an adult macaque hippocampus, whereas dcx expression in the dentate gyrus neurons in an adult rodent lasts only several weeks. a report by spalding, frisen and colleagues confirmed that neurogenesis continues to occur in the adult human hippocampus and to a much greater extent than previously estimated. they showed that hippocampal neurogenesis in adult humans is robust and continues through at least the fifth decade of life. while these data suggest that the age - related decline in adult neurogenesis is significantly slower in humans than in rodents, a direct comparison requires further validation because of the impoverished and sedentary environment of rodents reared under typical laboratory conditions. an emerging idea is that newborn neurons may be playing significantly larger roles in hippocampal function and human cognition than previously thought. previous models utilized rodent neurogenesis data for proposing computational roles for newborn neurons in learning and memory. given that the study by spalding and colleagues suggests that most human dentate gyrus neurons are subject to turnover compared with 10% of mouse dentate granule neurons, new models may be required for predicting the contribution of adult - born neurons in human hippocampal function. the next leap in understanding the role of newborn neurons in human health and disease will require studying hippocampus - dependent behaviors in humans. development of novel imaging techniques to visualize newborn neuron activity in the dentate gyrus of humans would lead to breakthroughs. one example of a funding initiative that would encourage development of necessary technologies is the recently announced brain research through advancing innovative neurotechnologies (brain) funding initiative by the obama administration, which encourages development of these transformational techniques. about 20 years ago, the dogma that no new neurons are born in adulthood began to crumble. the advances in the last few years have shown why neurogenesis may be important (theme 1), revealed a mechanism by which it may work (theme 2), suggested that newborn neuron function is heterogeneous along its axes (theme 3), and hinted at how what we know may be modified in the case of humans (theme 4). although these themes may at first appear to concern separate aspects of neurogenesis, it could be that the inter - relationships between these themes will be critical for understanding the importance of newborn neurons in humans. for example, the longer developmental time window in humans compared with rodents suggests a relatively prolonged state of hyperexcitability in newborn neurons. this extended period may impact the human dentate gyrus circuitry such that pattern separation may function qualitatively or quantitatively differently in humans than in rodents. does the presence of a different / larger ventral dentate gyrus have implications for whether newborn neurons function differently there? and , as noted, the larger scale of human adult neurogenesis may mean that we need to modify our computational models. recent work has sharpened our view of the regulation and function of adult hippocampal neurogenesis. new techniques and advancements will literally and figuratively present a progressively higher - resolution image of this striking example of neural plasticity.

the birth of new neurons in the adult mammalian brain once thought impossible is now a well - accepted phenomenon that takes place in the subventricular zone of the lateral ventricles and the hippocampus. this review focuses on the recent work that has sharpened our views of how hippocampal newborn neurons are regulated and function. areas of study include (a) how neurogenesis contributes to behavioral pattern separation, (b) how pattern separation may be influenced by the properties and circuitry of newborn neurons, (c) differences along the dorsal - ventral axis of how neurogenesis is regulated and functions, and (d) adult neurogenesis in primates, including new human data. these current avenues of research reveal new details of adult neurogenesis and foreshadow what we may learn about this exciting phenomenon in the near future.

the prevalence of chronic kidney disease in the adult cystic fibrosis (cf) population is estimated to be between 27 and 42%. there are a number of cf - associated factors contributing to renal impairment in this population, including aminoglycoside toxicity, diabetic nephropathy, non - steroidal anti - inflammatory drug toxicity, renal amyloidosis, nephrocalcinosis and iga nephropathy. despite the multitude of factors impairing renal function in the cf population, we present a case of a potential novel cf renal phenotype in this cohort of patients. in september 2005, a 32-year - old male patient with cf (f508/f508) presented to the cork adult cystic fibrosis centre with a 6-month history of peripheral oedema. past medical history was otherwise unremarkable with infrequent previous infective pulmonary exacerbations and no courses of intravenous antibiotics over the previous 10 years. there was no past history of hypertension, smoking, hearing impairment or diabetes mellitus. forced expiratory volume in 1 second (fev1) % predicted was stable at 57% predicted (2.36 l / min) and serum (creatinine) was 169 a spot urine protein - to - creatinine ratio revealed severe proteinuria at 691 mg / mmol (estimated at 6.1 g protein/24 h) with an associated hypoalbuminaemia (24 the patient repeatedly had normal oral glucose tolerance tests ( ogtt) and normal haemoglobin a1c (all < 6.2%). findings were consistent with a nephrotic syndrome, and treatment with an angiotensin - converting enzyme inhibitor and natriuretic agent was instigated and a renal biopsy performed. renal biopsy findings revealed nodular glomerulosclerosis (ngs) (figure 1a), identical to the classic kimmelstiel congo red staining for amyloid was negative, immunofluorescence was negative for immunoglobulin deposition (polyclonal, igg, iga, igm, c3c) and electron microscopy excluded fibrillary glomerulonephritis, amyloidosis and monoclonal immunoglobulin deposition disease. (a) haematoxylin and eosin - stained section demonstrating increased mesangial matrix with kimmelstiel wilson - like mesangial nodules. (c) periodic acid methenamine silver - stained section again highlighting the nodular lesion. (d) electron microscopy of glomerulus demonstrating absence of amyloid fibrils and absence of evidence for monoclonal immunoglobulin deposition disease and fibrillary glomerulonephritis. despite standard conservative management, he remained proteinuric, with a progressive deterioration in estimated glomerular filtration rate. an intercurrent growth of a mucoid strain of pseudomonas aeruginosa led to the initiation of alternate monthly nebulized tobramycin 300 mg bis in die (bid) in july 2007 at his annual assessment, in keeping with best international standards of care for patients with cf. this led to a sharp decline in his renal function, reverting back to a steady baseline rate of renal function decline on discontinuation of same after september 2007 (figure 2). no other changes to his therapies were made around this period and he was not on any other nephrotoxic medications. it is anticipated that he will require renal replacement therapy over the next 6 months and has been prepared for pre - emptive renal transplantation. creatinine over time plot demonstrating the progressive decline in renal function and intercurrent reversible dip following the temporary initiation of nebulized tobramycin 300 mg bid in july and september 2007 (circled area). this is the first reported case of ngs in a cf patient without diabetes mellitus in a european population and highlights the importance of multidisciplinary consultation in this cohort of patients. the differential diagnosis for proteinuria in cf has, until recently, been predominantly limited to the identification of diabetic nephropathy, renal amyloidosis, iga nephropathy, fibrillary glomerulonephritis and immunoglobulin deposition disease. historically, the presence of ngs on renal biopsy was considered pathognomonic of diabetic nephropathy, designated the kimmelstiel wilson lesion. ngs - like lesions have subsequently been described in a wide variety of conditions, most prominently amyloidosis and fibrillary glomerulonephritis. our findings are consistent with a previous australian report describing ngs in cf patients in the absence of diabetes mellitus and other recognized causes of ngs, supporting the existence of a cf - related ngs. all patients described to date are male cf patients, > 30 years of age, presenting clinically with nephrotic syndrome in the setting of repeatedly normal ogtts. interestingly, all patients progressed towards end - stage renal disease over the subsequent 4 years (personal communication with authors). progressive renal failure is of increasing importance for patients with cf given that a creatinine clearance < 50 ml / min/1.73 m is frequently viewed as a contraindication to lung transplantation. it is postulated that the pathogenesis of cf - related ngs in normoglycaemic, non - diabetic cf patients is similar to that of classic diabetic ngs, and may be mediated by specific binding to the receptor for advanced glycosylation end products (rage). in cf, chronic pulmonary infection / inflammation, in combination with reduced glutathione levels, contributes to an oxidative state; this oxidative state could in the formation of advanced glycosylation end products (age) in conditions of normoglycaemia. patients with cf have a number of other potential mechanisms for rage activation in the absence of hyperglycaemia, including an increase in serum levels of the s100/calgranulin pro - inflammatory cytokines. there is also emerging evidence that high - mobility group box 1 (hmbg-1), a potent inflammatory mediator known to be elevated in the serum of patients with cf, can cause an epithelial to mesenchymal transition; this has previously been shown to contribute to the renal accumulation of matrix protein by its action on rage. we speculate that the pathogenesis of cf - related ngs is consequent to ligand binding of rage by s100/calgranulin, hmbg-1 and age. additionally, this is the first reported case of nebulized tobramycin - induced toxicity in a patient with cf with pre - existing chronic kidney disease. it is important to highlight the rapid decline in renal function on administration of nebulized tobramycin to our patient (figure 2). previous studies in non - cf patients with renal impairment describe nephrotoxic serum trough tobramycin levels up to 2.5 g / ml following the administration of nebulized tobramycin 300 mg bd. another study reports acute renal failure secondary to nebulized tobramycin in a patient with cf without pre - existing renal disease. the bioavailability of nebulized tobramycin is known to vary widely between patients, which is related to a number of factors including incomplete delivery of the aerosolized drug to the lung, possible systemic absorption from the oropharynx, the method of nebulization used and the pre - existing renal function. we suggest that trough serum level monitoring should be considered for patients with pre - existent renal disease. in summary, this report highlights the importance of renal biopsy in proteinuric renal disease in adult cf patients. as the management of patients with cf continues to improve, the increasing life spans may lead to an increased identification of proteinuric renal disease, the aetiology of which can include cf - related ngs. in addition, the potential for nephrotoxicity secondary to nebulized tobramycin in patients with pre - existing renal dysfunction is highlighted. it may be that, in these patients, monitoring of serum levels of tobramycin post - nebulization is required.

cystic fibrosis (cf) is a multisystemic disease but without a classical disease - specific renal phenotype. a 32-year - old male patient with cf (f508/f508) presented with a nephrotic syndrome. renal biopsy revealed nodular glomerulosclerosis (ngs) occurring in the absence of diabetes mellitus, amyloidosis and any other known common cause of ngs. he had a progressive decline in estimated glomerular filtration rate (egfr) to chronic kidney disease stage v (egfr < 15 ml / min/1.73 m2) over a 3-year period despite optimal medical management. this is the fourth reported case of ngs in a patient with cf without diabetes and is the first to originate from a european country. this case supports the concept of a cf - related ngs.

different ways exist to conceptualize executive functioning. for research that has applied latent statistical models, the focus is on classification and documenting both the diversity and unity of efs. although related, efs can be classified into three categories, including (i) working memory and updating, (ii) executive attention or cognitive control, and (iii) inhibition (e.g., miyake et al ., 2000 ; friedman et al ., 2006 ; miyake and friedman, 2012). but the three aspects also form an integrated mechanism for processing information. this broader mechanism underlies the ability to manipulate and maintain tasks, plans, and goals (i.e., their mental representations) in an active state while monitoring performance and inhibiting distracting stimuli, whether from the environment or internally (kane and engle, 2002). executive functioning is a central topic in many areas of psychology (posner and rothbart, 2007). various theoretical frameworks emphasize that while some cognitive processes can run efficiently on limited cognitive resources, many others engage executive functioning. within social psychology, efs are important for a wide array of sub - fields that inform the nature of social behavior, including persuasion, attitudes and prejudice, social perception, self - control, and emotion regulation, for example (e.g., smith and decoster, 2000 ; von hippel, 2007). in all these areas of social functioning, automatic processes are thought to help create a first - pass, working model of the event (e.g., perceiver readily infers a speaker seems competent due to style of dress). assuming the perceiver is motivated and has the requisite cognitive resources (efs), they then can integrate more information about the event to enrich the working model and arrive at a deeper social understanding (for reviews, see chaiken and trope, 1999 ; smith and decoster, 2000). one central question about efs in day - to - day life is their malleability. except for changes across the lifespan (e.g., von hippel, 2007) , people s executive functioning has long been viewed as relatively static perhaps because of robust individual differences in efs. however, starting with recent reports of successful cognitive interventions, this view has begun to change and now efs are seen as much more open to both short- and long - term training, warm - up, and exhaustion effects. research has shown, for example, that working memory training not only leads to improvements on distinct measures of executive functioning but also to transfer effects ing in improvement on measures of fluid intelligence (e.g., jaeggi et al ., 2008, 2011). in young children , cognitive training has been shown to improve subsequent attention control (e.g., thorell et al ., 2008). other findings indicate efs benefit from meditation training (e.g., tang and posner, 2009). however, some of the most intriguing evidence comes from research showing that social engagement enhances performance on standard ef tests. this is important since the majority of people s life takes place in the social world it has long been argued that communicating with others, taking others perspectives, and following social rules sets the stage for the development and maintenance of efs, thus helping structure general mental functioning (e.g., vygotsky, 1978 ; buttelmann et al ., 2009). as previously mentioned, the developmental literature has long linked efs to performance on some aspects of tom (hughes and ensor, 2007). selection pressures related to the complex, dynamic and mixed - motive nature of social life have also been posited to partly underlie the evolution of primate intelligence (e.g., jolly, 1966 ; humphrey, 1976 ; dunbar and shultz, 2007). there also are intriguing suggestions regarding the overlap of the neural underpinnings of ef and social reasoning processes (von hippel, 2007 ; adolphs, 2009). and there are now several reports of positive correlations of some brain structures with social network size. for example , amygdala size correlates with the size and complexity of real social networks (bickart et al ., 2011), whereas gray matter correlates with the number of facebook friends (kanai et al ., 2011). more impressively, it appears that, at least in macaques, social network size causally influences brain structure and function (sallet et al ., 2011). with humans, much cross - sectional and longitudinal research has found positive relationships between social participation / engagement and executive functioning and related mental health outcomes (e.g., fratiglioni et al ., 2004). some findings with distinct populations thus, for example, a vygotskian based curriculum (tools of the mind) which requires much social interaction and taking others perspective boosted not only preschoolers social skills but also their executive functioning (e.g., diamond et al ., 2007). although most of the available work examining the effects of social engagement relies on correlational designs, a few studies are beginning to show positive causal effects of social processes on executive functioning, and the most direct evidence comes from social psychological research investigating the effect of on - line social interaction on executive functioning. in one experiment , we had participants interact by having a discussion of a social topic (ybarra et al ., 2008). participants were given 10 min total, with a few minutes to prepare for the discussion. following the interaction the participants evaluated the activity and then completed tests of cognitive functioning, in particular a test of working memory which is a critical component of executive functioning. we also tested participants assigned to either an intellectual activities condition or a control condition that involved watching a 10-min video. another aspect of this mental exercising through simple socializing research that should be highlighted is that participants assigned to the video condition were presented with social content involving several human characters that were engaged in social interaction (ybarra et al ., 2008). in a sense, the video content represents a rich stimulus compared to many of the stimuli used to study social cognitive processes from a neuroscientific perspective. still, the video condition did not in any boosts to efs; it was the real, on - line social interaction that ed in ef boosts, which were equivalent to those ing from engaging in difficult intellectual activities. thus, benefits to efs are selectively seen in on - line social cognition, where participants dynamically construct a model of another person, and less in off - line social cognition where participants deal with static or impoverished and less engaging representations of others. regarding the above research, the argument could be made that the discussion that comprised the social interaction was atypical in some way, in that participants had to take positions, make arguments, and discuss their point of view. but as recent theorizing on the evolution of reasoning suggests, this aspect of social interaction evaluating arguments and proffering others may have been an important pressure on the evolution of our distinctive human cognition (mercier and sperber, 2011). further, as we will discuss later, recent research indicates ef boosts also can from basic get - to - know - you interactions if structured in a particular, mind - engaging way (ybarra et al . so the positive effects on efs can occur from other types of on - line social interactions . however, evidence also exists for the detrimental effects of some social interactions on efs . most of these findings come from research on intergroup social interactions ( e.g., richeson and trawalter, 2005). in one study, for example , participants who underwent an interracial interview compared to a same race interview subsequently exhibited more interference on a stroop task, a measure of ef. further, this effect was greater the stronger participants associations between concepts denoting african - americans and negative personality characteristics, as measured with the iat test. studies found that after interacting with an attractive female, men showed worse performance on a subsequent ef task, and the decline was related to the degree men tried to manage their impression in the interaction (karremans et al ., 2009). interactions can in reduction of cognitive functioning (finkel et al ., 2006). these investigators had participants coordinate on a task with a confederate, who in some cases made scripted errors (high maintenance) and in some cases did not (low maintenance). participants in the high maintenance condition performed worse on various subsequent tests, although in this research the tests were indirectly tied to efs. note that most, if not all, studies showing ef reductions invoked self - presentation concerns (e.g., about appearing non - prejudiced, unintelligent, etc .). first, it constitutes a working memory load (what was the interaction about ? second, it triggers effortful attempts to self - regulate, which, if extensive, could deplete cognitive resources ( muraven and baumeister, 2000). at a broader level this reasoning also applies to some of the findings dealing with performance decrements under conditions of stereotype threat, for example. in our opinion , however, one key difference between studies showing reductions and boosts in efs has to do with the nature of the interaction. in particular , it is critical whether participants mentally engage with others and attempt to build a rich model of their minds, that they toggle between self and other perspectives, and that they communicate and create meaning during the social interaction versus disengage from the interaction. we argue that this is often, though not always (see below), determined by whether the interaction is cooperative or competitive. however, the default in a cooperative setting is often to engage with the other person, build a model of their mind, figure out whether or not they are trustworthy, and convey to them that they can trust us. in fact, under these trust - building conditions neuroimaging work has reported some of the most robust effects of mentalizing on activity in the medial prefrontal cortex (for reviews, see frith and singer, 2008). further, children s ability to mentalize (i.e., perform tom tasks) has been shown to be positively correlated to the likelihood of cooperating, for example, in prisoner s dilemma games and also to making fair offers in the ultimatum game (sally and hill, 2006 ; also see buttelmann et al ., 2009). in contrast, the default under competitive goals, as realized in the above research on intergroup interactions, is often to become self - protective and withdraw from engaging the other person. this may occur because the interaction is ambiguous and not well structured, which inclines people to back away from the situation as a general way of deterring interpersonal costs (ybarra et al . this may also occur because people have a tendency to dehumanize outgroups and thus attribute less mind to their competitors ( harris and fiske, 2006). finally, it is possible that competition (but also other high - stake situations) may sometimes usurp the cognitive resources necessary for careful and effortful mentalizing. so, people focus on themselves and their own interests rather than the other person, and they do not engage in attempts to take perspective and create a rich model of the other and the event. a key aspect then of whether or not social interaction creates subsequent ef boosts rests on people engaging each other. we propose that doing so invokes processes that exercise underlying efs, such as working memory and executive attention. thus, when people engage with others in social interaction, versus withdraw into themselves, they can exercise or warm - up these core cognitive processes, whose influence is then transferred (far) to executive functioning tasks (ybarra et al ., if such social cognitive processes underlie the cognitive boosts, disrupting them should eliminate the cognitive benefits . consistent with this idea, our recent experiments found that interaction goals ( competition) that disengage participants from perspective taking and mentalizing eliminate the cognitive benefits that can from social interaction (ybarra et al . importantly, follow - up work has shown that getting people to engage others during interaction, even when the interaction is competitive, helps counter the loss in cognitive benefits ( ybarra et al . this is consistent with other research in social cognition suggesting that skepticism, suspiciousness, and other competitive approaches can sometimes improve mental performance ( schul et al ., 2004, 2008). these findings provide evidence that engaging the other during interaction along with concomitant social cognitive processing (perspective taking, mind - reading) may partly underlie the boosts to executive functioning following social interaction. the above findings also help inform, at least in some small measure, the assumption that competition in social contexts played an important role in the evolution of primate cognition and the more intense varieties of social cognition and mentalizing (e.g., byrne and whiten, 1988 ; whiten and byrne, 1997). dealing with competitors can of course implicate the understanding of others behavioral tendencies and psychological states (e.g., tomasello et al ., 2003 ; decety et al ., 2004), but as we have shown in our experiments, people who expect to compete during social interaction, if not given an explicit goal to read the other person and form a model of what they are like, will disengage and behave evasively (ybarra et al ., 2010) and not receive cognitive benefits from the interaction (ybarra et al ., 2011). these findings suggest that people when competing have diverse options they can undertake, such as to try to hide or foil prediction. only when no, low - risk option is available will they engage or confront the opponent. this, however, does not mean some level of social understanding is not sought or created under competitive circumstances, but it may be that the working model of the other is of a generic, stereotypic nature that relies less on efs, similar to the social perception differences found when people judge members of outgroups versus ingroups (neuberg and fiske, 1987 ; brewer, 1988). when interdependence is called for meaning you have to interact on - line with a person and that your behavior is to some degree yoked to theirs the situation should instigate more intense social cognition and mentalizing to build a richer model of the other party. the discussion of competition helps highlight the role we assign to mentalizing and understanding others psychological states during social interaction. we propose that mental engagement with others leads to cognitive benefits from social interaction especially when the involved parties are taking perspective and dynamically building a model of what the other person is like. as we noted in the introduction , some social inferential processes can occur quite efficiently with little reliance on efs (e.g., winter and uleman, 1984 ; trope, 1986). a similar theme comes up in research on theory of mind, with certain processes (e.g., the calculation of what another sees) thought to be carried out efficiently and automatically (e.g., moll and tomasello, 2006 ; onishi and baillargeon, 2005 ; qureshi et al ., 2010). related ideas on the role of efficient social understanding also have been discussed from the point of view of embodied and situated social cognition (leudar et al ., 2004 ; however, some tom and mentalizing processes such as the selection of information for further processing, though, are thought to require limited cognitive resources or ef ( e.g., leslie et al ., 2005 ; bull et al ., 2008 ; qureshi et al 2010), and engagement with others in social interaction is considered the real domain in which minds are known (reddy and morris, 2004). thus, there are important elements of building a model of what another person is like, what they are thinking, and what they might do next that rely on efs. our proposal is that during real social interaction both low - level and high - level mentalizing and behavior prediction processes interact and inform each other. the automatic processes serve as input that feed into richer representations that are shaped and updated by processes requiring limited cognitive resources in real time. as noted with competition, it is not that people who are dealing with antagonistic parties fail to attempt to understand their foes but it is during social interaction when the parties are actively engaged with each other that richer representations of the other and of the interaction are created and dynamically updated, which entails the participation of efs. in addition to the issues discussed above, further work is needed to address the following questions: what specific types of social interactions benefit ef and what are the underlying processes that underlie theses boosts? given both the unity and diversity of efs, does social interaction affect some ef elements more than others, or does this also depend on the type of interaction? how long do cognitive boosts last and what is there time course, and does this depend on the task to which the cognitive processes are applied? are the neural correlates underlying on - line social interaction, in particular those underlying efs, similar or different from the correlates unearthed for off - line social interaction? how do short - term training effects of social interaction translate into long - term cognitive reserve, which may be captured in the cross - sectional and longitudinal studies on social engagement and cognitive functioning? and , what are the longer - term consequences of repeated interactions that in ef reductions? if people do not avoid others different from the self, can they learn to cope with such challenging interactions? it has long been argued that communicating with others, taking others perspectives, and following social rules sets the stage for the development and maintenance of efs, thus helping structure general mental functioning (e.g., vygotsky, 1978 ; buttelmann et al ., 2009). as previously mentioned, the developmental literature has long linked efs to performance on some aspects of tom (hughes and ensor, 2007). selection pressures related to the complex, dynamic and mixed - motive nature of social life have also been posited to partly underlie the evolution of primate intelligence (e.g., jolly, 1966 ; humphrey, 1976 ; dunbar and shultz, 2007). there also are intriguing suggestions regarding the overlap of the neural underpinnings of ef and social reasoning processes (von hippel, 2007 ; adolphs, 2009). and there are now several reports of positive correlations of some brain structures with social network size. for example , amygdala size correlates with the size and complexity of real social networks (bickart et al ., 2011), whereas gray matter correlates with the number of facebook friends (kanai et al ., 2011). more impressively, it appears that, at least in macaques, social network size causally influences brain structure and function (sallet et al ., 2011). with humans, much cross - sectional and longitudinal research has found positive relationships between social participation / engagement and executive functioning and related mental health outcomes (e.g., fratiglioni et al ., 2004). some findings with distinct populations thus, for example, a vygotskian based curriculum (tools of the mind) which requires much social interaction and taking others perspective boosted not only preschoolers social skills but also their executive functioning (e.g., diamond et al ., 2007). although most of the available work examining the effects of social engagement relies on correlational designs, a few studies are beginning to show positive causal effects of social processes on executive functioning, and the most direct evidence comes from social psychological research investigating the effect of on - line social interaction on executive functioning. in one experiment , we had participants interact by having a discussion of a social topic (ybarra et al ., 2008). participants were given 10 min total, with a few minutes to prepare for the discussion. following the interaction the participants evaluated the activity and then completed tests of cognitive functioning, in particular a test of working memory which is a critical component of executive functioning. we also tested participants assigned to either an intellectual activities condition or a control condition that involved watching a 10-min video. another aspect of this mental exercising through simple socializing research that should be highlighted is that participants assigned to the video condition were presented with social content involving several human characters that were engaged in social interaction (ybarra et al ., 2008). in a sense, the video content represents a rich stimulus compared to many of the stimuli used to study social cognitive processes from a neuroscientific perspective. still, the video condition did not in any boosts to efs; it was the real, on - line social interaction that ed in ef boosts, which were equivalent to those ing from engaging in difficult intellectual activities. thus, benefits to efs are selectively seen in on - line social cognition, where participants dynamically construct a model of another person, and less in off - line social cognition where participants deal with static or impoverished and less engaging representations of others. regarding the above research, the argument could be made that the discussion that comprised the social interaction was atypical in some way, in that participants had to take positions, make arguments, and discuss their point of view. but as recent theorizing on the evolution of reasoning suggests, this aspect of social interaction evaluating arguments and proffering others may have been an important pressure on the evolution of our distinctive human cognition (mercier and sperber, 2011). further, as we will discuss later, recent research indicates ef boosts also can from basic get - to - know - you interactions if structured in a particular, mind - engaging way (ybarra et al . so the positive effects on efs can occur from other types of on - line social interactions . . however, evidence also exists for the detrimental effects of some social interactions on efs . most of these findings come from research on intergroup social interactions ( e.g., richeson and trawalter, 2005). in one study, for example, participants who underwent an interracial interview compared to a same race interview subsequently exhibited more interference on a stroop task, a measure of ef. further, this effect was greater the stronger participants associations between concepts denoting african - americans and negative personality characteristics, as measured with the iat test. studies found that after interacting with an attractive female, men showed worse performance on a subsequent ef task, and the decline was related to the degree men tried to manage their impression in the interaction (karremans et al ., 2009). interactions can in reduction of cognitive functioning (finkel et al ., 2006). these investigators had participants coordinate on a task with a confederate, who in some cases made scripted errors (high maintenance) and in some cases did not (low maintenance). participants in the high maintenance condition performed worse on various subsequent tests, although in this research the tests were indirectly tied to efs. however, the findings are also puzzling. why do some interactions lead to ef impairments but other interactions to benefits? note that most, if not all, studies showing ef reductions invoked self - presentation concerns (e.g., about appearing non - prejudiced, unintelligent, etc .). first, it constitutes a working memory load (what was the interaction about ? second, it triggers effortful attempts to self - regulate, which, if extensive, could deplete cognitive resources ( muraven and baumeister, 2000). at a broader level this reasoning also applies to some of the findings dealing with performance decrements under conditions of stereotype threat, for example. in our opinion , however, one key difference between studies showing reductions and boosts in efs has to do with the nature of the interaction. in particular, it is critical whether participants mentally engage with others and attempt to build a rich model of their minds, that they toggle between self and other perspectives, and that they communicate and create meaning during the social interaction versus disengage from the interaction. we argue that this is often, though not always (see below), determined by whether the interaction is cooperative or competitive. however, the default in a cooperative setting is often to engage with the other person, build a model of their mind, figure out whether or not they are trustworthy, and convey to them that they can trust us. in fact, under these trust - building conditions neuroimaging work has reported some of the most robust effects of mentalizing on activity in the medial prefrontal cortex (for reviews, see frith and singer, 2008). further, children s ability to mentalize (i.e., perform tom tasks) has been shown to be positively correlated to the likelihood of cooperating, for example, in prisoner s dilemma games and also to making fair offers in the ultimatum game (sally and hill, 2006 ; also see buttelmann et al ., 2009). in contrast, the default under competitive goals, as realized in the above research on intergroup interactions, is often to become self - protective and withdraw from engaging the other person. this may occur because the interaction is ambiguous and not well structured, which inclines people to back away from the situation as a general way of deterring interpersonal costs (ybarra et al ., 2010). this may also occur because people have a tendency to dehumanize outgroups and thus attribute less mind to their competitors (harris and fiske, 2006). finally, it is possible that competition (but also other high - stake situations) may sometimes usurp the cognitive resources necessary for careful and effortful mentalizing. so, people focus on themselves and their own interests rather than the other person, and they do not engage in attempts to take perspective and create a rich model of the other and the event. a key aspect then of whether or not social interaction creates subsequent ef boosts rests on people engaging each other. we propose that doing so invokes processes that exercise underlying efs, such as working memory and executive attention. thus, when people engage with others in social interaction, versus withdraw into themselves, they can exercise or warm - up these core cognitive processes, whose influence is then transferred (far) to executive functioning tasks (ybarra et al ., 2008). if such social cognitive processes underlie the cognitive boosts, disrupting them should eliminate the cognitive benefits. consistent with this idea, our recent experiments found that interaction goals (competition) that disengage participants from perspective taking and mentalizing eliminate the cognitive benefits that can from social interaction (ybarra et al ., 2011, study 1). importantly, follow - up work has shown that getting people to engage others during interaction, even when the interaction is competitive, helps counter the loss in cognitive benefits (ybarra et al . this is consistent with other research in social cognition suggesting that skepticism, suspiciousness, and other competitive approaches can sometimes improve mental performance ( schul et al . these findings provide evidence that engaging the other during interaction along with concomitant social cognitive processing ( perspective taking, mind - reading) may partly underlie the boosts to executive functioning following social interaction. the above findings also help inform, at least in some small measure, the assumption that competition in social contexts played an important role in the evolution of primate cognition and the more intense varieties of social cognition and mentalizing (e.g., byrne and whiten, 1988 ; whiten and byrne, 1997). dealing with competitors can of course implicate the understanding of others behavioral tendencies and psychological states (e.g., tomasello et al ., 2003 ; decety et al ., 2004), but as we have shown in our experiments, people who expect to compete during social interaction, if not given an explicit goal to read the other person and form a model of what they are like, will disengage and behave evasively (ybarra et al ., 2010) and not receive cognitive benefits from the interaction (ybarra et al ., 2011). these findings suggest that people when competing have diverse options they can undertake, such as to try to hide or foil prediction. only when no, low - risk option is available will they engage or confront the opponent. this, however, does not mean some level of social understanding is not sought or created under competitive circumstances, but it may be that the working model of the other is of a generic, stereotypic nature that relies less on efs, similar to the social perception differences found when people judge members of outgroups versus ingroups (neuberg and fiske, 1987 ; brewer, 1988). when interdependence is called for meaning you have to interact on - line with a person and that your behavior is to some degree yoked to theirs the situation should instigate more intense social cognition and mentalizing to build a richer model of the other party. the discussion of competition helps highlight the role we assign to mentalizing and understanding others psychological states during social interaction. we propose that mental engagement with others leads to cognitive benefits from social interaction especially when the involved parties are taking perspective and dynamically building a model of what the other person is like. as we noted in the introduction , some social inferential processes can occur quite efficiently with little reliance on efs (e.g., winter and uleman, 1984 ; trope, 1986). a similar theme comes up in research on theory of mind, with certain processes (e.g., the calculation of what another sees) thought to be carried out efficiently and automatically (e.g., moll and tomasello, 2006 ; onishi and baillargeon, 2005 ; qureshi et al ., related ideas on the role of efficient social understanding also have been discussed from the point of view of embodied and situated social cognition ( leudar et al ., 2004 ; barrett and henzi, 2005 ; iacoboni et al ., 2005). however, some tom and mentalizing processes such as the selection of information for further processing, though, are thought to require limited cognitive resources or ef (e.g., leslie et al ., 2005 ; bull et al ., 2008 ; qureshi et al ., 2010), and engagement with others in social interaction is considered the real domain in which minds are known (reddy and morris, 2004). thus, there are important elements of building a model of what another person is like, what they are thinking, and what they might do next that rely on efs. our proposal is that during real social interaction both low - level and high - level mentalizing and behavior prediction processes interact and inform each other. the automatic processes serve as input that feed into richer representations that are shaped and updated by processes requiring limited cognitive resources in real time. as noted with competition, it is not that people who are dealing with antagonistic parties fail to attempt to understand their foes but it is during social interaction when the parties are actively engaged with each other that richer representations of the other and of the interaction are created and dynamically updated, which entails the participation of efs. in addition to the issues discussed above, further work is needed to address the following questions: what specific types of social interactions benefit ef and what are the underlying processes that underlie theses boosts? given both the unity and diversity of efs, does social interaction affect some ef elements more than others, or does this also depend on the type of interaction? how long do cognitive boosts last and what is there time course, and does this depend on the task to which the cognitive processes are applied? are the neural correlates underlying on - line social interaction, in particular those underlying efs, similar or different from the correlates unearthed for off - line social interaction? how do short - term training effects of social interaction translate into long - term cognitive reserve, which may be captured in the cross - sectional and longitudinal studies on social engagement and cognitive functioning? and , what are the longer - term consequences of repeated interactions that in ef reductions? if people do not avoid others different from the self, can they learn to cope with such challenging interactions? in addition to theoretical implications regarding the difference between real, on - line social cognition versus off - line social cognition, the processes discussed here have several practical implications. the major one is that certain social interactions can be an effective way of keeping mentally fit. sharpbrains, a company that tracks the mental fitness industry, estimated that worldwide revenue associated with cognitive training programs (e.g., computer software) surged to $ 850 million in 2008, up from $ 250 million in 2005. unfortunately, many of these training programs are not only expensive, but few have been scientifically evaluated (jaeggi et al ., 2011). more germane to this proposal, what is important to emphasize is that what also matters is engagement and taking an active and not a passive role to the technology or the social interaction. as reviewed earlier, some forms of social interaction in no boosts, whereas those in which the parties were engaged and actively tried to form a working model of the other yielded cognitive benefits. however, we would add that given that social connections are at the core of primate life (jolly, 1966), are central to the human survival strategy (barash, 1986 ; dunbar, 1992, 1998 ; baumeister, 2005), and yield various benefits to health and well - being (e.g., house et al . , 1988 ; ybarra et al ., 2008), engaged socializing with others in cooperative interaction may not only strengthen people s brains and minds but possibly their social relations as well, allowing them to reap the various benefits that flow from such bonds. in short, our review highlights the essential role of studying on - line social interactions for understanding the operation of fundamental cognitive processes. although we focused on how executive functioning can change due to task context, especially the on - line social context, it is also important to appreciate the role of people s beliefs and strategies in social navigation. most of us probably know people who seem quite intelligent but still do many dumb things in the cpu is a work of exquisite engineering, but if you try to use such a computer, task performance will be suboptimal and frustrating. the point here is simple: raw executive functioning matters for many social tasks, but so does the content of people s beliefs and strategies their rationality and match to the environment (both in terms of controlled and automatic mental processes). but social life, especially who people interact and associate with, also plays a central role in the beliefs and values that end up populating people s minds. 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.

a successful social interaction often requires on - line and active construction of an ever - changing mental - model of another person s beliefs, expectations, emotions, and desires. it also requires the ability to maintain focus, problem - solve, and flexibly pursue goals in a distraction - rich environment, as well as the ability to take - turns and inhibit inappropriate behaviors. many of these tasks rely on executive functions (ef) working memory, attention / cognitive control, and inhibition. executive functioning has long been viewed as relatively static. however, starting with recent reports of successful cognitive interventions, this view is changing and now efs are seen as much more open to both short- and long - term training, warm - up, and exhaustion effects. some of the most intriguing evidence suggests that engaging in social interaction enhances performance on standard ef tests. interestingly, the latest research indicates these ef benefits are selectively conferred by certain on - line, dynamic social interactions, which require participants to mentally engage with another person and actively construct a model of their mind. we review this literature and highlight its connection with evolutionary and cultural theories emphasizing links between intelligence and sociality.

insulin dependent diabetes mellitus (iddm), type 1 diabetes, is a form of diabetes mellitus that from autoimmune destruction of insulin - producing -cell of the pancreas. the deficiency or complete lack of insulin secretion leads to elevated blood glucose level. patients with type 1 diabetes present lipid disorders or hyperlipidemia, including elevated levels of total serum cholesterol (tc), triglycerides (tg), low - density lipoprotein (ldl - c), apolipoprotein a (apoa), apolipoprotein b (apob), malondialdehyde (mda) , very low - density lipoprotein (vldl), and low level of high density lipoprotein (hdl - c). it is well known that the liver is a central organ in lipogenesis, gluconeogenesis, and cholesterol metabolism. hepatic lipid metabolism is influenced by the balance between the degradation and synthesis and/or import and export of triglyceride (tg) and fatty acids (fa). generally, fatty acids are degraded through -oxidation or esterified and then stored as tg. hepatic tg accumulation finally ing in hepatic steatosis.moreover, the fa transport process appears to be disturbed in obesity and diabetes mellitus. accumulating evidences proved that free fatty acids are taken up by the hepatocytes in a facilitated fashion rather than by passive processes. it is well known that fatty acid translocase is abundantly expressed in tissues with high metabolic capacity for fatty acids. a number of studies have shown that fatty acid translocase (fat / cd36), fatty acid bounding protein (fabp), and fatty acid transporter (fatp) are membrane glycoproteins present on mononuclear phagocytes, adipocytes, and hepatocytes with multiple functions, which have also been identified to facilitate fa uptake and -oxidation. several studies have demonstrated that fat / cd36 as a shared transcriptional target is regulated by liver x receptor (lxr), pregnane x receptor (pxr), and aryl hydrocarbon receptor (ahr). overexpression of fat / cd36 in an increased rate of fa uptake and increased rate of fa metabolism. fatty acids are taken up into the cells and temporarily stored in a triglyceride pool. fa will be finally oxidized in mitochondria by means of carnitine palmitoyl transferase 1 (cpt1) and peroxisomal acyl - coenzyme a oxidase 1 (acox1). the insulin - mimetic properties and antidiabetic effects of vanadium compounds have been widely documented both in vivo and in vitro. vanadium compounds stimulate glycogen synthesis and lipogenesis and inhibit lipolysis. recently , various organic vanadium compounds with dipic, dipic - oh, or dipic - nh2 as organic ligand were reported as antidiabetic agents with little side effects and higher absorption than the simple salts. moreover, it was observed that vanadate can restore the altered lipogenic enzyme activities to the normal level. our previous studies showed that vanadium compounds treatment potentially ameliorate lipid metabolism in diabetes. therefore, the aim of the study was an attempt to elucidate the hypolipidemic effect of v4dipic - cl, if any, in regulating hepatic fat / cd36-induced fa uptake and tg accumulation in stz - induced diabetic rats. moreover, serum biochemical parameters and histopathological examination were used to evaluate the side effect of v4dipic - cl on hepatic functions in diabetic rats. stz was purchased from sigma (sigma - aldrich, usa) h2dipic - cl and v4dipic - cl were gifts from dr. tissue triglyceride (tg) kit was purchased from pplygen (pplygen, china). rna isolation reagent, ultrasybr mixture, and -actin antibody were purchased from beijing cowin bioscience (china). radio immunoprecipitation assay lysis buffer (ripa), hrp - labeled goat anti - rabbit igg, hrp - labeled goat anti - mouse igg, and electrochemiluminescence (ecl) reagent were purchased from beyotime (china). male wistar rats (220 10 g) were purchased from beijing academy of military medical sciences. the animals were maintained under standard conditions (12 h light / dark cycle, 22 2c) and had free access to standard laboratory chow and water. the animals were cared for in accordance with the principles of the guide for care and use of experimental animals. diabetes was induced by a single intravenous injection of freshly prepared stz (40 mg / ml ; 55 mg / kg body weight) in 0.1 mol / l citrate buffer (ph 4.5). animals with a fasting blood glucose level higher than 13.3 mm were considered to be diabetic rats. normal and diabetic rats were randomly divided into four groups: control group (c, n = 5), diabetic group (d, n = 5), h2dipic - cl - treated group (l, n = 5), and v4dipic - cl - treated group (v4, n = 5). v4dipic - cl was orally administrated to diabetic rats in drinking water at a concentration of 50 g v / ml daily for 28 days. we have selected this concentration of vanadium on the basis of earlier reports and the same has also been standardized in our laboratory to exhibit the glucose - lowering effects in stz - induced diabetic animals. in this present study, fresh solutions of h2dipic - cl and v4dipic - cl were prepared every day and were given to the animals through drinking bottles. at the end of the treatment schedules, the livers were perfused in situ with saline and then were immediately removed, collected, and stored in liquid nitrogen. biochemical parameters in serum, including tc, tg, hdl - c, ldl - c, alanine transaminase (alt), and aspartate aminotransferase (ast) were determined using an olympus au400 chemistry analyzer. sections measuring approximately 0.2 cm 0.2 cm were taken from the liver of each rat. they were dehydrated through graded solutions of alcohol ending in two changes of absolute alcohol for 2 h each. they were cleared in 2 changes of xylene, infiltrated in 2 changes of paraffin wax for 2 h each, and embedded in molten paraffin wax. sections were cut at 4 m with rotary microtome and stained with hematoxylin and eosin (h&e). futher, the stained slides was observed under light microscope at 10x and 40x magnifications for histopathological examination. real - time pcr was carried out using the method described by xue et al.. the cdna was used as a template to examine the mrna levels of fat / cd36, fatp5, fabp1, cpt1, acox1, apob, lxr, pxr, and ahr by using ultrasybr mixture. the pcr cycle was as follows: initial denaturation at 95c for 10 min, followed by 40 cycles of denaturation at 95c for 30 s, annealing at 60c for 30 s, and extension at 72c for 30 s. the primers for target genes are shown in table 1. liver tissues were lysed in 1 ml of radio immunoprecipitation assay lysis buffer (ripa) and then centrifuged at 14000 rpm for 5 min. the lysates were subjected to sodium dodecyl sulfate polyacrylaminde gel electrophoresis (sds - page). the gels were transferred to polyvinylidene fluoride (pvdf) membrane by semidry electrophoretic transfer at an electric current 1 ma / cm for 90 min. the pvdf membrane was blocked with 5% no - fat milk for 1 h at the room temperature and then incubated with the primary antibody (1 : 1000) overnight at 4c in a table concentrator. the membrane was washed per 5 min for 4 times prior to incubation in the secondary antibody (1 : 1000) solution for 1 h at the room temperature. immunoreactive bands were detected with electrochemiluminescence (ecl) reagents according to the manufacturer's instructions. the statistical analysis was performed by one - way anova followed by tukey's test. as shown in figures 1(a)1(c), serum tg, tc, and hdl - c levels in diabetic group were higher than those in control group. however, the level of serum tg was significantly decreased after treatment with v4dipic - cl. the concentration of tc in diabetic rats remained unchanged after treatment with v4dipic - cl. the ldl - c levels were not significantly different among the four groups of rats. moreover, the activities of serum alt and ast were markedly increased in diabetic rats. however, the alt and ast activities were decreased in v4dipic - cl - treated diabetic rats (figures 1(e) and 1(f) ). the hepatic tg level in diabetic group was higher than that in normal rats, which was significantly decresed after treatment with v4dipic - cl (figure 2). in comparison with the control group (figure 3(a) ), inflammatory cells infiltrate of liver lobules and dilated congested central vein were observed in figure 3(b). however, the pathological alterations were ameliorated after treatment with v4dipic - cl (figure 3(d) ) compared to those of h2dipic - cl - treated diabetic rats (figure 3(c) ). the mrna expression levels of fat / cd36, fabp1, and fatp5 in diabetes group were higher than those in control group (figure 4). however, the mrna expression level of fat / cd36 was significantly decreased after treatment with v4dipic - cl. moreover, the mrna expression levels of fat / cd36, fabp1, and fatp5 were significantly decreased in the h2dipic - cl - treated group. in contrast, treatment with v4dipic - cl did not affect the mrna expression levels of fabp and fatp in stz - induced diabetes. the mrna expression levels of lxr and pxr in diabetic group were significantly higher than those in control group. moreover, the mrna expression levels of lxr, pxr, and ahr were significantly elevated after treatment with v4dipic - cl (figure 5). however, the mrna expression levels of lxr, pxr, and ahr were significantly decreased in the h2dipic - cl - treated group as compared with the diabetic group (figures 5(a)5(c) ). carnitine palmitoyltransferase i(cpt1) is located on the outer membrane of mitochondria and participates in fatty acid transportation into mitochondria. as shown in figure 6(a), the mrna expression level of cpt1 was increased in diabetic group. however, the mrna expression level of cpt1 was significantly decreased after treatment with v4dipic - cl and h2dipic - cl. in contrast, the mrna expression levels of peroxisomal acyl - coenzyme a oxidase 1 (acox1) and apolipoprotein b (apob) were not significantly different among the four groups of rats (figures 6(b) and 6(c) ). accumulating evidences have demonstrated that stz - induced diabetes mellitus and insulin deficiency lead to hyperglycemia and dyslipidemia. it has been previously reported that hyperglycemia and dyslipidemia are associated with specific diabetic complications and disturbances in various tissues, such as diabetic nephropathy and cardiovascular diseases, but only limited data is available on the possible association between diabetic complications and liver function. the present study was designed to evaluate the effects of v4dipic - cl on lipid metabolism disorders in the liver of stz - induced diabetic rats. it has been recognized that dyslipidemia is a frequent complication in all types of diabetes which can range from hypercholesterolemia to hyperlipoproteinemia. hyperlipidemia could be a factor for fatty liver formation. in the present study, as expected serum tg and tc as well as hepatic tg levels were elevated in the diabetic group compared to those in the control group, which is consistent with other studies. however, the elevated level of serum tg in diabetic rats was significantly decreased after treatment with v4dipic - cl. this is in agreement with the evidence that vanadium compounds decrease the high levels of tg in serum and liver. moreover, the altered expression of genes involved in lipid biosynthetic pathways in diabetes returned to normal level after treatment with vanadium compounds. elevated activities of serum aminotransferases are a common sign of liver diseases. typical serum biochemical parameters, such as alt and ast, are often examined to evaluate whether the liver is damaged or diseased. in the present study, our findings of elevated serum alt and ast levels are in agreement with the findings of zafar et al.. the increase in alt and ast activities may be due to the cellular damage in the liver caused by stz - induced diabetes. after 28 days of treatment with v4dipic - cl, the activities of both alt and ast were significantly decreased. the suggests that v4dipic - cl may be capable of ameliorating the impaired liver function in stz - induced diabetic rats, which is consistent with previously reported for treatment with vanadium complexes. described the fatty liver and hyperlipidemia in idmm of treated shrews. in the present study, the histopathology of liver showed a development of the lesions which seems to be due to stz treatment. most liver sections showed inflammatory cells infiltrate of liver lobules and dilated congested central vein. zafar et al. who showed dilatation of veins and liver fibrosis in their study. however, we found that treatment with v4dipic - cl dramatically improved pathologic lesions seen in the liver. free fatty acids are a major component of blood lipids and plays a key role in regulating blood lipid levels, especially in triglyceride metabolism. in addition, elevated plasma fa is a risk factor for metabolic syndrome, which can lead to hyperlipidemia, fatty liver, and insulin resistance. fat / cd36 is a rate - limiting enzyme in high - affinity peripheral fa uptake in the liver. thus, fat / cd36 is an important regulator in the uptake of fatty acids in the liver and the pathogenesis of fatty liver disease. it was reported that fa uptake is reduced in fat / cd36 null mice and is reconstituted when fat / cd36 is reexpressed. luiken et al. reported that fat / cd36 mrna expression is increased in streptozotocin - induced diabetes. thus, fat / cd36 may participate in the pathogenesis of liver ectopic fat deposition. in the present study , we found that the mrna expression level of fat / cd36 in diabetes group was significantly higher than that in control group. however, v4dipic - cl treatment significantly ed in decrease in the mrna and protein expression levels of fat / cd36 in v4dipic - cl - treated group treatment. in addition to fat / cd36, goldberg and ginsberg described that fatty acid - binding protein (fabp) and fatty acid transport protein (fatp) can mediate fatty acid uptake in the liver. in the present study, the mrna expression levels of fabp1 and fafp5 were increased in diabetic group, which is consistent with the previous report that the expression of fabp is increased in stz - induced diabetes. however, treatment with v4dipic - cl did not affect the expression of fabp and fatp in stz - induced diabetes. thus, we propose that v4dipic - cl can mainly regulate fatty acid transporter fat / cd36 in liver. zhou et al. described that nuclear acceptors ahr, pxr, and lxr cooperate to promote hepatic steatosis by increasing the expression of fat / cd36. more recently, cheng et al. demonstrated that the mrna expression level of lxr was markedly increased in diabetic rats. reported that fenofibrate, ppar agonist, dramatically reduced hepatic triglyceride levels by activating expression of acox1 and cpt1 involved in fatty acid turnover. in the present study, we also found that the mrna expression levels of lxr, pxr, and cpt1 were increased in diabetic group. however, treatment with v4dipic - cl did not decrease the mrna expression levels of lxr, pxr, and ahr as well as the fa oxidation - related cpt1 and acox1 as compared to h2dipic - cl - treated diabetic rats. it is possible that some other mechanisms may contribute to regulation of fat / cd36 expression through modulating nuclear receptors by vanadium compounds. this study showed that v4dipic - cl ameliorates stz - induced hepatic inflammatory infiltration, liver disfunction, and hepatic tg accumulation. this effects were likely associated with the modification of lipid metabolism - related fat / cd36 in liver. these together with the previous observations suggest that v4dipic - cl can be used as a therapeutic agent for treatment of metabolic disorder in diabetes mellitus.

vanadium complexes are potent antidiabetic agents for therapeutical treatment of diabetes. in the present study, we investigated the hypolipidemic effect of vivo(dipic - cl)(h2o)2 (v4dipic - cl) in liver of streptozotocin- (stz-)-induced diabetic rats. we found that diabetic animals exhibited hepatic inflammatory infiltration and impaired liver function along with triglyceride (tg) accumulation in the liver. v4dipic - cl treatment not only ameliorated liver pathological state but also reduced hepatic tg level. moreover, the upregulation of fatty acid translocase (fat / cd36) mrna (4.0-fold) and protein (8.2-fold) levels in the liver of diabetic rats were significantly reversed after v4dipic - cl treatment. however, no significant effects of v4dipic - cl on the mrna expression of fatty acid metabolism - related fatty acid bounding protein 1 (fabp1) and fatty acid transporter 5 (fatp5) were observed. these suggest that the modification of lipid metabolism - related fat / cd36 in the liver of diabetic rats is likely involved in the hypolipidemic effects of v4dipic - cl.

over the last few decades, minimally invasive surgery has been widely applied, particularly in gynaecology, where laparoscopic surgery has assumed an increasingly central role. data regarding minimally invasive surgery have shown more benefits compared to abdominal surgery in terms of surgical outcomes, cost, quality of life, reduced morbidity, shorter hospital stay and a rapid return to daily activities. moreover, recent advances in laparoscopic instruments and techniques have allowed completion of a great number of complex surgical procedures including in gynaecologic oncology surgery. recently, the laparoscopic approach has been revised and laparoendoscopic single - site surgery (less) has been introduced. the term single - site describes the use of a single small skin incision instead of the multiple accesses created in conventional laparoscopy. the less port is a multichannel port that allows the insertion of up to three instruments. according to many authors, reducing number of ports on the abdominal wall may improve the cosmetic and decrease postoperative pain and complications such as hernias, infection and nerve injuries. the first application of less was a tubal sterilisation in 1969, while the first hysterectomy with salpingo - oophorectomy was performed in 1991. thereafter, less became effective, safe and feasible for performing complex surgical procedures in gynaecology and urology and in abdominal surgery. the aim of this study was to assess the surgical and oncological outcome in the management of endometrial cancer (ec) by less. all patients diagnosed with clinical international federation of gynecology and obstetrics (figo) stage i ii ec, both endometrioid and non - endometrioid, and confirmed at the definitive histological examination, who underwent less were included. before surgery, all patients underwent a clinical and instrumental evaluation, including taking medical history, undergoing a physical examination, a vaginal - pelvic examination, a chest x - ray, an ultrasound scan and pelvic magnetic resonance imaging scan. an intraoperative histological examination was required during all the surgeries and a pelvic lymphadenectomy was performed only when risk factors (myometrial invasion more than 50%, high grading and non - endometrioid histotype) were detected. para - aortic lymphadenectomy is not routinely performed unless pelvic lymph nodes are confirmed to have metastatic disease on frozen section evaluation in order to determine the field of postoperative radiation. informed consent for less was obtained from all patients in accordance with local and international legislation (declaration of helsinki). patient characteristics were recorded, including age, body mass index (bmi), histotype, figo stage, grading and prior abdominal surgery. subsequently, the intraoperative parameters were recorded: median skin incision, operative time, blood loss, transfusions, conversion rate and intraoperative complications. blood loss was evaluated by the difference in the total amounts of suction and irrigation fluids. postoperative parameters included early postoperative complications (in the first 30 days after surgery) and later postoperative complications (more than 30 days after surgery), type of adjuvant therapy (radiotherapy and/or chemotherapy), median follow - up in months, recurrence, time to recurrence and median follow - up. postoperative pain assessment was performed in all patients using a validated numeric rate score (nrs) and scored from 0 to 10 (0 = no pain and 10 = agonising pain). nrs was recorded at regular intervals: t0 (end of surgery), t1 (after 1 h), t2 (after 6 h), t3 (after 12 h), every 24 h starting from the second postoperative day. analgesic therapy with tramadol 100 mg plus ketorolac 60 mg by continuous infusion for the first 24 h after the end of surgery was administered. adjuvant therapy was tailored to the pathologic findings at primary operation after multidisciplinary tumour board (gynaecologic oncology, pathology, radiation oncology, medical oncology) discussion. treatment was based on the of prospective randomised clinical trials and national comprehensive cancer network guidelines. follow - up was performed every 3 - 4 months with physical and gynaecological examination for the first 2 years, and then with a 6-month interval until 5 years. the patient was positioned in the dorsal lithotomic trendelenburg position, with both legs supported by straps. no uterus manipulator devices were used, but the cervix was closed with a modified tenaculum called simple nebs arising incision landmark (snail, snail is obtained by the modification of an instrument named uterine tenaculum forceps, model schroder, code 32 - 622 - 25 of martin catalogue . currently the snail was patented but not yet marketed). a medical - grade silicone balloon, named colpo - pneumo occluder (cooper surgical, coopersurgical, inc . , 95 corporate drive, trumbull, ct 06611, usa) was also emplaced in the vagina in order to preserve an adequate pneumoperitoneum during colpotomy. by, using the hasson technique, it was possible to place the single - incision laparoscopic surgery (sils) port (covidien, 710 medtronic parkway, minneapolis, usa) through a 2 - 2.5 cm umbilical incision. the sils port is a multichannel port that allows the insertion of up to three instruments. after emplacing the co2 insufflation through one of the three available channels, it was possible to introduce a 5-wmm trocar for the insertion of a 30-degree laparoscope. in the remaining two channels, two 5-mm trocars were placed for the introduction of additional instruments, such as as roticulator endo dissect or mini - shears or grasp - all 5 mm (covidien). a careful inspection of the entire abdominal cavity was performed as the first surgical step in order to identify any suspicious peritoneal lesions that would exclude the patient from having the procedure completed by less. after cauterising the fallopian tubes, the right round ligament was sectioned, entering into the retro - peritoneal space. it was possible to cauterise the uterine vessels. finally, a sufficient margin of vagina was exposed, allowing its section. all patients underwent type a or b1 hysterectomy plus salpingo - oophorectomy (querleu morrow classification). in the end , the uterus and adnexa were extracted through the vagina and the vaginal cuff was sutured through the vagina, while the site of introduction of the port was closed and the fascia of the rectus muscles sutured using vicryl 1/0 (ethicon endo surgery, somerville nj). pelvic lymphadenectomy was performed based on frozen section analysis according to the technique of escobar et al. (a) less device inserted into the umbilicus (b) less device with three 5-mm trocars all patients diagnosed with clinical international federation of gynecology and obstetrics (figo) stage i ii ec, both endometrioid and non - endometrioid, and confirmed at the definitive histological examination, who underwent less were included. before surgery, all patients underwent a clinical and instrumental evaluation, including taking medical history, undergoing a physical examination, a vaginal - pelvic examination, a chest x - ray, an ultrasound scan and pelvic magnetic resonance imaging scan. an intraoperative histological examination was required during all the surgeries and a pelvic lymphadenectomy was performed only when risk factors (myometrial invasion more than 50%, high grading and non - endometrioid histotype) were detected. para - aortic lymphadenectomy is not routinely performed unless pelvic lymph nodes are confirmed to have metastatic disease on frozen section evaluation in order to determine the field of postoperative radiation. informed consent for less was obtained from all patients in accordance with local and international legislation (declaration of helsinki). patient characteristics were recorded, including age, body mass index (bmi), histotype, figo stage, grading and prior abdominal surgery. subsequently, the intraoperative parameters were recorded: median skin incision, operative time, blood loss, transfusions, conversion rate and intraoperative complications. blood loss was evaluated by the difference in the total amounts of suction and irrigation fluids. postoperative parameters included early postoperative complications (in the first 30 days after surgery) and later postoperative complications (more than 30 days after surgery), type of adjuvant therapy (radiotherapy and/or chemotherapy), median follow - up in months, recurrence, time to recurrence and median follow - up. postoperative pain assessment was performed in all patients using a validated numeric rate score (nrs) and scored from 0 to 10 (0 = no pain and 10 = agonising pain). nrs was recorded at regular intervals: t0 (end of surgery), t1 (after 1 h), t2 (after 6 h), t3 (after 12 h), every 24 h starting from the second postoperative day. analgesic therapy with tramadol 100 mg plus ketorolac 60 mg by continuous infusion for the first 24 h after the end of surgery was administered. adjuvant therapy was tailored to the pathologic findings at primary operation after multidisciplinary tumour board (gynaecologic oncology, pathology, radiation oncology, medical oncology) discussion. treatment was based on the of prospective randomised clinical trials and national comprehensive cancer network guidelines. follow - up was performed every 3 - 4 months with physical and gynaecological examination for the first 2 years, and then with a 6-month interval until 5 years. the patient was positioned in the dorsal lithotomic trendelenburg position, with both legs supported by straps. no uterus manipulator devices were used, but the cervix was closed with a modified tenaculum called simple nebs arising incision landmark (snail, snail is obtained by the modification of an instrument named uterine tenaculum forceps, model schroder, code 32 - 622 - 25 of martin catalogue . currently the snail was patented but not yet marketed). a medical - grade silicone balloon, named colpo - pneumo occluder (cooper surgical, coopersurgical, inc . , 95 corporate drive, trumbull, ct 06611, usa) was also emplaced in the vagina in order to preserve an adequate pneumoperitoneum during colpotomy. by, using the hasson technique, it was possible to place the single - incision laparoscopic surgery (sils) port (covidien, 710 medtronic parkway, minneapolis, usa) through a 2 - 2.5 cm umbilical incision. the sils port is a multichannel port that allows the insertion of up to three instruments. after emplacing the co2 insufflation through one of the three available channels, it was possible to introduce a 5-wmm trocar for the insertion of a 30-degree laparoscope. in the remaining two channels, two 5-mm trocars were placed for the introduction of additional instruments, such as as roticulator endo dissect or mini - shears or grasp - all 5 mm (covidien). a careful inspection of the entire abdominal cavity was performed as the first surgical step in order to identify any suspicious peritoneal lesions that would exclude the patient from having the procedure completed by less. after cauterising the fallopian tubes, the right round ligament was sectioned, entering into the retro - peritoneal space. then, the ureter and uterine artery were visualised and adnexal resection was performed. subsequently, vescicouterine, vascicovaginal and rectovaginal spaces were developed. in this way, it was possible to cauterise the uterine vessels all patients underwent type a or b1 hysterectomy plus salpingo - oophorectomy (querleu morrow classification). in the end, the uterus and adnexa were extracted through the vagina and the vaginal cuff was sutured through the vagina, while the site of introduction of the port was closed and the fascia of the rectus muscles sutured using vicryl 1/0 (ethicon endo surgery, somerville nj). pelvic lymphadenectomy was performed based on frozen section analysis according to the technique of escobar et al. (a) less device inserted into the umbilicus (b) less device with three 5-mm trocars between july 2009 and june 2013, 50 women underwent laparoendoscopic single - site hysterectomy with or without pelvic lymphadenectomy for the treatment of ec at the gynecological oncology unit at the regina elena national cancer institute, rome, italy. median age was 45 years (range, 39 - 84 years) and median bmi was 21.8 kg / m (range, 19 - 48 kg / m). in 15 patients (29.7%), previous abdominal surgical procedures were found. port placement, taking a median time of 1.5 min (range, 1 - 3 min), was successfully performed in all cases without accidental port removal. median operative time was 100 min (range, 50 - 240 min) and median blood loss was 90 ml (range, 10 - 300 ml). pelvic lymphadenectomy was performed in 6 patients, and the median numbers of pelvic lymph nodes retrieved was 14 (range, 5 - 20). no intraoperative complications occurred, but there were 4 postoperative complications: a vaginal cuff dehiscence, an umbilical wound infection and a lymphorrhoea and bladder fistula with the vagina in the same patient who underwent hysterectomy plus pelvic lymphadenectomy on the 13th postoperative day. all procedures were successfully completed but there were 2 patients who required a laparoscopic conversion due to extensive adhesions from previous surgery. it was strongly influenced by the prolonged hospital stay of a patient who had a length of stay of 9 days due to an umbilical wound infection. in fact, without this patient, the median length of stay was 2 days. all patients had a good cosmetic and postoperative pain control. regarding this last point, the median nrs at t0 was 3.5 (range, 1 - 5), at t1 was 2 (range, 1 - 4), at t2 was 1.5 (range, 1 - 3), and at t3 and during patient discharge was 0.5 (range, 0 - 1). (a) umbilical appearance at the end of surgery (b) umbilical appearance 6 months after surgery adjuvant therapy was performed in 10 patients: 9 patients underwent to radiotherapy (6 patients figo stage ib g3 and 3 figo stage ii) and only 1 underwent radio - chemotherapy (figo stage iiia). the median follow - up was 36 months (range, 16 - 62 months) and only 1 patient died due to the occurrence of a new cancer after 11 months from surgery. our report shows that less could be a safe and reliable technique in terms of surgical and oncological outcomes in patients with ec. during the last decade, minimally invasive surgery has been widely introduced in the management of the gynaecological malignant disease. even though the role of less in gynaecological oncology is well established, it is in constant development. in particular, over the last few years, published studies have shown the excellent application of the less in the treatment of early - stage ec. our are similar to those reported in the literature, except for a few aspects. undoubtedly, any previous abdominal surgery could limit the application and render the performance of this surgery difficult. moreover, surgery in our cases was performed by a surgeon with extensive skill and experience in laparoscopic surgery, which makes the application of less easier. in a review of literature on laparoendoscopic single - site hysterectomy with or without pelvic lymphadenectomy for the treatment of ec between 2009 and 2014 the operating time was 35 - 342 min, blood loss was 0 - 700 ml, pelvic and para - aortic lymph nodes removed were 2 - 43 and 2 - 28, respectively, while hospital stay was 1 - 6 days. the number of conversions reported was 21 (4.8%), with intraoperative and postoperative complications of 22 (5%) and 14 (3.2%), respectively. literature review of less procedure in only ec fagotti et al. reported the widest series of early ec treated with less described in the literature. among the 100 cases, total hysterectomy and bilateral salpingo - oopherectomy were performed, while pelvic and para - aortic lymphadenectomy in 48 and 27 patients, respectively. the median number of pelvic and para - aortic lymph nodes was 16 and 7, respectively, similar to those reported in studies utilising traditional laparoscopy. two patients undergoing lymphadenectomy required conversion, and 4 intraoperative and 4 postoperative complications occurred overall. overall, less entailed good in terms of surgical outcomes, postoperative pain and cosmetics. moreover, operative time seemed to decrease with surgeon's experience, increasing the number of performed surgeries. furthermore, escobar et al. in 2010 described single - port laparoscopic pelvic and para - aortic lymphadenectomy in 21 patients. the median number of pelvic and para - aortic lymph nodes retrieved was 14 and 6, respectively, and all procedures were successfully completed with only one conversion to traditional laparoscopy. in literature, oncological outcomes are not often investigated, and we actually report a higher median follow - up. in fact, with a median follow - up of 36 months (range, 16 - 62 months), we had only 1 patient die due to the occurrence of a new cancer 11 months after surgery. given the important role of minimally invasive surgery in gynaecological surgery, several studies have compared the perioperative outcomes of less with other, different minimally invasive techniques. escobar et al. compared less, conventional laparoscopic and robotic surgery in the treatment of ec, but no differences were found in terms of operative time, blood loss, hospital stay and complication rates. subsequently, another study compared less and robotic surgery, showing that less had a shorter operative time (122 min vs 175 min) and lower blood loss (50 ml vs 80 ml) than robotic surgery. furthermore, a case - control study compared sils (38 patients) and robotic single - port surgery (19 patients), showing no clinical relevant differences. in 2012, fanfani et al. , compared less and mini - laparoscopic surgery in a series of patients, including cases with malignant disease, and did not find any significant differences in terms of surgical outcomes except for operative time, which was longer in less groups. less is widely applied in gynaecological surgery with the same absolute contraindications of conventional laparoscopy: metastatic disease, heart or pulmonary disease, and severe abdominal adhesions. moreover, the surgeon should have advanced skills in laparoscopic and single - port surgery. obesity and previous abdominal surgery should no longer be considered absolute limits in the performance of less. one of the most important technical challenges is the instrument crowding due to the restricted space available to make necessary movements. the lack of triangulation is another important problem that can be easily overcome by applying articulating and flexible instruments or curved instruments. however, extensive experience in laparoscopic surgery might be enough to overcome the challenges in single - port surgery. the latest innovation in minimally invasive surgery is the introduction of the robotic system da vinci (intuitive surgical inc, 1266 kifer road, building 101 sunnyvale, ca). the combination of robot and single port offers new advantages as well as in allowing many challenges to be overcome in single - port surgery. despite being an expensive surgical technique, the robotic single port could be a promising new surgical approach, including in morbidly obese women. in a recent study, robotic surgery was confirmed to be a good surgical approach in the treatment of ec in obese women. the application of the da vinci robotic system in gynaecological surgery may make single - site surgery feasible and safe for many including morbidly obese women. in , less is a safe and feasible surgical approach in the treatment of ec. further studies are needed to define the long - term outcomes, the disease - free survival and overall survival. robotic single - port surgery is a potential area of development, but it is still not clear whether the benefits of robotic surgery will justify the high costs of this new approach.

: the aim of this study was to assess the surgical and oncological outcome for the management of endometrial cancer (ec) by laparoendoscopic single - site surgery (less).patients and methods: we performed a retrospective chart review of patients who underwent a less for ec. all the patients were treated by the same surgical team between july 2009 and june 2013 at the gynaecologic oncologic unit, regina elena national cancer institute, rome, italy.:a total of 50 women were included, with a median age of 45 years (range, 39 - 84 years) and a median body mass index (bmi) of 21.8 kg / m2 (range, 19 - 48 kg / m2). median operative time was 100 min (range, 50 - 240 min), median blood loss was 90 ml (range, 10 - 300 ml) and median hospital stay was 3 days (range, 2 - 9 days). the median number of pelvic lymph nodes retrieved was 14 (range, 5 - 20). no intraoperative complications occurred, but there were 4 postoperative complications. two patients required a laparoscopic conversion. the median follow - up was 36 months (range, 16 - 62 months) and no recurrence occurred.:our report showed that the less approach in the treatment of early ec can be a safe and reliable technique in terms of surgical and oncological outcomes.

g - protein - coupled receptors (gpcrs) constitute the largest family of membrane proteins and are known to respond to a plethora of different stimuli by activating numerous intracellular signaling pathways. because of their involvement in a variety of biological processes, gpcrs are the target of more than 50% of currently marketed drugs. to date, most of the drugs targeting gpcrs are known to interact with the orthosteric site - that is, the endogenous ligand - binding pocket. these orthosteric ligands need, however, to overcome many limitations, such as decreased selectivity, insufficient clinical efficacy, and undesirable effects on receptor regulation. the past decade witnessed the discovery of new pharmaceutical compounds that modulate receptor function by targeting allosteric sites. allosteric sites are, by definition, domains topographically distinct from the orthosteric binding pocket. these allosteric modulators are very promising pharmaceutical drugs devoid of many of the disadvantages of orthosteric ligands. in fact, these allosteric compounds show higher subtype selectivity (allosteric sites are generally less conserved than orthosteric sites), often have a safer pharmacological profile (exert their effects only in the presence of endogenous ligands) and, in some cases, do not induce receptor desensitization. there are three general categories of allosteric modulators (figure 1a): first, modulators affecting the binding affinity of orthosteric ligands, such as amno82, which is responsible for the incomplete inhibition of orthosteric antagonist binding to the metabotropic glutamate (mglu) 7 receptor, mglu7r; and second, modulators affecting orthosteric ligand efficacy. one example is cgp7930, which is known to enhance gamma - aminobutyric acid receptor b (gabab)-mediated gtp s (guanosine 5-o-triphosphate) binding. third, allosteric agonists and inverse agonists, known to engender a unique gpcr conformation that alters receptor signaling in the absence of orthosteric ligands. for example, mpep (2-methyl-6-(phenylethynyl)pyridine ) acts as a negative allosteric modulator of mglu5r by inhibiting inositol phosphate production in the absence of an agonist. another example is ac42, which was first identified as a selective agonist of the m1 muscarinic acetylcholine receptor (m1achr); however, mutation studies have implied that this ligand activates the receptor via an allosteric site. (a) an allosteric ligand may modulate binding and signaling of orthosteric ligands or induce signal transduction by its own (allosteric agonists) (adapted from langmead and christopoulos). (b, c) comparison of different binding modes of orthosteric (red circle) and allosteric (red triangle) ligands as monovalent ligands (b) or bivalent bitopic ligands (c). binding of orthosteric ligands induces signal 1, which can be modulated by allosteric ligands binding to sites that are close to or distant from the orthosteric binding site, generating signal 1 * or signal 1**. alternatively, the signal generated by the orthosteric ligand in one protomer can be allosterically modulated by the other protomer within a gpcr dimer, generating signal 1***. allosteric agonists can induce signaling by their own in the absence of orthosteric ligands (signal 2). signals 1 *, 1 * *, and 1 * * * can also be generated by bitopic ligands. in addition, bitopic ligands could induce signals (signals 1, 1, and 1) that are specific for these ligands and not observed upon simultaneous stimulation with monovalent orthosteric and allosteric ligands. recently, a new class of ligands, termed bitopic or dualsteric ligands, which simultaneously target orthosteric and allosteric sites, emerged (figure 1b). the development of bitopic ligands is based on the idea of combining high affinity (via orthosteric sites) with high selectivity (via allosteric sites). until now, muscarinic acetylcholine receptors (machrs) have proved to be a particularly fruitful receptor model for the development and characterization of bitopic ligands. in 2004, tahtaoui et al. synthesized seven fluorescent derivatives of the bodipy - labeled pirenzipine, an m1achr antagonist, and used fluorescent resonance energy transfer technology to study receptor - ligand interactions. the authors showed that these analogs might interact with both the acetylcholine (orthosteric) and brucine (allosteric) binding domains of the m1achr, therefore behaving as potential bitopic ligands. the literature also reports the design of hybrid molecules from allosteric modulators of w84-type compounds and orthosteric antagonists or agonists of machrs to obtain subtype selective muscarinic ligands with agonistic or antagonistic properties. in a recent publication, christopoulos and colleagues elegantly showed that mcn - a-343, a selective m2achr partial agonist, is actually a bitopic ligand. using several different binding and functional assays, the authors showed that mcn - a-343 is composed of an orthosteric agonist coupled to an allosteric modulator (3-chlorophenylcarbamate moiety). truncated derivatives of mcn - a-343 retaining the chlorophenylcarbamate moiety were positive modulators of the orthosteric antagonist n-methylscopolamine in radioligand binding assays, but in functional assays, such as m2achr - mediated erk1/2 (extracellular signal - regulated kinase 1/2) phosphorylation, they acted as negative modulators of agonist efficacy. finally, in 2009, antony et al. presented a novel approach to pharmacologically design subtype and signaling selective receptor agonists, once again using the machrs as templates. their paper reports the synthesis of two hybrids fusing a highly potent oxotremorine - like orthosteric activator with m2-selective bis(ammonio)alkane - type allosteric fragments. radioligand binding in wild - type and mutant receptors supplemented by receptor docking simulations showed an m2-selective and true allosteric / orthosteric binding. moreover, g - protein activation, in this context, was mediated by the orthosteric moieties. due to the unique features of bitopic ligands, their number is likely to expand in the near future. this may include the re - examination of already existing ligands, as was the case with mcn - a-343, and the de novo design of bitopic ligands as shown by antony et al.. where can we expect to see the fastest progress? the number of already existing ligands that might turn out to be bitopic is difficult to estimate and will strongly depend on the degree of information available for these putative bitopic molecules. the design of new bitopic ligands requires extensive knowledge about orthosteric and allosteric ligands available for a given gpcr as well as its ligand binding sites. this may explain the fact that bitopic ligands have predominantly been described for muscarinic receptors, for which detailed knowledge on allosteric binding sites and ligands is indeed available. once allosteric ligands have been identified, the choice of an optimal linker has to be considered. the extensive literature on the design and synthesis of bivalent ligands intended to target two orthosteric ligand binding sites within a gpcr dimer will be a rich source of inspiration for the synthesis of bitopic ligands. as illustrated by compound mcn - a-343 and hybrid compounds described by antony et al. , the better the structural knowledge about orthosteric and allosteric binding sites, the easier the rational design of the linker (chemical nature and length of linker, linker attachment point to pharmacophores) will be. recently solved gpcr structures will assist the design and synthesis of bitopic ligands that bind to identified orthosteric and allosteric binding pockets. such gpcr structures have not only provided detailed knowledge about orthosteric binding pockets, but also revealed the important role of extracellular receptor domains in guiding small molecular weight ligands to their high affinity ligand binding pockets within the transmembrane domain. these ligand entrance channels are interesting new targets for allosteric modulation by bitopic ligands. finally, the concept of bitopic ligands may also be extended to gpcr dimers, where the orthosteric and allosteric pharmacophores bind to two different protomers in the receptor dimer (figure 1b).

natural ligands of g - protein - coupled receptors interact with the orthosteric ligand binding site, as do most of the classical synthetic ligands. the discovery of ligands targeting different, allosteric binding sites considerably expanded the repertoire of g - protein - coupled receptor ligands. more recently, bitopic ligands have been described that target both orthosteric and allosteric sites at the same time.

anisakiasis is one of the fishborne zoonotic diseases, especially in localities, where people have a custom of eating raw marine fish and squids like in japan and korea. the concept of this disease was first raised by van thiel et al. in the netherlands. after then, human infection cases were consequently reported in england, japan, and the republic of korea (korea). nowadays, this zoonotic nematode disease is a clinical concern worldwide, including japan, korea, the netherlands, spain, france, italy, germany, and the united states. especially in japan , it has been known that more than 2,000 cases occurred annually. since the first human case was reported by kim et al. in 1971, until now numerous patients have increasingly occurred in korea. especially the increasing tendency of cases was accelerated by the common use of endoscopy in the diagnosis and treatment of gastric anisakiasis. recently, lee et al. analyzed total 141 cases diagnosed by the endoscopy in a local clinic in jinju - si, gyeongsangnam - do. clarified the etiologic agents of 16 human anisakiasis in korea, 15 of which were confirmed as anisakis pegreffii larvae and only 1 was anisakis simplex larva using a molecular analysis. in this study, we report an additional 15 gastric anisakiasis cases that occurred in gyeongsangnam - do, and then analyzed the epidemiologic and clinical characteristics of korean anisakiasis cases through a literature review. total 15 anisakiasis cases were diagnosed with the gastric endoscopy in the gyeongsang national university hospital and a local clinic located in jinhae - gu, changwon - si, gyeongsangnam - do from october 1999 to january 2010. most of the cases (80%) were men, and only 3 were women. total a single worm was detected from 12 cases, 2 worms from 1 case, and even 9 larvae were found from 2 cases. epigastric pain was the most common symptom in almost all cases, and nausea and vomiting were accompanied in some cases. symptom manifestations began at 10 - 12 hr after eating fish in most cases (73.3%). endoscopy was performed in 1 - 2 days after symptom onset in most cases (66.7%). as the probable source of infection, 7 (46.7%) and 2 (13.3%) cases recalled that they had eaten raw common conger, conger myriaster, and anchovy, respectively. however, 6 cases could not remember the exact species of fish; several kinds of fish mixed or uncertain. the case information is summarized in table 1. among the total 32 anisakis type i larvae (either the 3rd or 4th - stage), 7 were morphologically observed and measured with a micrometer (table 2). the articles of korean anisakiasis cases were basically searched through pubmed site using the internet. the references in all articles were strictly seen to prevent missing cases. until now, a total of 645 cases have been reported in 64 articles since the first case recorded by kim et al. in 1971. during 1990 - 1999, a total of 249 cases (38.6%) were reported in 25 papers (39.1%). the number of anisakiasis cases reported in the korean literature is shown in table 3. the incidence rate was the highest in jeju - do (34.4%), followed by in gyeongsangnam - do (22.8%), seoul (20.0%), busan (10.1%), incheon (6.5%), gyeongsangbuk - do (3.2%), and other 9 administrative regions (0.16 - 0.79%) (table 4). among a total of 404 cases which revealed the age and sex of patients, 185 (45.8%) were men and the remaining 219 (54.2%) were women. the age prevalence was the highest in forties (34.7%), followed by thirties (32.9%), fifties (14.9%), twenties (9.7%), over sixty (6.2%), and teens (1.7%) (table 5). as the probable source of human infections, the common conger was most frequently mentioned by patients (38.6%), followed by croaker (11.6%), squid (10.3%), yellowtail (9.7%), flatfish (flounder : 5.5%), and the other 12 species of marine fish (table 6). the etiologic larval type was designated in 203 cases in the literatures. among them, 165 cases (81.3%) were due to anisakis type i larvae, 24 (11.8%) were by terranova type a, and 8 cases (3.9%) were by anisakis type ii. the predilection site was the stomach (82.4%), followed by small intestines (11.4%), gastro - esophageal junction (3.5%), large intestines (1.5%), esophagus (0.5%), and others including the palatine tonsil, ileo - cecal region, and mesocolic lymph node (table 8). in most cases (65.7%), symptoms were manifested before 12 hr from eating raw flesh of fish (table 9). the seasonal prevalence was the highest in winter (38.8%), followed by spring (23.1%), autumn (19.9%), and summer (18.2%) (table 10). the articles of korean anisakiasis cases were basically searched through pubmed site using the internet. the references in all articles were strictly seen to prevent missing cases. until now, a total of 645 cases have been reported in 64 articles since the first case recorded by kim et al. in 1971. during 1990 - 1999, a total of 249 cases (38.6%) were reported in 25 papers (39.1%). the number of anisakiasis cases reported in the korean literature is shown in table 3. the incidence rate was the highest in jeju - do (34.4%), followed by in gyeongsangnam - do (22.8%), seoul (20.0%), busan (10.1%), incheon (6.5%), gyeongsangbuk - do (3.2%), and other 9 administrative regions (0.16 - 0.79%) (table 4). among a total of 404 cases which revealed the age and sex of patients, 185 (45.8%) were men and the remaining 219 (54.2%) were women. the age prevalence was the highest in forties (34.7%), followed by thirties (32.9%), fifties (14.9%), twenties (9.7%), over sixty (6.2%), and teens (1.7%) (table 5). as the probable source of human infections, the common conger was most frequently mentioned by patients (38.6%), followed by croaker (11.6%), squid (10.3%), yellowtail (9.7%), flatfish (flounder : 5.5%), and the other 12 species of marine fish (table 6). the etiologic larval type was designated in 203 cases in the literatures. among them, 165 cases (81.3%) were due to anisakis type i larvae, 24 (11.8%) were by terranova type a, and 8 cases (3.9%) were by anisakis type ii. the predilection site was the stomach (82.4%), followed by small intestines (11.4%), gastro - esophageal junction (3.5%), large intestines (1.5%), esophagus (0.5%), and others including the palatine tonsil, ileo - cecal region, and mesocolic lymph node (table 8). in most cases (65.7%), symptoms were manifested before 12 hr from eating raw flesh of fish (table 9). the seasonal prevalence was the highest in winter (38.8%), followed by spring (23.1%), autumn (19.9%), and summer (18.2%) (table 10). by the present study, 15 cases of gastric anisakiasis have been added among the korean anisakiasis cases. like in the previous studies, epigastric pain was the chief complain and most commonly manifested in almost all cases. it has been known that severe bleeding can occur due to anisakid larva infection in patients with gastric ulcer. time intervals from fish eating to symptom onset and from symptom onset to endoscopy were 10 - 12 hr and 1 - 2 days in most cases. the common conger, c. myriaster, is suspected as the most probable source of infection in 7 patients (46.7%). until now , in korea, a total of 645 cases have been reported in 64 papers since the first case reported by kim et al.. accordingly, as 15 cases have been added by this study, the total number of anisakiasis cases in korea became 660 cases in the korean literature. among 32 anisakis type i larvae detected, 7 were in the fourth - stage (l4), which had molted in the stomach of the patients. they were similar in size with the third stage larvae (l3), but they can be morphologically differentiated from l3 by the following characteristic features: absence of the boring tooth at the anterior end and the mucron at the posterior end; the presence of the 3 well - defined lips; the appearance of transverse striations on the cuticular body surface; the appearance of altered ventriculus and intestine (table 2). the number of human anisakiasis cases (38.6%) and papers (39.1%) published in korea were the highest during 1990 - 1999, while 163 cases (25.3%) were reported in 10 articles (15.6%) during 2000 - 2009, and only 77 (11.9%) were recorded in 18 papers (28.1%) during 1980 - 1989. during 1980 - 1989, each anisakiasis case was meaningful and could be easily published in journals. however, recently, anisakiasis case reports were done after gathering large numbers of cases to demonstrate something different and unique findings. so, the number of articles published was not so many during the recent years. the higher incidence rate was found in coastal areas, such as jeju - do, jinju, (gyeongsangnam - do), busan, incheon, and pohang (gyeongsangbuk - do), where people favorably consume raw flesh of marine fish. moreover, the presence of medical authorities, who are able to produce manuscripts describing case reports, is also an important factor. among total 404 cases that revealed the age and sex of patients, the number of women was slightly more than that of men, with the ratio of women to men of 1.18:1. , who analyzed 141 gastrointestinal anisakiasis cases in a local clinic in jinju - si, gyeongsangnam - do. in their study, the number of women (91 cases) was much more than that of men.. reported total 107 cases in jeju - do, the number of women (47 cases) was lesser than that of men, with the ratio of women to men of 0.78:1. regarding the age prevalence, most of the cases (67.6%) were patients in their thirties and forties. various species of marine fish and cephalopods have been known as the sources of human infections. in european countries, especially in the netherlands and spain, raw or pickled fish, such as herring, hake, anchovy, and cod, are known to be important infection sources. in japan, some species of flounders, japanese common squid, the pacific cod, and the bluefin tuna are the main sources in hokkaido, and the spotted sardine, the chub mackerel, the horse mackerel, the oceanic bonito, and the chum salmon are important infection sources in kyushu. however in korea, the common conger was the most frequently mentioned by the patients as the probable source of infections, followed by the croaker, squid, yellowtail, flatfish, and the other 12 species of marine fish. it has been known that the majority of human infections were caused by the larvae of a. simplex (anisakis type i), and rarely or occasionally by those of anisakis physeteris (anisakis type ii), and pseudoterranova decipiens (terranova type a). in the present review, 81.3% cases were due to anisakis type i larvae, and 11.8% and 3.9% were by terranova type a and anisakis type ii larvae, respectively. recently, the etiologic agents of human anisakiasis in korea, which were morphologically anisakis type i larvae, were molecularly confirmed to be a. pegreffii larvae. moreover, most of the anisakis type i larvae detected in 7 fish species from the yellow sea and the south sea in korea were also identified as a. pegreffii. although only 15 human cases were enrolled by lim et al., it seems that most of the korean ansakiasis cases may be caused by a. pegreffii based on the above 2 molecular studies. molecular methods should be more frequently adopted to identify the causative agent of human anisakiasis in the future. however, anisakid larvae were found rarely in other digestive organs, i.e., esophagus, small intestines, large intestines, and mesocolic lymph node. in most cases, the seasonal prevalence was the highest in winter, followed by in spring, autumn, and summer. this seasonality seemed directly related to the frequency of eating raw fish flesh in each season. the consumption of raw fish meat is more popular in winter season rather than in summer season in korea because of the anxiety for food poisoning.

the present study was performed to report 15 anisakiasis cases in korea and to review the korean cases reported in the literature. total 32 anisakis type i larvae were detected in the stomach of 15 patients by the endoscopy. single worm was detected from 12 cases, and even 9 larvae were found from 2 cases. epigastric pain was most commonly manifested in almost all cases, and hemoptysis and hematemesis were seen in 1 case each. symptom manifestations began at 10 - 12 hr after eating fish in 73.3% cases. endoscopy was performed 1 - 2 days after the symptom onset in most cases. the common conger, conger myriaster, was the probable infection source in 7 cases. in the review of korean anisakiasis cases, thus far, total 645 cases have been reported in 64 articles. anisakis type i larva was the most frequently detected (81.3%). the favorable infection site of larvae was the stomach (82.4%). the common conger was the most probable source of human infections (38.6%). among the total 404 cases which revealed the age and sex of patients, 185 (45.8%) were males, and the remaining 219 (54.2%) were female patients. the age prevalence was the highest in forties (34.7%). the seasonal prevalence was highest in winter (38.8%). by the present study , 15 cases of gastric anisakiasis are added as korean cases, and some epidemiological characteristics of korean anisakiasis were clarified.

the purpose of lowering high blood pressure is to prevent morbidity and mortality from hypertensive complications, such as heart attack, stroke, renal failure, and heart failure. the veterans administration studies in the 1960s proved beyond doubt that treating hypertension with thiazides and -blockers (the drugs available at that time) significantly diminished the rates of strokes, renal failure, and heart failure, although the decrease in the rate of myocardial infarcts did not reach statistical significance. it has been estimated that a 1015 mmhg fall in systolic blood pressure should lead to a 15% reduction in relative risk for heart attack and to a 40% reduction for stroke. with the advent of new classes of antihypertensive agents, the emphasis shifted from efficacy in lowering blood pressure, which is taken for granted, to potential to protect against end - organ damage. controlled clinical trials have indicated that drugs from different classes have different neurohumoral and metabolic profiles, which might enhance or partially offset the benefits from blood pressure lowering per se. for example, thiazides and -adrenergic blockers have been reported to further increase insulin resistance, and hence to accentuate the dysmetabolic syndrome that commonly accompanies essential hypertension. on the contrary, 1-adrenergic blockers and angiotensin - converting enzyme (ace) inhibitors have been reported to improve insulin sensitivity and the lipid profile, whereas calcium - channel blockers were found to be metabolically neutral. in terms of neurohormonal changes, the stimulation of the renin angiotensin and sympathetic systems associated with the use of diuretics and dihydropyridines should be detrimental to end organs, whereas angiotensin blockers and sympathetic blockers should be beneficial to them. several such effects that are theoretically considered to be beneficial have been used as' surrogate endpoints' in the absence of firm data on morbidity and mortality. improvement in surrogate endpoints may be encouraging but is not always predictive of real endpoints, and should not be sufficient to influence clinical decisions. this was shown repeatedly by recent trials (e.g. with estrogen replacement or various antioxidants), where amelioration in various markers did not in improved cardiovascular outcomes. nevertheless, clinical trials on selected subpopulations as well as meta - analyses of pooled data suggest that, at levels producing a similar blood pressure lowering effect, -blockers were cardioprotective and ace inhibitors were both cardioprotective and nephroprotective, while calcium - channel blockers might offer better protection from stroke. these newer classes have not strictly speaking been proven to reduce morbidity and mortality from hypertension, as they could not ethically be tested against placebo. they could, however, be tested against' the gold standard', a thiazide that has been proven to reduce morbidity and mortality in the placebo - controlled trials. this is what led to the antihypertensive and lipid lowering treatment to prevent heart attack trial (allhat). the double - blind, active - controlled component of the allhat was designed to determine whether the rate of the primary outcome (a composite of fatal myocardial infarcts and nonfatal coronary events) would be different in high - risk older patients treated with a drug from each one of three classes of antihypertensives, an ace inhibitor (lisinopril), a calcium blocker (amlodipine), or an 1-adrenergic blocker (doxazosin), compared with patients treated with a thiazide diuretic (chlorthalidone). the trial started in february 1994 and, after an interim analysis in january 2000, an independent review committee recommended that the doxazosin arm be discontinued. this was because, compared with chlorthalidone, doxazosin had a significantly higher relative risk of stroke (1.19) and of combined cardiovascular disease (relative risk = 1.25), and a more than double risk of congestive heart failure (relative risk = 2.04). there was dismay that, once more, improvement in surrogate endpoints (blood pressure, lipid profile, and other parameters of the dysmetabolic syndrome) did not translate into favorable outcomes. there was speculation on what concurrent changes might have overridden the benefits of those improvements. we would like to add our own plausible, although speculative, explanation for these findings. in addition to the renin - angiotensin and the sympathoadrenal systems, arginine vasopressin (avp) is the third major systemic pressor hormone. its pressor function is partly offset by its sensitizing influence on baroreflexes, not fully apparent until the other two systems have been impaired. the importance of avp to systemic or regional vascular resistance can not necessarily be predicted from the circulating levels, as it is markedly increased after effective sympathetic inhibition even in the absence of a further increase in plasma levels. it can only be accurately estimated from the response to a selective antagonist of the v1 type receptors of avp. using such a pharmacologic probe, we have found that the pressor action of avp is maximized after 1-adrenergic blockade. under certain conditions, avp becomes an important vasopressor factor in patients with hypertension and/or congestive heart failure. its pressor influence is most apparent in patients with autonomic insufficiency, such as diabetics or elderly individuals. severe coronary constriction in response to avp has been proposed as the mechanism underlying a number of acute ischemic events reported in the earlier literature. although avp does not seem to cause myocardial ischemia under normal conditions, it may well do so under chronic 1-adrenergic receptor blockade, especially in older patients and in those with various degrees of autonomic insufficiency. the experimental evidence suggests that, in the presence of functional baroreflexes, the small elevation in avp following 1-adrenergic receptor blockade produces an increase in systemic resistance with a strong reflex suppression of cardiac output. in the absence of an intact sympathetic system , the vascular sensitivity to the vasoconstrictor effect of avp is enhanced by several orders of magnitude. accordingly, a combination of these factors could explain the increased rates of ischemic cardiomyopathy and/or heart failure in these patients. what are the implications of the allhat findings? one implication is obviously that 1-adrenergic antagonists should not be first choice antihypertensives, and this message has already been widely disseminated. these agents are, however, used extensively for their urodynamic properties in patients with benign prostatic hypertrophy, many of whom are older hypertensives (possibly with ischemic heart disease) who prefer to use one drug as monotherapy for both purposes. indeed, doxazosin and terazosin are probably much more popular for the treatment of prostatic symptoms than for hypertension, and in this setting any cardiovascular adverse effects from their widespread use would most probably go unappreciated. ace = angiotensin - converting enzyme; avp = arginine vasopressin. allhat = antihypertensive and lipid lowering treatment to prevent heart attack trial.

the lowering of high blood pressure is supposed to protect target organs from hypertensive damage. the antihypertensive and lipid lowering treatment to prevent heart attack trial was designed to compare the cardioprotective properties of three antihypertensives from different classes (lisinopril, amlodipine and doxazosin) with chlorthalidone. despite effective blood pressure lowering and a favorable metabolic profile, the doxazosin arm of the trial had a significantly higher relative risk of cardiovascular disease and heart failure compared with the chlorthalidone arm. this article speculates on possible causes for this unexpected and suggests that the culprit may be accentuation of the vascular effects of vasopressin, which are maximized under -adrenergic blockade. these findings may have implications for the large number of older men who receive monotherapy with -blockers for treatment of prostatic symptoms.

estrogens play crucial roles in the development and maintenance of normal sexual and reproductive function. 17-estradiol (e2) is the predominant and most potent sexual hormone during the reproductive stage in females. its main functions are associated with reproduction, although it is also involved with different pathologies, such as cancer, autoimmune diseases, and infectious processes, in which the innate immune response plays a key function. in bovines, during the period around parturition, cows experience an increased susceptibility to inflammatory disorders in the mammary gland and uterus, for example, mastitis. this increased susceptibility has been correlated with the decreased functionality of the components of the innate immune response and with abrupt changes in the levels of sex steroids. e2 levels rise abruptly in the last week before parturition, peak in the last 3 days before delivery, and fall rapidly after calving and regain basal values. accordingly, lavon et al. reported that cows with subclinical mastitis (without apparent signs) exhibit low circulating e2 levels. in addition, the immunomodulatory effects of e2 in cows with mastitis are associated with a reduction of neutrophil migration. most of the genomic actions of estrogens, including e2, are mediated by two estrogen receptors (ers), er and er, and bovine mammary epithelial cells express both. however, there is little information concerning the immunomodulatory effects of e2 on epithelial cells from the bovine mammary gland. it is well known that e2 is required for mammary epithelial cell proliferation and ductal development in the growing animal. bovine mammary epithelial cells (bmecs) play a relevant role during intramammary infections because they are in intimate contact with the pathogens responsible for mastitis and are a target for intracellular bacteria causing chronic and subclinical infections, such as staphylococcus aureus. this pathogen can persist for long periods of time in cows and is internalized by a zipper - type mechanism depending on the presence of fibronectin - binding proteins (fnbps) on the bacterial surface, as well as the interaction of fibronectin and the host - cell 51 integrin, in which integrin is taken up together with its ligands. in addition, bmecs participate in the innate immune response of the bovine mammary gland producing pro- and anti - inflammatory mediators, as well as antimicrobial peptides, nitric oxide, and so forth , but the role of e2 on this response has not been explored. considering the immunomodulatory effects of e2 in different tissues and that one of its targets is the mammary epithelium, the aim of this work was to analyze whether this hormone modulates the innate immune response of bmecs and its implications during s. aureus internalization. 17-estradiol was acquired from sigma, and working solutions were dissolved in 1% ethanol (1500 pg / ml). for all of the experiments, 1% ethanol (vehicle) was used as a control. the live / dead baclight bacterial viability kit (thermo scientific) the monoclonal blocking antibodies anti-51 integrin (mab2514) and anti - tlr2 (tlr2.1) were obtained from millipore and abcam, respectively. the anti - phospho - er (2511s, ser118) was obtained from cell signaling and the anti - er was acquired from life technologies. the fitc - conjugated secondary antibodies against mouse and rat iggs were purchased from invitrogen and thermo scientific, respectively. this strain was isolated from a case of bovine clinical mastitis and has the capacity to invade bmecs. s. aureus were grown at 37c overnight in luria - bertani broth (lb bioxon), and the cfus were adjusted by measuring the optical density at 600 nm (od 0.2 = 9.2 10 cfu / ml). bmecs were isolated from the alveolar tissue of the udders of healthy lactating cows as previously described. the bmecs were cultured in growth medium (gm) that was composed of a dmem medium / nutrient mixture f12 ham (dmem / f12k, sigma) supplemented with 10% fetal calf serum (equitech bio), 10 g / ml insulin (sigma), 5 g / ml hydrocortisone (sigma), 100 u / ml penicillin, 100 g / ml streptomycin, and 1 g / ml amphotericin b (invitrogen). the cells were grown in a 5% co2 atmosphere at 37c. to perform the e2 and/or s. aureus challenge, polarized monolayers of bmecs (dishes covered with 610 g / cm rat - tail type i collagen, sigma) were cultured in serum - free dmem / f12k without phenol red (sigma) and antibiotics (incomplete medium) for 24 h, and then they were treated with the hormone and/or infected with the bacteria. for the invasion assays, bmec polarized monolayers were used (~10,000 cells were cultured onto 96-well flat - bottom dishes ( corning) ), which were incubated with different concentrations of e2 (1, 5, 10, 50, 150, 300, and 500 pg / ml) in dmem / f12k (sigma) without antibiotics, serum, and phenol red (incomplete medium) for 24 h and then were infected with s. aureus (moi 30 : 1 bacteria per cell). for this, the bmecs were inoculated with bacterial suspensions from a broth of 9.2 10 cfu / ml and incubated for 2 h in 5% co2 at 37c. then, the cells were washed three times with pbs (ph 7.4) and incubated with incomplete medium that was supplemented with 80 g / ml gentamicin for 1 h at 37c to eliminate extracellular bacteria. finally, the bmec monolayers were detached with trypsin- (0.05%) edta (0.02%) (sigma) and lysed with 250 l of sterile distilled water. the bmec lysates were diluted 100-fold, plated on lb agar in triplicate, and incubated overnight at 37c. the number of bmecs cultured in each well plate was calculated for each invasion assay using an automated cellular counter (bio - rad, tc20). the data are presented as the ratio of the cfu recovered per bmec. for the invasion assays in the presence of the blocking antibodies, one hour previous to the addition of s. aureus to the bmecs , the blocking antibodies anti-51 integrin or anti - tlr2 were added separately (10 and 5 g / ml, resp .) to triplicate wells. rat iggs (purified from normal rat serum with protein a - sepharose beads ( sigma) ) or mouse iggs (purified from normal mouse serum and acquired from pierce) were used as the negative controls. the invasion assays were performed using gentamicin protection assays as previously described. to determine the effect of e2 on bmec viability , 10,000 cells were incubated with different concentrations of the hormone in incomplete medium for 24 h at 37c in 96-well plates. then , 10 l of a 5 mg / ml of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h - tetrazolium bromide (mtt, sigma) solution in pbs was added to each well and incubated for 4 h at 37c. finally, 100 l of acid isopropanol (95% isopropanol and 5% 1 n hcl) were added to dissolve the formazan crystals. the optical density was measured with a microplate reader (bio - rad) at 595 nm. the bmec viability was also tested using the trypan blue exclusion assay and the cells were counted in an automated cell counter (bio - rad, tc20). to analyze the effect of e2 on s. aureus growth, 9.2 10 cfu / ml were cultured at 37c in lb broth. the bacterial suspensions were treated with different concentrations of the hormone (1, 5, 10, 50, 150, 300, and 500 pg / ml) and growth was monitored turbidimetrically (600 nm) over 24 h (measuring the absorbance at 2, 4, 6, 8, 12, and 24 h). to evaluate the s. aureus viability in the presence of e2, bacteria were grown (as previously described) in lb broth and were treated with e2 for 24 h at 37c overnight. later, the bacteria were plated on lb agar in triplicate and were incubated overnight at 37c. to do this, the bmecs were treated with e2 and infected with s. aureus as described above. after 2 h of infection, the media were recovered and the bacteria pellet was obtained by centrifugation at 10,000 rpm for 10 min at 4c and was washed three times with pbs. the s. aureus viability was measured using a live / dead baclight bacterial viability kit (thermo scientific). the pellet was incubated with equal volumes (1.5 ml) of component a (syto 9 dye, live indicator) and component b (propidium iodide, dead indicator) at room temperature in the dark for 15 min. after staining the bacteria, the fluorescent signals of 10,000 events were measured and evaluated using a bd accuri c6 cytometer. in addition, s. aureus growth was also analyzed using the e2-treated bmec conditioned media. to do this, the bacteria were grown at 37c overnight, and a s. aureus suspension containing 9.2 10 cfu / ml (od 0.2 at 600 nm) was incubated at 37c for 2 h with the conditioned media. then, the bacteria were plated on lb agar in triplicate and were incubated overnight at 37c. the bmec polarized monolayers were cultured in 24-well dishes (corning) and were treated with e2 (50 pg / ml) for 24 h and/or s. aureus as described above. after the treatment, the cells were washed three times with pbs, detached with trypsin- (0.05%) edta (0.02%) (sigma), centrifuged at 2,500 rpm for 10 min at 4c, and washed with pbs. the bmec pellet was blocked with normal goat serum (5% in pbs, pierce) for 30 min at 4c with shaking, and then the cells were centrifuged and the pellet was incubated with the primary antibodies anti - tlr2 or anti - integrin separately. to analyze the presence of the ers (er or er), nuclei from the bmecs were obtained using the proteojet kit (fermentas), and the procedure continued as described above. to measure the tlr2 membrane abundance (ma) or the nuclear presence of the ers , the cells were incubated with the antibody at a dilution of 1: 50 (pbs containing bsa 0.1%) for 1 h at 4c with shaking. for the determination of integrin ma, invasion assays were performed using different infection times: 30, 60, and 120 min in control bmecs or e2-treated (50 pg / ml, during 24 h) bmecs. the cells were incubated with the anti-51 integrin at a concentration of 10 g / ml for 2 h at 4c with shaking. in all cases, after the primary antibody incubation, the bmecs were washed three times with pbs and incubated with the respective secondary antibody (diluted 1 : 50) (fitc - conjugated anti - mouse iggs for tlr2 and er and fitc - conjugated anti - rat iggs for er and 51 integrin) for 1 h at 4c with shaking in the dark. the pellet was recovered by centrifugation, washed, and fixed with paraformaldehyde (4%) for 10 min at 4c and finally washed three times with pbs. the fluorescent signals of 10,000 events were measured and evaluated using the bd accuri c6 cytometer. the polarized bmec monolayers were cultivated in 96-well flat - bottom plates that were coated (corning - costar) with 610 g / cm rat - tail type i collagen (sigma). the bmecs were incubated with e2 (50 pg / ml for 24 h). prior to the invasion assay (30 min, 1 or 2 h), pharmacological inhibitors of p38 (5 m, sb203580), jnk (20 m, sp600125), or erk1/2 (2.5 m, u0126) were added separately to the bmecs. the cells lysates were plated on lb agar in triplicate and incubated overnight at 37c. the cfu numbers were determined with the standard colony counting technique or s. aureus viability was measured using the live / dead baclight bacterial viability kit as described above. 0.1% dmso (vehicle) was used as a control. to evaluate the map kinase activation levels by flow cytometry, the bmecs were treated with e2 (50 pg / ml), s. aureus, or both, and the samples (30 g of protein) were prepared according to the manufacturer's protocol for adherent cells (becton dickinson, germany). pp38 (t180/y182), pjnk1/2 (t183/185), and perk1/2 (t202/y204) were quantitatively determined using antibodies from a flex set cytometric bead array (becton dickinson) according to the manufacturer's protocol. the flow cytometric analyses were performed using the bd accuri c6 and the cba analysis fcap software (becton dickinson). the minimum detection levels for each phosphoprotein were 0.38 u / ml for pjnk and 0.64 u / ml for pp38 and perk. monolayers of bmecs were cultured in 6-well dishes (~5 10) and then were incubated with 50 pg / ml of e2 (24 h) and/or s. aureus (moi 30 : 1) for 2 h, as described above. the rna was extracted with trizol reagent (invitrogen) according to the manufacturer's instructions. the rt - qpcr assay was performed using the comparative ct method (ct) with a stepone plus real - time pcr system (applied biosystems) according to the manufacturer's instructions. the reactions were carried out with a sybr green pcr master mix (applied biosystems). the specific primer pairs were obtained from invitrogen; their sequences and pcr conditions used to amplify the different bovine mrnas are detailed in table 1. for the measurement of the tnf-, il-1, il-6, and bovine -defensin 1 (defb1) concentrations in the medium, the conditioned media from bmecs treated with e2 (50 pg / ml) and/or s. aureus were collected. the concentrations of tnf- were measured using the duoset elisa development kit (r&d systems) to quantify the bovine tnf- levels according to the manufacturer's instructions, and the concentrations of il-1 and il-6 were assessed using the bovine il-1 and il-6 screening kits, respectively (thermo scientific). the measurement of bovine defb1 in bmecs culture medium was analyzed using the elisa kit for defb1 (mybiosource). the nitric oxide (no) secreted by bmecs into culture medium was evaluated by measuring the nitrite concentration (no) in cell - free media using the griess reaction. after the invasion assays, the medium was filtered through 0.22 m membranes (millipore) to eliminate bacteria. the data were obtained from three independent experiments, each of which was performed in triplicate (except for the elisa of defb1, which was performed in duplicate) and compared by analysis of variance (anova). the are reported as the means the standard errors (se) and the significance level was set at p < 0.05, except for rt - qpcr analysis where standard deviations are shown and fold - change values greater than 2 or less than 0.5 were considered as significantly differentially expressed mrnas according to morey et al.. the from internalization assays, gene expression analyses, and receptor membrane abundance experiments are shown normalized to the cells treated with vehicle (1% ethanol). to evaluate the effect of e2 on s. aureus growth and bmec viability, we incubated bacteria or bmecs in the presence of different concentrations of this hormone (1500 pg / ml). the showed that e2 did not have effect on bmec viability after 24 h of culture (figures 1(a) and 1(b) ), which was determined by a trypan blue exclusion analysis and the mtt assays. in the same way , we showed that e2 did not affect the bacterial viability and growth (figures 1(c) and 1(d) ). to determine the effect of e2 on s. aureus internalization into the bmecs bmecs were treated with different concentrations of the hormone (1500 pg / ml) 24 h before the challenge with the bacteria. according to the number of cfus recovered, a concentration of 50 pg / ml of e2 significantly inhibited s. aureus internalization into the bmecs by 50% (figure 2). the concentrations of 1, 10, and 300 pg / ml also reduced internalization by 20%. the other concentrations did not affect the internalization rate of s. aureus into the bmecs. considering these , in the following experiments related to the regulation of the innate immune response of bmecs by e2, we analyzed only the concentration of 50 pg / ml. because 51 integrin is one of the main extracellular receptors of epithelial cells employed during s. aureus internalization, we wondered whether the specific functional blocking of 51 integrin would modify the number of bacteria internalized into the bmecs. to test this, primary cultures of bmecs treated for 24 h with e2 were incubated with a specific antibody against 51 integrin (figure 3(a) ). e2 treatment (50 pg / ml) reduced the number of cfus internalized into the bmecs, but the blockade of 51 integrin slightly reduced this number (~20%). in addition, the bmecs treated with vehicle reduced ~60% of the s. aureus internalization when 51 integrin was blocked. this finding suggests that, during the internalization of s. aureus into the e2-treated bmecs, integrin 51 does not participate. to determine whether this effect is correlated with 51 integrin gene expression s. aureus induced the expression of the 5 subunit (~8-fold), and e2 upregulated the expression of both subunits. however, the effect of both treatments was similar to the effect of s. aureus (figure 3(b) ). to analyze whether this effect is correlated with the 51 integrin ma in the bmecs the mean fluorescence intensity was used as an indicator of the level of integrin expression per cell in the population. e2 significantly reduces the level of 51 integrin ma (~40%); however, after 2 h of challenge with s. aureus, the level of 51 integrin ma was reduced in a similar manner as that of the e2-treated cells. a similar effect was also observed when the e2-treated bmecs were infected, indicating that integrin is taken up together with bacteria. typical population profiles from the flow cytometry analysis are also shown in figure 3(c). we additionally evaluated the level of 51 integrin ma at different times of challenge with s. aureus in the e2-treated bmecs. the presence of 51 integrin at the membrane of the bmecs was reduced starting from 30 min of s. aureus challenge, showing a similar behavior both in the control and e2-treated cells. despite the data showing that e2 reduces integrin ma (24 h treatment), these suggest that bacteria are able to induce integrin trafficking. thus, 51 integrin ma does not contribute to the reduction in internalization detected in the e2-treated bmecs (figure 2). to explore whether tlr2 participates in the reduction of s. aureus internalization into the bmecs induced by e2, we investigated whether the blockade of this receptor with a specific antibody would modify bacterial endocytosis. as shown in figure 4(a), the blockade of tlr2 in a significant reduction in the amount of s. aureus internalized (~50%); however, a slight reduction was detected in the e2-treated cells (~15%). surprisingly, e2 induces tlr2 gene expression (~6-fold) (figure 4(b) ). as we previously reported, s. aureus induces tlr2 mrna expression (~3-fold); however, the effect of both treatments was similar to the effect of s. aureus alone. the analysis of the mean fluorescence intensity (figure 4(c) ) shows that only s. aureus (~1.4-fold) increased the tlr2 ma, but there is no difference in the tlr2 ma in the presence of e2 and bacteria compared with that obtained with infection alone. thus, tlr2 could not be involved in s. aureus internalization, and it is probably inactivated in e2-treated bmecs. next, we evaluated whether e2 (50 pg / ml) induces the activation of mapks (p38, jnk, or erk1/2), which are activated by tlr2. initially, we investigated whether these kinases participate in s. aureus internalization into bmecs using pharmacological inhibitors prior to bacterial invasion. bmecs that were incubated (for 30 min or 1 or 2 h) with pharmacological inhibitors of p38 (5 m, sb203580), jnk (20 m, sp600125), or erk1/2 (2.5 m, u0126) showed a considerable reduction in s. aureus internalization (~5060% reduction), indicating that these kinases are involved in this process (figure 5(a) ). s. aureus internalization was also analyzed by cfu counting and flow cytometry, which obtained similar (figure 5(b) ). interestingly, these kinases do not participate in the reduction of bacterial internalization induced by e2 because in the presence of inhibitors this reduction persists. furthermore, we evaluated p38, jnk, and erk1/2 phosphorylation to relate their activation states with the e2-mediated reduction of s. aureus internalization into bmecs. when the bmecs were s. aureus - challenged (2 h), the basal activation of p38 was not modified, phosphorylated jnk1/2 was augmented, and erk1/2 activation was reduced (figure 5(c) ), as we previously reported. the vehicle employed for the mapk dilutions was 0.1% dmso, which did not have an effect on internalization assays (data not showed), as was previously reported. interestingly, the bmecs that were treated with e2 demonstrated reduced p38 phosphorylation (~5-fold), and the erk1/2 activation levels were also reduced by ~1-fold, but jnk1/2 remained unchanged in relation to the control cells. when the e2-treated cells were s. aureus - challenged, the p38 activation level was augmented; however, the jnk1/2 and erk1/2 activation levels were essentially not changed. the mapk indicated that e2, prior to bacterial invasion, inhibits the bmec inflammatory response via p38 and erk1/2 inactivation. estrogens act through the estrogen receptors er and er, which belong to the nuclear receptor superfamily of transcription factors. to determine whether these receptors are activated in bmecs treated with e2 and/or infected with s. aureus, we performed a flow cytometry analysis. we used an anti - er antibody, which recognizes its phosphorylated isoform, as an indicator of its activation. to perform these analyses , we isolated bmec nuclei. in figure 6(a), we show that e2 slightly induces the activation of er (~20%), which was returned to basal levels during s. aureus infection. regarding the expression of er mrna, we observed that e2 also induces the expression of its gene (~5-fold), but after infection this induction is higher (~25-fold) (figure 6(b) ). with regard to er activation, we performed flow cytometry analysis on the bmec nuclei. in figure 6(c), we show that only the bmecs infected with s. aureus induce the activation of this receptor in both vehicle- or e2-treated cells in addition, the expression of er mrna was induced both in the e2-treated and the s. aureus - challenged bmecs (~3.5-fold) (figure 6(d) ). the infection of the bmecs treated with the hormone maintained the upregulated levels of er (~2.5-fold). according to these , we hypothesized that 50 pg / ml e2 activates the bmecs via er. next, we explored whether e2 modified the inflammatory response of the bmecs before and during s. aureus infection. we focused on (i) proinflammatory cytokines, such as tnf-, il-1, and il-6; (ii) an anti - inflammatory cytokine (il-10); and (iii) a chemokine, il-8 (figure 7). regarding the proinflammatory cytokines, we performed rt - qpcr for mrna expression and elisas for the evaluation of cytokine secretion. the bmecs treated with e2 (50 pg / ml, 24 h) significantly increased tnf- and il-1 mrna expression (~7- and 3-fold, resp .) (figure 7(a) ). s. aureus infection alone also induced the expression of both cytokines (~5-fold for tnf- and ~3-fold for il-1). in the presence of s. aureus, interestingly, in the e2-treated cells the secretion of these proinflammatory cytokines was diminished in relation to bmecs treated with vehicle (figure 7(c) ). furthermore, 50 pg / ml of e2 also reduced the secretion of tnf- and il-6 in the infected bmecs. only the secretion of il-1 was increased when the e2-treated cells were infected with s. aureus. in figure 7(b), we show that e2 also induces the expression of il-10 and il-8 mrnas (~2.5 and 4.5-fold, resp .). the infection induced the expression of il-10 but together with the hormone increased its expression (~8-fold). the expression of il-8 was not modified in the presence of infection both in the vehicle- and e2-treated cells. altogether, these point to the anti - inflammatory effects of e2 on the bmecs infected with s. aureus. to explore if some antimicrobial mediators are being regulated by e2, which could explain the reduction in s. aureus internalization, we measured the viability of s. aureus in the infection assays. in figure 8(a), we show the viability of s. aureus from the supernatants obtained from the invasion assays (2 h). s. aureus employed in the invasion assays from the e2-treated bmec cultures showed a reduced viability rate (60% of reduction) compared to the bacteria recovered from the supernatants of the vehicle - treated bmecs. this suggests that antimicrobial components are present in the culture media from the bmecs treated with 50 pg / ml of e2. with the purpose of reinforcing these data, we incubated s. aureus for 2 h with 1 ml of conditioned media obtained from the vehicle- or e2-treated bmecs. according to figure 8(b), the conditioned medium from the e2-treated bmecs reduces the number of cfu of s. aureus (a 60% of reduction). next, we explored whether the gene expression of some antimicrobial elements was induced in the e2-treated bmecs. figure 8(c) shows the rt - qpcr analysis of different antimicrobial peptides, including several -defensins and 1 psoriasin (s100a7). (defb1, ~5-fold) and bovine neutrophil beta defensin 5 (bnbd5, ~3-fold). in addition, e2 induces the expression of psoriasin s100a7 (~6.5-fold). in the presence of infection, a similar behavior was observed in the cells treated with e2 and infected with s. aureus, where the expression of psoriasin was upregulated by ~9-fold. considering these , we measured the accumulation of defb1 in the bmec medium by elisa. in figure 8(d), we show that e2 does not induce the secretion of defb1 in the culture medium, which is stimulated by infection. s. aureus infection in the e2-treated cells reduces the concentration of defb1 secreted in relation to infection alone. thus, this is not the mechanism by which bacterial internalization is reduced in the e2-treated cells. in addition, the secretion of no was not modified in the presence of e2 in the bmecs (figure 8(e) ), and thus it is not involved in the reduced internalization detected in the e2-treated bmecs. it is well known that, during the postpartum period, dairy cows experience an increased incidence of mammary infectious diseases, such as mastitis. this augmented frequency has been correlated with a decreased functionality of the innate immune system. during this period, e2 levels rise abruptly in the last week before parturition, peak in the last 3 days before delivery, and fall rapidly after calving and regain basal values. it has been described that estrogens have both anti - inflammatory and proinflammatory functions, and their role in the modulation of the innate immune response of epithelial cells during infection has been documented. it has been shown that e2 is required for mammary epithelial cell proliferation and ductal development in the growing animal. the goal of this work was to study the immunomodulatory functions of e2 on bmecs during s. aureus infection, which have not been explored to date. according to previous reports showing immunomodulatory functions of e2 on bovine neutrophils , we evaluated e2 in a range of concentrations between 1 and 500 pg / ml. thus, it is important to analyze whether e2 alters s. aureus viability or growth. at the incubation times and the range of e2 concentrations analyzed we did not detect a bacteriostatic or a bactericidal effect of e2 on s. aureus (figure 1). in agreement, hosoda et al. showed that different steroid hormones were not bactericidal for s. aureus. in addition, at the range of concentrations tested, e2 was not toxic to the bovine mammary epithelial cells. have reported that similar concentrations of e2 and progesterone exert stimulatory effects on autophagy in bmecs after 48 h, which is implied during the involution of the bovine mammary gland in the dry period. in the present study, we can not discard the possibility that longer incubation times with e2 could be cytotoxic for these cells. in this work , we demonstrated that 50 pg / ml of e2 significantly inhibits ~50% s. aureus internalization into the bmecs (figure 2). because e2 does not have antibacterial activity , this reduction might be the of the immunomodulatory effects that have been associated with this hormone. according to our , the effect of e2 on s. aureus internalization shows a nonmonotonic dose - response curve. this behavior requires further investigation, however; nonmonotonic effects of e2 have been described on the development of mammary gland in in vivo models, as well as in human breast cancer cells. a possible explanation could be related to the versatility of e2 actions, which include the classic genomic functions, as well as nongenomic or membrane - initiated estrogen - signaling pathway. the inhibitory effect of e2 on s. aureus internalization can not be attributable to the reduction in integrin 51 ma because this receptor does not participate in the internalization of s. aureus in e2-treated bmecs. in addition, although e2 reduces 51 ma, the presence of bacteria promotes its traffic at early times of infection (figure 3), as we reported previously. considering this , the inhibition on s. aureus internalization in the e2-treated bmecs could be a consequence of the activation of immunomodulatory pathways. to explore this possibility accordingly, the shown in figure 4 suggest that e2 does not induce the ma of this receptor despite the fact that it upregulates the expression of its gene. mammary epithelial cells play an essential role in the surveillance of mammary tissue during infections because they help in immune cell recruitment and bacterial recognition via the tlr signaling pathways. we have previously demonstrated that s. aureus induces tlr2 ma and activation in bmecs in the same way that the lactogenic hormone prolactin stimulates s. aureus internalization in the bmecs. e2 shows differential effects on tlr2 expression and/or activation; for example, in thp-1 cells, it increases the expression of tlr2 mrna, but, in a bovine oviduct epithelial cell culture, it reduces the lps - induced tlr2 mrna expression. thus, the activation of the tlr2 pathways by e2 depends on the tissue and the physiological condition of the organism. in agreement with the absence of tlr2 activation in e2-treated bmecs , we detected that the mapk p38 and erk1/2 are inactivated in these cells (figure 5). in addition, these kinases are not employed during the inhibition of s. aureus internalization induced by e2 (figure 5). we have previously reported that these three mapks are required during s. aureus internalization into the bmecs. we next explored whether e2 or infection regulates ers activation because the direct effects of this hormone are exerted via intracellular ers. in figure 6 , we showed that e2 induces the intracellular activation of er, as well as upregulating the expression of its gene. likewise, s. aureus infection increases er mrna expression in the e2-treated bmecs, but this induction is not related with er activation. in agreement, yart et al. reported that in a cell line of bmecs (mac - t) the expression of er protein increases in response to e2 treatment. however, the activation of er by infection has not been extensively explored to date. the presence of er in the nuclei from infected bmecs (figure 6(c) ) indicates the participation of this receptor during s. aureus infection, which has not been reported. nevertheless, flow cytometry analysis reveals that e2 does not activate er. taken together, these suggest that e2 activates er in bmecs, which might be directly or indirectly implicated in the reduction of s. aureus internalization. because we did not detect the activation of mapks in the e2-treated bmecs (figure 5(c) ) it is probable that e2 triggers the direct activation of er in bmecs, which up- and/or downregulates the transcription of various genes by binding to the estrogen response element of the genes or by interacting with other transcription factors. to address this hypothesis, we analyzed some inflammatory genes in the e2-treated bmecs in the absence or presence of s. aureus infection. we previously reported that, with the exception of tnf-, s. aureus inhibits the innate immune response of bmecs, as it is unable to induce the gene expression of some proinflammatory cytokines, such as il-1 or il-6. however, we also reported that, in the presence of ethanol as vehicle (2%), s. aureus slightly induces il-1 mrna expression. interestingly, e2 at 50 pg / ml significantly increases proinflammatory cytokine mrna expression such as tnf- and il-1, prior to s. aureus infection. however, upon infection, the levels of both cytokine mrnas were downregulated in e2-treated bmecs. the differential influence of e2 on the expression of innate immune response genes depends on multiple factors, such as the hormone concentration or the physiological condition of the individuals. these coincide with other reports using urinary epithelial cells, where e2 reduces the lps - induced cytokine expression. steroids have the ability to suppress (and/or resolve) an inflammatory response. in agreement with the anti - inflammatory function of e2 on bmecs , we detected a significant reduction in the secretion of proinflammatory cytokines (figure 7(c) ) in the e2-treated cells. this effect was maintained in the presence of s. aureus, with exception of il-1, which was induced in the infected e2-treated cells. this effect can be the consequence of the activation of other mechanisms, such as inflammasome activation, which requires further research. interestingly, and in agreement with the anti - inflammatory actions of the hormone, e2 also induces the mrna expression of il-10, an anti - inflammatory cytokine, which was upregulated by the hormone in the e2-treated cells challenged with s. aureus. in addition to the immunomodulatory role of e2, it has been reported that this hormone also induces the production of antibacterial compounds in uterine epithelial cells; a similar effect in the present model could explain the reduction in the internalization of s. aureus. our data suggest that e2-treated bmecs produce an antibacterial compound, which is secreted into the culture medium (figures 8(a) and 8(b) ). to determine the origin of this activity, we analyzed the mrna expression of different antimicrobial peptides (-defensins and psoriasin). we also analyzed the secretion of defb1 into the culture medium, which was only induced by bacteria and not by e2. it is necessary to analyze the concentrations of bnbd5 or psoriasin in the culture medium to resolve if they are implicated in the reduction of s. aureus internalization in the e2-treated bmecs. accordingly, fahey et al. determined that in uterine epithelial cells e2 exerts anti - inflammatory effects and enhances the production of antimicrobial peptides, which display direct defense actions during the infection of this tissue. it is possible that a similar effect could be occurring in bmecs, but further research is necessary to corroborate this possibility. in addition, the induction of psoriasin expression by e2 has been demonstrated in the human mammary epithelial cell line mcf-7. according to our altogether, these indicate that e2 at 50 pg / ml reduces s. aureus internalization in the bmecs by modulating the innate immune response through the activation of er. because we did not detect er activation in s. aureus - challenged bmecs, we can hypothesize that 50 pg / ml of e2 during 24 h activates er in bmecs, which promotes a slightly anti - inflammatory response that is enhanced during infection. it is possible that during infection other mechanisms (i.e., transcription factors) are participating because the activation of er is turned off. in this process e2 (50 pg / ml) induces the anti - inflammatory response of the bmecs during s. aureus internalization through the participation of er. this leads to the increased production of antimicrobial molecules, favoring s. aureus elimination.

17-estradiol (e2), the predominant sexual hormone in females, is associated with the modulation of the innate immune response (iir), and changes in its levels at parturition are related to intramammary infections, such as mastitis. in bovine mammary epithelial cells (bmecs), e2 regulates differentiation and proliferation, but its immunomodulatory functions have not been explored. staphylococcus aureus is the predominant pathogen causing mastitis, which can persist intracellularly in bmecs. the aim of this work was to analyze whether e2 modulates the iir of bmecs during s. aureus internalization. bmecs treated with e2 (50 pg / ml, 24 h) reduced bacteria internalization (~50%). the host receptors 51 and tlr2 do not participate in this reduction. however, e2 activates er and modulates the iir reducing the s. aureus induced - mrna expression of tnf- (~50%) and il-1 (90%). e2 also decreased the secretion of these cytokines as well as il-6 production; however, in infected bmecs, e2 induced the secretion of il-1. furthermore, e2 upregulates the expression of the antimicrobial peptides defb1, bnbd5, and psoriasin s100a7 (~5-, 3-, and 6-fold, resp .). in addition, e2 induced the production of antimicrobial compounds in bmec culture medium, which, together with the modulation of the iir, could be related to the reduction of s. aureus internalization.

renal transplantation is the best available renal replacement therapy for end - stage renal disease. kidney transplant recipients have a better quality of life and consume fewer health care resources compared with patients on dialysis. the number of patients with end - stage renal disease is rising rapidly, while those who can undergo a kidney graft are limited because of the donor organ shortage. the organs supplied by living donors are superior to those from cadaveric sources. improvements in the use of immunosuppression and advances in tissue typing have been associated with better patient and graft survivals in recent years. despite studies that compared the outcome of related and unrelated living donation worldwide, an evaluation of the impact of live unrelated kidney donor (lurd) as a source for renal transplantation has not been adequately studied in egypt. concerning this hypothesis, we decided to study the donor - recipient relationship and its impact on both graft and patient survival among egyptian patients. in egypt, there are no cadaveric kidney transplantations and the only source for renal transplantation is through living donation. so, we tried to encourage all types of living donation participants to answer the question whether unrelated donation is inferior to related donation. this study was comprised of 2,485 kidney transplant recipients who received their grafts between march 1976 and december 2013 at our center. out of this total, 2,075 received their grafts from living related donors (related group a parent, a child, or a sibling of the recipient), while 410 received their grafts from live unrelated kidney donors (unrelated group - spouses, friends, or altruistic individuals). we compared demographic characteristics, acute rejection episodes, chronic rejection, complication rates and long - term graft, and patient survivals among the groups. rejection was diagnosed on the basis of an increase in serum creatinine, confirmed by examination of a graft biopsy sample. all donors and recipients were evaluated by standard biochemical, serological, and radiological evaluation and they received immunosuppressive therapy. a total of 2,075 living related donor (lrd) and 410 living unrelated donor (lurd) transplants were performed during the period. our showed high statistical significance regarding both donor and recipient age (p < 0.001); the mean age of donors was higher in the related group (lrd 36.2 10.5 years versus lurd 31.4 6.4 years), while the mean age of recipients was higher in the unrelated group (lurd 34.8 11.1 years versus lrd 28.8 9.8 years). the percentage of male donors was significantly higher in the unrelated group (p < 0.001). hematological workup showed that blood grouping had a high statistical significance (p = 0.002), however, no significant difference regarding blood transfusion could be observed between both groups (p = 0.71). the percentage of couples with one dr matched locus was higher in the unrelated group (lurd 94.9% versus lrd 85.5%), while the percentage of couples with two dr matched loci was higher in the related group (lrd 14.5% versus lurd 5.1%). the percentages of couples with zero, one, and two hla matching were higher in the related group (8.8%, 12.8%, 64.5%), respectively, while the percentages of couples with three and four hla matching were higher in the unrelated group (39.8% and 21.9%) (table 1). (%) or mean sd. the most common causes of end - stage kidney disease (eskd) in the lurd group were glomerulonephritis and polycystic kidney disease (n = 66, 16.1% and n = 43, 10.5%), while in the lrd were due to unknown causes and obstructive uropathy (n = 1,406, 67.7% and n = 98, 4.7%) (table 2). to induction and maintenance immunosuppressive protocols, atg induction had the highest percentage in the unrelated group (lurd 16.4% versus lrd 6.9%) (table 3). the percentages of recipients maintained on steroid - azathioprine or mycophenolate mofteil (mmf) and tacrolimus (tac)-mmf were significantly higher in the related group (lrd 13.5% versus lurd 7.1%, and lrd 17.1% versus lurd 11.4%), respectively, while the percentages of recipients maintained on steroid - cyclosporine azathioprine or mmf were significantly higher in the unrelated group (lurd 61.2% versus lrd 52.2%) with comparable percentages in both groups regarding other protocols (table 4). there were no significant differences between both groups regarding hypertension, diabetes mellitus, hepatic problems, infections, or malignancy (table 5). the rate of acute vascular rejection was significantly higher in the unrelated group (lurd n = 26, 6.3% versus lrd n = 71, 3.41%), while the rate of cases without acute rejection was significantly higher in the related group (lrd n = 960, 46.3% versus lurd there was no statistical significance between both groups in regards to creatinine clearance and serum creatinine for one, three, and five years post transplantation ( p = 0.684, 0.579, 0.201, and 0.107), respectively (table 7). the graft and patient survival of each group is shown in tables 8 and 9. kaplan - meier graft and patient survival curves for each group are shown in figures 1 and 2. there were no significant differences regarding graft and patient survival between both groups (p = 0.071 and p = 0.386, respectively). kidney donation by biologically unrelated persons has been attempted in different areas of the world, including the middle and far east. these donations have received adverse publicity because of multiple factors, including the following: unresolved ethical issues like donor payment and possible coercion, unacceptably high donor and recipient morbidity and mortality, and poor allograft survival rates. with these points in mind, our center allows transplantation from living unrelated donors under certain circumstances, like hereditary nephritis, polycystic renal diseases and in the case of re - transplantation. renal transplantation from living unrelated donors is successful, but has been met with some opposition due to poor tissue antigen compatibility and fear of commercialization. in the present study, living unrelated recipients tend to be more elderly with younger donors, and a high percentage of male donors; however, in the living related group there are a high percentage of female donors, which was observed in live - donor programs in most countries, including the united states and australia. in australia, female donors accounted for 53% and 62% of overall lrd and lurd donors, respectively; the latter likely reflects the growth in spousal donation. the reason for the greater proportion of female donors remains unclear, although some contributing factors could be medical (higher rates of cardiovascular disease in men) or psychosocial (financial issues and differing perception towards donation between genders). our immunological work agrees with fuller tf et al. and humar a et al. , since the number of live related transplants (lrt) with 3 & 4 hla & 2dr matching are significantly higher than in the live unrelated transplants (lurt). we started our transplantation program in the mansoura urology and nephrology center (unc) moving from one immunosuppressive protocol to another by starting with steroid and azathioprine and moving to the use of mmf, tac, and sirolimus. our study revealed no significant differences between lrt and lurt regarding immunosuppressive protocols, apart from the protocols steroid - azathioprine or mmf and tac - mmf where a higher percentage of lrd group (p < 0.001) and (p = 0.004) respectively and this correlated with better hla matching that encouraged less immunosuppressive drugs, like a steroid - free regimen (tac and mmf protocol), while the protocols steroid - cyclosporine azathioprine or mmf were significantly higher in the unrelated group (p < 0.001). for induction immunosuppression, we considered the poorer hla matching in the unrelated group and used anti - thymocyte globulin (atg) and this correlated with the kdigo guidelines that recommend the use of atg, which is a potent immunosuppressive agent, rather than interleukin-2 receptor antibodies principally for groups at high - risk for allograft rejection. although the incidence of early graft loss because of acute rejection has decreased steadily over the past decades, acute rejection is considered a major risk factor for chronic rejection and a strong predictor of long - term graft survival in both cadaveric and living donor kidney transplants. in the present study, the percentages of patients with acute vascular rejection were significantly higher in the unrelated group (p = 0.005). , who reported that the incidence of acute rejection was not higher for lurd recipients after comparing 595 lrds with 116 lurds; these mismatching could be explained by the difference in immunosuppression protocols or the difference in hla matching. surprisingly in our study, there was no difference in the biopsy proved chronic rejection between both groups (p = 0.07), despite the higher incidence of acute vascular rejection in lurd. we found a higher incidence of early rejection in lurd compared to lrd and this agrees with fuller et al , who reported higher percentages of early and severe rejections in lurt than lrt. there are some important factors that might impede the use of lurd sources, such as the elderly age of donors and the higher number of hla mismatches compared with lrd. our study is not in agreement with previous studies, since the lurd ages were significantly younger than lrd ages; this may be due to most lurd recipients in that study being friends and spouses, which is not the same as in our study. we reported no significant differences in regards to creatinine clearance and serum creatinine for one, three, and five years post transplantation between the two groups. the one-, five-, and ten - year graft survival rates were 97%, 86.6%, and 67.9%, respectively, for recipients of lrd, while that for recipients of lurd were 95.4%, 83.6%, and 66.7%, respectively (figure 1) (tables 8 and 9). the one-, five-, and ten - year patient survival rates were 97.1%, 95.1%, and 80.8%, respectively, for recipients of lrd, while that for recipients of lurd were 95%, 88.8%, and 67%, respectively (figure 2) (tables 8 and 9). for example, in the 2008 annual report of the scientific registry of transplant recipients, the unadjusted five - year survival of lurd kidneys was the same as that of living related donor kidneys (approximately 80 %). in italy, graft survival rates of 172 lurt recipients were 87% in one year, 79% in five years, and 69% in nine years. 19 ), reported that patient survival in lurd recipients was worse than in lrd recipients; however, this study included a high percentage of diabetic patients. second, there were many changes in immunosuppressive protocols over the last few decades, but we should consider that the study was comprised of live matched donors and the majority were related donors with insignificant immunological risks in the unrelated group. graft survival is affected by factors like age of the donor, degree of hla compatibility, original kidney disease, number and severity of acute rejection episodes, despite that kidney transplant recipients who received their grafts either from live related donors or live unrelated donors had a comparable patient and graft survival. kidney donation by volunteers who are genetically unrelated to their recipients is medically successful, socially valuable, and ethically acceptable provided that donors are healthy, competent, and well - informed. second, there were many changes in immunosuppressive protocols over the last few decades, but we should consider that the study was comprised of live matched donors and the majority were related donors with insignificant immunological risks in the unrelated group. graft survival is affected by factors like age of the donor, degree of hla compatibility, original kidney disease, number and severity of acute rejection episodes, despite that kidney transplant recipients who received their grafts either from live related donors or live unrelated donors had a comparable patient and graft survival. kidney donation by volunteers who are genetically unrelated to their recipients is medically successful, socially valuable, and ethically acceptable provided that donors are healthy, competent, and well - informed.

renal transplantation is the ideal method for management of end - stage renal disease. the use of living donors for renal transplantation was critical for early development in the field and preceded the use of cadaveric donors. most donors are related genetically to the recipients, like a parent, a child, or a sibling of the recipient, but there are an increasing percentage of cases where donors are genetically unrelated like spouses, friends, or altruistic individuals. donor shortages constitute the major barrier for kidney transplantation, and much effort has been made to increase the supply of living donors. the impact of donor source on the outcome of renal transplantation is not adequately studied in our country.objectivesthe aim of the study was to evaluate the impact of donor source on the outcome of live donor kidney transplantation.patients and methodsfrom march 1976 to december 2013, the number of patients that underwent living renal transplantation sharing at least one hla haplotype with their donors was 2,485. we divided these patients into two groups: 2,075 kidney transplant recipients (1,554 or 74.9% male and 521 or 25.1% female) for whom the donors were living related, 410 kidney transplant recipients (297 or 72.4% male and 113 or 27.6% female) for whom the donors were living unrelated. all patients received immunosuppressive therapy, consisting of a calcineurin inhibitor, mycophenolate mofetil, or azathioprine and prednisolone. we compared acute rejection and complication rates, as well as long - term graft and patient survival of both groups. demographic characteristics were compared using the chi - square test. graft survival and patient survival were calculated using the kaplan - meier method.the percentages of patients with acute vascular rejection were significantly higher in the unrelated group, while percentages of patients with no rejection were significantly higher in the related group, but there were no significant differences regarding patient and graft survivals between both groups.kidney transplant recipients who received their grafts either from live related donors or live unrelated donors had comparable patient and graft survival outcomes.

a patient with massive exposure to tetrachloroethylene fumes presented with coma and severe pulmonary edema. sequential blood gases, chest x - rays, and clinical findings showed dramatic improvement with conventional but aggressive management and the patient recovered completely. there was no evidence of permanent renal, hepatic, or central nervous system damage.imagesfigure 1.figure 2.figure 3.

it covers a vast array of devices that are derived from the areas of engineering, biology, physics and chemistry. these devices include vectors for the targeted delivery of anticancer drugs, imaging contrast agents and arrays for the early detection of tumors. these, and other nano - devices, may support the future diagnosis and treatment of common conditions such as cancer. nanotechnology was first proposed in 1867 by james clarke maxwell, a scottish physicist who developed electromagnetic theory and statistical physics. there is plenty of room at the bottom , given at caltech in 1959, he spoke of building factories that maneuvered things atom by atom. later in 1974, this concept was defined by professor norio taniguchi of tokyo university of science as the separation, consolidation, and deformation of materials by one atom or one molecule. k. eric drexler, who suggested that it would be possible to build computers and robots far smaller than a single cell. nanotechnology typically refers to structures up to 200 nanometers in size. as a system s size diminishes to nano - scale quantum realm. in this realm, particle behavior and processes are governed by quantum and statistical mechanics and atoms are manipulated by covalent, electromagnetic and van der walls forces. structures made up of a monolayer (a sheet that is one atom thick), have properties that differ markedly from conventional experience. opaque and stable metals, such as gold, are transformed into monolayers that are transparent, inflammable catalysts. nanotechnology is being used not only in the development of such catalysts, but additionally in semi - conductor technologies, such as memory storage, construction and household products such as sunglasses. relating to cancer, nanotechnology is being applied in the following two broad areas: the development of nanovectors - nanoparticles which can be loaded with drugs or imaging agents and then targeted to tumor cells.nanosensors - devices for the detection of cancer cells. the development of nanovectors - nanoparticles which can be loaded with drugs or imaging agents and then targeted to tumor cells. nanovectors may eventually become a powerful tool for the delivery of anticancer drugs. usually have a tripartite constitution; a core constituent material, a therapeutic and / or imaging payload, and biological surface modifiers which enhance the bio - distribution and tumor targeting of the nanoparticle. through the process of targeted biorecognition , nanovectors have the potential to deliver large amounts of a therapeutic or imaging agent to a tumor. for example, a nanovector may selectively bind to the neovascular endothelium of a tumor and release a penetration enhancer. this may then deploy a track of molecular motor molecules such as actin. the nanovector, or another, can then release a therapeutic agent, bound to a conjugate molecule, such as myosin. this agent could then travel along the molecular track, reaching deep into the cancer lesion and overcoming opposing oncotic and osmotic pressures. in addition to the delivery of therapeutic agents, nanovector s formulations can be designed to reduce the clearance time of small peptide drugs by providing protection of active agents from enzymatic or environmental degradation and avoiding obstacles to the targeting of the active moiety. this includes gadolinium or iron oxide - based nanoparticles and multiple - mode imaging contrast nano - agents, which combine magnetic resonance with biological targeting and optical detection. low - density lipid nanoparticles also have been used to enhance ultrasound imaging. many polymer - based nanovectors have been investigated and some appear more promising for clinical translation. for different imaging modalities, it is possible to develop nanoparticles, which can provide signal enhancement combined with biomolecular targeting capabilities. these nanovectors have typically been developed as components of a multi - functional unit with generic core components, a nanoplatform. this is coupled to a generic image enhancing agent, a generic therapeutic agent (such as a cell toxin) and a generic reporter agent which enables sensors to detect the successful delivery of the therapeutic agent to its target. a vector has been engineered at the atomic level to deliver a magnetic core and an optical probe to cells with receptors for the luteinizing hormone (lh - rh). they consist of three major architectural components: core, branches, and end groups. one of the most appealing aspects of technologies based on dendrimers is that it is relatively easy to precisely control their size, composition, and chemical reactivity. dendrimers can serve as versatile nanoscale platforms for creating multifunctional devices capable of detecting cancer and drug delivery. nanoparticles can be designed to extravasate into the tumour stroma through the fenestrations of the angiogenic vasculature and demonstrate targeting by enhanced permeation and retention. the particles carry multiple antibodies, which direct antitumor action by targeting epitopes on cancer cells. when irradiated by external energy, nanoparticles become activated and release their cytotoxic action. through this , they could provide anti - angiogenic therapy by preferentially adhering to cancer neovasculature and causing it to collapse. cantilevers are constructed as parts of a larger diagnostic device and can provide rapid and selective detection of cancer - related molecules. cancer specific molecules bind to molecules that act as sensors. this movement is exaggerated as the sensor molecule acts like a single lever cantilever bridge. dna - coated gold nanoparticles (nps) form the basis of a system that also uses larger magnetic microparticles (mmps) to detect femtomolar (10 molar) concentrations of serum proteins. in this case, a monoclonal antibody to prostate specific antigen (psa) is attached to the mmp, creating a reagent to capture free psa. a second antibody to psa is attached to the nps and two particles create a sandwich of the captured protein which can be easily separated using a magnetic field. this technique has the potential to quantify an antigen at concentrations that would not be possible using a conventional enzyme linked imunosorbant assay (elisa). in an ideal scenario, the onset of the transformational processes leading towards malignancy would be detected early. this could exist as routine screening by non - invasive means such as proteomic pattern analysis from blood samples, or the in vivo imaging of molecular profiles and evolving lesion contours. the biology of the host and the disease would be accurately determined, and dictate choices for targeting and barrier - avoiding strategies for an intervention plan. transforming cellular populations would be eradicated or at least contained, without collateral effects on healthy tissues, in a routine that could be repeated many times. if fully integrated with the established cancer research enterprise, nanotechnology might help this vision become reality. some of the principal challenges along this path are as follows: developing approaches for the in vivo detection and monitoring of cancer markersimprovement of the targeting efficacy of therapeutic or imaging agents to cancer lesions and their microenvironmentrefining technology platforms for early detection of cancer biomarkers ex vivoengineering nanoparticles to avoid biological and biophysical barriers. developing approaches for the in vivo detection and monitoring of cancer markers improvement of the targeting efficacy of therapeutic or imaging agents to cancer lesions and their microenvironment refining technology platforms for early detection of cancer biomarkers ex vivo engineering nanoparticles to avoid biological and biophysical barriers. nanotechnology is expected to play an important role in the early detection of transforming cell populations by in vivo imaging or ex vivo analysis. this new and highly sophisticated technology allows the appropriate combination of agents, based on accurate tumour specific biological information, targeting them to the cancer cells, whilst avoiding biological barriers. however, their approval perspectives, biodistribution, reliability of production protocols, as well as safety for patients and the health - care workers should not be ignored.

nanotechnology is the engineering of functional systems at the molecular scale which may exert a revolutionary impact on cancer diagnosis and therapy. nanotechnology is being applied to cancer in two broad areas: i ) the development of nanovectors such as nanoparticles which can be loaded with drugs or imaging agents and then targeted to tumours, and ii ) high - throughput nanosensor devices for detecting the biological signatures of cancer. combined, such technologies could lead to earlier diagnosis and better treatment for cancer patients.

using a hierarchical framework, tissue engineering can be subdivided into different strategies or concepts (fig . 1). one strategy is purely cell - based involving the transplantation of autologous or allogeneic cell suspensions or cell - sheets that are injected and/or transplanted to a defect site or an injured tissue. another strategy utilizes biomolecules (growth factors, completely lyophilized cell fractions, peptides, polysaccharides, etc .) aimed at delivering cues to the cells of the host tissue. other methods are based on the use of different types of matrices (hydrogels, microspheres / beads, etc .) in combination with cells and/or biomolecules. the fourth and most frequently applied strategy focuses on seeding and culturing specific cell types in 3-d environments that closely mimic natural extracellular matrix. such 3-d environments are specifically configured as cellular solids and referred to as' scaffolds' (fig . a paradigm shift is taking place in orthopaedic and reconstructive surgery from using medical devices and tissue grafts to engineering a tissue engineered construct ( c) that uses biodegradable scaffolds (a) combined with cells (b) or biological molecules (b #) to repair and/or regenerate tissues. the tissue engineering laboratory at nus did design, fabricate and characterize composite scaffolds made of mpcl- cap by fdm in vitro (ho st, hutmacher dw . ( zhou yf, chou am, li zm, hutmacher dw, sae - lim v, lim tm . combined marrow stromal cell sheet techniques and high strength biodegradable composite scaffolds for engineered functional bone grafts . biomaterials . 2007 ; 28:81424) polymer / cap composites confer favourable mechanical and biochemical properties, including strength viathe ceramic phase, toughness and plasticity viathe polymer phase, more favourable degradation and resorption kinetics, and graded mechanical stiffness. in these scaffolds, ceramic phase was homogenously distributed in the matrix (arrows, inset left corner) as well as exposed on the surface (figure a, arrows highlight cap particles on surface). contact angle measurements showed that such composite surfaces were more hydrophilic and that the degradation kinetics were accelerated 3 to 4 times compared to pcl alone. it can be argued that the' scaffold - based tissue engineering concept' was introduced in the mid- 1980s when dr joseph vacanti of the children's hospital approached dr robert langer of mit with an idea to design scaffolds for cell delivery as opposed to seeding cells onto or mixing cells into naturally occurring matrices with physical and chemical properties that are difficult to manipulate. today's concepts of scaffold- and matrix - based tissue engineering involve the combination of a scaffold with cells and/or biomolecules that promote the repair and/or regeneration of tissues. these tissue- engineered constructs (tec) are under intense investigation and various approaches and strategies are continually being developed. however, despite intense efforts, the ideal scaffold / cell or scaffold / neotissue construct (even for a specific tissue type) has yet to be developed. certain minimum requirements are essential when developing tecs that must address the biochemical as well as chemical and physical properties of native tissue. of these requirements, biocompatibility, vascularization and chemotaxis are vital. the scaffold must also be nonimmunogenic and free from prions involved in disease transmission and it must also possess suitable architectural qualities that are easily reproducible. lastly, one must consider the temporal and spatial variations in some of these factors both in vitro and/or in vivo. despite rapid advancements in the fabrication of tecs, tissue engineering is still a long way off matching natures' ability to grow and repair tissues and organs. hence, tissue engineering still has room for improvement, nevertheless, from an engineering perspective the past decade has seen significant advances in scaffold design and fabrication. starting with simple foams and fibers, porosity and pore interconnectivity has quickly become a central theme, with pore dimensions and material properties being vital in promoting cell seeding, migration, proliferation and the de novo production of extra - cellular matrix. significant advances have also been made in the incorporation of bioactive molecules into scaffolds, specifically in the area of bone engineering (figs 3 and 4). constructs with bmsc sheet - scaffolds (mpcl - tcp) were implanted into nude rat, harvested after 28, 56 and 84 days. (a) gross appearance of bmsc sheet - scaffolds constructs with osteogenic induction (right) and non - induction (control) (left) after 4 weeks. (b) x - ray detected bone like tissue formation in 28 day (c) 56 day (d) 84 day implants. (e) x - ray image of constructs without cell seeding (control). (f) micro - ct demonstrated the overall highly mineralized tissue similar to cortical (golden) and cancellous (red) bone in implanted constructs after 28 day. mineralized tissue with similar density with cancellous bone was detected (darker areas) while the lighter colour represented cortical bone. (h) fluorescence was detected on the formed bone tissue after 28 days; the fluorescence came from the cfda labeled bmsc. (i) micro - ct quantification of implant tissue compositions depicted substantial bone formation in the induction group, accounting for 40% total volume for the 28, 56 and 84 day implantation, while the control group formed only connective tissue. (j) h&e staining shows lamellar bone - like tissue formed in both outer part and interior of constructs after 28 days. (k) high magnification image shows well organized lamellar bone like tissue (bo) with distinct osteocytes located within bone tissue. (l) the typical osteoblasts (ob, black arrow) located on the surface of neo mineralized tissue with marrow cavities and blood vessels in 56 days implants. (m) safranin - o staining demonstrated that hypertrophic chondrocytes (white arrow) were observed in 56 day implants in very low numbers. high magnification shows the chondrocytes (white arrow) with weak safranin - o staining surrounded by the osteocyte (black arrow). (n) ocn staining. strong signals (arrow head) were detected on neo mineralized tissue while very weak to no - signals were detected on chondrocyte like cells (o). limited signals (arrow head) were detected on chondrocyte like cells while no staining for the mineralized tissues. bv: blood vessel + red blood cells; ma: marrow; ob: osteoblast; bo: bone; oc: osteocyte; cy: chondrocyte. defects of load - bearing long bones, for instance, require constructs with high mechanical stability whereas initial plasticity is not essential. on the other hand for craniofacial applications, e.g. orbital floor fractures, moldable scaffolds (a d) are favorable. depending on the implantation site, initial vascularization is essential for enhanced engraftment and prevention of infections. mechanical stability, osteoconductivity by attracting and stimulating bone - forming cells of the host bone, and ease of handling have been well balanced in the design of mpcl scaffold sheets in order to properly meet the clinician's needs. the clinical follow up 2.5 years postsurgery (lower ct image) of a patient receiving a mpcl scaffold (defect site shown in upper ct image) for the reconstruction of a orbital floor fracture defect showed complete bone regeneration of the defect site (arrow). native bone tissue is a major storage site for growth factors, especially for those characterized by their high affinity to heparin (heparin - binding growth factors), such as fibroblast growth factors (fgfs), transforming growth factor-s (tgf-s), and bone morphogenic proteins (bmps). bmps are biologically active molecules capable of inducing new bone formation and have shown significant potential for clinical use in the repair of bone defects. although there have been several successful studies using bmps, there remain many unanswered questions such as patient site - specificity, for example, the ideal dose of bmp in an anterior cervical spine fusion may be quite different to that of a cranial defect. carriers for bmp play an equally important role when it comes to the optimal delivery leading to predictable and functional bone regeneration. for instance, a non - compressible scaffold has been shown to promote better fusion in the posterolateral spine than the clinically used collagen sponge. in addition to the delivery of growth factors scaffold design is focusing on supporting the adhesion, growth and function of anchorage - dependent cell types by mimicking the interaction between cell surface receptors and extracellular matrix (ecm) molecules. this interaction is vital in regulating cellular functions including adhesion, survival, proliferation, migration and differentiation. the major focus in the literature has been on the integrin family of receptors that interact with a wide variety of ecm proteins. more recently, heparan sulphate proteoglycans (hspgs) have been identified as having a role in cell adhesion. these interactions occur mainly through electrostatic interactions between the negatively charged hspg and the cluster of positively charged amino acids within the binding domains of ecm proteins. hspgs are abundant cell surface and ecm molecules that consist of a defining core protein (such as syndecan, glypican or perlecan) to which are attached highly sulphated glycosaminoglycan (gag) side - chains of heparan sulphate (hs) (fig . 5). a ) heparan sulfate proteoglycans can be broadly classified as glypicans, perlecans and syndecans, depending on their distribution within and around the cell. glypicans are attached to the cell membrane by gpi anchors, whereas syndecans are transmembrane proteins. perlecans are actively secreted into the pericellular matrix and are predominantly found in the basement membrane. (b) heparan sulfate is composed of repeating disaccharide units of glucuronic acid and glucosamine of varying length that can be modified at irregular intervals along the chain. these modifications cluster the sulfation patterns on the hs chain into discrete protein - binding domains and ultimately lead to different growth factor - binding specificities of different hs species. copyright permission granted from nature. the importance of hs in mediating cell responses has also been shown with heparinase / heparitinase and sodium chlorate treatments. heparinase cleaves hs chains into inactive disaccharide and tetrasaccharide components that have been shown to inhibit fgf2-mediated smooth muscle cell proliferation in injured carotid arteries. several in vitro studies have also cultured cells in media supplemented with chlorate to study the role of sulphated gags in different cell types. sodium chlorate (naclo3) imparts its effects by inhibiting atp - sulphurylase, the first enzyme in the synthesis of 3-phosphoadenyl 5-phosphosulphate (paps), a high - energy sulphate donor in biological reactions. the mitogenic response of cells to fgf2 is inhibited when cells are cultured in the presence of 30 mm naclo3. such data has helped establish the idea that hs does not only bind a ligand for delivery to its cognate receptor, but must also itself bind to the receptor, in order to initiate signal transduction; a powerful molecule indeed. numerous growth and adhesive proteins contain heparin - binding domains that specifically interact with hs. in all cases, the binding to hs modifies their biochemical properties and biological activity. thus understanding the specific nature of the cell surface receptor - ecm interactions may provide a foundation for developing functional biomaterials designed to promote cell adhesion and growth. indeed, the attachment of adhesive - peptide ligands mimicking heparin - binding domains to biomaterial surfaces has been shown to promote cell attachment. based on the above , the authors of this review began a collaboration in 2004 with the aim of developing a bone engineering platform technology that combines novel composite scaffolds that mimic the host tissue matrix with growth factor potentiating hs sugar isoforms (fig . the ability of hs to regulate a suite of endogenous factors involved in the repair process of bone makes it an attractive therapeutic agent compared to the application of a single growth factor, e.g. bmp . we studied the bone regeneration potential of mpcl / tcp - col1 scaffolds doped with 5 ( left) and 30 (right) microgram hs in critical sized rat cranial defects. (a) mpcl - tcp scaffold (5 mm diameter and 1 mm thick, 0 - 90o lay - down pattern, 70% porosity) before treatment, left, and after lyophilization of 350 mg of rat tail collagen type 1, right. (b) light microscopy showing the scaffold, left, and immunofluorescence showing the distribution of the hs within the lyophilised collagen. (c) two full - thickness critical bone defects (5 mm in diameter) were created in the rat parietal bone. implantation of mpcl / tcp - col1 scaffolds doped with 5 and 30 microgram hs into the defects. (a) macroscopically observation of the defects sites shows excellent integration of the constructs with the host tissue (arrows). a smooth and non- fibrous integration could be observed for all groups at all time points. ct scanning of the skull defect showing the progressive bone formation at 1 month (b) and 2 month (c) months with 5 microgram (hsl) and 30 microgram hs (hsh). (d) antibody staining with a bone specific marker, namely osteocalcin (arrows show interface between host bone and newly formed bone) shows a stronger signal from the hsh group when compared to hsl group. as discussed above, a conceptual shift is taking place in orthopaedic and reconstructive surgery from using synthetic implants and tissue grafts to a tissue engineering approach that uses biodegradable scaffolds integrated with biological cells or molecules to regenerate tissues. this new paradigm requires scaffolds that balance temporary mechanical function with morphological properties (pore architecture, size and interconnectivity) to aid biological delivery and tissue regeneration. complex scaffold architecture designs generated using hierarchical image - based or cad techniques usually can not readily be built using conventional techniques. instead, such scaffold architectures must be built using layer - by - layer manufacturing processes known collectively as solid free - form fabrication (sff). a number of review articles and book chapters have reviewed and compared sff scaffold fabrication methods so this review will concentrate on how scaffolds have per- formed clinically in bone engineering, and future directions for their use in regenerative medicine. melt extrusion - based sff systems provide a powerful instrument for the generation of scaffold platforms. recent advances in both computational scaffold design and sff have made it possible for tissue engineers to design and fabricate a whole range of new types of scaffolds. one of the major benefits is the flexibility to create scaffolds with highly reproducible architecture and compositional morphological variation across the entire matrix, due to the computer controlled fabrication process (fig . 7). the applications of sff technologies in scaffold fabrication are wide and varied, however, only a small number are have reached the clinical arena. an interdisciplinary group at the national university of singapore has evaluated and patented the parameters necessary to process medical grade polycaprolactone (mpcl) and mpcl composites by fused deposition modelling (fdm). these socalled first - generation scaffolds have been studied for more than 5 years in a clinical setting and gained food & drug administration (fda) approval via a 510k application in 2006. has used mpcl scaffolds as burr whole plugs in a pilot study for cranioplasty. the clinical outcome after 12 months was positive, with all patients tolerating the implants with no adverse side effects reported. more then 200 patients have received burr hole plugs, scaffolds for orbital floor reconstruction (fig . the majority of current tissue engineering approaches rely on the so called extrinsic mode of neovascularization . the neovascular bed originates from the periphery of the construct which should be implanted into a site of high vascularisation potential . here recites a core limitation for transfer of tissue engineering models from the in vitro to the in vivo environment diffusion is the initial process involved, but it can only provide for cell nutrient supply and waste transport within a maximum range of 200 m into the matrix . the survival of cells in the center of large cell containing constructs is therefore often limited by suboptimal initial vascularization . cell labeling experiments have disclosed a considerable loss of osteoblasts within the first week following transplantation in porous cancellous bone matrices . hence, reconstruction of small to moderate sized bone defects using extrinsic engineered bone tissues is technically feasible, and some of the currently developed concepts may represent alternatives to autologous bone grafts for certain clinical conditions, the reconstruction of large volume defects however remains challenging . hence, reconstructive surgeons aim to generate so called axially vascularized tissues that can be transferred to the defect site using microsurgical techniques of vascular anastomosis . these tissues are immediately vascularized upon implantation into the defect as free flaps do erol and spira were the firast to report prefabrication of skin flaps by using an arteriovenous vessel which introduced this technique to the tissue engineering community . most recently, the horch / kneser laboratory started collaboration with the hutmacher laboratory to use the by av - loop model ( figure c, small arrows) in combination with a composite scaffold (figure c, long arrow shows with contrast agent filled loop, highly vascularized composite scaffold is found quadrant) ) which is manufactured by a melt extrusion based sff technique. micro ct analysis (figure b) of the with a construct agent perfused vascular network (figure d, arrow show perfusion site with microfil filled needle, double line arrow shows with microfil filled vascular loop with caplillary network) of the engineered construct revealed a highly vascular zed construct. the second generation scaffolds produced by fdm for bone engineering are based on composites and have been evaluated in vitro and in vivo. mpolymer / cap composites confer favourable mechanical and biochemical properties, including strength via the ceramic phase and toughness and plasticity via the polymer phase, they also possess more favourable degradation and resorption kinetics, and graded mechanical stiffness. in these scaffolds, the ceramic phase was homogenously distributed in the matrix as well as exposed on the surface. contact angle measurements have shown that such composite surfaces were more hydrophilic and that the degradation kinetics were seen to be accelerated three to four times compared to pcl alone. biochemical advantages include improved cell seeding, and enhanced control and/or simplification of the incorporation and immobilization of biological factors, such as bmp's. most recently the group at the national university of singapore have undertaken several studies utilizing mpcl / cap composite scaffolds in large animal models (fig . implantation of a msc loaded mpcl - cap / pcl scaffold ( long arrow) into a high load - bearing osteochondral defect (inset small arrows, defect size in the medial condyle diameter 5 mm, depth 8 mm) in a pig model. implantation of a msc loaded mpcl - cap / scaffold (14125 mm3) into a spinal fusion model (inset, disks were removed on level l1/l2 and l4/l5 and tissue engineered constructs were combined with internal fixation devices) in a 6 month old domestic pig. orthopaedic, plastic and reconstructive surgery is beginning to use bone engineering as a preferential route to autologous or allogenic bone grafting. one treatment concept involves either doping a scaffold with growth factors (bmp, fgf, vascular endothelial growth factor , etc .) or other biological molecules (hs, etc). in growth factor therapy , the drug delivery system should not only promote effective biological activity but should also limit spatial spread of the factor. many of the growth factors currently being investigated for fracture healing are hbgfs that are produced by the osteoblasts and stored within the matrix of bone. of these, fgf-1 and fgf-2 have been extensively investigated as candidates for fracture healing. fgf-1 has been shown to aid in the bridging of a parietal bone critical defect and to increase the bone - implant interface when used in combination with titanium - based scaffolds. fgf-2 has been more extensively studied for its use in fracture healing, and it has been shown that daily, intravenous injections of fgf-2 for up to 2 weeks can enhance bone formation in rats. members of the tgf- superfamily have also been shown to be successful in enhancing osteogenesis. both tgf-1 and 2 are known to increase differentiation of committed osteoprogenitor cells, and are potent stimulators of bone repair in calvarial and long bone defects when administered alone or in combination with either igf - i or igf - i / growth hormone (gh). in addition, growth factors can directly regulate the availability of other growth factors; fgfs have been shown to regulate the expression of vegf and hgf, factors that are also mitogenic for osteoprogenitor cells. likewise, combined local delivery of igf - i in combination with tgf-1 in a hydrogel scaffold has been shown to significantly enhance bone formation in a rat tibial segmental defect over igf - i alone suggesting that perhaps the mode of delivery also affects the potential for growth factor - mediated bone healing. importantly, these findings clearly demonstrate that a universally applicable combination of growth factors has yet to be established for fracture therapy. hs by virtue of its binding to a multitude of growth factors normally present in the fracture haematoma, may represent an exciting avenue to augment fracture healing. indeed, we have shown that a single application of hs at the time of injury is sufficient to enhance fracture repair, and is responsible for the up - regulation of endogenous growth factors, notably fgf-1, igf - ii, tgf- 1 and vegf. in addition to effective dose, incompatibility with sterilization procedures, poor thermal and ph tolerance and delivery regimes , growth factor efficacy in orthopaedics is complicated by virtue of their inherent instability. growth factors are rapidly cleared in vivo as well as being highly susceptible to proteolytic degradation. as such, large quantities of growth factors furthermore, the number of growth factors that are proto - oncogenic is increasing that is becoming an important translational problem. thus, the long - term use of growth factors as a therapy is still being debated. as such , an alternative, bioactive agent that can protect, localize and/or enhance the effects of exogenously applied growth factors in vivo would be a great advantage in orthopaedics as it would reduce the dosage of exogenous growth factors required. furthermore, an agent that can be used independently to augment in vivo bone formation by harnessing the potential of endogenously produced growth factors would be even more therapeutically desirable. recently, the use of bmp inhibitors together with fgf2 in the culture medium of human embryonic stem cells (he s) has been shown to facilitate the longterm maintenance of these cells in a pluripotent state. hence, the balance between fgf- and bmpmediated signalling is thought to impose a primary control over cell - fate decisions. as immunological rejection limits the use of allogenic or xenogenic bone grafting, there has been a significant shift within the field of orthopaedics towards bone engineering and its potentially more sophisticated techniques for promoting osteogenesis. one particular avenue our group is exploring is better harnessing the unique properties of growth factors by presenting them together with specific co - factors, such as the hs molecules together with osteoconductive scaffolds (fig . 4a and b). bmp / heparin mimetics have shown promise when delivered on collagen sponges.now what is needed is the coupling of bioactive molecules to the next generation of scaffolding materials that will provide the key elements mentioned previously in this review.we are thus developing methodologies to engineer scaffolding devices that sustain tissue growth and maturation in the presence of functionalized growth factor / hs combinations. this is an important paradigm shift that we belief is achievable due to the resilience of hs to sterilization techniques, ph change, temperature and chemical solvents required for scaffold processing. therefore, successful bone tissue engineering could be achieved through the delivery of bioactive growth factors in bioresorbable scaffolding matrices functionalized with hs. this method is particularly attractive as it can boost local osteoprogenitor cell recruitment, proliferation and differentiation. hs - like molecules have been tested over the last decade for their ability to augment bone formation, as well as in the healing of other tissue types. dextran derivatives substituted with carboxymethyl benzylamide sulfonate (cmdbs) have been shown to mimic heparin / hs by providing a somewhat similar protection and stabilization capability for growth factors as imparted by hs; albeit with less affinity. otherwise known as' regenerating agents' (rgtas), these hs - like molecules have been shown to stimulate tissue repair in skin, bone, muscle and the cornea. in all cases, these hslike molecules are thought to exert their effect by increasing both cell proliferation and differentiation within the wound site, presumably by enhancing growth factor - mediated signals. although hs - like molecules have been shown to have the potential to accelerate fracture repair, hs molecules themselves are only now being used as a treatment therapy in bone. recently demonstrated that hs harvested from osteoblasts can increase trabecular bone volume by 20% following a once - off application into a rat middiaphyseal femoral fracture. furthermore, hs supplementation was shown to increase the expression of alkaline phosphatase (alp) and runx2, as well as the expression of a number of hbgfs present within the callus.together, these findings show that hs and hs - like molecules appear to have the remarkable potential to create an environment favourable to fracture repair. what is needed now is a better understanding of how best to deliver these potent molecules. due to the temporal pattern of growth factor expression during bone repair, we hypothesize that prolonged localized delivery of hs over the period of healing may further improve the effects of hs. we recently developed two techniques to prolong the delivery of hs. in the first study, hs was incorporated into electrospun poly(- caprolactone) (pcl) nanofibre mats. this produced fibres with smooth surfaces and no bead defects and was spun from polymer solutions with 8% w / v pcl in 7:3 dichloromethane: methanol solvent. assessment of hs loading and imaging of fluorescently labelled hs showed a homogenous distribution throughout the fibre mats and a sustained release for up to 14 days. importantly, the hs fibres did not induce an inflammatory response in macrophage cells in vitro and the released hs had sustained biological activity. in the later study, we encapsulated hs by the water - in oil - in water (w1/o / w2) technique using polycaprolactone (pcl) microcapsules and achieved similar biocompatibility and bioactivity with a prolonged delivery of up to 40 days. whilst promising in terms of hs release and bioactivity, these material constructs require extensive in vivo testing. do they induce ectopic bone formation, can they be used to co - deliver important growth factors? sff techniques are experiencing increasing application in many biomedical fields, including regenerative medicine and tissue engineering. in general, scaffold - based tissue engineering requires a welldefined internal structure with interconnected porosity and sff techniques allow us to design and fabricate such scaffolds. from a biological point of view , the designed scaffold should serve several functions, including the ability to act as an immobilization site for transplanted cells, the formation of a protective space to prevent unwanted tissue growth into the wound bed and allow healing with differentiated tissue, directing the migration or growth of cells via surface properties of the scaffold and directing migration or growth of cells via release of soluble molecules, such as growth factors, hormones and/or cytokines. future work has to provide further compelling evidence that other sff methods next to fdm offer the right balance of capability and practicality to be suitable for fabrication of materials in sufficient quantity and quality to move holistic tissue engineering technology platforms into a clinical application. in addition to considerations of scaffold performance based on tissue engineering strategies, practical considerations of manufacture also arise. from a clinical point of view, it must be possible to manufacture scaffolds under good manufacturing practice (gmp) conditions in a reproducible and quality controlled fashion at an economic cost and speed. to move the current tissue engineering practices to the next frontier, some manufacturing processes will be required to accommodate the incorporation of cells and/or biological molecules during the scaffold fabrication process. these approaches are working towards the enablement of the tissue - engineered construct to not only have a controlled spatial distribution of cells and molecules, but also to possess a versatility of scaffold material and microstructure within one specifically designed and fabricated construct, for implantation in an intended anatomical site. the development of an' hs approach' for the control of progenitor phenotype has become more feasible in recent years as our understanding of hs cell biology improves. hss are particularly attractive, as they potentiate the powerful effects of growth factors on cell recruitment, proliferation and differentiation, and do not depend on the synthesis of other co - factors or other specific activating agents. we would anticipate that hs, by virtue of its modulation of the biological activities of the fgfs and the bmps, offers the possibility of intervening not just in embryonic growth, and stem and progenitor cell self - renewal, but also in stem cell - dependent wound healing and scaffold - based tissue engineering. unlike other nucleic acid - based clinical approaches (e.g. virus and sirna), therapeutic intervention may be particularly advantaged because hss are chemically stable. realization of the potential of hs for regenerative medicine will depend, however, on suitable delivery systems with its own intrinsic properties that mimic the host tissue architecture.

abstracta paradigm shift is taking place in orthopaedic and reconstructive surgery from using medical devices and tissue grafts to a tissue engineering approach that uses biodegradable scaffolds combined with cells or biological molecules to repair and/or regenerate tissues. one of the potential benefits offered by solid free - form fabrication technology (sff) is the ability to create scaffolds with highly reproducible architecture and compositional variation across the entire scaffold, due to its tightly controlled computer - driven fabrication. in this review, we define scaffold properties and attempt to provide some broad criteria and constraints for scaffold design in bone engineering.we also discuss the application - specific modifications driven by surgeon's requirements in vitro and/or in vivo. next, we review the current use of sff techniques in scaffold fabrication in the context of their clinical use in bone regeneration. lastly, we comment on future developments in our groups, such as the functionalization of novel composite scaffolds with combinations of growth factors; and more specifically the promising area of heparan sulphate polysaccaride immobilization within the bone tissue engineering arena.

osseointegrated dental implants have proven to be predictably successful when appropriate guidelines are followed. traditionally, implant treatment of edentulous patients was based on a two - stage surgical protocol, with a healing period of three to six months, during which the implants were submerged to achieve osseointegration. this approach was considered to be an essential step for successful implant treatment, as it was believed that the micromovement of the implants, due to functional forces at the bone - implant interface during wound healing, could induce the formation of fibrous tissue rather than bone, leading to failure. several aspects of the implant morphology have been studied for years. among these, a submerged implant was thought necessary to prevent infection and epithelial downgrowth. more recently, several reports have demonstrated that one - stage surgery, with immediate loading, is possible, with good clinical . although zdis have been used since the nineties, there are few reports focusing on the clinical outcome of these fixtures. consequently, we decided to perform a retrospective study on a large series of zdis, to identify the variables statistically associated with the clinical outcome. in the period between january 2007 and june 2011, 125 patients were treatetd with zdis by two surgeons (cms and ez). the last check - up was performed in june 2012, with a mean follow - up period of 17 9 months (minimummaximum, 8 - 64 months). controlled oral hygiene, the absence of any lesions in the oral cavity, and sufficient residual bone volume to receive implants at least 3.25 mm in diameter and 8.0 mm in length. in addition, the patients had to agree to participate in a post`-operative check - up program. insufficient bone volume, a high degree of bruxism, smoking more than 20 cigarettes / day and excessive consumption of alcohol, localized radiation therapy of the oral cavity, antitumor chemotherapy, liver, blood, and kidney diseases, immunosupressed patients, patients taking corticosteroids, pregnant women, patients with inflammatory and autoimmune diseases of the oral cavity, and poor oral hygiene. before surgery, radiographic examinations were done with the use of an orthopantomograph and computed tomography (ct) scans. in each patient, the peri - implant crestal bone levels were evaluated by a calibrated examination of the ortopantomograph x - rays. measurements were recorded before surgery; after surgery, and at the end of the follow - up period. the measurements were carried out mesially and distally to each implant, calculating the distance between the edge of the implant and the most coronal point of contact between the bone and the implant. the bone level recorded just after the surgical insertion of the implant was the reference point for the following measurements. a peak scale loupe with a magnifying factor of seven times and a scale graduated in 0.1 mm was used. peri - implant probing was not performed because controversy still existed with regard to the correlation between the probing depth and implant success rates. the implant success rate (scr i.e., good clinical, radiological, and esthetic outcomes) was evaluated according to the following criteria: absence of persisting pain or dysesthesia; absence of peri - implant infection with suppuration; absence of mobility; and absence of persisting peri - implant bone resorption greater than 1.5 mm during the first year of loading and 0.2 mm / year during the following years. in 125 patients a total of 556 plants were inserted: two hundred and ninety - five (53.1%) in the maxilla and 261 (46.9%) in the mandible. there were 480 (86.3%) screw - vents , 51 (9.2%) swiss - plusses , and 25 (4.5%) splines. sixteen, 355, 34, 90, 55, and 6 fixtures had a diameter of 3.25, 3.7, 3.75, 4.1, 4.7, and 4.8 mm, respectively. twenty - eight, 145, 5, 217, 8, 141, and 12 implants had a length of 8, 10, 11, 11.5, 12, 13, and 14 mm, respectively. the implants were inserted to replace 136 (24.5%) incisors, 80 (14.4%) cuspids, 198 (35.6%) premolars, and 142 (25.5%) molars. two surgeons inserted the implants: cms 324 (58.3%) and ez 232 (41.7%). three hundred and twenty - seven (58.8.%) were inserted in females and 229 (41.2 %) in males. seven (1.3%) implants were inserted in diabetic patients and 80 (14.4%) in subjects treated with anti - hypertensive drugs. thirty - four (6.1) were inclinated as they were used for an all - on - four technique and 179 (32.2%) were inserted in smokers (less than 20 cigarettes per day). an antimicrobial prophylaxis was administered with 1 g amoxycillin twice daily for five days, starting one hour before surgery. local anesthesia was induced by infiltration of articaine / epinephrine and postsurgical analgesic treatment was performed with 100 mg nimesulid, twice daily, for three days. the sutures were removed seven days after surgery. in case of a two stage procedure, following 24 weeks of implant insertion, the provisional prosthesis was provided, and the final restoration was usually delivered within an additional eight weeks. the number of prosthetic units (i.e., implant / crown ratio) was about 0.6. the mean age was 62 10 years (minimummaximum 23 - 88 years). two hundred and thirty - two (41.7%) implants were insered after the use of piezo, laser, and platelet - rich plasma derivates. fourteen (2.5%) were covered with resorbable membranes and 244 (43.9%) were inserted in post - extractive sockets. as no implants were lost (i.e., srv = 100%) and no statistical differences were detected among the studied variables, no or reduced crestal bone resorption was considered as an indicator of scr, to evaluate the effect of several host-, implant-, and occlusion - related factors. the difference between the implant abutment junction and the bone crestal level was defined as the implant abutment junction (iaj) and calculated at the time of operation and during follow - up. delta iaj was the difference between iaj at the last check - up and iaj recorded just after the operation. the pearson chi - sqaure test was used to detect the variables most associated with implant success. in the period between january 2007 and june 2011, 125 patients were treatetd with zdis by two surgeons (cms and ez). the last check - up was performed in june 2012, with a mean follow - up period of 17 9 months (minimummaximum, 8 - 64 months). controlled oral hygiene, the absence of any lesions in the oral cavity, and sufficient residual bone volume to receive implants at least 3.25 mm in diameter and 8.0 mm in length. in addition, the patients had to agree to participate in a post`-operative check - up program. insufficient bone volume, a high degree of bruxism, smoking more than 20 cigarettes / day and excessive consumption of alcohol, localized radiation therapy of the oral cavity, antitumor chemotherapy, liver, blood, and kidney diseases, immunosupressed patients, patients taking corticosteroids, pregnant women, patients with inflammatory and autoimmune diseases of the oral cavity, and poor oral hygiene. controlled oral hygiene, the absence of any lesions in the oral cavity, and sufficient residual bone volume to receive implants at least 3.25 mm in diameter and 8.0 mm in length. in addition, the patients had to agree to participate in a post`-operative check - up program. insufficient bone volume, a high degree of bruxism, smoking more than 20 cigarettes / day and excessive consumption of alcohol, localized radiation therapy of the oral cavity, antitumor chemotherapy, liver, blood, and kidney diseases, immunosupressed patients, patients taking corticosteroids, pregnant women, patients with inflammatory and autoimmune diseases of the oral cavity, and poor oral hygiene. before surgery, radiographic examinations were done with the use of an orthopantomograph and computed tomography (ct) scans. in each patient, the peri - implant crestal bone levels were evaluated by a calibrated examination of the ortopantomograph x - rays. measurements were recorded before surgery; after surgery, and at the end of the follow - up period. the measurements were carried out mesially and distally to each implant, calculating the distance between the edge of the implant and the most coronal point of contact between the bone and the implant. the bone level recorded just after the surgical insertion of the implant was the reference point for the following measurements. a peak scale loupe with a magnifying factor of seven times and a scale graduated in 0.1 mm was used. peri - implant probing was not performed because controversy still existed with regard to the correlation between the probing depth and implant success rates. the implant success rate (scr i.e., good clinical, radiological, and esthetic outcomes) was evaluated according to the following criteria: absence of persisting pain or dysesthesia; absence of peri - implant infection with suppuration; absence of mobility; and absence of persisting peri - implant bone resorption greater than 1.5 mm during the first year of loading and 0.2 mm / year during the following years. in 125 patients a total of 556 plants were inserted: two hundred and ninety - five (53.1%) in the maxilla and 261 (46.9%) in the mandible. there were 480 (86.3%) screw - vents , 51 (9.2%) swiss - plusses , and 25 (4.5%) splines. sixteen, 355, 34, 90, 55, and 6 fixtures had a diameter of 3.25, 3.7, 3.75, 4.1, 4.7, and 4.8 mm, respectively. twenty - eight, 145, 5, 217, 8, 141, and 12 implants had a length of 8, 10, 11, 11.5, 12, 13, and 14 mm, respectively. the implants were inserted to replace 136 (24.5%) incisors, 80 (14.4%) cuspids, 198 (35.6%) premolars, and 142 (25.5%) molars. two surgeons inserted the implants: cms 324 (58.3%) and ez 232 (41.7%). three hundred and twenty - seven (58.8.%) were inserted in females and 229 (41.2 %) in males. seven (1.3%) implants were inserted in diabetic patients and 80 (14.4%) in subjects treated with anti - hypertensive drugs. thirty - four (6.1) were inclinated as they were used for an all - on - four technique and 179 (32.2%) were inserted in smokers (less than 20 cigarettes per day). an antimicrobial prophylaxis was administered with 1 g amoxycillin twice daily for five days, starting one hour before surgery. local anesthesia was induced by infiltration of articaine / epinephrine and postsurgical analgesic treatment was performed with 100 mg nimesulid, twice daily, for three days. the sutures were removed seven days after surgery. in case of a two stage procedure, following 24 weeks of implant insertion, the provisional prosthesis was provided, and the final restoration was usually delivered within an additional eight weeks. the number of prosthetic units (i.e., implant / crown ratio) was about 0.6. the mean age was 62 10 years (minimummaximum 23 - 88 years). two hundred and thirty - two (41.7%) implants were insered after the use of piezo, laser, and platelet - rich plasma derivates. fourteen (2.5%) were covered with resorbable membranes and 244 (43.9%) were inserted in post - extractive sockets. as no implants were lost (i.e., srv = 100%) and no statistical differences were detected among the studied variables, no or reduced crestal bone resorption was considered as an indicator of scr, to evaluate the effect of several host-, implant-, and occlusion - related factors. the difference between the implant abutment junction and the bone crestal level was defined as the implant abutment junction (iaj) and calculated at the time of operation and during follow - up. delta iaj was the difference between iaj at the last check - up and iaj recorded just after the operation. the pearson chi - sqaure test was used to detect the variables most associated with implant success. the mean peri - implant bone resorption was 0.7 0.6 mm (minimum - maximum 0 - 5.7 mm). twenty - seven fixtures had a peri - implant bone resorption greater than the cut - off values (scr = 95.1%), and therefore, were used to detect those variables statistically related to an augmented peri - implant bone resorption. among the studied variables (i.e., implant type, diameter, length, gender, smoke, diabetes, drugs, platelet - rich plasma, sinus augmentation, membrane, immediate loading, fixture inclination, laser, piezo, post - extraction socket, surgeon, tooth position, and jaws) only jaws was statistically significant, with a better outcome for implants inserted in the maxilla (p = 0.017). zdis have been used since the nineties, but few reports analyzed the clinical outcome of these fixtures. in 2001, khayat et al. reported a study on a wide diameter screw - vent. one hundred and eleven implants were evaluated at the recall examination. almost all implants supported a fixed partial prosthesis. no implants were lost during the loading period. in 2002, arlin analyzed 435 screw - vent implants. the focus group was compared to a mixed implant design group, with a variety of abutment connections and surfaces from several other manufacturers. the cumulative survival rates were 94.2% (n = 435) for the focus group and 90.1% (n = 2339) for the reference group. in 2005, minichetti et al. reported the of implants placed into extraction sites grafted with particulate mineralized bone allograft (puros). a total of 313 extraction sites were grafted with mineralized bone graft during a 36-month period. a total of 252 screw - vent implants were placed into the grafted extraction sites after a four- to seven - month healing period. a total of 244 implants were restored with fixed prosthesis and six with removable overdentures, for a total of 250 loaded implants. a total of six implants failed, which required their removal (two implants before load and four after loading), ing in a 97.6% implant success rate. in 2007, khayat et al. a total of 835 implants, with diameters of 3.7 mm (9%), 4.7 mm (76%), and 6.0 mm (15%) were placed in 328 patients, using a single - stage, delayed - loading protocol. the implants were restored with a variety of prostheses and monitored over two years of functional loading. cumulative implant survival was 99.4% (n = 835); differences between mandibular (99.0%, n = 408) and maxillary (99.8%, n = 427) implants were not statistically significant. six implants failed to meet the success criteria by sustaining mesial and distal bone loss below the first implant thread; however, they remained stable and continued functioning without pain or inflammation. cumulative implant success was 98.6% (n = 835); the differences between the maxillary (98.6%) and mandibular (98.8%) implants were not statistically significant. the success rates by implant diameter were 98.6% (3.7 mm), 98.4% (4.7 mm), and 100% (6 mm). the authors concluded that after two years of functional loading, the survival and success rates for screw - vent implants placed in a non - submerged protocol, equaled or surpassed those of single - thread, straight - walled implant historical controls. one year later, minichetti et al. reported the of implants placed in the maxillary sinuses grafted with particulate mineralized cancellous bone allograft alone or in combination with resorbable hydroxyapatite, over a three - year period. a total of 56 sinuses were grafted, and 136 dental implants were placed into the grafted sites after a four- to eight - month healing period. all re - entries revealed a bony hard structure acceptable for osteotomy preparation. of these implants, 124 had been restored with fixed prosthesis and 12 with removable overdentures for a total of 136 loaded implants. a total of three implants required removal (failure) ing in a 97.7% implant success rate (2.3% failure rate). the authors concluded that a mineralized human allograft placed in a lateral window sinus elevation, was a clinically predicable method, acceptable for implant placement and restoration. evaluated the immediate loading implant and possible effects correlated with peri - implants. in 2008, ormianer et al. , performed a non - randomized, uncontrolled, retrospective study to evaluate the clinical outcomes of treatment with tapered, multithreaded implants, with a special emphasis on peri - implant crestal bone status. chart reviews were conducted of 60 patients who had been treated with 267 implants for the placement of one or more missing and/or unsalvageable teeth, and who met the general inclusion criteria for dental implant therapy. in all cases, marginal bone changes were calculated from the cementoenamel junction or the implant neck to the crestal bone level, with standardized radiographs taken at the implant placement (baseline) and during annual follow - up. after a mean follow - up of 7.5 years , the implant survival was 98.5% (263/267) for all implants placed, and the implant success was 96.2% (253/263) for all surviving implants. crestal bone loss was observed in 25% (15/60) of the total study subjects and in 12% (32/263) of all surviving implants: twenty - nine implants exhibited 1 mm of bone loss and three implants lost 2 mm of bone. low - density maxillary jawbone and more extensive bone remodeling, which were required around the implants immediately placed into extraction sockets, were the probable causes of the observed bone loss in this study. the implants exhibited excellent long - term outcomes, with little or no bone loss. most of the studied variables (i.e., implant type, diameter, length, gender, smoke, diabetes, drugs, platelet - rich plasma, sinus augmentetion, membrane, immediate loading, fixture inclination, laser, piezo, post - extraction socket, surgeon, and tooth position) have no impact on the clinical outcome. implants inserted in the mandible have a slight, but a statistically significant worse outcome, with a higher peri - implant bone resorption, compared to those inserted in the maxilla. in , zdis are reliable devices to use in implantology.

: one - stage surgery with immediate loading is possible, with good clinical . many types of dental implants are available in the market. zimmer dental implants (zdis) have been used since the nineties, but few reports have analyzed the clinical outcome of these fixtures. we planned a retrospective study on a series 566 zdis, to evaluate their clinical outcome.materials and methods: in the period between january 2007 and june 2011, 125 patients were treatetd with zdis. the last check - up was performed in june 2012, with a mean follow - up period of 17 9 months (minimum maximum, 8 - 4 months). zdis were inserted as follows: 295 (53.1%) in the maxilla and 261 (46.9%) in the mandible. there were 480 (86.3%) screw - vents, 51 (9.2%) swiss plusses, and 25 (4.5%) splines. sixteen, 355, 34, 90, 55, and six fixtures had a diameter of 3.25, 3.7, 3.75, 4.1, 4.7, and 4.8 mm, respectively. twenty - eight, 145, 5, 217, 8, 141, and 12 implants hade a length of 8, 10, 11, 11.5, 12, 13, and 14 mm, respectively. the implants were inserted to replace 136 (24.5%) incisors, 80 (14.4%) cuspids, 198 (35.6%) premolars, and 142 (25.5%) molars.:no implants were lost (i.e., srv = 100%). among the studied variables, only those for the jaws were statistically significant, with a better outcome for implants inserted in the maxilla (p = 0.017).: zdis are reliable devices to be used in implantology, althougth a higher marginal bone loss has to be expected when these implants are inserted in mandible.

aneurysm formation after pancreatitis is well documented in the literature, caused by the digestion of arterial walls by the enzymes such as elastase released by the pancreas. rupture is generally considered to be a rare complication after pancreatitis, but the formation of aneurysms themselves is relatively common, occurring in 10% of patients following pancreatitis. endoscopic retrograde cholangiopancreatography (ercp) is a common procedure used both as a diagnostic and therapeutic tool in patients with biliary tree obstruction. it is considered a safe procedure and serious complications are rare; with the most common complications being pancreatits, cholangitis, bowel perforation, and bleeding from the ampulla of vater following sphincterotomy. however, although far rarer than any of the above the following case illustrates life - threatening and therefore significant complication following ercp. we present the case of a 75 year old female admitted with acute pancreatitis, deranged liver function and gram - negative rod septicaemia. she was admitted to intensive care and magnetic resonance cholangiopancreatography carried out to determine the aetiology of the pancreatitis. this showed a 2 cm filling defect in the dilated lower end of the common bile duct, suggestive of an impacted stone. endoscopic retrograde cholangiopancreatography (ercp) was performed 10 days later, but showed an undilated biliary system with no filling defects; despite trawling the common bile duct several times. subsequently the patient had several episodes of haematemesis and malaena, with the patient s haemoglobin repeatedly dropping despite transfusion. oesophogastroduodenal (ogd) endoscopy and computerised tomography (ct) scan failed to identify a source. the only positive finding on ct scan was pneumobilia, most likely due to sphincterotomy during the ercp. the haematemesis continued and emergency laparotomy was performed. on external examination of the stomach and duodenum pyloroduodenotomy identified a small healed duodenal ulcer and fresh clots in the first part of duodenum. on palpation of the third part of duodenum, a periampullary diverticulum was identified, although it was not bleeding. a second urgent ogd endoscopy showed more blood clots and fresh blood in the fundus, but once again no source for the bleeding was seen. this showed a 1 cm pseudoaneurysm of the right hepatic artery in the right lobe close to the porta hepatis. the pseudoaneurysm arose from the anterior branch of the right hepatic artery and communicated with the bile duct. the 2 part of the duodenum was opacified, showing haemorrhage from the lesion into the duodenum via the bile duct (figure 1). initially , selective embolisation of the anterior right hepatic artery was performed using small coils (cook uk) and small particles of grated gelatin sponge soaked in 0.5ml of 5% ethanolamine. however this proved to be ineffective (figure 2), and so the posterior branch of the right hepatic artery was embolised, successfully controlling the haemobilia. there were signs of recurrent haemorrhage such as fluctuating serum haemoglobin levels and some ongoing malaena. however, these ceased spontaneously after a few days, and the patient subsequently made an uneventful recovery. the formation of pseudoaneurysms after pancreatitis is very common, although most of these will not rupture and therefore remain asymptomatic. cases of haematemesis following spontaneous fistulation into the biliary tree after pancreatitis are described in the literature; although often they have presented after a more severe necrotising pancreatitis. similar presentations have also been described with a different pancreatic source, such as a bleeding pancreatic pseudocyst. given that the haematemesis commenced following ercp in this case, it is likely that it was the instrumentation of the common bile duct which led to the fisulation of the aneurysm into the biliary tree. this theory is feasible as the branches of the hepatic artery and portal vein are in very close proximity to the bile duct allowing concurrent injury to occur. we hypothesize that during the ercp procedure the walls of the intra - hepatic bile duct and paralleling artery were injured during steel guide wire maneuvering, causing the false aneurysm. it is important to recognize that iatrogenic causes are the most common cause for haemobilia (52%). we also assume the bleeding in the acute recovery phase post embolisation was mainly due to the dissolving nature of the gelatin sponge. however, even though a fraction of the particles may have dissolved, the bleeding subsequently stopped as low enough perfusion pressures were maintained to stabilize the lesion. ogd endoscopy is of course the first line investigation in any patient presenting with haematemesis, but in this case was unhelpful because there was no blood discharging from the ampulla of vater at the time of investigation. ct scanning lacked the sensitivity required to detect the 1 cm pseudoaneurysm described in this case, though it has been successfully used in the diagnosis of larger aneurysms in other cases. angiography is considered the gold standard in the diagnosis of this condition, and it is important learning point to remember this as the next line of investigation of haematemesis, particularly in patients with a history of pancreatitis. in this case there was a delay before an angiogram was done, and this delay was very nearly fatal for the patient. although this case report describes a rare complication of pancreatitis, it is a complication that is readily treatable provided the diagnosis is made in time. therefore we publish it in the hope that clinicians will think of it in patients with upper gastrointestinal bleeding of unknown origin.

we present a case of pancreatitis with secondary pseudoaneurysm formation in a branch of the hepatic artery, which fistulated into the common bile duct following endoscopic retrograde cholangiopancreatography. this aneurysm manifested itself clinically with recurrent haematemesis, and was treated by embolisation of the anterior and posterior branches of the right hepatic artery.

oropharyngeal cancers account for a major proportion of cancers of the upper aerodigestive tract. in 1995, the standard treatments for these cancers can be either surgery followed by radiotherapy eventually with concomitant chemotherapy or exclusive radiotherapy eventually with concomitant chemotherapy. in spite of the lack of methodologically solid comparative studies, no difference in terms of survival between these two options seems to exist. a new challenge recently appeared, certainly less important than survival, but important all the same: the quality of life of patients among whom dysphagia seems to be a very important criterion. some treatments may be too detrimental in particular with regard to swallowing. in the same way, the recently developed concept of therapeutic utility (measurement of the preference of patients for a health condition) is raised: will a patient prefer to live for three years with normal eating ability or for four years with exclusive enteral feeding? the aim of our study was to evaluate the short-, medium-, and long - term (five to 10 years) feeding abilities of patients treated for oropharyngeal cancer to determine prognostic factors for feeding difficulties. after consulting the medical information department database of the croix - rousse hospital, 252 consecutive patients treated between january 1st, 1998 and december 31st, 2003 for oropharyngeal squamous cell carcinoma were selected. if patients were operable, they were offered surgical treatment (tumor and lymphadenectomy) followed by radiotherapy (5054 gy in 5 to 6 weeks) sometimes potentiated by cisplatin according to the pathology (more than 3 metastatic nodes, extracapsular extension, endovascular metastases, or perineural infiltration). the surgery procedures were transoral approach, submandibular approach (lateral oropharyngectomy, hyosubglossoepiglottectomy), or transmandibular approach (mandibular swing or pharyngectomy with mandibulectomy). during surgery, a nasogastric tube was inserted, and tracheotomy was performed except in the case of small tumors treated by the transoral approach. most of the patients benefited from swallowing rehabilitation during their hospitalization, which in certain cases continued after return home or after admission to a rehabilitation institution. unfortunately, this variable could not be exploited because of the lack of data. if operable patients refused surgery, they received exclusive radiotherapy (70 gy in 6 - 7 weeks) generally potentiated by cisplatin. in the event of an inoperable tumour, and sometimes after induction chemotherapy, the patient received exclusive radiotherapy (70 gy in 6 to7 weeks) generally potentiated by cisplatin. radiotherapy fractionation was standard (2 gy per session, 5 sessions a week); 2 lateral ports with 4 to 8 mv x - rays split at 40 gy due to the spinal cord tolerance limit to 2 anterior lateral ports with x - rays and 2 posterior ports with 8 to 10 mev elecron beams. the level iv regions were treated with mixed 46 mv x - rays and 8 to 10 mev elecron beams. additional boosts were given for high - risk nodal areas (in cases of node involvement with extracapsular extension) either with 8 to 10 mev electron beams. the following data were retrospectively collected: weight, swallowing ability (solids, pastes, liquids) according to doctors' and speech pathologists' notes, and the use of a nasogastric tube during the posttherapeutic followup (five to ten years). after exclusion of the patients initially treated with palliative care or palliative chemotherapy (including metastatic patients), functional tolerance to treatments was analyzed in order to determine prognostic factors for post - therapeutic swallowing difficulties and their repercussion on body weight. factors which were thought to have a significant impact on swallowing were the sex, the age, the american society of anaesthesiology (asa) score, previous cancer of the head and neck, the tumour, location in the oropharynx, the t stage, the n stage, the use of induction chemotherapy, the type of treatment, the reconstruction type, the existence of a postoperative complication, the dose of radiation to the tumour and the dose of radiation to the nodes. for the study of weight, a linear regression according to time was used with nested mixed models on repeated data. the binary variables of liquids, pastes, and solid foods were studied using a multivariate model of survival censored by intervals. the reference patient was by definition a 55-year - old man, asa 1, with no past history of cancer who developed t1n0 cancer of the base of tongue who received no induction chemotherapy and was treated using pharyngectomy with mandibulectomy, without postoperative complications or postoperative radiotherapy. such a patient does not exist but changing one (or more) parameters of the model gives a hazard ratio that allows comparisons to be made. sixty - two patients had previously at least one cancer of the head and neck (24.6% of the patients). one hundred and thirty - five patients still drank alcohol at the time of the diagnosis (68.5% of the data collected on this item), while 133 patients still smoked at the time of the diagnosis (65.6% of the data collected on this item). four patients presented pulmonary metastases, and one patient had osseous metastases at the time of the diagnosis. eighty - one patients (32.1%) had induction chemotherapy, the majority with 5-fluorouracil / cisplatin because of inoperable tumours. (i) surgery for the tumourone hundred and twenty - two patients (48.4% of the patients) did not have tracheotomy, 25 patients (9.9% of total patients, 15.8% of operated patients) underwent prepared tracheotomy, and 70 patients (27.7% of total patients, 44.3% of operated patients) had tracheotomy. the mean duration of the tracheotomy was 2.2 weeks (es 3.3 weeks), the mean time before postsurgical recovery of feeding was 1.8 weeks (es 1.8 weeks), and the mean duration of postsurgical nasogastric tube was 2.7 weeks (es 1.8 weeks). one hundred and twenty - two patients (48.4% of the patients) did not have tracheotomy, 25 patients (9.9% of total patients, 15.8% of operated patients) underwent prepared tracheotomy, and 70 patients (27.7% of total patients, 44.3% of operated patients) had tracheotomy. the mean duration of the tracheotomy was 2.2 weeks (es 3.3 weeks), the mean time before postsurgical recovery of feeding was 1.8 weeks (es 1.8 weeks), and the mean duration of postsurgical nasogastric tube was 2.7 weeks (es 1.8 weeks). (ii) reconstructive surgerythe loss of substance due to tumor surgery required a pectoralis major flap for 23 patients (18 mandibular swings, 4 pharyngectomies with mandibulectomy, 1 lateral oropharyngectomy) and a sternocleidomastoid flap for seven patients, all after lateral oropharyngectomy. the loss of substance due to tumor surgery required a pectoralis major flap for 23 patients (18 mandibular swings, 4 pharyngectomies with mandibulectomy, 1 lateral oropharyngectomy) and a sternocleidomastoid flap for seven patients, all after lateral oropharyngectomy. (iii) complications of the surgeryforty - one patients (25.9% of the operated patients) had a surgery - related complication: one death from an unknown cause, cervical infection, bleeding, pharyngostoma, cervical hematoma, pneumonia, orostoma, fever of an undetermined cause, flap disunion, hepatic failure, acute delirium, and lymphorrhea. the last nine patients were transferred to the intensive care unit during the postoperative phase: five transfers were scheduled (two for dialysis for chronic renal failure, three patients with a heavy cardiac past history requiring close surveillance), and four whose stay was unscheduled (one pneumonia, one hemorrhagic hypovolemic shock, one hepatic failure, one cardiac failure). forty - one patients (25.9% of the operated patients) had a surgery - related complication: one death from an unknown cause, cervical infection, bleeding, pharyngostoma, cervical hematoma, pneumonia, orostoma, fever of an undetermined cause, flap disunion, hepatic failure, acute delirium, and lymphorrhea. the last nine patients were transferred to the intensive care unit during the postoperative phase: five transfers were scheduled (two for dialysis for chronic renal failure, three patients with a heavy cardiac past history requiring close surveillance), and four whose stay was unscheduled (one pneumonia, one hemorrhagic hypovolemic shock, one hepatic failure, one cardiac failure). table 2 presents the different types of radiotherapy and table 3 the doses of radiation. fifteen patients had local brachytherapy during postoperative external radiotherapy, four during exclusive external radiotherapy, and one patient without external radiotherapy (postoperative or exclusive). 167 patients (87.7%) were operated on by the same surgeon who also ensured the medical followup of 242 patients (96.0%). the data collecting is therefore very uniform. figure 1 presents the evolution of swallowing ability with time by type of treatment, and figure 2 shows the evolution of the use of a nasogastric tube by type of treatment. the mean weight at diagnosis was 64.9 kg. in the event of a history of cancer of the upper aerodigestive tract, the mean weight at diagnosis was significantly lower (3.6 kg ; p = .0173). patients with a post - operative complication were significantly heavier (+ 1.2 kg, p < 10). age, location of the oropharyngeal tumor, t stage, and n stage had no significant impact on weight at the time of diagnosis (p = .2282,.7536,.4524, resp .). the average weight loss was 1.56 kg / yr (p < 10) throughout the followup, and the type of treatment did not have a significant impact on weight loss (p = .634). table 4 presents the of the models' parameters. because of the small numbers of patients in some categories (e.g. pharyngectomy with mandibulectomy), the confidence interval was large and did not allow us to prove any significant differences. in the international literature, patients' weight is seldom taken into account in the estimate of the functional of treatments for cancer of the upper aerodigestive tract. however, denutrition is a predictive factor of tolerance to the treatment. according to our analyses, the treatments for oropharynx cancers, whatever they are and whatever the site of the oropharyngeal lesion is, caused a continuous, considerable (1.56 kg / yr), significant and long - term (60 to 120 months of followup) weight loss in patients in an often already precarious nutritional state. however, correct feeding was rapidly possible for the majority of patients and remained possible with time. significantly, factors influencing the ability to swallow liquids, pastes, and normal foods were generally the same: the initial t stage, induction chemotherapy, and the type of treatment. because of the absence of anatomophysiological explanations for the harmful impact of induction chemotherapy on swallowing and the fact that this finding has not been described in other studies, it needs to be interpreted with caution, and other studies need to be conducted to rule out or to confirm this hypothesis. putting aside the possible role of induction chemotherapy, the data of our study are in agreement with those already published. indeed, for several authors , the functional outcomes of surgery followed by radiotherapy and radiochemotherapy were not significantly different except for those reported by allal et al. for whom the functional quality of life and abilities after exclusive radiotherapy were similar for t1t2 but significantly better for t3t4. other authors showed the existence of predictive factors of good functional performances, but these factors varied from one study to another. for zuydam et al. , swallowing was significantly better in the absence of radiotherapy (p < .001) and in the event of direct closure (p = .003), and speech was also significantly better in the event of direct closure. for zelefski et al. , the predictive factors of good swallowing were the type of surgery (in ascending order towards better swallowing : pharyngectomy with mandibulectomy, mandibular swing, and lateral oropharyngectomy) and location (in ascending order tonsil then base of tongue). this study should probably not be retained because of the illogical physiological character and the fact it is isolated from its . , swallowing depended especially on the volume of tissue removed. , only postoperative radiation decreased swallowing ability. in of the analysis of the literature , the functional seemed better in the absence of postoperative radiation and of reconstructive surgery and in the event of small tumour size, which is perfectly logical from a physiological point of view. we could not show that certain treatments (in particular exclusive radiotherapy and transmandibular approaches) caused more serious swallowing disorders in the medium and long term. in our everyday clinical experience, the principal factor that has an impact on function, which proved to be relevant for all locations of upper aerodigestive tract cancers, is radiation and there seems to be a threshold level of radiation (60 gy) above which deterioration of functional abilities is accentuated. however, this hypothesis could not be proved in this study probably because of the high doses of postoperative radiation, more often closer to 6064 gy than to 5054 gy (figure 2). patients had a dose of radiation that was higher than the requested dose, and it is advisable to carefully discuss doses of radiation with the radiotherapists. whatever the possible and selected treatment was, the impact on the functional capacities and, thus, the quality of life of the patients was considerable. even though we could not significantly demonstrate that exclusive radiotherapy caused more long - term undesirable effects than surgery followed by radiotherapy, our daily practice has shown that we should favour the latter. further prospective studies about long - term side effects of treatments of oropharyngeal malignancies should be conducted.

objective. to analyze the functional impact of the various possible treatments of oropharyngeal squamous cell carcinomas to find the main prognostic factors of dysphagia induced by these treatments. patients. clinical data from 254 patients treated for squamous cell carcinoma of the oropharynx between 1998 and 2003 were retrospectively analyzed. a multivariate model enabled us to evaluate the role of each potentially harmful factor on swallowing. main outcome measures. the significant factors influencing the consumption of liquid, pasty, and normal food were the same: the initial t stage and the type of treatment. . whatever the possible and selected treatment was, the impact on the functional capacities, and thus, the quality of life of the patients was considerable. even though we could not significantly demonstrate exclusive radiotherapy caused more long - term undesirable effects than surgery followed by radiotherapy, our daily practice has shown that we should favour the latter.

the receptor - binding cancer antigen expressed on siso cells (rcas1) was initially recognized by the mouse monoclonal antibody, 22 - 1 - 1, which was raised by the immunization of mice with the human uterine cervical adenocarcinoma cell line siso. it is a 40-kd type ii membrane protein that forms homo - oligomers through its c - terminal coiled - coil structures, whereas it also exists in the soluble form probably by alternative splicing. rcas1 has currently been considered as a novel tumor - associated antigen that can function as a ligand for a putative receptor that is present on various human cell lines, such as erythroid leukaemia and normal peripheral lymphocytes, inhibiting cell growth and inducing apoptosis. thus, rcas1 upregulation is considered to play a crucial role in tumor progression by enabling cancer cells to evade immune surveillance. gastric cancer is the second largest cause of cancer - related death worldwide, presenting the higher incidence in japan and china, lower in europe, and the lowest in the usa. studies on gastric cancer development suggest that genetic predisposition, infection, and diet are parts of a complex interaction. helicobacter pylori (hp) infection and to a lesser extent smoking have been identified as the main environmental risk factors for gastric cancer. chronic inflammation plays important roles in the development of various cancers of the digestive tract, including hp - associated gastric cancer. notably, several inflammatory mediators have been associated with tumor progression, angiogenesis, and metastasis in gastric cancer. in the last few years , rcas1 protein expression has been described in a variety of human malignancies, including oral, esophageal, breast, thyroid, lung, neurological, pancreatic, colorectal, urothelial, endometrial, cervical, and ovarian carcinomas. notably, rcas1 expression was shown to be associated with important clinicopathological parameters for patients' management and prognosis, being considered as an informative biomarker in several types of tumour malignancy. however, the available studies so far assessing the clinical significance of the rcas1 expression in gastric neoplasia remain scarce, and they are restricted to t3 gastric carcinoma cases or they do not concern patients' prognosis. in view of the previous considerations, the present study aimed to assess immunohistochemically rcas1 expression in 54 gastric adenocarcinoma specimens and to evaluate the association of rcas1 expression levels with clinicopathological parameters, tumor proliferative capacity, and patients' survival. fifty - four gastric tumoral samples obtained from an equal number of patients, who underwent surgical resection due to gastric cancer at the 2nd department of propedeutic surgery, medical school, university of athens, greece, were consecutively included in this study. the study was approved by the hospital ethics committee, and informed consent was obtained from all participants. forty - three (79.6%) of the patients were men and 11 (20.4%) were women. the mean age of the patient cohort was 67.33 8.47 years (median : 67 years, range : 3988 years). tumors were typed according to lauren classification as intestinal in 24 (44.4%) and diffuse in 30 (55.6%) gastric adenocarcinoma cases. three levels differentiation were used to classify grading as: well in 1 (1.9%), moderately in 24 (44.4%), and poorly differentiated in 29 (53.7%) patients. tumors staging was assessed using the 5th edition of the tumor, node, and metastasis (tnm) system according to the union internationale contra la cancrum (uicc) and the american joint committee on cancer (ajcc). the resected tumors were staged as: t1 in 6 (11.1%); t2 in 15 (27.7%); t3 in 27 (50.0%), and t4 in 6 (11.1%) cases. nineteen (35.2%) patients were lymph node negative (n0) and 35 (64.8%) lymph node positive; n1 in 32 (59.3%) and n2 in 3 (5.6%) cases. patients were followed up for a time interval between 1 to 148 months (28.21 22.63 months). rcas1 immunostaining was performed on formalin - fixed, paraffin - embedded gastric adenocarcinoma tissue sections using a mouse monoclonal anti - rcas1 antibody (mbl international co, nagoya, japan). briefly, 4 m thick tissue sections were dewaxed in xylene and were brought to water through graded alcohols. antigen retrieval was performed by microwaving slides in 10 mm citrate buffer (ph 6.1) for 20 minutes (min) at high power, according to the manufacturer's instructions. to remove the endogenous peroxidase activity, sections were treated with freshly prepared 0.3% hydrogen peroxide in methanol in the dark, for 30 min, at room temperature. nonspecific antibody binding was blocked using sniper, a specific blocking reagent for mouse primary antibodies (sniper, biocare medical, walnut, creek, ca, usa) for 5 min. the sections were incubated for 1 hour (h), at room temperature, with the primary antibody against rcas1, diluted 1: 750 in phosphate buffered saline (pbs). after washing three times with pbs, sections were incubated at room temperature with biotinylated linking reagent (biocare medical) for 10 min, followed by incubation with peroxidase - conjugated streptavidin label (biocare medical) for 10 min. the ant immune peroxidase activity was developed using a dab substrate kit (vector laboratories, usa) for 10 min. sections were counterstained with harris' hematoxylin and mounted in entellan (merck, darmstadt, germany). appropriate negative controls were performed by omitting the primary antibody and/or substituting it with an irrelevant antiserum. pancreatic and thyroid cancer tissue sections with known increased rcas1 immunoreactivity were used as positive control. immunohistochemical evaluation was performed by counting at least 1000 tumor cells in each case by two independent observers (s.t . and p.a . the immunoreactivity of the tumor cells for rcas1 was scored according to the percentage of rcas1 positive tumor cells as 0 : < 5% of tumor cell positive ; 1 : 524% of tumor cells positive ; 2 : 2549% of tumor cells positive ; 3 : 5074% of tumor cells positive ; 4 : and 75100% of tumor cells positive and according to its intensity as 0 : negative staining, 1 : mild staining ; 2 : intermediate staining ; and 3 : intense staining . finally, the expression of rcas1 was classified as low ; if the total score was 5 and high ; if the total score was 6 . in this way , we ensure that each group has a sufficient and more homogeneous number of cases in order to be comparable with the other groups . chi - square test was used to assess the associations of rcas1 protein expression with clinicopathological variables . survival curves were constructed using the kaplan - meier method and the differences between the curves were compared by the log - rank test . a cox proportional - hazards regression model was developed to evaluate the association between the potential prognostic marker and patients ' survival . cox regression analysis was conducted at both univariate and multivariate levels . a p value less than 0.05 was considered the limit of statistical significance . rcas1 was abundantly expressed in the gastric adenocarcinoma cases examined, presenting both a cytoplasmic and membraneous pattern of staining ( figure 1). all cases were found positive for rcas1, while the mean percentage rcas1 expression value was 64.5%. the intensity of rcas1 immunostaining was classified as mild in 4 (7.4%), moderate in 19 (35.2%), and intense in 21 (57.4%) out of 54 gastric adenocarcinoma cases. by applying the immunohistochemical score, 25 (46.3%) cases were found to present low rcas1 expression, and the remaining 29 (53.7%) cases showed high expression. weak cytoplasmic rcas1 expression was also noted in less than 5% in normal (nonmalignant) gastric tissues. in cross - tables, high rcas1 expression was significantly more frequently observed in gastric adenocarcinoma cases with advanced histopathological stage and presence of organ metastasis (table 1, p = 0.0327 and p = 0.0084, resp .). rcas1 expression did not show statistical significant associations or trends of correlation with the other clinicopathological variables examined (table 1, p > 0.05). univariate analysis was performed to assess the strength of the association of each clinicopathological parameter and rcas1 expression (low versus high) with overall patients' survival. patients' age, histopathological type, grade of differentiation, disease stage, tumor size, presence of lymph node and organ metastasis, as well as rcas1 expression were identified as significant prognostic factors of overall patients' survival (table 2, p = 0.0031, p = 0.0003, p < 0.001, p = 0.001, 0.023, 0.0021, 0.09, and p = 0.021, resp .). kaplan - meier survival curves indicated that patients with high rcas1 expressing tumors had significantly shorter survival times compared to those with low rcas1 expression (figure 2(a), log - rank test, p = 0.016 ). the mean survival time in patients presenting high rcas1 expression was 33.88 months (23.19 months), whereas in those presenting low rcas1 expression was 23.32 months (17.73 months). in multivariate analysis, tumor histopathological grade of differentiation, stage, and rcas1 expression proved to be independent prognostic factors of overall patients' survival (table 3, p = 0.003, p = 0.020 and p = 0.008, resp .). statistical analysis for rcas1 expression was further performed in each gastric adenocarcinoma histopathological type, separately. in diffuse - type gastric adenocarcinoma, rcas1 expression was significantly associated with the presence of organ metastasis (p = 0.025) and borderlined with histopathological stage (p = 0.078). kaplan - meier survival curves indicated that diffuse - type gastric adenocarcinoma patients with high rcas1 expression presented significantly shorter survival times compared to those with low rcas1 expression (figure 2(b), log - rank test, p = 0.033 ). histopathological stage and rcas1 expression were identified as significant predicting factors for patients prognosis in multivariate analysis (cox regression analysis, p = 0.018 and p = 0.048, resp .). in intestinal - type gastric adenocarcinoma, intestinal - type gastric adenocarcinoma patients presenting high rcas1 expression levels showed shorter survival times compared to those with low levels at a no significant level, though (figure 2(c), log - rank test, p = 0.225 ). rcas1 has been documented to be overexpressed in various tumors, affecting many aspects of cancer biology, such as differentiation, proliferation, invasion, and angiogenesis. elevated rcas1 expression has been associated with the malignant state of several tissue types and may play crucial role in tumor progression by enabling cancer cells to evade immune surveillance. the expression of rcas1 in tumors of the gastrointestinal tract has already been documented; however, its associations with clinicopathological parameters and patients' prognosis in gastric neoplasia remains scarce. in the present study moreover, 29 (53.7%) out of the 54 gastric cancer cases showed high rcas1 expression. notably, current substantial evidence supported that the fact rcas1 may be considered as a promising target for future (gene) therapeutic approaches. in this aspect, the increased frequency of high rcas1 positivity in the examined gastric adenocarcinoma cases reinforces the therapeutic utility of this receptor in gastric cancer chemoprevention and treatment. importantly, the recent evidence that rcas1 sirna suppressed rcas1 mrna and protein expression and delayed tumor growth, in vivo, has already unfolded new perspectives for the development of future rcas1 receptor target inhibitors. antiestrogens may also employ rcas1 as a molecular switch to activate immune cytotoxicity against tumor cells. the present study further showed that rcas1 expression was significantly elevated in gastric cancer patients with advanced histopathological stage and presence of organ metastasis. elevated rcas1 expression was also significantly associated with poor patients' prognosis in both univariate and multivariate analysis. these findings are in accordance with previous substantial evidence in carcinomas of other anatomic sites of the gastrointestinal tract, such as oral, esophageal and colorectal carcinomas. concerning gastric cancer, nakamura et al. documented that rcas1 staining pattern (diffuse or not) correlated with tumor size, depth of tumour invasion, histological type, and lymph node metastasis. however, rcas1 positivity was not associated with the clinicopathological variables examined, while no data concerning the patients' survival was reported. in a study by fukuda et al, which was performed on 129 t3 gastric carcinoma cases, rcas1 expression was also significantly correlated with histological type and lymph node metastasis, as well as patients' prognosis. in another study applying a semiquantitative rt - pcr analysis, the perinuclear pattern of rcas1 expression was more frequently observed in well differentiated gastric adenocarcinoma compared to moderate ones. the diffuse pattern of rcas1 expression was more frequently recognized in gastric carcinomas which invaded beyond the submucosa compared to intramucosal carcinomas. in contrast to the previous studies, we did not find any significant association with the presence of lymph node metastasis and the grade of differentiation; however, diffuse type gastric cancer cases showed an increased frequency of enhanced rcas1 expression compared to intestinal ones without, though, reaching statistical significance. currently, substantial evidence has suggested that rcas1 expression upregulation may play a crucial role in tumor progression by enabling cancer cells to evade immune surveillance. interestingly, rcas1 induced apoptosis in both cultured human lymphoma cell lines and normal peripheral lymphocytes, which express rcas1 receptor. this apoptotic effect was supported to be mediated through induction of genes or caspase molecules, which strongly abrogated rcas1-induced apoptosis, in vitro. moreover, rcas1 facilitated tumor cell invasion of connective tissue in uterine cervical cancer via the enhancement of invasive potency by induction of stromal tissue remodeling, as well as through evasion of antitumor immune surveillance by an apoptotic counterattack mechanism against lymphocytes. rcas1 may also modulate surface expression of tumor - associated o - linked glycan structures, which are considered to participate in cell adhesion, invasion, and metastasis of cancer cells. taking into account the previous considerations, further studies should be performed in order to delineate whether rcas1 positive tumor cells may induce apoptosis to their surrounding tumor infiltrating lymphocytes. as rcas1 is easily detectable in biofluids further research effort should also be orientated to the determination of rcas1 levels in serum or saliva during and after the treatment of gastric cancer patients with the aim of recognizing patients' recurrence. rcas1 expression was associated with clinicopathological parameters crucial for patients' management and prognosis. of even more clinical significance are the data supporting the contribution of rcas1 in the pathophysiological aspects of the disease that affect patients' survival. these findings may suggest an important potential role of rcas1 in the biological mechanisms underlying the carcinogenic evolution of gastric adenocarcinoma. further research conducted on larger cohorts and focused on each histopathological type separately that additionally concerned more sensitive techniques is strongly recommended in order to assess whether rcas1 may be considered as a useful biomarker in gastric adenocarcinoma, evaluating also its therapeutic utility in gastric cancer chemoprevention and treatment.

. the receptor - binding cancer antigen expressed on siso cells (rcas1) is a human tumor - associated antigen that has been considered to play a crucial role in tumor progression by enabling cancer cells to evade immune surveillance. the present study aimed to evaluate the clinical significance of the rcas1 expression in gastric adenocarcinoma. material and methods. rcas1 protein expression was assessed immunohistochemically on 54 gastric adenocarcinoma tissue samples and was analyzed in relation to clinicopathological parameters, tumor proliferative capacity, and patients' survival. . enhanced rcas1 expression levels were significantly associated with advanced histopathological stage and presence of organ metastasis (p = 0.0084 and p = 0.0327). gastric cancer patients with elevated rcas1 expression levels showed significantly shorter survival times compared to those with low rcas1 expression (log - rank test, p = 0.0168). in multivariate analysis, histopathological stage and grade of differentiation as well as the rcas1 expression were identified as independent prognostic factors (cox regression analysis, p = 0.0204, p = 0.0035, and p = 0.0081). . our data support the evidence that rcas1 upregulation may contribute to gastric malignant progression, representing a useful biomarker to predict the biological behaviour and prognosis in gastric neoplasia.

with the growth of the internet use over the last two decades, there has been an increase in its usages as well as in the frequency of experienced dysfunctions related to its overuse. users report loss of control over their internet use, social problems as well as school and/or occupational difficulties. public health concerns are emerging concerning the propensity of compulsive internet use developing into pathological behaviors. about 20% and 33% of internet users engage in some form of online sexual activity. nearly 80% of online gamers are losing out at least one element of their lives, such as sleep, work, education, socializing with friends, family, and interaction with a partner. the younger the players, the longer the time they dedicated to playing online games, leading to further functional impairment in their lifestyle. poor coping and cognitive expectations also mediate the development of the excessive use of internet if other risk factors are present such as depression, social anxiety, low self - esteem, low self - efficacy, and high stress. depression, social phobia, hostility, and symptoms of adhd are seen as comorbid condition to problematic internet use. individuals with social anxiety reported a greater feeling of comfort and self - disclosure when socializing online compared to face - to - face communication. about 8% of the pathological users used the internet to meet new people for emotional support and to play interactive games. about 9% of the clinical subjects (n = 300) have problematic usage of social networking sites. in previous studies conducted in the indian context has shown problematic to the addictive use of technology. the majority of the subjects had psychological distress as the comorbid condition. users were also using information technology to manage their psychological distress, to avoid a stressful situation, and way of managing boredom. there is a dearth of information about the pattern of technology use among the psychiatric population as well as its relationship with other sociodemographic variables. survey method was used to recruit 75 subjects (male / female) from the in - patient and out - patient psychiatric setting of national institute of mental health and neurosciences, bengaluru, karnataka with inclusion criteria of age range of 16 years and above, using internet for the minimum duration of 1 year and ability to read and write english. subjects with active psychopathology, illiterate, and unwillingness to participate were excluded from the study. data sheet developed by the investigator to record sociodemographic details which covers age, sex, socioeconomic status, education, occupation religion, marital status and type of family, details of the psychiatric illness (as per file diagnosis as per international classification of diseases-10 or diagnostic and statistical manual of mental disorders criteria) such as duration of illness, nature and course of illness, treatment taken, and premorbid personality traits. information related to technology use, the age at which individual starts using it, type of information technology used, reason to start using information technology, frequency of use, sites accessed, currently accessed sites, individual / group activities, duration of use, having smart phone with internet, availability at home, purpose of using information technology, situation associated with the use of information technology, any history of attempt to reduce the usage of information technology, perception about the usage, relationship of coping (to manage boredom, emotional state etc.)/psychiatric condition with technology use as well as for seeking health information, type of activity; impact of technology use on one's life, care giver perspective and need for change. internet addiction impairment index is a twenty items questionnaire based on 5-point likert scale to assess addiction to internet. internet addiction impairment index can be utilizing to help classify the behavior regarding mild - moderate and severe impairment. the scale covering the degree to which their internet use affecting their daily routine, social life productivity, sleeping pattern, and feelings video game use patterns, to assess individuals video game use pattern in 9-item scale with two self - reported assessment of video game using pattern, and the emotional distress associated with it. pornography addiction screening tool is a twenty items questionnaire based on 5-point likert scale to assess addiction to pornography and online sexual behavior. screening for mobile phone use evolved screening questions developed for icmr funded behavioral addiction project will be used. subjects were taken from the in - patient / out - patient psychiatric setting of nimhans bengaluru, karnataka. prior consent was obtained from the concerned treating team as well as from the user. the process and objectives of the study were explained to the patients and informed consent was sought. the sociodemographic information were filled as per the information given by the patient and care givers as well as from the case file. the internet addiction questionnaire, video game use pattern questionnaire, facebook intensity questionnaire, pornography addiction test, and screening questionnaire for mobile phone addiction were administered in individual setting. the data were coded for the computer analysis and statistical package for social science 16.0 version was used to carry out the analysis of the quantitative data. descriptive statistics such as mean, standard deviation percentage, and frequencies were used to analyze the demographic data as well as the details of psychiatric condition. pearson's chi - square test was computed to examine the significance of the relation among the variables. all the figures have been rounded off to two decimal places and for the level of significance probability level of 0.05 and 0.01 are used. survey method was used to recruit 75 subjects (male / female) from the in - patient and out - patient psychiatric setting of national institute of mental health and neurosciences, bengaluru, karnataka with inclusion criteria of age range of 16 years and above, using internet for the minimum duration of 1 year and ability to read and write english. subjects with active psychopathology, illiterate, and unwillingness to participate were excluded from the study. data sheet developed by the investigator to record sociodemographic details which covers age, sex, socioeconomic status, education, occupation religion, marital status and type of family, details of the psychiatric illness (as per file diagnosis as per international classification of diseases-10 or diagnostic and statistical manual of mental disorders criteria) such as duration of illness, nature and course of illness, treatment taken, and premorbid personality traits. information related to technology use, the age at which individual starts using it, type of information technology used, reason to start using information technology, frequency of use, sites accessed, currently accessed sites, individual / group activities, duration of use, having smart phone with internet, availability at home, purpose of using information technology, situation associated with the use of information technology, any history of attempt to reduce the usage of information technology, perception about the usage, relationship of coping (to manage boredom, emotional state etc.)/psychiatric condition with technology use as well as for seeking health information, type of activity; impact of technology use on one's life, care giver perspective and need for change. internet addiction impairment index is a twenty items questionnaire based on 5-point likert scale to assess addiction to internet. internet addiction impairment index can be utilizing to help classify the behavior regarding mild - moderate and severe impairment. the scale covering the degree to which their internet use affecting their daily routine, social life productivity, sleeping pattern, and feelings video game use patterns, to assess individuals video game use pattern in 9-item scale with two self - reported assessment of video game using pattern, and the emotional distress associated with it. pornography addiction screening tool is a twenty items questionnaire based on 5-point likert scale to assess addiction to pornography and online sexual behavior. screening for mobile phone use evolved screening questions developed for icmr funded behavioral addiction project will be used. subjects were taken from the in - patient / out - patient psychiatric setting of nimhans bengaluru, karnataka. prior consent was obtained from the concerned treating team as well as from the user. the process and objectives of the study were explained to the patients and informed consent was sought. the sociodemographic information were filled as per the information given by the patient and care givers as well as from the case file. the internet addiction questionnaire, video game use pattern questionnaire, facebook intensity questionnaire, pornography addiction test, and screening questionnaire for mobile phone addiction were administered in individual setting. the data were coded for the computer analysis and statistical package for social science 16.0 version was used to carry out the analysis of the quantitative data. descriptive statistics such as mean, standard deviation percentage, and frequencies were used to analyze the demographic data as well as the details of psychiatric condition. pearson's chi - square test was computed to examine the significance of the relation among the variables. all the figures have been rounded off to two decimal places and for the level of significance probability level of 0.05 and 0.01 are used. 17 were married (22.67%), 57 were unmarried (76%), and 1 was divorced (1.33%). 36% were from the rural area and the 64% were from the urban area. sociodemographic information of the sample table 2 shows the diagnosis of the sample population and its frequency, 32 different diagnoses in different frequencies were taken. frequencies and percentages of subjects with psychiatric diagnosis according to international classification of diseases-10 (f - code) table 3 indicates the presence of addiction for mobile phone (18.67%), internet addiction (16%), pornography (46.67%), and video games (14.67%). pattern of information technology addiction among sample table 4 shows the duration of illness of the sample (n = 75), varies from 6 months to 21 years, and the mean is 6.4 years with the standard deviation of 4. pattern of duration of psychiatric illness and premorbid personality of sample table 5 shows that 58.7% of individuals in the overall sample reported that they were spending more time with information technology to 14.7% were using to avoid any negative emotions, 2.7% (2 people) were using to cope with the situations and the 24% of the total sample spending time for other purposes like to get general information or as the part of career and academics. use of information technology to avoid negative emotions / as a method of coping was more among users who had a 5 h or more usage per day. relationship between average time using for the internet per day and the situations associated with the usage of internet table 6 shows that disturbance of sleep was (delay in initiation of sleep) more in moderate to severe category of use. relationship between internet addiction and sleep (delay in the initiation of sleep) table 7 shows that age had negative correlation with the duration of the illness, the average time spending on internet, internet addiction, mobile addiction, video game use, and pornography addiction. average time spending per day on the internet showing a positive correlation with the mobile phone, internet, videogame, and pornography addiction. mobile phone addiction had significant positive correlation with internet, video game use, and pornography addiction. this study indicates the trend toward the presence of addiction to the mobile phone (18.67%), internet addiction (16%), pornography (46.67%), and video games (14.67%) among subjects seeking treatment for psychiatry problems. age has a negative correlation with internet addiction, video game addiction mobile addiction, and pornography. the mean age of the sample was 26.67 with the standard deviation of 6.5. duration of illness of the sample (n = 75), varies from 6 months to 21 years, and the mean is 6.4 years with the standard deviation of 4. 49.33% had personality characterized by difficulty in adjustment and personality traits. use of information technology was seen to avoid negative emotions / as a method of coping was more among users who had a 5 h or more usage per day. moderate to the severe use of information technology was associated with a delay in initiation of sleep. age had negative correlation with the duration of the illness, the average time spending on internet, internet addiction, mobile addiction, video game use, and pornography addiction. average time spending per day on the internet showing a positive correlation with the mobile phone, internet, videogame, and pornography addiction (vii). individuals belonging to the age group of 2029 used the internet more, while internet addiction scores of the individuals belonging to the group of 19 and below was higher than other groups and that this situation varied according to gender. problematic internet use showed the correlation of 75% with depression; 57% with anxiety, 100% with symptoms of adhd; 60% with obsessive - compulsive symptoms and 66% with hostility / aggression. the adolescents who play more than 1 h of console or internet video games may have more or more intense symptoms of adhd or inattention than those who do not. people with low self - esteem, self - efficacy, and vulnerability to stress are more prone to have a general internet addiction. sleep deprivation seems to be one of the major problematic effect of internet addiction and late night logins. the present work documents the presence of information technology addiction among subjects with psychiatric problems. addiction to internet and pornography is also associated with delay in the initiation of sleep. although the obtained prevalence is low in comparison to international prevalence, it can be addressed in a large sample study. the present communication gave trend toward association of age / average time spent per day with addiction to information technology; use of information technology as a coping method. the present work has implications in term of screening the technology addiction as comorbid condition among the psychiatric population. the future work can focus on exploring the psychosocial correlates among subjects with psychological problems, caregiver issues related to the handling of addictive use of information technology as well as evolving the intervention for the promotion of the healthy use of technology.

: technology usage has seen an increase among users. the usage varies from social, personal, and psychological reasons. users are frequently using to overcome mood states as well as to manage the other psychological states. this work is going to explore the information technology use among subjects with a psychiatric disorder.materials and methods: a total of 75 subjects were assessed using data sheet, internet addiction impairment index, video game use pattern, pornography addiction screening tool and screening for mobile phone use, from in - patient and out - patient setting of tertiary mental health setting.:it showed the presence of addiction to mobile, internet, video game, and pornography. age was found to be negatively correlated with this addiction. average usage time had been associated with management of mood states. the addiction to information technology had been associated with a delay in initiation of sleep.:this work has implication for screening technology addiction among subjects seeking treatment for psychological problems and motivate them to develop the healthy use of technology.

for a long time, plants represented one of the most important therapies for different diseases. nowadays, the popular use of plants as a way of treatment is still very important for human beings. pterospartum tridentatum (l.) willk, gomphrena globosa l., and cymbopogon citratus (dc .) stapf are examples of those plants that are widely used in folk medicine, mostly as infusions. p. tridentatum (family : fabaceae) is a european endemic species and its flowers infusion is used against liver, bladder, kidney, and rheumatism problems; it is also used for high blood pressure, cough, kidney stones, diabetes, and bronchitis. g. globosa (family : amaranthaceae) is native from panama and guatemala and its aqueous extract of its purple inflorescences is good to treat bronchial asthma, acute and chronic bronchitis, and whooping cough; the infusion of the flowers is used to treat oliguria and indigestion, also as expectorant and pertussis. c. citratus (family : poaceae) is native from the southwest asia and its aqueous extract (i.e., in the form of infusion) is used for the treatment of several inflammation - based pathologies, in digestive disorders, diabetes, nervous disorders, and fever. according to novais , the infusion is used as gastric analgesic, intestinal anti - inflammatory, and renal antispasmodic and for gall - bladder ailments, sea - sickness, and bladder ailments. there are several reports about the biological activity of the mentioned plants, especially concerning antioxidant activity , that have been related to their phenolic composition , but studies regarding the composition on primary metabolites and nutrients are scarce. moreover, many of the commercially available samples are wild and require chemical characterization. besides secondary metabolites / nonnutrients such as phenolic compounds, medicinal plants contain primary metabolites and nutrients (e.g., as sugars, organic acids, and tocopherols) that need to be profiled and quantified. mono- and oligosaccharides, with low molecular weight, and their derivatives such as sugar alcohols display a major role in the structure and function of all living cells. organic acids are involved in several biochemical pathways, including energy production and formation of precursors for amino - acid biosynthesis. vitamin e (including tocopherols) is known to be an essential micronutrient for maintaining the health and wellbeing of humans and other animals. several studies suggest that the use of vitamin e may contribute to help lowering the risks of specific chronic and degenerative diseases such as alzheimer's disease, some types of cancer, cataracts, and ischemic heart disease. all these molecules can be determined by high performance liquid chromatography (hplc) coupled to different detectors according to their chemical properties, namely, fluorescence for tocopherols, refraction for sugars, and uv absorption for organic acids. therefore, the objective of the present paper was to characterize tocopherols, sugars, and organic acids in three medicinal plants (p. tridentatum, g. globosa, and c. citratus), widely consumed as infusions. plant material of pterospartum tridentatum (l.) willk, gomphrena globosa l., and cymbopogon citratus (dc .) stapf was purchased from ervital, a portuguese company in castro daire (portugal). this company, settled in a high diverse mountain region (montemuro, the natura 2000 site), markets several certified plant materials with different origin, such as sustainable wild harvesting of spontaneous local species and organic farming of exogenous species. pterospartum tridentatum flowers were wildly gathered in spring 2012 (respecting plant phenology and abundance) and the other studied species were grown, also in 2012, with organic farming methods. harvested plants were processed using in - storage and low temperature drying methods (solar heated air, average daily temperature around 3032c in shade conditions, and controlled relative humidity). samples for analysis were prepared from dried plant materials provided by the company, and botanical identification was confirmed by ana maria carvalho, responsible for the medicinal plant collection of the herbarium of the escola superior agrria (bresa), of the polytechnic institute of bragana (trs - os - montes, portugal). hplc - grade acetonitrile, ethyl acetate, and n - hexane were purchased from fisher scientific (lisbon, portugal). l - ascorbic acid, tocopherol, sugar, and organic acid standards were purchased from sigma (st . louis, mo, usa). racemic tocol (50 mg / ml) was purchased from matreya (pleasant gap, pa, usa). water was treated in milli - q water purification system (tgi pure water systems, greenville, sc, usa). the equipment consisted of an integrated system with a pump (knauer, smartline system 1000, berlin, germany), degasser system (smartline manager 5000), autosampler (jasco as-2057, easton, md, usa), and a fluorescence detector (jasco fp-2020) programmed for excitation at 290 nm and emission at 330 nm. the chromatographic separation was achieved with a polyamide ii (250 mm 4.6 mm i.d .) normal - phase column from ymc waters (dinslaken, germany) operating at 30c (7971 r grace oven). the mobile phase used was a mixture of n - hexane and ethyl acetate (70 : 30, v / v) at a flow rate of 1 ml / min, and the injection volume was 20 l. the compounds were identified by chromatographic comparisons with authentic standards. quantification was based on calibration curves obtained from commercial standards of each compound using the internal standard (is) methodology; racemic tocol was used as is. free sugars were determined by high performance liquid chromatography coupled to a refraction index detector (hplc - ri), after an extraction procedure previously described. analysis was performed by hplc (equipment described above) using an ri detector (knauer smartline 2300, berlin, germany). the chromatographic separation was achieved with a eurospher 100 - 5 nh2 column (4.6 250 mm, 5 mm, knauer, berlin, germany) operating at 30c. the mobile phase was acetonitrile / deionized water, 70: 30 (v / v) at a flow rate of 1 ml / min. the analysis was performed using a shimadzu 20a series uflc (shimadzu corporation, kyoto, japan). separation was achieved on a sphereclone (phenomenex, torrance, ca, usa) reverse phase c18 column (5 m, 250 mm 4.6 mm i.d .) the elution was performed with sulfuric acid (3.6 mm) using a flow rate of 0.8 ml / min. detection was carried out in a pda (photodiode array detector), using 215 and 245 nm (for ascorbic acid) as preferred wavelengths. the organic acids found were quantified by comparison of the area of their peaks recorded at 215 nm with calibration curves obtained from commercial standards of each compound. all the assays were carried out in triplicate, and the are expressed as mean values and standard deviation (sd). the were analyzed using one - way analysis of variance (anova) followed by tukey's hsd test with = 0.05. the chemical composition of the three plant species in tocopherols, free sugars, and organic acids is presented in table 1. c. citratus gave the highest - and total tocopherols content but did not present -tocopherol that was found in the other two species (e.g., figure 1(a) ). tocopherols are lipid - soluble antioxidants, being -tocopherol the most active isoform, due to its role in lipid peroxidation inhibition. these molecules are widely used as functional ingredients in food, pharmaceutical, and cosmetic preparations. as far as we know this is the first report on tocopherols composition of c. citratus and g. globosa; otherwise, the values obtained for p. tridentatum were similar to the ones described by the authors for a wild traditionally shade - dried sample (8.8 g / g dw). furthermore, tocopherols have also been reported in other species of the fabaceae family, such as cicer arietinum, lathyrus sativus, cytisus multiflorus, cytisus scoparius, and cytisus striatus; and the amounts found ranged between 6.3 and 23.1 mg/100 g of dw, which is in the range of the sample studied herein, detecting all the isoforms. all isoforms of tocopherols have also been reported in poaceae and amaranthaceae families, but the quantities can not be compared due to the units in which they are expressed. regarding free sugars, p. tridentatum showed the highest levels of fructose and total sugars; nevertheless, the values obtained were much higher than the concentrations found in a wild sample previously studied (0.3 and 49.6 g/100 g dw for fructose and total sugars, resp .). this could be due to different growth conditions of the plants (e.g., variability of weather and soil characteristics are major factors affecting plant development) and to different drying processes applied, which influence moisture content and plant material quality. simulated consumers' traditional conditions of use (shade - drying, plant material being stored in a dark, dry place and at room temperature for 30 days); dried plant material used for analysis in this study was processed in five days under the best conditions of shade, daily temperature, and relative humidity as well as airflow rate. furthermore, the presence of sugars has also been reported in the poaceae family, namely, sucrose, glucose, fructose, trehalose, and raffinose, but the amounts obtained can not be compared to the ones studied herein. some species belonging to the fabaceae family (mentioned above) have also shown the presence of sugars, mainly fructose, glucose, sucrose, and trehalose, although some species revealed the presence of other sugars. the amounts found ranged between 2.56 and 18.67 g/100 g of dw; these are much lower than the one present in this study. it should be highlighted that fructose can display antioxidant properties due to its reducing capacity. furthermore, sugars are one of the molecules present in plant infusions that contribute to their energetic value. c. citratus gave the highest levels of glucose and sucrose (table 1 and figure 1(b) ). no reports were found considering sugars composition in the mentioned species or in g. globosa. concerning organic acids, g. globosa was the sample with the highest concentration of these compounds, mainly malic and oxalic acids (table 1 and figure 1(c) ). citric and succinic acids were found in higher levels in p. tridentatum and c. citratus, respectively. the latter also presented ascorbic acid, a powerful antioxidant phytochemical. besides their important role in the human metabolism, organic acids have other applications; for example, citric acid is a crystal thickener in bones, succinic acid is known to help in diabetes treatment, and malic acid is reported to have a bactericidal effect. among the three analyzed species, only p. tridentatum was previously studied regarding organic acids composition; despite the fact that similar total amount was found (8.1 mg / g dw), the profile then described was slightly different, reporting also the presence of quinic, succinic, and fumaric acids. as mentioned before, differences may be caused by distinct ecological conditions for plant development (plant material provenance is quite different) and also by different characteristics of the plant material used for analysis, as a consequence of the drying processes applied to each material. a five - day controlled in - storage process may produce a better quality plant material, in terms of color (visually confirmed), texture, and moisture content, than the traditional shade - drying techniques. all the organic acids, with the exception of quinic acid, have been previously reported in some species (mentioned above) belonging to the fabaceae family , although the quantities present have a large variation depending on the species. overall, c. citratus possessed the highest content of - and total tocopherols, glucose, sucrose, succinic, and ascorbic acids. otherwise, g. globosa showed the highest organic acids concentration, due to the highest content of oxalic and malic acids. this study is of great importance because it fills an existing void in relation to chemical characterization of these plants, more precisely its composition in sugars, organic acids, and tocopherols, that can be present in the consumed forms (mostly infusions).

pterospartum tridentatum (l.) willk, gomphrena globosa l., and cymbopogon citratus (dc .) stapf are medicinal plants that require a more detailed chemical characterization, given the importance of their consumption as infusions. therefore, the individual profiles in tocopherols, free sugars, and organic acids were obtained by high performance liquid chromatography (hplc) coupled to different detectors (fluorescence, refraction index, and photodiode array, resp .). c. citratus revealed the highest content of -, and total tocopherols, glucose, sucrose, succinic, and ascorbic acids. p. tridentatum presented the highest fructose and total sugars content. otherwise, g. globosa showed the highest organic acids concentration. as far as we know, this is the first study reporting the mentioned chemical compounds in g. globosa and c. citratus.

the worldwide incidence of cervical cancer is 15.3 / 100,000 women per year. cancer of the cervix uteri is the second most common cancer among women worldwide, with an estimated 529,409 new cases and 274,883 deaths in 2008. about 86% of the cases occur in developing countries, representing 13% of female cancers. the cause of this cancer is attributed to human papillomavirus in 99% of these cases. worldwide, mortality rates of cervical cancer are substantially lower than incidence with a ratio of mortality to incidence to 52%. the highest incidence rates are reported from eastern and western africa at 35.4% and 33.7% respectively. in the developing countries, there is however marked variation in the frequency of cervical cancer in the developing countries owing to the differences in screening programmes and the prevalence of risk factors. the lowest reported incidence rates are from middle east and are among muslims and jews, compared to other religious groups. in saudi arabia the most recent estimates from cancer registry 2007 report indicate that cervical cancer ranks as the 5 most frequent cancer among women in between 30 - 44 years of age representing an incidence of 3.1%. data is not yet available on hpv burden in the general population of saudi arabia. the overall age - standardized incidence rate is 2.2/100,000 with highest incidence of 8.1% in age group of 60 - 64 years. the age - standardized mortality rate is 0.8 /100,000 in women population. the highest incidence is reported in jouf region with 5.6% followed by hail region 3.5%, najran 3.2%, al baha 2.2 % and the lowest in jazan and mekkah with 2.2%. there are no reported rates for other regions such as riyadh, eastern and northern region, madinah, qassim, asir, and tabuk. cervical intraepithelial neoplasia incidence in saudi arabia is not registered because there are no well - developed screening programs nationwide. however recent studies from different regions of the kingdom show slight increase in the prevalence of epithelial cell abnormalities (eca) in pap smears (ps). a study from maternity and children's hospital, saudi arabia reported that cervical cancer among saudi nations represents 33.5% of all gynecological malignancies. the incidence and mortality of cervical cancer has declined dramatically ever since the introduction and wide spread utilization of ps screening test especially in countries with well - established cervical screening programs such as usa where it is considered the sixth most common cancer in women. cytological evidence of all eca can be easily, safely and economically obtained by the preparation and examination of cytology smears. identification of preventable, precursor lesions by cytology could prevent further progress of the disease by simple therapeutic procedures and continued surveillance and as such emphasizes the need for early detection of cervical intraepithelial neoplasia. over the years , the original ps classification system has been revised in response to our growing knowledge of cervical cancer precursors and our understanding of the role of hpv cervical carcinogenesis. standardization of ps reporting using the bethesda system (rbs) has unified various overlapping and confusing terminologies and has overcome the consequences of inter - observer subjectivity in interpretation and application of these terminologies to some extent. the current study was conducted to explore the changing pattern of epithelial cell abnormalities (eca) detected in ps in females of the western region of saudi arabia at king abdulaziz university hospital, jeddah using bethesda system (rbs). a retrospective study targeting 15805 pss performed during january 2000- october 2012 at king abdulaziz university hospital (kauh) jeddah was performed. the data was collected by conducting a computerized search through the cytopathology archives at kauh jeddah. this study included both saudi and non saudi females, since kauh is a teaching hospital of king abdulaziz university medical school. it is the only hospital at jeddah providing treatment to all nationalities thereby providing the unique opportunity of studying cross cultural variations and similarities.the data was filtered indicating the following essential parameters: date of undergoing ps, personal identity (medical record number, age, sex etc) and relevant clinical information (indicated for routine or for gynecological symptoms).the study included all pss performed at kauh during the period of study. repeated smears from the same patients with insignificant cytological changes and carrying the same diagnosis and unsatisfactory pss were excluded from this study. the data were rechecked manually to avoid duplications. statistical package for the social sciences, version 15.0 (spss inc ., chicago, il, usa) program was used for analysis. descriptive and frequency statistics were obtained for the variables studies. the total number of pap smears examined (tse) after excluding unsatisfactory smears and the total number and percentages of abnormal pap smears (taps) were analyzed and correlated with the patient's age. the procedures followed in the present study were approved by and were in accordance with the ethical standards of the hospital ethical committee on human experimentation and with the helsinki declaration of 1975, as revised in 2000. a smear was considered sufficient when it was appropriately labeled having adequate sampling of the endocervical transformation zone with optimal preservation of the cells. all sufficient cervical cytology smears were reviewed and reclassified according to the 2001 rbs of ps reporting (table 1), into negative for intraepithelial lesions, positive for epithelial cell abnormalities (eca) and sub classified into specific categories. quality indicators such as inflammation, hemorrhage and reactive cellular changes associated with repair, squamous metaplasia, follicular cervicitis, atrophy and organisms were also identified. in cases of ambiguous diagnoses, the cytology slides were reviewed on a multi - headed microscope by a senior cytotechnologist and two pathologists to resolve the discrepancy. a total of 15805 cervical pss were retrieved and reported in the department of cytopathology at kauh, jeddah in the period from january 2000 to october 2012. the study identified 15721 (99.46%) sufficient smears and 84 (0.53%) unsatisfactory smears (table 1). the latter cases were excluded from further analysis. of the 15721 cervical pss, 12797 (81.15%) smears were reported as negative for eca while 2295 (14.52%) were diagnosed as positive for eca. the smears positive for squamous and glandular cell abnormalities were further sub classified according to the 2001 rbs as shown in table 2. unsatisfactory smears were 0.53 % among other quality indicators and were withdrawn for further analysis as they were beyond the scope of this study. we compared our to the past studies from our institution in table 3 and other recent studies from the saudi arabia, arab world and other international studies in table 4. in comparison to the previous studies from our institution (table 3) there is a significant increase in the number of smears positive for eca from 4.7% in the study that was conducted in year 2006 to 17.3% in the study that was conducted in 2009. in the present study squamous cell abnormalities have also increased from 3.5% in the year 2006 to 17.3% in 2009 to 11.42 % at the time of this study and glandular cell abnormalities from 1.10 in the year 2006 to 3.35% in 2009 to 3.1% at the time of the study. cervical intraepithelial neoplasia and invasive cervical carcinoma are less common in saudi arabia compared to the western countries and early stages of cervical cancer are even less. the original ps reporting system contained several overlapping terminologies and in order to present a more comprehensive system it was updated in 2001. the observations in the present study imply an overall increase in the number of abnormal ps with a relative and significant increase in squamous cell abnormalities especially abnormal squamous cells of undetermined significance (asc - us). the published data of 2009 showing a marked increase in eca, squamous and glandular abnormalities raised concerns and led the cytopathologists at our institution to strictly adhere to the criteria for each diagnostic category thereby addressing issues of over interpretation with obvious improvement. the current study, however still seems to revalidate and compliment the previous studies as regards the increase in number of eca. although the comparison of the previous studies are not valid due to overlap of data but it is a crude method to indicate that there is a continuous increase in the number of abnormal pss. the increase in the total number of pss however is explained by a number of reasons, such as the increasing awareness among the females in the western region of saudi arabia regarding the importance of cervical cancer screening through campaigns from cervical cancer scientific chair in jeddah area. if we assess studies from other parts of saudi arabia (table 4) using bs in diagnosis of pss, we notice a gradual increase in the incidence of eca especially in the squamous cell category is being reported. furthermore studies from the arab world have also reported a significant increased trend in asc - us and agus over the years (table 4). abul el - all et al study from egypt reported eca of 7.8%, asc - us of 34.4%, low grade squamous intraepithelial lesion (lgsil) 41%, high grade squamous intraepithelial lesion (hgsil) 5.2%, squamous cell carcinoma (scc) of 0.5% and agus of 15.3%. most of these studies indicate that there has been a definite increase in the prevalence of eca in the current years. it is not clear whether it is due to a high level of awareness of the targeted population within these studies or it is related to the trend of acquiring a more open life style.these issues need further investigation and assessment. however the lowest reported incidence of eca is from the eastern region, al khobar, wherein ascus detected was 0.48%, abnormal squamous cells - high grade (acs - h) was 0.06%, lsil was 0.19%, hsil was 0.37%, scc was 0.10% and agus was 0.25%. other recent studies from asia report an eca between 5 - 10.2% (table 4) and although the asc - us category in these studies is within an acceptable range of 0.3 - 1 % but they show a higher incidence of lgsil and hgsil which could be partly explained by the variation in the population under consideration. also it could be partly due to subjective variation in application of asc - us criteria early in these lesions. there are 2 - 3 million asc - us, 1.25 million lgsil and 300,000 hsil in usa. approximately three fourth of all cervical cancers in usa are squamous cell and the remaining are adenocarcinomas. hpv 16 and 18 account for approximately 68% of squamous cell carcinomas and 83% of adenocarcinomas. this finding warrants the need for incorporating hpv - dna testing in the screening programme. in most developed countries the current prevalence of cytological abnormality is between 5 - 10%. hpv l1 capsid protein and hterc gene (human telomerase rna component) may serve as markers for the early diagnosis and prediction of cervical lesions. the increase in l1/hterc ratio reflects the progression of cervical lesions to a certain extent. currently there is no organized cervical screening programme in saudi arabia so information regarding the rate of ps testing in saudi arabia remains obscure. in the arab world a screening rate of 14.3% is reported. in a study from usa the prevalence of hpv16 was 13.3% among asc - us, 23.6% among lsil and 60.7% among hsil. in the developing countries, we , however face an important challenge to apply hpv - based technology. hpv - dna testing has higher sensitivity but lower specificity than thin layer pap screening. the age - specific incidence of cervical cancer peaks around the age of 40 years, which suggests that the specificity and efficacy of hpv screening is maximal when it is performed on women between 30 and 40 years of age. virus - like particles (vlps) containing hpv structural proteins are being used as vaccines and can induce genotype specific virus neutralizing antibodies for preventing hpv infections. however, hpv infection as the current concept of the etiology of cervical cancer lacks adequate validation in terms of effective screening especially in saudi arabia. no population based data is available to indicate the prevalence of hpv cervical infections or their age specific incidence in saudi arabia. in saudi population, co factors found contributory to cervical cancer are smoking and use of oral contraceptives. a recent study emphasizes the need to include patients more than 45 years of age for pap screening in developing countries suggesting that a negative history before the age of 50 years does not necessarily exclude the risk of having cervical cancer. the limitation of the current study is that the diagnosis of asc - us is much over the acceptable range although the asc - us / sil ratio of 2.3 is within the limit. the increased trend at our institution is partly explained by the fact that the criterion for asc - us and lsil is quantitative and cytopathologists prefer to use the diagnosis of asc - us rather than lsil. the possible rationale behind this is that patients with asc - us diagnosis are treated for inflammation followed by a repeat ps within 6 months, while patients with lsil diagnosis are subjected to colposcopy and biopsy, especially in the absence of routine hpv testing in asc - us or sil diagnosis. there is evidence indicating increase in the number of abnormal pss over the last decade. it may not be a significant increase to call for nationwide mass cervical cancer screening program, which is a continuous process that requires allocation of huge resources that should be justifiable. it should, however, be obligatory for all women in the age of 35 - 55 to undergo pss and those with abnormal ec findings to have a test for hpv subtype. the data from all provinces should be evaluated thoroughly and if the indicate considerable health problem with a definite increase in the incidence over successive years, proper documentation of such data should be enforced for more comprehensive analysis and decision on mass screening programme nationwide.

objective(s): in developing countries and worldwide cervical cancer is an important cause of female mortality. reports describing the frequency and pattern of abnormal pap smears in saudi arabia, using the revised bethesda system (rbs) are very few. the current study was conducted to explore the changing pattern of epithelial cell abnormalities (eca) detected in pap smears (ps) in females of the western region of saudi arabia at king abdulaziz university hospital, jeddah using the rbs.materials and methods: a retrospective study was designed to review all the pss from the archives of cytopathology department at king abdulaziz university hospital, starting from january 2000 to october 2012 using rbs. cytological aspects of pss were reviewed with age distribution.: of the 15805 ps, 84 (0.53%) unsatisfactory smears were excluded. there were 2295 cases (14.52%) with eca. in the abnormal squamous cell category the distribution of lesions was as follows: atypical squamous cells of indeterminate significance (asc - us) were 7.1%; atypical squamous cells, can not exclude high squamous intraepithelial lesion (asc - h) were 1.08%; low grade squamous intraepithelial lesion (lsil) including human papillomavirus was 2.2%, high grade squamous intraepithelial lesion (hsil) was 0.8% and high grade squamous intraepithelial lesion with suspicious invasion was 0.06% smears. the mean age (ma) incidence was 39,43,45,46 and 45 years respectively.: the percentage of abnormal ps is increasing (14.52%) over the last decade. this increase is evident by different studies conducted across saudi arabia. under present circumstances the need for mass screening.

the application of osseointegration, introduced by branemark, was initially limited to edentulous patients but is now widely used in both edentulous patients and patients with missing individual teeth. replacement of missing teeth by dental implant has become an integral part of modern dental health care1. there has been much technological development to enhance the survival rates of implants, particularly with regard to implant surface treatment. albrektsson et al.2 reported that, when an implant had a rough surface rather than a machined surface, more rapid bone growth and superior physical adhesion were observed, while osteoblasts were better attached to rough surface implants, thus influencing the maturation, differentiation, and bone - implant interface of attached cells. wennerberg et al.3 reported that a rough surface implant had larger bone - implant contact (bic) and higher removal torque compared with a machined surface implant. surface treatment methods, by which an implant surface is made rough to increase the bone - implant interface, include acid etching4, blasting5, hydroxyapatite coating6, titanium plasma spray7 and sandblasting with large grit, and acid etching (sla)8. buser et al.9 reported that the 10-year survival rate of implants with the sla surface was 98.8%, while the prevalence of peri - implantitis over 10 years was 1.8%. in the present study, the authors evaluated the five - year survival rate and the surrounding bony change of dental implants with the sla surface (snucone dental implant, daegu, korea). the subjects in the study are 54 patients who have been followed - up to date, of the patients who underwent implant surgery at chungnam national university hospital (daejeon, korea) from may 1, 2009 through april 30, 2011. medically compromised patients were sent them to physicians to undergo medical treatment prior to implant surgery. in all, 176 implant placements were performed. the implant surgeries were divided into first and the second surgeries and were carried out in two stages. the surgeries and prosthetic treatments were carried out by one skillful oral and maxillofacial surgeon. the first surgeries were performed according to general surgery principles and a protocol provided by the manufacturer. in the case of lateral window opening cases, horizontal and vertical incisions were made on the alveolar ridge and the lateral surface of the maxillary sinus, respectively, for exposure of the lateral surface of the maxillary sinus. in all lateral window opening cases, the bone graft material and the membrane were xenograft (bio - oss ; geistlich pharma ag, wolhusen, switzerland) and collagen membrane (bio - gide ; geistlich pharma ag), respectively. after the first surgeries, antibiotics and analgesics were prescribed for all patients for seven to ten days. all the patients were instructed to not apply any stimuli to the surgical site until the removal of the stitches. they were also instructed to perform a 0.12% chlorhexidine mouth rinse three or four times a day. , the second surgery was performed an average of 2.5 months after the first surgery. in the maxilla , however, the second surgery was performed five months after the first. for lateral window opening cases, dental impressions were usually performed two weeks after the second surgery, and temporary prostheses were installed one week after the impressions were created. panoramic radiographs were collected before and immediately after the first surgery, after the second surgery, immediately after the final prosthesis setting, three months after the final prosthesis setting, six months after the final prosthesis setting, and every year after that. bone loss surrounding an implant was evaluated in accordance with the method suggested by romanos and nentwig10. evaluation of bone loss was graded as follows: group 0, no bone loss; group m, minimal bone loss (less than 2 mm at the crestal aspect of the implant); group 1, bone loss involving 1/4 of the implant length; group 2, progressive bone loss between 1/4 and 1/2 of the implant length. bone loss measurements were made on the mesial and distal sides of the fixture - abutment junction to the cortical bone. the values from the mesial and distal sides were averaged to be used in the evaluation. bone loss was evaluated by calculation of the rates to the known length of the fixture. the radiographs taken right after the final setting of the prosthetic appliance were compared with the follow - up radiographs for evaluation. as the number of implants placed was not large, no statistical analysis was performed. the age range of the men was 24 years to 70 years, with an average of 54.8 years. the age range of the women was 39 years to 75 years, with an average of 57.4 years. among men, those in their 50s were the most common, at 20 patients. among women, those in their 60s were the most common, at 6 patients. of the 176 implants, 106 were placed in the maxilla, while 70 were placed in the mandible; 122 implants were performed in men and 54 in women.(table 1) fixtures 4.3 mm in diameter and 10 mm in length were used most often. the number of the fixtures used is shown in table 2. in lateral window opening cases, 30 implant placements were performed in 18 cases in 16 men, and 14 implant placements in eight cases in eight women. an osteotome technique with a crestal approach was used for 14 implant placements in 10 men, while no women were treated with an osteotome technique. as for the kinds of prosthetic appliances, the number of the implants manufactured as a bridge was 156, while the number of single prostheses was 20. in a total of 54 patients the range of the follow - up periods was four years to six years, with an average of 4.9 years. the bone loss evaluated by the method suggested by romanos and nentwig10 is shown in table 3. the numbers of implants included in group m were nine for molar teeth in the maxilla, three for molar teeth in a mandible, and two for premolar teeth in the maxilla.(fig . 1) of these, five teeth in molar areas in the maxilla and three teeth in molar areas in the mandible were single prostheses. the horizontal positions of the alveolar bone were maintained in a relatively stable state in the rest of the implants.(fig . 2) a total of six implants failed - four in the mandible and two in the maxilla; all of these were single - implant cases. in one case, a fixture placed in the mandible of a male patient experienced fracture. in three cases, the patients were receiving chemotherapy, while two patients were suffering from diabetes or chronic renal failure. in all five cases, except for the patient with the fractured fixture, no abnormalities were found in the radiograph tests performed right before the implant failures occurred. all the failed implants belonged to group 0, and no failed implants belonged to group m. the implant survival rate was 98.1% in the maxilla and 94.3% in the mandible, ing in an overall survival of 96.6%. studies are continually being performed to improve the survival rate of implants and to shorten the healing period through development of bone graft material and improvement of surgical methods, implant design, and surface treatment methods. fast healing of peri - implant bone after implant placement and continuous and stable maintenance of osseointegration are important factors. several studies have shown that a rough surface implant has more merits than a machined surface implant211121314. osteoblasts have greater binding affinity with a rough surface than with a machined surface, while a rough surface influences the maturation, differentiation, and bone - implant interface of attached cells. deposition of an inorganic component in bone tissues and the bone cell alkaline phosphatase activity have been found to be higher on a rough implant surface. the surface treatment method, which is widely used lately, uses either resorbable blasting media (rbm) or sla. the rbm method uses resorbable particles, such as calcium phosphate, in order to supplement the shortfall of blasted particles left on the implant surface5. i m et al.17 reported higher bic in sla implants (82.7%) than in rbm implants (78.3%) 12 weeks after implant placement in adult mutts. elkhaweldi et al.18, who compared the survival rate of rbm surface implants with that of sla surface implants, reported that the former was 95.2% and the latter 99.1%, with a higher survival rate observed in sla surface implants. they reported that sla surface implants produced better in the posterior maxilla with poor bone. for long - term success of an implant, stable maintenance of the peri - implant bone is an important indicator. in this study , we evaluated the bone loss in accordance with the method suggested by romanos and nentwig10. the implants included in group m were placed in areas experiencing high occlusal force, while they occurred between two and three years after the final placement of the prosthetic appliances, and many of them were in the maxilla. considering that most of the failed implants occurred within one year after placement, a somewhat longer follow - up period is thought to be needed. the reason for the higher number of failures in the maxilla is thought to be similar to the reason for the lower implant survival rate in the upper jaw than in the mandible1819. after about one year, peri - implant bone had become stable, and little subsequent change was observed. the use of panoramic radiographs, which have lower resolution than periapical radiographs, might have ed in a somewhat low accuracy; however, it is believed that there were no major errors because we performed the measurements in consideration of the rates to the known length of the fixtures. in the external type of implant, where the abutment and the fixture are connected with a butt joint connection system , sealing was not secured between the abutment and the fixture, allowing the possibility of microleakage of bacteria. in addition, most of the stress caused by occlusal force is concentrated on the fixture top, causing marginal bone loss20. therefore, in the external type of implant, bone loss of 1.5 mm or less, with exposure of one or two threads, one year after loading and bone loss of 0.2 mm of less per year after that have generally been accepted as normal21. lazzara and porter22 introduced a platform switching concept and demonstrated the maintenance of healthy soft tissue around an implant, with little marginal bone loss. an internal type of implant was used in this study, where the abutment and the fixture were connected onto a slip joint connection system, similar to the platform switching concept, and where, it is believed, the fixture and the abutment were so closely connected that no microleakage occurred and healthy biologic width was maintained, maintaining the marginal bone. except for the one fractured implant, all five failed implants occurred in patients suffering from serious systemic diseases. there were no abnormalities found in the panoramic view examinations of these patients, and clinical examinations were performed right before the failures. unless demineralization of bone exceeds 30% or more, it is difficult to evaluate the bony lesion; therefore, we used panorama radiographs, whose resolution is slightly lower than that of periapical radiographs. for this reason, we think radiographs, which are more accurate, are necessary in implant follow - up. there were no failed implants in the group m. the survival rate of sla - treated implants was 98.1% in the maxilla and 94.3% in the mandible, with an overall survival rate of 96.6%. within the limitations of this study, implants with the sla surface have a very superior survival rate. in particular, implants with the sla surface seem to be superior in areas of relatively poor bone such as the maxilla. it is necessary to evaluate marginal bone loss using more accurate radiographs, such as periapical radiographs or cone - beam computed tomography, throughout the treatment periods.

objectivesthe purpose of this study is to evaluate five - year radiographic follow - up of the korean sandblasting with large grit, and acid etching (sla)-treated implant system.materials and methodsthe subjects of the study are 54 patients who have been followed - up to date, of the patients who underwent implant surgery from may 1, 2009 to april 30, 2011. in all, 176 implant placements were performed. radiographs were taken before the first surgery, immediately after the first and second surgeries, immediately and six months after the final prosthesis installation, and every year after that. bone loss was evaluated by the method suggested by romanos and nentwig.a total of 176 implant placements were performed-122 in men and 54 in women. these patients have been followed - up for an average of 4.9 years. in terms of prosthetic appliances, there were 156 bridges and 20 single prostheses. nine implants installed in the maxillary molar area, three in the mandibular molar area and two in the maxillary premolar area were included in group m, with bone loss less than 2 mm at the crestal aspect of the implant. of these, eight implants were single prostheses. in all, six implants failed - four in the mandible and two in the maxilla. all of these failures occurred in single - implant cases. the implant survival rate was 98.1% on the maxilla and 94.3% on the mandible, with an overall survival of 96.6%.within the limitations of this study, implants with the sla surface have a very superior survival rate in relatively poor bone environments such as the maxilla.

the advent of next - generation sequencing (ngs) technologies has unveiled the entire picture of the cellular transcriptome, wherein various non - coding rna (ncrna) are widely expressed from a large part of the genome.1,2 the functional significance of ncrnas is particularly evident for the short 20- to 31-nucleotide (nt) ncrnas bound to the family of argonaute proteins.35 micrornas (mirnas), the best - studied class of such rnas, are approximately 22 nt in length and posttranscriptionally regulate the expression of complementary target mrnas. while nearly 2,800 species of human mirnas are currently annotated in the mirbase database,6 computational analyses of massive rna - sequencing (rna - seq) data has kept increasing the number of mirna species.7,8 mirnas are estimated to regulate the expression of most protein - coding genes,9 and a wide variety of other short and long functional ncrna species coexist in the cellular transcriptome,10,11 exhibiting a tremendous impact of ncrna - orchestrated regulatory mechanisms on normal developmental and physiological processes and diseases.12,13 ngs studies have accelerated findings on not only novel ncrna repertoires but also well - characterized fundamental rna species with canonical functions, such as transfer rnas (trnas), which stimulated us to revisit their significance and versatility. trnas are 70- to 90-nt ncrnas that fold into a cloverleaf secondary structure and l - shaped tertiary structure. since their discovery in the 1950s, trnas have been best known as fundamental adapter components of translational machinery, where they translate codons in mrna into amino acids in protein.14 the human genome encodes > 500 trna genes,15 along with numerous trna - lookalikes resembling nuclear and mitochondrial trnas.16,17 the multitude of trna genes in the genome, along with high stability,18 places trnas among the most abundant rna molecules in the cellular transcriptome. because of their abundance and well - defined role in translation, rna fragments derived from trnas, found in early ngs studies, were often disregarded as nonfunctional degradation products. however, recent biochemical and bioinformatic evidence has generated a previously unsuspected concept that trnas are not always end products but are further able to serve as a source for short ncrnas. in many organisms, specific trna - derived ncrnas are produced from mature trnas or their precursor transcripts, not as random degradation products, but as functional molecules involved in many biological processes beyond translation.1923 trna - derived ncrnas identified to date can be classified into two groups: trna halves and trna - derived fragments (trfs).1923 trna halves are composed of 3035 nt fragments derived from either the 5- or the 3-part of mature trnas. trfs are shorter than trna halves, ranging from 13 to 32 nt in length and are currently subclassified into four subgroups: 5-trfs, 3-cca trfs, 3-u trfs, and 5-leader - exon trfs. the 5-trfs and 3-cca trfs correspond to the 5- and 3-parts of mature trnas containing processed mature 5- and 3-cca termini, respectively (fig . the 3-u trfs are derived from the 3-leader sequence of precursor trnas ( pre - trnas) that harbor 3-terminal uridine stretches, which from transcriptional termination by rna polyme rase iii at thymidine tracts (fig . the 5-leader - exon trfs include the 5-leader sequence of pre - trnas and the 5-part of mature trnas . although several excellent reviews have already described the repertoire, biogenesis, and functional significance of each class of the trna - derived ncrnas,1923 in this review, we particularly focus on the trfs bound to argonaute proteins and summarize their conceivable biogenesis factors and emerging roles in gene expression regulation as mirnas . in humans, the argonaute protein family has eight members : four of the ago clade ( ago14), which are ubiquitously expressed in numerous tissues, and four of the piwi clade, whose expressions are restricted to the germline.35 all four ago members associate with mirnas to form effector complexes for their posttranscriptional regulatory roles, while only ago2 shows endonucleolytic cleavage activity of target mrnas. biochemical purification and identification of mirnas and their target mrnas have been accomplished by purifying ago proteins using immunoprecipitation (ago - ip) followed by investigation of the ago - bound rnas in the ip fraction.24,25 similar sizes of trfs with mirnas allowed researchers to reason that trfs may be ago - bound rna species. although early studies have already observed trna - derived sequences from ago - ip fraction, as described below, more recent reports have demonstrated an evident association of the trna - derived ncrnas with ago proteins. hereafter, we will use trf to denote a trf derived from the trna corresponding to the amino acid and anticodon sequences. the first focused detection of ago - bound trna - derived ncrnas in humans was reported in 2009.26 cole et al. identified numerous trfs in rna - seq data from hela cells.26 majority of the identified trfs were 5-trfs, and 5-trf, selected for the analyses, this suggests the loading of the trf in both of the ago proteins, although 5-trf was also found in the fraction where ago proteins were absent and therefore it seems that not all trfs are loaded onto the ago proteins. detected 3-cca trfs and 3-u trfs in human embryonic kidney 293 (hek293) cells by northern blots.27 interestingly, the identified trf was selectively loaded onto different ago proteins; 3-u trf was found to be loaded onto ago3 and ago4 but not to ago1 and ago2.27 burroughs and ando et al. performed ago1 - 3 ips using thp-1 cells followed by ngs of the rna contents of the ip fractions, and identified several 5-trfs that were specifically loaded onto ago1, but slightly or not at all onto ago2 and ago3.28,29 they also observed the presence of 3-cca trfs in their ago - bound rna libraries. li et al. identified numerous 5-trfs and 3-cca trfs in rna - seq libraries from bcp1 and hek293 cells and demonstrated that both of the selected trfs, 3-cca trf and 3-cca trf, were loaded onto ago2 but not onto ago1.30 maute et al. focused on 3-cca trf whose expression levels are regulated by differentiation stages in mature b cells.31 the trf was enriched in the ago4-ip fraction, followed by fractions from ago1- and ago3-ips, but less enriched in the ago2-ip fraction. analyzed many available short rna libraries and observed numerous 5-trfs, 3-cca trfs, and 3-u trfs, whose expression profiles varied in different cell lines and tissues.32 their analyses of hek293 ago - ip fractions obtained from photoactivatable - ribonucleoside - enhanced crosslinking and immunoprecipitation (par - clip)33 suggested that 5-trfs and 3-cca trfs associate with ago1, ago3, and ago4, but not with ago2,32 whereas 3-u trfs were absent in all four ago - ip fractions. positional analyses of crosslink - induced mutations in par - clip data indicated that trfs were loaded onto ago proteins in a manner similar to mirnas.32 taken together, it could be concluded that the attempts to connect trfs, at least partially, with the mirna pathway have been successful. as mirnas, some trf species of the three classes (5-trfs, 3-cca trfs, and 3-u trfs) are the interacting partners of ago proteins. the presence of trfs in the ago - ip fraction has also been reported for many other organisms such as mouse,34 drosophila,35 and bombyx,36 as well as in plant.37 along the same lines, piwi proteins, the other argonaute family subclade that associates with piwi - interacting rnas (pir - nas) for germline development,35 have also been reported to interact with trna - derived ncrnas in various organisms such as human,38 marmoset,39 drosophila,40 trypanosoma,41 and tetrahymena.42,43 functional significance of the piwi - bound trfs in rna metabolism has been shown in tetrahymena.43 these findings suggest that trna - derived ncrnas are widely - conserved functional factors across phyla that interact with argonaute family proteins. asymmetric loading of trfs onto the different ago proteins could be due to coupling of trf biogenesis with ago loading mechanism and/or preferences of the ago proteins and other biogenesis factors for the particular structures of trfs or their precursors, namely mature trnas and pre - trnas. mature trnas are produced from pre - trnas, which undergo several maturation steps44 (fig . pre - trnas are first transcribed by rna polymerase iii, and their 5-leader and 3-trailer sequences are removed by endonucleolytic cleavage of rnase p and rnase z, respectively . trinucleotides are then attached to the 3-termini by a cca - adding enzyme, and various different non - canonical base modifications are attached by modification enzymes . judging from the regions and sequences that the trfs originate from, both pre - trnas and mature trnas must serve as substrates for the production of trfs . in vertebrates, canonical mirna biogenesis ( fig . 1) starts from transcription of the mirna gene by rna polymerase ii to generate a primary transcript (pri - mirna), which is subsequently cleaved by drosha, an endonuclease belonging to the rnase iii superfamily, forming a complex with dgcr8.35 the ant 70-nt precursor mirna (pre - mirna) with a hairpin - shaped structure is further processed by dicer, another rnase iii endonuclease, leading to the production of rna duplex, one strand of which is then loaded onto ago proteins.35 considering the expression of trfs complexed with ago proteins, one may infer drosha and dicer endonucleases are involved in the production of such ago - loaded trfs. indeed, the biogenesis of some trfs belonging to 5-trfs, 3-cca trfs, or 3-u trfs have been reported to be dependent on dicer in humans,26,27,31 mice,45 and drosophila35 (fig . , dicer is also reported to cleave trna to generate 5-trf in vitro.26 although the cloverleaf secondary structure of trnas does not meet the criteria for canonical dicer substrates, some precursor and mature trna molecules may alternatively form a long hairpin structure, particularly before modifications are attached, which may make trna structures suitable for dicer processing as predicted in the case of mice trna.45 some trfs seem to be generated via dicer - independent biogenesis mechanism.27,32 because several non - canonical mirna biogenesis pathways are independent of drosha or dicer and utilize precursor rnas forming various structures other than canonical hairpin - loop structures, such as single - stranded rnas, ago - bound trfs may be produced through such non - canonical pathways.46,47 alternatively, the other enzymes, which are not involved in mirna biogenesis would be involved in the production of ago - bound trfs . rnase z cleavage has been shown to produce 3-u trfs from pre - trnas in a dicer - independent manner.27,48 moreover, angiogenin ( ang), a member of the rnase a superfamily, is the candidate enzyme responsible for ago - bound trfs. in mammalian cells , ang was first discovered to be the enzyme that cleaves the anticodon loops of mature trnas to produce trna halves upon stress stimuli.49,50 however, it has also been shown that ang is able to cleave the t - loop of trnas to produce 3-cca trf in vitro (fig . it remains to be elucidated how the biogenesis factors are able to access and process the rigid structure of mature trnas, because, in the cytoplasm, mature aminoacylated trnas form an l - shaped tertiary structure and are tightly bound by an elongation factor to be transported to the ribosome . because trfs and trna halves coexist ( e.g., 5-trfs were purified together with 5-trna halves in dicer - immunoprecipitates),36,5154 the trna halves may be direct precursors of trfs in some instances. because the anticodon loop is exposed and accessible in animoacylated trna elongation factor complexes, mature trnas may first be subjected to anticodon cleavage by ang or other nucleases and then the generated trna halves dissociated from an elongation factor may be further processed for the production of trfs. further analyses are required to fully unveil the biogenesis factors and mechanisms for ago - bound trfs. mirnas deposit ago proteins onto target rnas by recognizing complementary sequences generally located in their 3-utr.35 imperfect mirna base - pairing with target mrnas appears to induce translational silencing, whereas extensive base - pairing triggers exonucleolytic decay of the target mrnas. considering their association with ago proteins and their similar size to mirnas, it is not surprising that ago - bound trfs act as mirnas and silence the expression of their complementary target mrnas. research on viruses has shown the first demonstration of ago - bound trfs functioning as mirnas. it has long been known that retroviruses use host trna as a primer for reverse transcription during the first step of retroviral replication. the 3-part of such trnas is complementary and binds to the primer - binding site (pbs) of the virus sequence. in human mt4 t - cells infected with human immunodeficiency virus type 1 (hiv-1), yeung et al. observed the abundant expression of an 18-nt 3-cca trf which is complementary to the pbs of hiv-1.55 moreover, the 3-cca trf was loaded onto ago2 and induced ago2-mediated cleavage of the pbs, thereby silencing pbs - containing reporter gene and a hiv-1 gene. these suggest important roles of ago - loaded trfs on repression of virus replication. because the human genome contains many endogenous retroviral sequences and 3-trfs are highly complementary to these,30 trf - directed pathways may also play ubiquitous roles in silencing endogenous viral elements. as another example of the involvement of trfs in viral expression, wang et al. reported that infection with human respiratory syncytial virus (rsv) led to accumulation of 5-trfs derived from various cytoplasmic trnas.56 one chosen 5-trf, 5-trf, was shown to be generated by ang - mediated cleavage at sites adjacent to the 5-end of the anticodon loop. because the 5-trf showed activity in repressing complementary reporter gene expression, the trf was further shown to have functional significance in regulating rsv replication.56 in addition to the viral research, maute et al. reported a trf functioning as mirna in mature human b cells.31 a 22-nt ago - bound 3-cca trf showed activity in repressing the expression of target mrnas in a sequence - specific manner. rpa1, an essential gene for dna dynamics and repair, was identified as one of the endogenous targets containing complementary sequences of the trf in its 3-utr. stable expression of the trf suppressed cell proliferation and modulated the response to dna damage in an rpa1-dependent manner, indicating the clear biological importance of 3-cca trf.31 haussecker et al. used a reporter assay to confirm that ago - bound 3-cca trfs and 3-u trfs repressed complementary target rnas.27 these suggested that the trfs were able to function as mirnas, although further identification of endogenous trf targets using clip25,33 and degradome rna analysis57 will be required to fully understand their biological significance. one of the trfs used in their analysis, a 3-u trf, was reported to promote cell proliferation in the other study by lee et al.48, validating that at least this trf has an important cellular role. in addition to their roles in translational repression in the cytoplasm, ago proteins have been reported to play distinct roles in the nucleus, such as modulation of histone methylation, mrna splicing, and dna damage repair, which is guided by mir - nas.58,59 ago - bound trfs may be involved in such nuclear functions of ago proteins. accumulation of further evidence of the functionality and endogenous biological roles of ago - bound trfs would lead to clearcut designation of these trfs as regulatory rna molecules. the number of reports characterizing expression and function of trna - derived ncrnas has been growing, which enforces the conceptual consensus that cells utilize mature and precursor trnas as a source for short, functional ncrnas. although this review detailed argonaute - bound trfs, there are various other classes of trna - derived ncrnas that are functional through mechanisms without involvement of argonaute proteins. a representative example is the trna halves, termed trna - derived stress - induced rnas (tirnas),49 whose production through ang cleavage of the trna anticodon are triggered by various stress stimuli.1923 tirnas promote the stress response by stress granule formation60 and global inhibition of translation by yb-1-involved pathway,49,61,62 thereby associating with normal biological processes and diseases.1923,52 as another example of functional trna halves, sex hormone - dependent trna - derived rnas (shot - rnas) have been recently identified in breast and prostate cancers.63 shot - rnas are also produced by ang cleavage of the trna anticodon, which is promoted by sex hormones and their receptors, and these enhance cell proliferation.63 despite the findings described in this review, information regarding expression profiles, biogenesis pathways, and biological meanings of ago - bound trfs is still fragmented. one of the obstacles for comprehensive identification of the trfs would be the extensive presence of posttranscriptional modifications in trnas. over 100 posttranscriptional modifications are present in trnas, many of which play crucial roles in trna folding and function such as codon recognition.6467 because it is highly plausible that many trna - derived ncrnas are produced from modified trnas and because many modifications inhibit watson crick base paring and thus arrest reverse transcription,68 heavily modified trna - derived ncrnas would not be accurately captured by rt - pcr based sequencing methods. for example, the presence of base - pairing - inhibitory modification inside the ago - bound trf would affect the efficiency of targeting and regulation of mrnas. these points based on trna biological properties should be borne in mind for analyses of trna - derived ncrnas. as another considerable point genomes encode many identical trna genes and their slightly different variants, and identical trna fragment sequences can be present in non - trna regions in the genome.69 very recently, during revising this manuscript, exhaustive analyses of massive rna - seq data have newly identified the fifth class of trf, i - trfs, which are wholly derived from internal regions of mature trnas and are loaded onto ago proteins.70 trfs including i - trfs were reported to be differentially expressed in different cell types, tissues, disease states and human race.70 as in the effort to generate a trna fragment database by kumar et al.71, cataloging the expression of trna fragments in various cell types and tissues, which should be based on appropriate bioinformatics approach and uniform nomenclatures will accelerate future investigations of trna - derived ncrnas. trna abundance varies greatly among different human cells and tissues, and the translational regulation by trna heterogeneity has been suggested.7274 the expression of trna halves is also variable in different cells and tissues and further regulated by trna modification states, various stressors, and hormones.1923,63 the uniquely regulated mechanism in controlling the quality and quantity of trnas and trna halves may consequently in the opportunity for cells to gene rate, from these precursor rnas, specific class of ncrnas whose species and abundance would then also be uniquely controlled. in that regards, as shown in the case of various types of ncrnas,11 trna - derived ncrnas may emerge as a source of biomarkers and targets for therapeutics. indeed, use of trna - derived ncrnas as indicators for biological states has been suggested.63,70,75,76 research on trna - derived ncrnas is still in the initial stage and their biological functions may extend beyond our expectations. further efforts to understand the expression profiles, biogenesis, and function of trna - derived ncrnas will advance our knowledge regarding the expanding trna world and may provide significant insights into the pathophysiology of diseases.

the advent of next - generation sequencing technologies has not only accelerated findings on various novel non - coding rna (ncrna) species but also led to the revision of the biological significance and versatility of fundamental rna species with canonical function, such as transfer rnas (trnas). although trnas are best known as adapter components of translational machinery, recent studies suggest that trnas are not always end products but can further serve as a source for short ncrnas. in many organisms, various trna - derived ncrna species are produced from mature trnas or their precursor transcripts as functional molecules involved in various biological processes beyond translation. in this review, we focus on the trna - derived ncrnas associated with argonaute proteins and summarize recent studies on their conceivable biogenesis factors and on their emerging roles in gene expression regulation as regulatory rnas.

embolism of the pulmonary artery or one of its branches is the most striking and characteristic appearance of thromboembolic disease. according to textbooks and most of literature in most cases the starting point of the embolus is phlebothrombosis or thrombophlebitis of veins of lower extremities. pulmonary embolism is very common nowadays, and favoring factors for its occurrence are the chronic diseases and therefore a long lying (bed rest), which leads to the development of phlebothrombosis and a large number of different surgical operations phlebothrombosis and thrombophlebitis of lower extremities veins phlebothrombosis of pelvic and abdominal veins usually after surgery thrombophlebitis and phlebothrombosis of veins of lower extremities after trauma and various surgical operations intracardiac thrombosis of the right cardiac ventricle thrombosis of the pulmonary artery or some of its branches in situ there are other, less common types of emboli: e.g. tissue cells, fat, oil, gas embolism and the like phlebothrombosis and thrombophlebitis of lower extremities veins phlebothrombosis of pelvic and abdominal veins usually after surgery thrombophlebitis and phlebothrombosis of veins of lower extremities after trauma and various surgical operations intracardiac thrombosis of the right cardiac ventricle thrombosis of the pulmonary artery or some of its branches in situ there are other, less common types of emboli: e.g. tissue cells, fat, oil, gas embolism and the like factors dependent on the patient: age over 40 years obesity immobilization / bed rest/- lying in bed for longer than 4 days deep vein thrombosis or pulmonary embolism in history thrombophilia, deficiency of antithrombin(at) iii factor, protein c, protein s; lupus anticoagulant, resistance to activated protein c, hemocistinemia immobilization / bed rest/- lying in bed for longer than 4 days deep vein thrombosis or pulmonary embolism in history thrombophilia, deficiency of antithrombin(at) iii factor, protein c, protein s; lupus anticoagulant, resistance to activated protein c, hemocistinemia factors dependent on the type of illness or surgery trauma or surgery: especially the pelvis, hip, leg malignant processes - particularly in the pelvis, abdomen, primary or metastatic heart failure recent myocardial infarction hormonal therapy (estrogen, progesterone) using the contraceptive pills paraplegia severe infection intestinal inflammations polycythemia paraproteinemia behcet's disease paroxysmal nocturnal hemoglobinuria trauma or surgery: especially the pelvis, hip, leg malignant processes - particularly in the pelvis, abdomen, primary or metastatic recent myocardial infarction hormonal therapy (estrogen, progesterone) using the contraceptive pills intestinal inflammations paroxysmal nocturnal hemoglobinuria although etiology and common risk factors for pulmonary thromboembolism are well known there is little data about frequency of postoperative pulmonary thromboembolism after different surgical operations. phlebothrombosis and thrombophlebitis of lower extremities veins phlebothrombosis of pelvic and abdominal veins usually after surgery thrombophlebitis and phlebothrombosis of veins of lower extremities after trauma and various surgical operations intracardiac thrombosis of the right cardiac ventricle thrombosis of the pulmonary artery or some of its branches in situ there are other, less common types of emboli: e.g. tissue cells, fat, oil, gas embolism and the like phlebothrombosis and thrombophlebitis of lower extremities veins phlebothrombosis of pelvic and abdominal veins usually after surgery thrombophlebitis and phlebothrombosis of veins of lower extremities after trauma and various surgical operations intracardiac thrombosis of the right cardiac ventricle thrombosis of the pulmonary artery or some of its branches in situ there are other, less common types of emboli: e.g. tissue cells, fat, oil, gas embolism and the like factors dependent on the patient: age over 40 years obesity immobilization / bed rest/- lying in bed for longer than 4 days deep vein thrombosis or pulmonary embolism in history thrombophilia, deficiency of antithrombin(at) iii factor, protein c, protein s; lupus anticoagulant, resistance to activated protein c, hemocistinemia immobilization / bed rest/- lying in bed for longer than 4 days deep vein thrombosis or pulmonary embolism in history thrombophilia, deficiency of antithrombin(at) iii factor, protein c, protein s; lupus anticoagulant, resistance to activated protein c, hemocistinemia factors dependent on the type of illness or surgery trauma or surgery: especially the pelvis, hip, leg malignant processes - particularly in the pelvis, abdomen, primary or metastatic heart failure recent myocardial infarction hormonal therapy (estrogen, progesterone) using the contraceptive pills paraplegia severe infection intestinal inflammations polycythemia paraproteinemia behcet's disease paroxysmal nocturnal hemoglobinuria trauma or surgery: especially the pelvis, hip, leg malignant processes - particularly in the pelvis, abdomen, primary or metastatic recent myocardial infarction hormonal therapy (estrogen, progesterone) using the contraceptive pills intestinal inflammations paroxysmal nocturnal hemoglobinuria although etiology and common risk factors for pulmonary thromboembolism are well known there is little data about frequency of postoperative pulmonary thromboembolism after different surgical operations. our objective is to show the number of patients with postoperative pulmonary thromboembolism (ppte) treated in intensive care unit of clinic for pulmonary diseases an tb podhrastovi in three - year period: from june 1, 2011 - june 1, 2014 and to indicate the importance of various surgical operations in the development of pulmonary thromboembolism (pte). this is the retrospective study which shows the number of patients with postoperative pulmonary thromboembolism treated in intensive care unit of clinic for pulmonary diseases an tb it represents the number of these patients, per cent of patients with postoperative pulmonary thromboembolism of total patients with pulmonary thromboembolism, age and sex of patients, type of surgery, period expressed in days from surgery to clinical presence of pulmonary thromboembolism, presence of deep venous thrombosis (dvt) of lower extremities, massiveness of ppte e.g. level of pulmonary artery with embolus: segmental, lobar, main branches of pulmonary artery; unilateral, bilateral. in three - year period 232 patients with pte were treated in intensive care unit of clinic podhrastovi. there were 24 males or 40% middle - aged 58.5 years, and 36 females or 60% middle - aged 56.3 years. only one man had dvt (deep venous phlebothrombosis) (after orthopedic surgery), and five of them had anamnesis about previous dvt (one with abdominal, one with vascular, one with orthopedic and two with cardiology surgery). only one woman had dvt (after orthopedic surgery), and no one had anamnesis about previous dvt or pte. patients were subjected to different types of surgical operations which are presented on figure 1. pte developed in 15 patients with abdominal, 11with urologic, 8 with gynecologic, 15 with orthopedic, 4 with cardiologic. 2 with vascular, 3 with neurosurgical, 1 with glandular (breast) and 1 with orl operations the average period in days from surgery to the development of pte. pte developed in one to thirty days after operation, on average for abdominal operations it was 19 days, for urologic 5, for gynecologic 10, for orthopedic 15.5, for cardiologic 13, for vascular 4.5, for neurosurgical 18.5, for glandular 10 and for orl operations 10 days after operation. ppte developed on average 10 days in women, and 14 days after operation in men. massiveness of ppte e.g., the site or level of embolus was different - from segmental to main branches of pulmonary artery which is shown on figure 3 and figure 4. the level or site of pulmonary embolus according to the type of surgery in women. the level of pulmonary embolism is different, from segmental to main branches of pulmonary artery with different number of patients according to operation. the level or site of pulmonary embolus according to the type of surgery in men. the level of pulmonary embolism is different, from segmental to main branches of pulmonary artery with different number of patients according to operation there is notably increasing number of patients with pulmonary thromboembolism in recent years treated in clinic for pulmonary diseases and tb podhrastovi we are not yet sure whether it is real increasing or it is matter of better diagnostics. in three - year period 60 of them or 25.86% were patients where pte developed after different types of surgical operations. we intended with this study to point out the significance and frequency of postoperative pulmonary thromboembolism, and to indicate that surgical operations of different type are significant etiologic and risk factor for the development of pte. first of all we did not have real data about regularity of preoperative and postoperative prophylaxis with anticoagulants, early rising from bed after surgery (mechanical prophylaxis), we did not have data about intraoperative or early postoperative deaths caused possibly by pte. furthermore, not small number of patients with postoperative pte was treated in corresponding surgical clinic under the control of pulmonologist from our clinic, and released home to continue anticoagulant therapy under the control of pulmonologist in outpatient department. although common risk factors for the development of pte are well known, studied and confirmed in clinical practice there are no enough data in literature about postoperative dvt or pte out of controlled studies. many authors indicate the significance of preoperative anticoagulant prophylaxis. one big prospective study was done by group of authors on 75 771 patients with vascular and orthopedic operations from 1996 to 2001 in veteran health administration hospital. major comorbidities included diabetes mellitus, chronic obstructive pulmonary disease, and congestive heart failure. symptomatic pte was diagnosed in 805 patients (0.68%) and varied significantly with procedures: 0.14% for carotid endarterectomy to 1.34% for total hip arthroplasty. in our study we dealt with patients of both sex (60% of them women), average age was 58.5 years for men, and aged 56.3 years for women, and patients were subjected to different types of surgery. we dealt with patients not prospectively, but only with surgical treated patients with different operations with diagnosed ppte to indicate the importance of surgery to the development of pte. these authors indicate the postoperative importance of pneumonia and other infections, including urinary tract infection, myocardial infarction. only two patients in our study had dvt after operation (one man and one woman), but in all others were not be able to find the starting point of embolus. so we think that the preoperative and postoperative prophylaxis (either anticoagulant, or mechanical - early rise from bed) is of the most significance, especially in patients with risk factors for dvt or pte, even it is only the expected lying in bed (bed - rest) longer than 4 days. there is the need for one long prospective study in different surgical clinic for the assessment of more factors (age, sex, preoperative diseases, type and site of operation, postoperative complications, duration of bed - rest, anticoagulant and mechanical prophylaxis etc .) to give the more realistic picture of postoperative pulmonary thromboembolism. there is great significance of anticoagulant prophylaxis before surgery even in patients with no anamnesis of previous dvt or pte.

objective: our objective is to show the number of patients with postoperative pulmonary thromboembolism (ppte) treated in intensive care unit of clinic for pulmonary diseases an tb podhrastovi in three - year period: from june 1, 2011 - june 1, 2014 and to indicate the importance of various surgical operations in the development of pulmonary thromboembolism (pte).material and methods: this is the retrospective study which shows the number of patients with ppte treated in intensive care unit of clinic for pulmonary diseases an tb podhrastovi in three - year period: from 01.06.2011.-01.06.2014. it represents the number of these patients, per cent of patients with ppte of total patients with pte, age and sex of patients, type of surgery, period expressed in days from surgery to diagnosis of pte, presence of deep venous thrombosis (dvt) of lower extremities, massiveness of ppte e.g. level of pulmonary artery with embolus.:in three - year period 232 patients with pte were treated in intensive care unit of clinic podhrastovi. 60 of them or 25.86% were patients with 24 males or 40% middle - aged 58.5 years, and 36 females or 60% middle - aged 56.3 years. ppte developed in 15 patients with abdominal, 11 with urologic, 8 with gynecologic, 15 with orthopedic, 4 with cardiologic, 2 with vascular, 3 with neurosurgical, 1 with glandular and 1 with orl operations. the average period from operation to diagnosis of ppte was 10.5 days for women, and 13.8 days for men. only two patients had acute dvt after operation (one man and one woman), and five had amnesias of previous dvt or pte. the level or the site of pulmonary embolus was different from segmental to main branches of pulmonary artery.:different surgical operations are the big risk factor for the development of pte. there is great significance of anticoagulant prophylaxis before surgery even in patients with no anamnesis of previous dvt or pte.

systems biology is an emerging academic field aiming at system - level understanding of biological systems. recent progress in molecular biology has enabled us to gain information on the interactions among the underlying molecules from comprehensive experimental data sets. in general, a system - level understanding of a biological system can be derived from insight into four key properties: the system's structure, the system dynamics, the control method, and the design method. equivalently, identifying related components and their interactions, gathering qualitative and quantitative information about the system's evolution under different circumstances, achieving the desired outputs by controlling the input with appropriate definitions of inputs and outputs of the system, and reconstructing analogous systems by eliminating the undesired properties are four essential steps in systems biology done by collaboration among engineers, biologists, and doctors. systems biology is a cross - cutting research area connecting control engineering, biology, and medical science, as shown in figure 2. it aims at understanding the bare function and integration function of the cell to reconstruct the biological systems with desired features. control and automation play critical roles in this novel field not only by providing new technology and equipment for biologists to design and perform meticulous experiments, to take high - throughput measurements, and to analyze experimental data efficiently, but also by offering doctors new medical applications and improving the precision of medical manipulations. the wide range of aspects which control and automation have been applied to include, but are not limited to, gene regulation , drug delivery , and neuron networks. the equipment provided by control engineers includes, but is not limited to, nanodevices, biochips, cuvettes for electroporation, and gene guns. biologists perform various biological experiments, such as protein synthesis and virus dna modifications, to gather measurements for model revisions and verifications, to conclude theoretical and practical from evidence, and to help medical practice. doctors use both theoretical and practical from biologists to perform tissue engineering, such as organ transplants and artificial tissue construction. according to their scales , biological systems can be divided into three levels: the molecular level (nm), cellular level (m), and tissue level (cm), analogous to the part, individual, and group, respectively. molecular - level research focuses on how, when, where, and to what extent a gene is expressed. the essential goal is to sketch a complete blueprint of genes by identifying the control sequences of coding dna segments and their interactions. cellular level research, in general, treats one cell as a plant in classical control theory and investigates the reactions of the cell to the changing environment, for instance, concentration changes of related chemicals. state - of - the - art medical therapies are primarily based on experimental at the cellular level. tissue - level research mainly concerns tissue reconstruction, artificial tissue substitutes, or tissue function recovery. in contrast, understanding biological systems at the molecular level is crucial, since species have the same basic inheritance, dna macromolecules, and follow a common rule in gene expression, the central dogma in molecular biology. molecular level understanding of biological systems provides instrumental information about radical causes of many diseases and the genetic evidence of evolution. it also helps biologists to gain a better understanding of molecular level interactions, draw a complete blueprint of gene networks, improve existing means, create novel means to cure genetic diseases, and to elaborate on the theory of evolution. recent technology in gene sequencing makes it possible to conquer the difficulty in measurement at the molecular level and to identify the nucleotide sequences of a particular dna segment. targeted sequencing is the most promising step toward maximizing the efficiency of the next - generation sequencing technology using polymerase chain reaction. the availability of dna microarray makes it possible to accomplish tens of thousands of genetic tests for picomoles of a specific dna sequence. researchers have applied various methods to model, simulate, and control the gene regulation processes. early attempts to model and simulate gene regulatory systems are summarized in, including direct graphs, bayesian networks, boolean networks, ordinary and partial different equations, qualitative differential equations, quantative differential equations, stochastic equations, and role - base formalisms. other approaches include petri nets, transformational grammars , and process algebra. three important modeling methods in recent work are gene regulatory units viewed under compound control , logic network models , and base - to - base molecular - level formulation. the first modeling method quantitatively describes chemical concentration variations corresponding to external environmental changes at the cellular level. the last modeling converts dna segments to discrete vectors. in our paper, we adapt the base - to - base molecular level formulation to express state variables. most existing models are constructed by data - driven or hypothesis - driven methods, with only partial information available. due to the complexity of the systems and incomplete information, the mathematical models are usually formulated by modifying empirical equations or proposing heuristic equations. although those models can disclose significant details of the system's structure and dynamics, the inconsistency between theoretical and experimental creates difficulties for control engineers to verify the models, develop optimal controls, and reconstruct systems with desired properties. in this paper , we use a novel approach to build up abstract mathematical models at the molecular level in section 2.2, based directly on biological theory. with reasonable assumptions different from existing methods, focusing on a gene or changes in chemical concentration, we emphasize the base change in the nucleotide bases. the cost function, a summation of costs for applying mutagens and the off - trajectory penalty, together with the system equations, formulates the optimal control problem. section 3 shows simulation of the optimal control problem at different scales and is followed by several important propositions. the central dogma of molecular biology, first elaborated in and restated in, illustrates the detailed residue - by - residue transfer of genetic sequential information. nowadays, it describes the genetic information flow among three kinds of biopolymers: dna, rna, and protein. in most living organisms, this process is usually irreversible, thus protein always acts as the sink of information flow. a codon consists of three consecutive nucleotide bases, corresponding to one amino acid according to the genetic codes. since there are only 20 kinds of amino acids and 64 combinations of codons, there exists redundancy in genetic codes. we are particularly interested in mutations that happen during the process of dna replication, as dna serves as long - term genetic information storage and is the basis of genetic inheritance, the accuracy of which is particularly important to ensure the correct expression of genes. dna molecules consist of four kinds of nucleotide acids, adenine (a), thymine (t), guanine (g), and cytosine (c), and a backbone made of sugars and phosphate. in 1953, james d. watson and francis crick found the double helix structure of dna and the rule of basepairing, known as watson - crick basepairing. a always pairs with t, g always pairs with c, and vice versa. in nature, replication errors occur at a very low rate, one error for every 10 nucleotides added. the redundancy of information caused by the double - helix structure ensures the accuracy of dna replication. some dna self - repair mechanisms, listed in, such as proofreading, also help to eliminate errors during the replication process. point mutations can be further divided into transitions (ag or ct) and transversions (a / gc / t). transversions are theoretically expected to be twice as frequent as transitions, but transitions may be favored over transversions in coding dna because they usually in a more conserved polypeptide sequence. in this section, we first give the problem statement in section 2.1, and then we construct system equations for both deterministic and stochastic mutations in section 2.2. at last, in section 2.3, we formulate the optimal control problem and apply dynamic programming algorithm to solve it. figure 3 shows the system diagram of restoring an abnormal dna segment back to a normal sequence by applying mutagens during the process of dna replication. after we obtain a patient's genome, we compare the coding dna segments with normal dna segments in our database to figure out the possible range of mutated segments. due to the redundancy in genetic codes, as long as any two dna segments can be transcribed and then translated to the same amino acid sequence, the distance reference between them is considered to be zero. therefore, instead of having a predetermined final state or a neighborhood of a final state, our final state lies in a set where the distance reference between any sequence in this set and the desired sequence is zero. the prescription is then determined by comparing the current measurement and every sequence in the desired set. we treat dna sequences as state variables, the on / off controls of all available mutagens at every spot on the given dna segment as inputs, the measurements as the outputs, and one cell cycle as the step increment in our system equations. the objective function is defined as a summation of the costs (including risks) of applying mutagens and the off - trajectory penalty. the optimal control sequences are computed beforehand to let doctors make treatment plans according to the patient's condition. in general, the optimal control sequence and the corresponding optimal trajectory are not unique because the bases mutate independently in most cases and the order of mutating different bases does not matter if the number of medical treatment sessions is not under a tight restriction. additional measurements are taken before and after each treatment, if necessary. in deterministic cases, the purpose of taking additional measurements is to check the current sequence and to eliminate both internal and external disturbances. in stochastic settings, the measurements are taken to conquer the randomness caused by both mutagens and other noises. , our system is a discrete - time dynamic system with finite state space and output space, and a set of on / off switches as controls. our goal is to optimally drive this system from a given initial state to a desired final set at the lowest cost. we mainly focus on applying chemical mutagens and radiation to restore the original amino acid sequence during the process of dna replication. other factors that may affect the gene mutation, including temperature and electroporation, are not within our consideration. in addition, we assume that chemical mutagens or radiation target one and only one nucleotide base at any preset site, despite the technical limitation, and the of applying chemical mutagens and radiation are independent. for simplicity, we normalize the dose of mutagens to transfer one nucleotide base to another in one step to 1. in most cases, nucleotide bases mutate independently, therefore there is no chain effect caused by mutagens. to avoid reactions among different mutagens, we require that at most one chemical mutagen and one radiation be applied in each cell cycle. while constructing a generalized model, since the order of applying chemical mutagens and radiation does not affect the , without loss of generality, we require they be applied in the order shown in figure 4. that is, chemical mutagens are always applied before the duplication process starts, radiation is always applied in the middle of the cell cycle, and the measurements are taken before every replication starts. lastly, we assume that the measurements are always correct, and dna replication error, mutation rate, and other random noise can be eliminated from measurements by considering them as spontaneous mutation. denote the targeted dna segment with n nucleotide bases at kth step by a column vector x k, as shown in figure 5. x k is the ith element of x k. let p be the transfer matrix from x k to x k+1, for all k, k {0}, without mutation. then, the perfect dna replication process can be expressed as proposition 1 p = i. proofas no mutation occurs, x k+1 is completely complementary to x k by watson - crick base pairing rule, and x k+2 is completely complementary to x k+1. therefore, x k+2 is exactly the same as x k. thus, xk+2=pxk+1=p2xkp2=i. since every base mutates independently, every element of x k+1 only depends on the corresponding element of x k , thus p is diagonal. in addition, x k+1 x k, we conclude p = i. as no mutation occurs, x k+1 is completely complementary to x k by watson - crick base pairing rule, and x k+2 is completely complementary to x k+1. therefore, x k+2 is exactly the same as x k. thus, xk+2=pxk+1=p2xkp2=i. since every base mutates independently, every element of x k+1 only depends on the corresponding element of x k , thus p is diagonal. in addition, x k+1 x k, we conclude p = i. based on proposition 1, we assign values to nucleotide bases set {a, g, c, t, o}, where o is an artificial nonsense base. define an equivalence relationship between {a, g, c, t, o} and {1,2, 2, 1,0}, that is, {a, g, c, t, o}{1,2, 2, 1,0}, with xki={1,if a,2,if g,2,if c,1,if t,0,if o. proposition 2{1,2, 2, 1,0} is a field under proper definitions of addition and multiplication. {1,2, 2, 1,0} is a field under proper definitions of addition and multiplication. proofdefining the addition table and multiplication table as in tables 1 and 2, we check if the set {1,2, 2, 1,0} satisfies the definition of field. defining the addition table and multiplication table as in tables 1 and 2 , we check if the set {1,2, 2, 1,0} satisfies the definition of field. commutativity of addition and multiplicationsatisfied as tables 1 and 2 are symmetric according to the diagonal. additive and multiplicative inversesadditive inverses pair: 11, 22, 00.multiplicative inverses pair: 11, 22, 11. we conclude {0,1, 2, 2, 1} is a field under addition and multiplication defined by tables 1 and 2. from now on, we use to denote the field {0,1, 2, 2, 1}. and x k is the state vector representing a dna segment with n nucleotide bases, where is the set of -valued vectors of dimension n. we start with the simplest form of mutations, point mutation. suppose there is a point mutation, we write it mathematically as xk+1=(i+s)xk+w, where x k+1, x k , and i reduces to 1 as only one base is involved. the corresponding values of s and w, obtained by reverse engineering with all possible pairs of x k and x k+1, are listed in table 3. here , s represents the mutation from four normal nucleotide bases, and w corresponds to mutation from nonsense base, that is, w 0 only if x k = 0. rewriting by collecting all values of s and w in table 3, we get (5a)xk+1=(i+j=04ukjsj)xk+j=04ckjwj (5b)=(i+uks)xk+ckw, where {s 0, s 1, s 2, s 3, s 4} = {w 0, w 1, w 2, w 3, w 4} = {0,1, 2, 2, 1}, u k , c k {0,1}, representing the on / off controls, u k = , c k = , and s = w =. in (5a), s j and w j are constants for all k and j. u k and c k , the inputs of the system, are the on / off controls for chemical mutagens or radiation. clearly, j=0 c k = 1 only if x k = 0. equation (5b) is a simplified version of (5a) as we put u k , s j, c k , w j into vector form u k, s, c k, w. s and w serve as vector basis for base - to - base deterministic model. u k and c k are now multi - input controls; each of them contains 5 on / off controls, corresponding to all possible transfer patterns. for a particular k, at most one of u k s and c k s can be 1, as stated in proposition 3. this is consistent with the fact that every state can be transferred to only one of the five states in the state space with corresponding mutagens available. proposition 3it is always 1 1 transfer when mutation occurs, that is, one nucleotide base can only transfer to another one, thereforeif x k = 0 and c k = 0, or c k = , then x k+1 = 0,if x k 0, then c k = 0 and u k is either 0 or a unit row vector, if x k = 0, then u k = 0 and c k is either 0 or a unit row vector, u k + c k is either 0 or a unit row vector, for all k {0}. it is always 1 1 transfer when mutation occurs, that is, one nucleotide base can only transfer to another one, thereforeif x k = 0 and c k = 0, or c k = , then x k+1 = 0,if x k 0, then c k = 0 and u k is either 0 or a unit row vector, if x k = 0, then u k = 0 and c k is either 0 or a unit row vector, u k + c k is either 0 or a unit row vector, for all k {0}. if x k = 0 and c k = 0, or c k = , then x k+1 = 0, if x k 0, then c k = 0 and u k is either 0 or a unit row vector, if x k = 0, then u k = 0 and c k is either 0 or a unit row vector, u k + c k is either 0 or a unit row vector, for all k {0}. now, suppose for some reason we need to take an addition, measurement in the middle of the cell cycle, after the completion of the kth duplication and before the start of the (k + 1)th. we name this kind of measurement an intermediate state, and denote by it x k. then, we have xk+1=(i+s) xk+w, where the values of s and w, listed in table 4, are obtained in the same way as getting s and w in table 3. comparing tables 3 and 4 , we find the collection of s and s, w and w, form the same set, respectively. thus, we continue using s and w when rewriting in the form of (5a) and (5b), that is, xk+1=(i+vks)xk+ckw, where v k, c k are the counterparts of u k, c k, respectively, and s, w are the same as in (5b). similar to proposition 3, we get proposition 4. proposition 4 v k and c k in need to satisfy the following conditions.if x k = 0 and c k = 0, or c k = , then x k+1 = 0.if x k 0, then c k = 0 and v k is either 0 or a unit row vector.if x k = 0, then v k = 0 and c k is either 0 or a unit row vector. v k + c k is either 0 or a unit row vector, for all k {0}. v k and c k in need to satisfy the following conditions.if x k = 0 and c k = 0, or c k = , then x k+1 = 0.if x k 0, then c k = 0 and v k is either 0 or a unit row vector.if x k = 0, then v k = 0 and c k is either 0 or a unit row vector. v k + c k is either 0 or a unit row vector, for all k {0}. if x k = 0 and c k = 0, or c k = , then x k+1 = 0. if x k 0, then c k = 0 and v k is either 0 or a unit row vector. if x k = 0, then v k = 0 and c k is either 0 or a unit row vector. v k + c k is either 0 or a unit row vector, for all k {0}. now take, both chemical mutagens and radiative rays under our consideration and apply them in the order as shown in figure 4. then, we can express our system equation as (8a)xk=(i+uksmutations caused by chemicalmutagens from normal bases)xk + ckwmutations caused by chemicalmutagens from o, (8b)xk+1=(i+vksmutations caused by radiativerays from normal bases)xk + ckwmutations caused by radiativerays from o, (8c) yk = xk, where u k and v k are the inputs of the system and y k is the measurement. obviously, (8a) is modified from (5b) and (8b) from. the two - step mutation and the intermediate state x k avoid the case x k is changed to different bases by radiation and chemical mutagens simultaneously, which causes confusion. substituting (8a) and (8b), we get (9a)xk+1=(i+vks)(i+uks)xk+(i+vks)ckw+ckw, (9b)yk = xk. obviously, proposition 3 still holds for u k and c k, and proposition 4 holds for v k and c k for (9a). for point mutations , we have 20 on / off controls in total for every step k, 10 for chemical mutagens as described before, and the rest for radiation. in general, now, we show how to extend our model to large - scale systems. suppose we have a coding dna segment with length n, then x k. since a coding dna segment usually contains integer number of codons, which is made of three consecutive bases, n is a multiple of 3. let x k denote the ith component of x k. this notation is consistent with the one in section 2.2.1. initiated by the base - to - base deterministic model from section 2.2.1, we write our system equation for large - scale system as xk=(i+i=1nukiskimutations caused by chemicalmutagens from normal bases)xk + ikckiwkimutations caused by chemicalmutagens from o, xk+1=(i+i=1nvkiqkimutations caused by radiative raysfrom normal bases)xk + ikbkirkimutations caused by radiativerays from o, yk = xk, where u k , v k , c k , b k are on / off controls of the ith element, s k , q k are n n square matrices corresponding to the mutations between normal bases or from normal bases by chemicals and radiation, respectively, w k , r k are n - dimensional column vectors representing mutations from nonsense bases by chemicals and radiation, respectively, and k = {i : x k = 0,1 i n}, k = {i : x k = 0,1 i n}. s k and q k are diagonal matrices since each base mutates independently. the values in the first four rows of tables 3 and 4 correspond to the diagonal elements of s k and q k , respectively. the last rows of tables 3 and 4 are assigned to w k and r k , n - dimensional vectors, at nonsense base's spots for x k. define = {s j e i e i , i, j, 0 j 4,1 i n}, a collection of n n matrices, where s j is the same as in (5a) and (5b), e i is the unit column vector of length n with ith component equal to 1 and all other components equal to 0, and e i e i is the square matrix with only the ith element on the diagonal equals to 1, and 0 otherwise. then, s k , q k can be written as linear combinations of all elements from , with the coefficient of each element either 0 or 1 corresponding to the on / off control u k and v k , respectively. similarly, define = {w j e i, i, j, 0 j 4,1 i n}, where w j is the same as (5a) and (5b). w k , r k can be written as linear combinations of all components from , with coefficient of every component either 0 or 1 corresponding to the on / off control c k and c k , respectively. therefore, instead of using step - varying s k , s k , w k , r k , we find matrix basis for those four square matrices to make the controls to be the only variables depending on k, as we did for single - base cases. then, we can, write as (11a)xk=(i+i=1n j=04uk(i, j)sjeieit)xk+ik j=04ck(i, j)wjei, (11b)xk+1=(i+i=1n j=04vk(i, j)sjeieit)xk+ik j=04ck(i, j)wjei, (11c)yk = xk, where u k , v k , c k , c k {0,1}. as shown in (11a), (11b), and (11c), multisites mutations contain 20n controls in total for every step k, where n is the number of nucleotide bases on the targeted gene. similar to point mutations, every single site has 20 controls in each step, 10 for chemical mutagens and 10 for radiation. we can view u k, v k, c k, c k as binary matrices of dimension n 5, and u k , v k c k , c k are the corresponding element of ith row and jth column. use u k , v k , c k , b k , binary row vectors of dimension 5, to denote the ith row of u k, v k, c k, c k, respectively. again, s = w =. combining (11a) and (11b), and writing control variables in vector forms, we get xk+1=(i+i=1nvkiseieit)(i+i=1nukiseieit)xk + (i+i=1nvkiseieit)ikckiwei+ikbkiwei, yk = xk. proposition 5for large - scale deterministic system, u k, v k, c k, c k satisfy conditions below.if e i x k = 0, then i k.if e i x k = 0, c k = 0 or c k = , then i k.for all i k, u k is either 0 or a row unit vector and c k = 0.for all i k, c k is either 0 a row unit vector and u k = 0.for all i k, v k is either 0 or a row unit vector and b k = 0.for all i k, b k is either 0 or a row unit vector and v k = 0.for all i, k, 1 i n, k {0}, u k + c k is either 0 or a unit row vector and v k + b k is either 0 or a unit row vector. for large - scale deterministic system, u k, v k, c k, c k satisfy conditions below.if e i x k = 0, then i k.if e i x k = 0, c k = 0 or c k = , then i k.for all i k, u k is either 0 or a row unit vector and c k = 0.for all i k, c k is either 0 a row unit vector and u k = 0.for all i k, v k is either 0 or a row unit vector and b k = 0.for all i k, b k is either 0 or a row unit vector and v k = 0.for all i, k, 1 i n, k {0}, u k + c k is either 0 or a unit row vector and v k + b k is either 0 or a unit row vector. if e i x k = 0, then i k. if e i x k = 0, c k = 0 or c k = , then i k. for all i k, u k is either 0 or a row unit vector and c k = 0. for all i k, c k is either 0 a row unit vector and u k = 0. for all i k, v k is either 0 or a row unit vector and b k = 0. for all i k, b k is either 0 or a row unit vector and v k = 0. for all i, k, 1 i n, k {0}, u k + c k is either 0 or a unit row vector and v k + b k is either 0 or a unit row vector. the mathematical model is quite flexible and can be easily extended to many cases, such as transcription process, multiple spot mutations within one - step or broken dna strands. our system equation can represent this phenomenon by dividing the whole system into small subsystems. significant brokage of dna strands is simply eliminated by cell mechanism to ensure the accuracy to dna replication. equation shows the case of one single dna strand breaking into two segments by chemical mutagens (xkxk)=(im+i=1mukiseieit00inm+i = m+1nukiseieit) (xkxk)+ (ik,1imckiweiik, ( m+1)inckiwei),xk+1=(im+i=1mvkiseieit)xk+ik, 1imbkiwei, xk+1=(inm+i = m+1nvkiseieit)xk + ik, (m+1)inbkiwei. in reality, therefore, we need to derive the model for gene - to - gene stochastic mutations. introduce new random variables, h k, l1 , r k, l2 , h k, l3 , r k, l4 {0,1}, associated with probability p l1,j , p l2,j , p l3,j , p l4,j , for all i, k, 1 i n, k {0}, respectively, where k is the step index, l 1, l 2 are indices for chemical mutagens inducing mutation from normal bases and from o, respectively, l 3, l 4 are indices for radiation inducing mutation from normal bases and from o, respectively, i is the index of dna segment, and the value of j corresponds to the transfer pattern, which can be found in tables 3 and 4. note different mutagens have different probability assignments, the probability assignments are only related to the type of mutagens, and the probability associated with every kind of mutagens sums up to 1, that is, j=04pl1,j(h)=1, l1, 1l1l,j=04pl2,j(r)=1, l2, 1l2m,j=04pl3,j(h)=1, l3, 1l3l,j=04pl4,j(r)=1, l4, 1l4m. the controls are u k, l1 , c k, l2 , v k, l3 , b k, l4 {0,1}, with the fact that 1 representing mutagen with corresponding index is applied at ith spot of dna segment at kth generation, and 0 representing mutagen with corresponding index is not applied at spot i at kth step, similar to sections 2.2.1 and 2.2.2. the mutagen indices l 1, l 2, l 3, l 4 can be omitted in deterministic mutations since given the current state and control, the next state is unique. however, they are necessary for stochastic mutations, because there exist multiple possible states for the next stage given the control. in other words, the next state is determined by random variables h k, l1 , r k, l2 , h k, l3 , r k, l4 , given the values of u k, l1 , c k, l2 , v k, l3 , b k, l4 , and x k. suppose we have (l + m) kinds of chemical mutagens available, with l kinds to induce mutations from normal bases and m kinds to induce mutations from o. and we have (l + m) kinds of radiation available, with l kinds to induce mutations from normal bases and m kinds to induce mutations from o. therefore, we have total (l + m + l + m) controls for each spot i at each step k. we can write our system equation as (15a)xk=(i+l1=1l i=1nuk, l1ij=04hk, l1(i, j)sjeieitmutations caused by chemical mutagens from normal bases)xk + l2=1 m ikck, l2ij=04rk, l2(i, j)wjeimutations caused by chemicalmutagens from o, (15b)xk+1=(i+l3=1l i=1nvk, l3ij=04hk, l3(i, j)sjeieitmutations caused by radiative rays from normal bases)xk + l4=1m ikbk, l4ij=04rk, l4(i, j)wjeimutations caused by radiative rays from o, (15c) yk = xk. again, we define h k, l1 , r k, l2 , h k, l3 , r k, l4 the elements at ith row and jth column of n 5 binary matrices h k, l1, r k, l2, h k, l3, r k, l4, respectively. h k, l1 , r k, l2 , h k, l3 , r k, l4 , and binary row vectors of dimension 5 denote the ith row of h k, l1, r k, l2, h k, l3, r k, l4, respectively. then, we can simplify (15a), (15b), and (15c) and combine (15a) and (15b) as xk+1=(i+l3=1l i=1nvk, l3ihk, l3iseieit) (i+l1=1l i=1nuk, l1ihk, l1iseieit)xk + (i+l3=1l i=1nvk, l3ihk, l3iseieit)l2=1 m ikck, il2rk, l2iwei + l4=1m ikbk, l4irk, l4iwei, yk = xk. proposition 6for large - scale stochastic system, u k, l1 , h k, l1 , c k, l2 , r k, l2 , v k, l3 , h k, l3 , b k, l4 , r k, l4 follow the rules below.if e i x k = 0, then i k.if e i x k = 0 and l2=1 c k, l2 = 0, then i k.if e i x k = 0, l2=1 c k, l2 = 1 and r k, l2 = , then i k.for all i, k, l 1, 1 i n, k {0},1 l 1 l, if u k, l1 = 1, then h k, l1 is a unit row vector.for all i, k, l 2, 1 i n, k {0}, 1 l 2 m, if c k, l2 = 1, then r k, l2 is a unit row vector.for all i, k, l 3, 1 i n, k {0}, 1 l 3 l, if v k, l3 = 1, then h k, l3 is a unit row vector.for all i, k, l 4, 1 i n, k {0}, 1 l 4 m, if b k, l4 = 1, then rk, l4 is a unit row vector.for all i k, l1=1 u k, l1 = 0 or 1 and c k, l2 = 0, for all l 2, 1 l 2 m.for all i k, l2=1 c k, l2 = 0 or 1 and u k, l1 = 0, for all l 1, 1 l 1 l.for all i k, l3=1 v k, l3 = 0 or 1 and b k, l4 = 0, for all l 4, 1 l 4 m.for all i k, l4=1 b k, l4 = 0 or 1 and v k, l3 = 0, for all l 3, 1 l 3 n, k {0}, l1=1 u k, l1 + l2=1 c k, l2 = 0 or 1 and l3=1 v k, l3 + l4=1 b k, l4 = 0 or 1. for large - scale stochastic system, u k, l1 , h k, l1 , c k, l2 , r k, l2 , v k, l3 , h k, l3 , b k, l4 , r k, l4 follow the rules below.if e i x k = 0, then i k.if e i x k = 0 and l2=1 c k, l2 = 0, then i k.if e i x k = 0, l2=1 c k, l2 = 1 and r k, l2 = , then i k.for all i, k, l 1, 1 i n, k {0},1 l 1 l, if u k, l1 = 1, then h k, l1 is a unit row vector.for all i, k, l 2, 1 i n, k {0}, 1 l 2 m, if c k, l2 = 1, then r k, l2 is a unit row vector.for all i, k, l 3, 1 i n, k {0}, 1 l 3 l, if v k, l3 = 1, then h k, l3 is a unit row vector.for all i, k, l 4, 1 i n, k {0}, 1 l 4 m, if b k, l4 = 1, then rk, l4 is a unit row vector.for all i k, l1=1 u k, l1 = 0 or 1 and c k, l2 = 0, for all l 2, 1 l 2 m.for all i k, l2=1 c k, l2 = 0 or 1 and u k, l1 = 0, for all l 1, 1 l 1 l.for all i k, l3=1 v k, l3 = 0 or 1 and b k, l4 = 0, for all l 4, 1 l 4 m.for all i k, l4=1 b k, l4 = 0 or 1 and v k, l3 = 0, for all l 3, 1 l 3 l.for all i, k, 1 i n, k {0}, l1=1 u k, l1 + l2=1 c k, l2 = 0 or 1 and l3=1 v k, l3 + l4=1 b k, l4 = 0 or 1. if e i x k = 0, then i k. if e i x k = 0 and l2=1 c k, l2 = 0, then i k. if e i x k = 0, l2=1 c k, l2 = 1 and r k, l2 = , then i k. for all i, k, l 1, 1 i n, k {0},1 l 1 l, if u k, l1 = 1, then h k, l1 is a unit row vector. for all i, k, l 2, 1 i n, k {0}, 1 l 2 m, if c k, l2 = 1, then r k, l2 is a unit row vector. for all i, k, l 3, 1 i n, k {0}, 1 l 3 l, if v k, l3 = 1, then h k, l3 is a unit row vector. for all i, k, l 4, 1 i n, k {0}, 1 l 4 m, if b k, l4 = 1, then rk, l4 is a unit row vector. for all i k, l1=1 u k, l1 = 0 or 1 and c k, l2 = 0, for all l 2, 1 l 2 m. for all i k, l2=1 c k, l2 = 0 or 1 and u k, l1 = 0, for all l 1, 1 l 1 l. for all i k, l3=1 v k, l3 = 0 or 1 and b k, l4 = 0, for all l 4, 1 l 4 m. for all i k, l4=1 b k, l4 = 0 or 1 and v k, l3 = 0, for all l 3, 1 l 3 l. for all i, k, 1 i n, k {0}, l1=1 u k, l1 + l2=1 c k, l2 = 0 or 1 and l3=1 v k, l3 + l4=1 b k, l4 = 0 or 1. we close this section with the definition of controllability to the system equations proposed above. dna replication systems with system equations proposed as (9a), (9b),, and are completely controllable if and only if for all x 0, x 2k1, x 2k2 + 1 , k 1, k 2 {0}, at least one path from x 0 to x 2k1 and at least one path from x 0 to x 2k2 + 1 by applying proper mutagens in the correct order, with k 1, k 2 finite. we first define our objective function that can be adapted to all kinds of systems proposed in section 2.2 with minor changes. mathematically, in systems where the controllable parameters of interest are discrete, the objective function is usually a weighted sum representing the number of times that a piece of equipment is turned on or off or the number of resources needed to execute certain tasks in the frequent cases. in our case , this summation is the total number of times that different mutagens are applied weighted by the corresponding cost (including the risk). another key factor of objective function is the off - trajectory penalty. designing a trajectory if the measurement indicates that the current state is off the predefined trajectory, we include a distance reference between current state and desired state as penalty and change the treatment plan accordingly. therefore, our objective function can be expressed as j0(x0)=minu, c, v, c h, h,r, r, with x 0, x k , 1 k n, n 0 (mod 3) given. the physical meaning of u k, l1 , c k, l2 , v k, l3 , b k, l4 , l 1, l 2, l 3, l 4 is the same as in section 2.2.3. l1, l2, l3, l4 , for all l 1, l 2, l 3, l 4, 1 l 1 1 l 2 m, 1 l 3 l, 1 l 4 m, are the corresponding cost of applying chemical mutagens and radiative rays indexed l 1, l 2, l 3, l 4, respectively. {x k}: {0} denotes the desired set at kth stage, generated by the dna sequences representing the same amino acid sequence as x k , the desired state at kth step. and d(x k, { x k} ) is the distance reference of the current state x k to the desired set {x k} at kth step. the final penalty, the distance reference from the final state to the desired set at k = n, is included in the last term. in general, l2, l4 l1, l3, for all l 1, l 2, l 3, l 4, 1 l 1 l, 1 l 2 m, 1 l 3 l, 1 l 4 m, because physically o is a set of nonsense bases and more details are necessary to convert an o back to normal bases, for instance, the cost to identify the exact element in the set o. our goal is to drive our system optimally from initial state x 0 to the desired final set {x n} by applying a sequence of mutagens indexed with {l 1, l 2, l 3, l 4}, at problematic positions i, and in a correct order k. in, the first four terms inside the expectation do not depend on random variables h k, l1 , r k, l2 , h k, l3 , and r k, l4 , for all i, k, l 1, l 2, l 3, l 4 as the treatment plan is computed based on the initial state x 0. given y k, the last term inside expectation, k=0 d(x k, { x k} ), is the only term in summation that depends on the distribution of the random variables. the constraint of the optimal control problem, in general, is the system equation. we choose multidimensional stochastic system equation as the generalized constraints as it can be degenerated to one - dimensional and multidimensional deterministic cases by proper modifications. therefore, we can rewrite our objective function and formulate our optimal control problem as j0(x0)=min{u, c, v, c}0,1, ,n1 ], subject to xk+1=(i+l3=1li=1nvk, l3ihk, l3iseieit) (i+l1=1li=1nuk, l1ihk, l1iseieit)xk + (i+l3=1li=1nvk, l3ihk, l3iseieit)l2=1mikck, l2irk, l2iwei + l4=1mikbk, l4irk, l4iwei, yk = xk. we need to choose a proper distance reference to quantitatively describe the relationship between dna segments of same length. we first define the distance reference between codons, and the distance reference between dna segments is a weighted sum of distance reference between every pair of codons. the distance reference between codons, d(1, 2), 1, 2 , needs to fulfill the biological requirements as below. mathematically, d: {0}, d(1, 2) 0. symmetry: the distance reference from codon 1 to codon 2 equals the distance reference from codon 2 to codon 1, that is, d(1, 2) = d(2, 1). the distance reference between two codons corresponding to different amino acids should reveal the chemical and physical differences between two amino acids. the distance reference from stop codons to all other codons is much larger than those between other codons as early termination of amino acid sequences is more harmful than other forms of mutations. all the existing metric defined on the finite field can not achieve all the requirements above. the second requirement violates the identity of indiscernible, that is, d(1, 2) = 0 if and only if 1 = 2. the redundancy in genetic codes implies d(1, 2) = 0 if those two amino acids, 1 and 2, are translated into the same amino acids. in addition, the triangular inequality is not necessarily true, according to the underlying physical meanings. we take the assumption that the stop codons are of the same distance reference from and to all other codons. we ignore codons containing o since their chemical and physical properties can not be found in literature. from table 5, we can see all codons are divided into different sets with each set corresponding to one amino acid. the size and this implies that the costs of driving one codon to the desired final set generated by the desired final state might be different from the costs of driving the complementary codon to the desired final set generated by the complementary of desired final state. more discussions about this issue are presented in section 3. the distance reference between any two codons can be defined by a weighted sum of the differences between physical and chemical properties or other reasonable functions. and the distance reference between two dna sequences is defined as the sum of distance reference between the corresponding pair of codons. an example of the distance function can be expressed as d=polarity polarity + ph ph+size size,polarity = {0if 1,2 are both polar or non - polar,1if one of 1,2 is polar, and the other non - polar, ph = |ph value of 1ph value of 2|,size = {0,if 1,2 are both tiny, small, or normal,1,if one of 1,2 is tiny, and the other small,2,if one of 1,2 is tiny, and the other normal,3,if one of 1,2 is small, and the other normal, where 1, 2 are two amino acids . d( 1, 2) is then assigned to d(1, 2) with 1, 2 corresponding to amino acids 1, 2, respectively. since the generalized optimal control problem in and is a multistage problem that can be broken down into simpler steps at different time points. for dynamic programming, the optimal control policy is constructed backward. and bellman's principle of optimality states that the optimal policy for x 0 to {x n} is also the optimal policy for the tail problem, from x q to {x n}. the tail problem is defined as jq(xq)=min{u, c, v, c}q, q+1, ,n1{k = qn1l1=1li=1nl1uk, l1i+k = qn1l2=1mi=1nl2ck, l2i + k = qn1l3=1li=1nl3vk, l3i+k = qn1l4=1mi=1nl4bk, l4i + k = qn{h, r, h,r}q, q+1, ,n1}. the iterative update equation to find optimal policy can be expressed by, according to the dynamic programming algorithm in. jn(xn)=d(xn,{xnd}),jq(xq)=minuq, cq, vq, cq hq, rq, hq,rq=minuq, cq, vq, cq{l1=1li=1nl1uq, l1i+l2=1mi=1nl2cq, l2i+l3=1li=1nl3vq, l3i + l4=1mi=1nl4cq, l4i + hq, rq, hq,rq}, q=0,1, ,n1. in the following examples, we consider applying chemical mutagens only because the randomness of applying radiation is much larger and more difficult to control. we also omit the mutations between a normal base and o because of high - cost l2 and the unavailable chemical and physical properties for codons containing o. the distance reference between codons used in sections 3.2 and 3.3 is computed by with polarity = 8, ph = 3, size = 1, 1 = 2, 2 = 5 and 3 = 3. we only keep the final penalty but omit the off - trajectory penalty along the trajectory. we define the distance reference between bases as d={0,if xn = xnd,,if xnxnd, with 1, 2 {0}, where {0} denotes the set excluding the element 0. our optimal control problem for point mutations is j0(x0)=minuk, l1, 0kn, 1l1l{k=0n1 l1=1ll1uk, l1}, subject to xk+1=(i+l1=1luk, l1s)xk, xn = xnd, with x 0 given, x k {0}. suppose that there are 12 kinds of mutagens (l 1 = 12), each corresponding to a specific transfer pattern as in table 6, all available controls and the respective costs can be immediately listed as in tables 7 and 8. the elements along the antidiagonal of table 7, u at, u gc, u cg, and u ta, are artificially added, because the complementary transfers naturally happen and no mutagen is necessary. thus, the costs along the antidiagonal of table 8 are all zero, that is, at = gc = cg = tc = 0. the equivalence relationship between subscription in two nucleotide bases and subscription in integer l 1(1 2): {a, t, g, c} {a, t, g, c} {integers from 1 to 12} is defined by table 6. under the above assumptions, we can write update equation for optimal policy explicitly as jq(xq)=minuq, l1{xq+jq+1,{a, t, g, c}{0}}. proposition 7for the same x n , jqjq+1, for all q, 0 q n 1, for all {a, t, g, c}, where denotes the complementary base of. if, in addition, the system is completely controllable, m, s.t. j m is the global minimum and for all q m, jq=jm if m q 1 (mod 2), and j q = j m if m q 0 (mod 2). in our example, m n 6. for the same x n , jqjq+1, for all q, 0 q n 1, for all {a, t, g, c}, where denotes the complementary base of. if, in addition, the system is completely controllable, m, s.t. j m is the global minimum and for all q m, jq=jm if m q 1 (mod 2), and j q = j m if m q 0 (mod 2). in our example, proofthis first part is due to the zero cost for the transfers between complementary bases in the consecutive steps. for any 0 q n 1 , the relationship between minimal costs in consecutive steps is shown in. since {a, t, g, c}, +jq+1 is one of the four elements in the set from which the j q is picked. moreover, =0. therefore, jq+1 is one of the four elements in the set. since j q is the minimum picking for a set containing jq+1, we conclude that jqjq+1.the m value in our example is proved by brute force method, that is, j n6 is a guaranteed global minimum. for completely controllable systems the existence of m implies that for without limitation in the number of steps, we can reach the global optimal in n m steps, 6 steps in our example.suppose that q = m, j m is the global minimum, thus jm-1jm. jm-1 is also a global minimum.by backward induction, suppose for q = q 1, the statement is true, that is, jq1 - 1=jq1 is the global optimal either from xq1 - 1= or x q1 = to x n. obviously, for q = q 1 1, the statement is still true. therefore, jq=jq-2=jq-1, {a, t, g, c}, for all q, 2 q m. this first part is due to the zero cost for the transfers between complementary bases in the consecutive steps. for any 0 q n 1, the relationship between minimal costs in consecutive steps is shown in. since {a, t, g, c}, +jq+1 is one of the four elements in the set from which the j q is picked. since j q is the minimum picking for a set containing jq+1, we conclude that jqjq+1. the m value in our example is proved by brute force method, that is, j n6 is a guaranteed global minimum. for completely controllable systems, this m always exists. the existence of m implies that for without limitation in the number of steps, we can reach the global optimal in n m steps, 6 steps in our example. suppose that q = m, j m is the global minimum, thus jm-1jm. suppose for q = q 1, the statement is true, that is, jq1 - 1=jq1 is the global optimal either from xq1 - 1= or x q1 = to x n. obviously, for q = q 1 1, the statement is still true. therefore, jq=jq-2=jq-1, {a, t, g, c}, for all q, 2 q m. in the proof of global minimum that can be reached in the finite step in proposition 7, we also discover proposition 8. here, j q(x q, x n) denotes the optimal cost from x q to x n. proposition 8given two single base mutation optimal control problems, with the same fixed n, with and desired final states complementary to each other. if j m(, x n) is the global minimum, then jm(,xnd) is also the global minimum, that is, the global minimum of both systems is reach at the same stage m. moreover, for all q, 0 q m, jq(,xnd)=jq(,xnd), ,xnd{a, t, g, c}. given two single base mutation optimal control problems, with the same fixed n, with and desired final states complementary to each other. if j m(, x n) is the global minimum, then jm(,xnd) is also the global minimum, that is, the global minimum of both systems is reach at the same stage m. moreover, for all q, 0 q m, jq(,xnd)=jq(,xnd), ,xnd{a, t, g, c}. physically, proposition 8 states that the optimal can be achieve at the same step from a pair of complementary bases to another pair of complementary bases at the same cost. however , this fact is true only for base - to - base deterministic mutations, because the distance reference is well defined by. since we apply mutagens before the replication starts, u aa actually transfer a to t and then to a by replication. for simplicity , we just use the kth and (k + 1)th step states as subscripts to represent the corresponding control and cost. therefore, ac, ca, gt, tg is smaller than other mutagens, except artificial ones. if we use to denote the costs of mutagens as listed in table 9, then ==. running the dynamic programming for every pair of (x q, x n) {a, t, g, c} {a, t, g, c}, n = 9. here, we sightly modify our notation. we use j q(x q, x n) to denote the optimal cost from x q to x n. then, jq=. the path to reach the optimal cost is denoted by pq=, where p q(x q, x n) is the (q + 1)th state from x q to x n , that is, x q+1 = p q(x q, x n). the simulation are shown as below, including optimal costs for all possible transfer pairs (x q, x n) {a, t, g, c} {a, t, g, c}, j q, 0 q 8, graphical representation in figure 6, and optimal path p q, 0 q 7. j0=,j1=,j2=,j3=,j4=,j5=,j6=,j7=,j8=, p0=,p1=,p2=,p3=,p4=,p5=,p6=,p7=. for simplicity , we use 1 to represent a, 2 to g, 3 to c, and 4 to t in graphical interpretation. from figure 6 , we can see clearly that the optimal cost decreases as q decreases in the first few steps, and then optimal cost remains at the global minimum. this phenomenon obeys proposition 7. in this example, global optimal is reached at m = 5 for all pairs of initial and final states as j 7 j 5 = j 3 and j 6 j 4 = j 2. so, the global minimum is achieved before we reach n 5 = 4 in this particular case. this also implies that with n free we can reach desired final state in 4 steps from given initial state. observing closely to j q, 0 q 5, j q1 equals to j q by exchanging the first and the last columns, and the second and the third columns, which is consistent with proposition 8. or we can exchange the first and the last rows, and the second and the third rows of j q to obtain j q1. j q1 and j q2 are the same for q 1, q 2 m = 5 for q 1 q 2 = 0 (mod 2). the optimal trajectories are generated from p q(x q, x n). for example, given x 2 = t, and the final state x 9 = g, we want to generate the optimal trajectories. x 3 = p 2(t, g) = a, g. if x 3 = a, x 4 = p 3(a, g) = t; if x 3 = g, x 4 = p 3(a, g) = c. if x 4 = t, x 5 = p 4(t, g) = a, g; if x 4 = c, x 5 = p 4(c, g) = g. if x 5 = a, x 6 = p 5(a, g) = t; if x 5 = g, x 6 = p 5(g, g) = c. if x 6 = t, x 7 = p 6(t, g) = a, g; if x 6 = c, x 7 = p 6(c, g) = g. if x 7 = a, x 8 = p 7(a, g) = t; if x 7 = g, x 8 = p 7(g, g) = c. so, the optimal routes are tatatattgutgg; tatattgutggcg; tattgutggcgcg; ttgutggcgcgcg. consequently, the optimal cost is j 2(t, g) = 0.64 = tg. optimal trajectories for other pairs of initial and final states can be obtained in the same manner. it takes less than 1 second to generate optimal trajectories for all pairs of initial and final states with n = 9 on a regular desktop. since we have already proven by the brute force method that the global optimal can be achieved with m 6, the computation time can be further reduced by taking n = 6 with all the necessary for this example. for codon - to - codon deterministic mutations, we formulate our optimal control problem as j0(x0)=minuk, l1i, 0kn,1l1l, 1i3{k=0n1l1=1li=13l1uk, l1i+d(xn,{xnd} ) }, subject to xk+1=(i+l1=1li=13uk, l1iseieit)xk, with x 0, x n {0} given, x k {0} , for all k, 0 k n, and d(1, 2), 1, 2 {0} as defined in section 2.3. if we take the same assumption on available mutagens as in section 3.1, then we can write update equation for optimal control policy explicitly as jq(xq)=minuq, l1i, 1l1l, 1i3{xq11+jq+1, xq22+jq+1,xq33+jq+1, 1,2,3{a, t, g, c}{0} }, with x q {a, t, g, c} {0}, 1 i 3 denotes the ith element of x q {0} , and xqi denotes the complementary base of x q. the optimal control sequences depend on the numerical values of l1s and d(1, 2), 1, 2 {0}. though we do not have real values of l1s and d(1, 2) , we can always obtain simulation to compare the differences by assigning different numerical values to those parameters. therefore, we use three different assignments of l1s and the same d(1, 2) to generate our simulation . those three assignments of l1s are , 5, and 0.5, respectively, with the same as assigned in section 3.1. in every particular example, it takes approximately 2 seconds on a regular desktop to generate the optimal path table for all pairs of initial and final states with n = 19, and the dynamic programming algorithm ensures that the optimal control for tail problems is generated at the same time. the graphical interpretation of three assignments are shown in figures 7, 8, and 9, respectively. the x - axis and y - axis denote x q and x n , respectively. for a codon , 1, 2, 3 {a, t, g, c} {0}, its index is calculated by 42+4+3,where i = 1 if a, i = 2 if g, i = 3 if c, and i = 4 if t, 1 i 3, for the simplicity of graphical interpretation. thus, there are 64 pairs of initial and final desired states, and there are 64 21 pairs of initial state and final desired set. j q is calculated following the same procedure as in base - to - base deterministic cases. the value of optimal cost can be read directly from graphical interpretation, and the optimal path can be generated from path matrix p q, similar to base - to - base deterministic case. both j q and p q, for all q, 0 q n, are of 64 64 dimension. from the graphical interpretation and table 10, we find that the value of q where the global minimum is reached at the first time decreases as l1 decreases. and j 0 is more similar to j 18 with = 5 than with = or = 0.5. this implies that if d(1, 2) are the deterministic term in our objective function, then the treatment plan is made to drive the final state as close to the desired set as possible; if the costs of applying mutagens is the deterministic term in the objective function, then the treatment plan tends to stay in the original state and applies less mutagens; if they are of equal weight, then the treatment plan deals with this tradeoff. moreover, no matter how the numerical values of final penalty and the costs of applying mutagens changes in our objective function as shown in, there is always a m, m n 18, j m(x m) is global minimum. proposition 7 can be extended to codon - to - codon deterministic mutations as stated in proposition 9. proposition 9given an optimal control problem with objective function in the form of, constraints in the form of, and all available chemical mutagens and their corresponding transfer pairs and costs as listed in tables 6, 7, and 8, jqjq+1, {0}3. if, in addition, the system is completely controllable, m, s.t. j m is the global minimum and for all q m, jq=jm if m q 1 (mod 2), and j q = j m if m q 0 (mod 2). in our example, m n 18. given an optimal control problem with objective function in the form of, constraints in the form of, and all available chemical mutagens and their corresponding transfer pairs and costs as listed in tables 6, 7, and 8, jqjq+1, {0}3. if, in addition, the system is completely controllable, m, s.t. j m is the global minimum and for all q m, jq=jm if m q 1 (mod 2), and j q = j m if m q 0 (mod 2). in our example, m n 18 n 18 here since the rest is similar to the proof of proposition 7. the objective function in can be written as the summation of three separate single - base mutation systems and the distance reference between final states and the final desired set, that is, jq(xq)=minn1,n2,n302nn1+n2+n33n1{jn1(xn1)(xq1,1) optimal costs of base - to - basedeterministic optimal controlproblem formed by the 1st base + jn2(xn2)(xq2,2)optimal costs of base - to - base deterministic optimal controlproblem formed by - the 2nd base + jn3(xn3)(xq3,3)optimal costs of base - to - basedeterministic optimal control problemformed by the 3rd base + d(,{xnd} ) }, where n q = (n n 1) + (n n 2) + (n n 3).according to proposition 7, j n6(x n6) is guaranteed to be the global optimal for single - base mutations. therefore, optimal costs corresponding to three single - base mutation systems, j n1(x n1)(x q , 1), j n2(x n2)(x q , 2), j n3(x n3)(x q , 3) are guaranteed to reach their own global optimal at n 1 = n 2 = n 3 = n 6 with all possible combinations of 1, 2, 3 {a, t, g, c}. therefore, q = n 18 is a guaranteed global optimal. n 18 here since the rest is similar to the proof of proposition 7. the objective function in can be written as the summation of three separate single - base mutation systems and the distance reference between final states and the final desired set, that is, jq(xq)=minn1,n2,n302nn1+n2+n33n1{jn1(xn1)(xq1,1) optimal costs of base - to - basedeterministic optimal controlproblem formed by the 1st base + jn2(xn2)(xq2,2)optimal costs of base - to - base deterministic optimal controlproblem formed by - the 2nd base + jn3(xn3)(xq3,3)optimal costs of base - to - basedeterministic optimal control problemformed by the 3rd base + d(,{xnd} ) }, where n q = (n n 1) + (n n 2) + (n n 3). according to proposition 7, j n6(x n6) is guaranteed to be the global optimal for single - base mutations. therefore, optimal costs corresponding to three single - base mutation systems, j n1(x n1)(x q , 1), j n2(x n2)(x q , 2), j n3(x n3)(x q , 3) are guaranteed to reach their own global optimal at n 1 = n 2 = n 3 = n 6 with all possible combinations of 1, 2, 3 {a, t, g, c}. therefore, q = n 18 is a guaranteed global optimal. indeed, the m value where the first global optimal is reached at earlier stage as listed in table 10. graphically, the indices of complementary codons 1 = and 2=t sum up to 65, that is, (16+4+3 ) + (16+4+3 ) = (16+4+3 ) + (16(1)+4(1)+ ) = 65. therefore, j q and j q1, 1 q m are symmetric about the plane x = 32.5, j q2 and j q, 2 q m are the same, as shown in figures 7, 8, and 9. however, proposition 8 can not be extended to codon - to - codon deterministic case due to the redundancy of genetic codes, that is, the set of codons translated to the same amino acid varies from one amino acid to another as shown in table 5. the simulation show that the costs, a pair of complementary codons, to two final desired set generated by a pair of complementary final desired codons are different, that is, jq(xq,{xnd})jq(xq,{xnd} ), in general, for any q. graphically, the optimal cost profile j q is not symmetric about the plane y = 32.5 for j q1, 1 q m. therefore, the doctors need to pick the strand with lower cost to make the treatment plan. this also implies that in large - scale cases, for instance, a gene containing hundreds of nucleotide bases, the doctors should make the treatment plan based on the strand the total cost of which is lower than the other. the optimal control problem of codon - to - codon stochastic mutations can be written as j0(x0)=minuk, l1i, 0kn11l1l, 1i3{k=0n1l1=1li=13l1uk, l1i + hk, l1i, 0kn11l1l, 1i3}, subject to xk+1=ixk+l1=1li=13uk, l1ihk, l1iseieitxk, with x 0, x n {0} given, x k the major difference between deterministic and stochastic systems is that we impose the random binary vector, h k, l1 , in our system equation. we denote the probability associated with h k, l1 to be p l1,12 with 1, 2 {a, t, g, c}. the equivalence relationship between j and 1 2 can be found in table 3. again, we assume that we have l 1 = 12 kinds of mutagens, each corresponding to one major transfer pattern, associated with probability assignments, as listed in table 11. then, we can write updated formula for optimal control policy explicitly as jq(xq)=minuq, l1i, 1l1l, 1i3{xq11+hq, l1(xq11)1 ) ], xq22+hq, l1(xq22)2 ) ], xq33+hq, l1(xq33)3 ) ], 1,2,3{a, t, g, c}{0} } where hq, l1(xq11)1)]=pl1(xq11), xq1a(h) ) ] + pl1(xq11), xq1g(h) ) ] + pl1(xq11), xq1c(h) ) ] + pl1(xq11), xq1t(h) ) ], where x q {a, t, g, c} {0}, 0 q n 1, 1 i 3 denotes the ith element of x q {0} , xqi denotes the complementary base of x q , and l 1: 1 2 {a, t, g, c} {a, t, g, c} {integers from 1 to 12}, the mapping from major transfer pattern 1 2 to mutagen index, as shown in table 11. the mathematical expression of hq, l1(xq22)2t ) ] and hq, l1(xq33)3t ) ] is similar to hq, l1(xq11)1t ) ] as shown above. in order to run the simulation, we assign numerical values to probabilities in table 11, as illustrated in table 12. as in section 3.2, we use three different assignments for l1s, , 5, and 0.5, respectively. the optimal cost profile j q with selected q values, for every pair of (x q, x n) , is graphically interpreted in figures 10, 11, and 12, respectively, with n = 29, with computation time of approximately 7 seconds on a regular desktop. the profile of j 0 is more similar to j 29 when l1s are assigned 5 than or 0.5. this implies in codon - to - codon stochastic mutations; the optimal control sequence behaves as codon - to - codon deterministic cases, that is, the system tends to getting as close as possible to the final desired set if l1s are much smaller than d(1, 2), and the system tends to remain in the same state with minor mutations when l1s are relatively larger than d(1, 2), 1, 2 {0}. and jqjq+1, {0} is still valid in codon - to - codon stochastic case. however, for stochastic cases, we can not reach a global minimum because of the randomness caused by mutagens. since, in usual cases, there exists no stationary global minimum , we need to define error tolerance , that is, if |jq-jq-1| with the same {x n}, then we can stop at j q(x q). however, we can still observe figures 10, 11, and 12 to conclude that j 0 and j 2 are almost of the same shape in all three different parameter assignments. higher dimensional optimal control problems, gene - to - gene stochastic mutations, can be solved as a series of cascade codon - to - codon stochastic problems. in this paper, we present a mathematical model to deal with mutations in the process of dna replication in the view of control systems. different from the existing models, our model is constructed directly from the basic biological theories, the central dogma in molecular biology, and the complementary base pair for dna molecules with double helix structure. it precisely describes how the induced mutations affect the targeted dna segments at molecular level. it provides instrumental information of molecule interactions in gene mutation for biologists and doctors to gain a better understanding of cellular and tissue level systems' behavior. though we emphasize that we target at induced mutations during the process of dna replication in our work, this model can be extended to other biological processes at molecular level, such as transcription process and dna brokage. in our optimal control problem, the objective function includes two factors: the risk / cost of applying mutagens and the off - trajectory penalty. under optimal control policy, the summation of those two factors are minimized, by dynamic programming, to propose a low - risk treatment plan. we define the distance reference following the chemical and physical properties of amino acids, representing the penalty. our objective is to drive the system from given initial state to the final desired set generated by the final desired state at the lowest cost. we define the final desired set since redundancy in genetic codes gives us additional options of final desired state to further lower the cost. we also discuss three different small - scale system, and show the simulation of examples. the optimal control problems of base - to - base deterministic mutations and codon - to - codon deterministic mutations are of theoretical importance. as shown in the propositions, the global optimal if the step limit is larger than the number of steps that global optimal can achieve, then we have some flexibility in our treatment plan. in addition, there exist multiple optimal paths with the same total cost, given the initial state and the final desired set. the optimal control problem of codon - to - codon stochastic mutations is of practical importance, since codon is the basic component forming long dna sequences. the step limit n is decided by doctors according to patients' conditions, and the treatment plan is made according to the initial state, the final desired set, and the step limit. since the doctors constantly take measurement to see the of treatments at current stage, the treatment plan is updated accordingly. solving codon - to - codon stochastic optimal control problem is a key step to realize the optimal control to gene - to - gene stochastic mutations in the real world. the optimal control sequences generated by dynamic programming make it possible for biologists and doctors to mutate certain sections of genes on purpose in laboratory, at a relative low cost and low risk, which is an essential step to identify the functional units, to examine the interactions among different segments, and to find healthy, harmful, and lethal nucleotide sequences. all those are beneficial in gene network construction. in addition, the fundamental details of gene mutations at molecular level help biologists to elaborate on the biological theories at the cellular and tissue levels, such as the theory of evolution. moreover, by our method, biologists can distinguish the harmful and beneficial mutations and induce beneficial mutations during the evolution process in a proper way, which greatly helps to save rare species in danger. furthermore, our solution to the optimal control problem proposed provides a new medical intervention to genetic diseases. compared to the existing gene therapy, treatments by mutagens are safer by avoiding the side effect of virus infection. calculation errors, the mispairings in the process of two single - stranded dna segments, can be corrected at lowest cost by applying a correct mutagen sequence. further work can be done by extending codon - to - codon stochastic optimal control problem to gene - to - gene stochastic mutations. the distance reference between dna segment with equal length can be defined as a weighted sum of the distance references between codons. since certain combinations of amino acids this goal can be achieved by either defining a preset trajectory or eliminating high - risk sequences in the state space. under this situation, the spontaneous mutations can be modeled as an additional random factor in our state updating equations, and another random noises should be added to the output equation.

we propose a molecular - level control system view of the gene mutations in dna replication from the finite field concept. by treating dna sequences as state variables, chemical mutagens and radiation as control inputs, one cell cycle as a step increment, and the measurements of the ing dna sequence as outputs, we derive system equations for both deterministic and stochastic discrete - time, finite - state systems of different scales. defining the cost function as a summation of the costs of applying mutagens and the off - trajectory penalty, we solve the deterministic and stochastic optimal control problems by dynamic programming algorithm. in addition, given that the system is completely controllable, we find that the global optimum of both base - to - base and codon - to - codon deterministic mutations can always be achieved within a finite number of steps.

we defined cholera decedents as persons who died of suspected cholera (acute watery diarrhea in persons > 5 years of age) with illness onset after october 16, 2010, three days before the first case - patients were seen at the hospital (reflecting the 3-day average incubation period). to locate decedents, we obtained reports of cholera - related deaths from 2 sources: admission records from 2 hospitals in artibonite that had cholera treatment centers (hpital albert schweitzer and hpital charles colimon) and verbal reports from community health workers (chws). we attempted to locate households of all decedents from hospital records and verbal reports. logistic and time constraints limited case finding to communities within 2 hours travel from the hospitals. we visited decedents households; obtained informed consent; and interviewed families about demographics, symptoms, health - seeking behavior, treatment, type of health facility, and knowledge about cholera. we also asked decedents household members and local chws about other cholera - related deaths. the centers for disease control and prevention institutional review board (atlanta, ga, usa) and mspp determined that this emergency response activity was nonresearch. of 28 decedents identified from hospital records, we found homes of 22 (79%); homes of 6 decedents could not be located or were too remote for inclusion. illness onset ranged from october 16 through november 14; a total of 29 (33%) persons died during the first week of the epidemic (figure 2). median age of decedents was 50 years (range 5100 years); 58 (67%) were male. forty - eight (55%) decedents died in a health facility (health facility decedents) and 39 (45%) died at home or en route to a facility (community decedents). we identified 17 (35%) health facility decedents from hospital records and 31 (65%) from community interviews; we identified 5 (13%) community decedents from hospital records and 34 (87%) from community interviews. number of persons who died of cholera, artibonite department, haiti, october 16november 14, 2010. twenty - three (48%) health facility decedents and 9 (23%) community decedents had used oral rehydration solution (ors) at home before seeking care (table 1). ors use at home was lower for persons who died during the first week of the outbreak than during the second or third weeks. we observed ors sachets in homes of 17 (35%) health facility decedents and 14 (36%) community decedents. health facility decedent, cholera case - patient who died in a health facility; community decedent, cholera case - patient who died at home or en route to a health facility; ors, oral rehydration solution. median time from illness onset to death was 20 hours (range 3 hours7 days) for health facility decedents and 12 hours (range 2 hours8 days) for community decedents. twenty - two (46%) health facility decedents died on day of admission and 26 (54%) died after spending > 1 night in the facility (table 2). twenty - three (59%) community decedents never sought care, 8 (21%) died en route to care, and 8 (21%) died after discharge. of those who sought care, 29 (60%) health facility decedents and 7 (44%) community decedents waited < 2 hours to visit a health facility. family members of community decedents reported the following reasons for not seeking care: no need for care, long distance to the health facility, too ill to travel, lack of transport, unsafe to travel at night, and cost of transport. * health facility decedent, cholera case - patient who died in a health facility; community decedent, cholera case - patient who died at home or en route to a health facility; na, not applicable. of 48 health facility decedents, 38 (79%) were treated in hospital and 10 (21%) at a health center or dispensary. decedents received intravenous fluids, ors, both, or neither. household members of 33 (69%) health facility decedents and 30 (81%) community decedents reported receiving information about cholera after the outbreak started. the most common information sources for families of health facility and community decedents, respectively, were radio (26 vs. 26), friend (6 vs. 8), cellular telephone text message from mspp (4 vs. 4), community meeting (2 vs. 2), and chws (1 vs. 3). fewer than half of family members of health facility and community decedents believed cholera was treatable. of these, 16 (70%) health facility decedents and 17 (90%) community decedents knew to seek care at a health facility. our findings suggest that, early in the cholera epidemic in haiti, death occurred rapidly, and care was either inadequate or nonexistent. global deficiencies in the distribution and use of ors in recent years have impeded the ability of chws to initiate treatment. second, chws probably lacked sufficient information, experience, and resources to provide proper treatment early in the outbreak. deaths in health facilities in haiti might have ed from problems commonly observed elsewhere: overwhelming patient load, inadequate supplies, and health worker shortages. third, decedents relatives identified several commonly observed barriers to care: distance to health facility, lack of transport, and unaffordable transport. research suggests that the effect of distance and lack of transport on cholera - related death can be mitigated by local treatment with ors by chws. finally, the epidemic strain, which was particularly virulent, might have contributed to deaths. this finding suggests that cholera - related deaths might have been underreported, particularly in more remote communities. first, time and logistics limited our ability to visit remote communities where more deaths might have occurred. second, our geographically circumscribed convenience sample might not have been representative of all cholera deaths. findings from this assessment suggested several practical actions that could mitigate the risk for death from cholera. chws, particularly in remote settings, should receive training in cholera treatment and referral and adequate supplies of ors; similar efforts for hiv and tuberculosis in haiti have been promising. longer term efforts to increase health facility staffing and improve access to care should be prioritized. in response to the epidemic, training and supplies have been provided to health workers in all 10 departments of haiti. by april 2011

we evaluated a high (6%) cholera case - fatality rate in haiti. of 39 community decedents, only 23% consumed oral rehydration salts at home, and 59% did not seek care, whereas 54% of 48 health facility decedents died after overnight admission. early in the cholera epidemic, care was inadequate or nonexistent.

breast cancer is one of the most common cancers affecting women worldwide and a primary cause of cancer - related death in women. despite high morbidity and mortality, therapeutic options for breast cancer include both targeted (e.g., anti - estrogens and her2 antagonists) and non - targeted therapies (e.g., radiation therapy). if diagnosed at an early stage, breast conservation surgery can often be performed; this involves surgical removal of the cancer cells with minimal disturbance of normal breast tissue. , patients often receive adjuvant radiation therapy, which helps kill any remaining cancerous cells that may not have been removed during the operation. to date , an optimal fractionation schedule for breast irradiation has not been universally accepted, and many studies have examined the benefits and drawbacks of various treatment regimens. previous studies have shown that breast - conserving surgery in conjunction with irradiation has a similar outcome as a radical operations such as a full mastectomy. the current standard for radiation treatment involves whole - breast tangential irradiation with a subsequent boost to the tumor bed; this has been proven to decrease locoregional recurrence. however, this standard regimen occurs over a long period of time and can potentially interfere with post - operative chemotherapy treatment. radiation treatment following breast - conserving surgery for early - stage disease typically occurs over the course of 67 weeks. previous work has shown that breast cancer has a low / ratio (34 gy) and that hypofractionated radiation therapy (> 2 gy / fraction) may be effective without significantly increasing adverse effects. interestingly, a study showed that implementation of a cost - minimization strategy was effective on a patient - by - patient basis and significantly reduced medical expenses. here, we further examine the influence of radiotherapy scheduling on patients undergoing breast conservation surgery. specifically, we examined whether there were any outcome differences between early - stage breast cancer patients receiving standard radiotherapy compared to those receiving whole - breast hypofractionated irradiation with a concomitant boost. patients met the inclusion criteria were enrolled the study in the department of radiation oncology, tianjin medical university cancer institute and hospital between january 2011 and december 2011. patients were randomly divided into an experimental group (24-day group) and a control group (44-day group) using a random number table. written informed consent was obtained from all patients and the ethics committee of tianjin medical university cancer institute and hospital approved the study protocol. patients were enrolled in the study if they met the following inclusion criteria: patients were diagnosed at an early disease stage (pt1 - 2n0 - 1m0) and underwent breast - conserving surgery. all patients were 18 years of age or older, with a karnofsky performance status (kps) score greater than or equal to 70. silver clips were used to denote tumor volume and help localize radiation, which had to be administered within 1 month of surgery. patients who met any of the following exclusion criteria were not enrolled in the study: any patient who had received radiation therapy in the ipsilateral breast, chest wall, lung, or lymph nodes was excluded from the study. additionally, women diagnosed with either inflammatory breast cancer or bilateral breast cancer or who had received prior breast - conserving surgery or breast reconstruction surgery were excluded. finally, those who experienced breast cancer recurrence were prevented from enrolling in the study. patients had undergone various treatment modalities based on their presentation and tumor characteristics. here, we outline the primary treatment regimens used for all patients in the study. breast - conserving surgery was performed by surgical excision of the primary tumor, with a 23 cm margin of macroscopically normal tissue and an axillary dissection. when the sentinel node biopsy was positive, the axillary lymph nodes were dissected; in all other cases, no dissection of axillary lymph nodes was performed. the dissection of axillary lymph nodes was conducted at either a level i ii or a level i iii, depending on the patient s risk level. during surgery, patients received postoperative chemotherapy if they were axillary lymph node positive or if they were axillary lymph node negative but at a high risk of recurrence (i.e. < 35 years old with a tumor diameter 2 cm, a histological grade of ii iii, signs of vascular invasion, her-2 positive, er / pr negative). a 2-drug chemotherapy regimen was used in this study either pharmorubicin combined with cyclophosphamide or pharmorubicin combined with paclitaxel. there was no difference in the proportion of different chemotherapy regimens between the 2 groups. radiotherapy was started within 1 month of surgery and 3 weeks after the first cycle of chemotherapy. patients in experimental group underwent 24 days of whole - breast 2-field tangent radiotherapy of 43.2 gy in 18 fractions (dose / fraction=2.4 gy) with a concomitant boost to the tumor bed of 50.4 gy in 18 fractions (dose / fraction=0.4 gy). those in control group underwent 44 days of whole - breast 2-field tangent radiotherapy of 45 gy in 25 fractions with a dose / fraction of 1.8 gy and a subsequent boost to the tumor bed of 59 gy in 7 fractions (dose / fraction=2 gy). irradiation was administered to the entire breast tissue and to lymph nodes between the pectoral muscles and lymphatic drainage area of the pectoral wall under the breast. we used 6 mv of x - ray, which was 95% of the prescribed isodose x - ray. the irradiated range of the tumor bed was 12 cm outside of the silver clip. the photon beam energy was set between 6 and 9 mv based on tumor bed depth. patients with tumors that were hormone receptor - positive (er & pr) received endocrine therapy after chemotherapy. patients were followed until september 2013. specifically, follow - up was performed immediately at the end of radiation and 6 weeks, 6 months, 12 months, and 24 months after this time. follow - up observations included a review of patient medical history, physical examination, bilateral breast anteroposterior x - ray examination, chest radiography, mammography, and breast ultrasound. secondary endpoints included acute skin reactions, advanced skin reactions, aesthetic outcome, and hematological toxicity. locoregional recurrence was defined as the area of the breast and the supraclavicular lymph drainage area within the radiation field; definite diagnosis was confirmed by both clinical and imaging examinations. skin adverse reactions (levels l4) were assessed according to american acute and late radiation tumor tissue radiation injury grading standards. aesthetic , including breast edema, skin sag, fibrosis, telangiectasia, scarring, pigmentation, breast size, nipple level, and bilateral symmetry were graded as excellent , good , fair , or poor according to guidelines established by the joint center for radiation therapy (jcrt) and as previously described. data for comparability, adverse reactions, and aesthetic outcomes in the 2 groups were compared and analyzed by chi - square test. all statistical analyses were performed using spss version 16.0 (spss, inc ., chicago, il). patients met the inclusion criteria were enrolled the study in the department of radiation oncology, tianjin medical university cancer institute and hospital between january 2011 and december 2011. patients were randomly divided into an experimental group (24-day group) and a control group (44-day group) using a random number table. written informed consent was obtained from all patients and the ethics committee of tianjin medical university cancer institute and hospital approved the study protocol. patients were enrolled in the study if they met the following inclusion criteria: patients were diagnosed at an early disease stage (pt1 - 2n0 - 1m0) and underwent breast - conserving surgery. all patients were 18 years of age or older, with a karnofsky performance status (kps) score greater than or equal to 70. silver clips were used to denote tumor volume and help localize radiation, which had to be administered within 1 month of surgery. patients who met any of the following exclusion criteria were not enrolled in the study: any patient who had received radiation therapy in the ipsilateral breast, chest wall, lung, or lymph nodes was excluded from the study. additionally, women diagnosed with either inflammatory breast cancer or bilateral breast cancer or who had received prior breast - conserving surgery or breast reconstruction surgery were excluded. finally, those who experienced breast cancer recurrence were prevented from enrolling in the study. patients had undergone various treatment modalities based on their presentation and tumor characteristics. here, we outline the primary treatment regimens used for all patients in the study. breast - conserving surgery was performed by surgical excision of the primary tumor, with a 23 cm margin of macroscopically normal tissue and an axillary dissection. when the sentinel node biopsy was positive, the axillary lymph nodes were dissected; in all other cases, no dissection of axillary lymph nodes was performed. the dissection of axillary lymph nodes was conducted at either a level i ii or a level i iii, depending on the patient s risk level. during surgery patients received postoperative chemotherapy if they were axillary lymph node positive or if they were axillary lymph node negative but at a high risk of recurrence (i.e. < 35 years old with a tumor diameter 2 cm, a histological grade of ii iii, signs of vascular invasion, her-2 positive, er / pr negative). a 2-drug chemotherapy regimen was used in this study either pharmorubicin combined with cyclophosphamide or pharmorubicin combined with paclitaxel. there was no difference in the proportion of different chemotherapy regimens between the 2 groups. radiotherapy was started within 1 month of surgery and 3 weeks after the first cycle of chemotherapy. patients in experimental group underwent 24 days of whole - breast 2-field tangent radiotherapy of 43.2 gy in 18 fractions (dose / fraction=2.4 gy) with a concomitant boost to the tumor bed of 50.4 gy in 18 fractions (dose / fraction=0.4 gy). those in control group underwent 44 days of whole - breast 2-field tangent radiotherapy of 45 gy in 25 fractions with a dose / fraction of 1.8 gy and a subsequent boost to the tumor bed of 59 gy in 7 fractions (dose / fraction=2 gy). irradiation was administered to the entire breast tissue and to lymph nodes between the pectoral muscles and lymphatic drainage area of the pectoral wall under the breast. we used 6 mv of x - ray, which was 95% of the prescribed isodose x - ray. the irradiated range of the tumor bed was 12 cm outside of the silver clip. the photon beam energy was set between 6 and 9 mv based on tumor bed depth. patients with tumors that were hormone receptor - positive (er & pr) received endocrine therapy after chemotherapy. breast - conserving surgery was performed by surgical excision of the primary tumor, with a 23 cm margin of macroscopically normal tissue and an axillary dissection. when the sentinel node biopsy was positive, the axillary lymph nodes were dissected; in all other cases, no dissection of axillary lymph nodes was performed. the dissection of axillary lymph nodes was conducted at either a level i ii or a level i iii, depending on the patient s risk level. during surgery, patients received postoperative chemotherapy if they were axillary lymph node positive or if they were axillary lymph node negative but at a high risk of recurrence (i.e. < 35 years old with a tumor diameter 2 cm, a histological grade of ii iii, signs of vascular invasion, her-2 positive, er / pr negative). a 2-drug chemotherapy regimen was used in this study either pharmorubicin combined with cyclophosphamide or pharmorubicin combined with paclitaxel. there was no difference in the proportion of different chemotherapy regimens between the 2 groups. radiotherapy was started within 1 month of surgery and 3 weeks after the first cycle of chemotherapy. patients in experimental group underwent 24 days of whole - breast 2-field tangent radiotherapy of 43.2 gy in 18 fractions (dose / fraction=2.4 gy) with a concomitant boost to the tumor bed of 50.4 gy in 18 fractions (dose / fraction=0.4 gy). those in control group underwent 44 days of whole - breast 2-field tangent radiotherapy of 45 gy in 25 fractions with a dose / fraction of 1.8 gy and a subsequent boost to the tumor bed of 59 gy in 7 fractions (dose / fraction=2 gy). irradiation was administered to the entire breast tissue and to lymph nodes between the pectoral muscles and lymphatic drainage area of the pectoral wall under the breast. we used 6 mv of x - ray, which was 95% of the prescribed isodose x - ray. the irradiated range of the tumor bed was 12 cm outside of the silver clip. the photon beam energy was set between 6 and 9 mv based on tumor bed depth. patients with tumors that were hormone receptor - positive (er & pr) received endocrine therapy after chemotherapy. patients were followed until september 2013. specifically, follow - up was performed immediately at the end of radiation and 6 weeks, 6 months, 12 months, and 24 months after this time. follow - up observations included a review of patient medical history, physical examination, bilateral breast anteroposterior x - ray examination, chest radiography, mammography, and breast ultrasound. secondary endpoints included acute skin reactions, advanced skin reactions, aesthetic outcome, and hematological toxicity. locoregional recurrence was defined as the area of the breast and the supraclavicular lymph drainage area within the radiation field; definite diagnosis was confirmed by both clinical and imaging examinations. skin adverse reactions (levels l4) were assessed according to american acute and late radiation tumor tissue radiation injury grading standards. aesthetic , including breast edema, skin sag, fibrosis, telangiectasia, scarring, pigmentation, breast size, nipple level, and bilateral symmetry were graded as excellent , good , fair , or poor according to guidelines established by the joint center for radiation therapy (jcrt) and as previously described. data for comparability, adverse reactions, and aesthetic outcomes in the 2 groups were compared and analyzed by chi - square test. all statistical analyses were performed using spss version 16.0 (spss, inc ., chicago, il). in total, 80 patients met the inclusion criteria and were enrolled in the study (experimental group, n=40 ; control group, n=40) (figure 1). the clinical characteristics between the 2 groups were similar, and detailed information is provided in table 1. at a median follow - up of 27 months (range : 2032 months) the 2-year survival rate of both groups was also 100%, and there was no locoregional recurrence. adverse skin reactions (levels 12) experienced by patients in the 2 groups were similar. no skin toxicities higher than grade 3 were detected in any patient during the follow - up period. patients in both groups experienced overall good aesthetic ; the only problems encountered were breast fibrosis and alteration in pigmentation in both groups (3 cases in experimental group and 2 cases in control group for breast fibrosis, 7 cases in both of the 2 groups for alteration in pigmentation). finally, we did not detect any significant differences in hematological toxicity (i.e., neutropenia, levels 12 or platelet decline, level 1). overall, there were no statistically significant differences in any of the examined categories between the 2 groups (table 2). in this study, we examined whether there were any outcome differences between early - stage breast cancer patients receiving standard radiotherapy compared to those receiving whole - breast hypofractionated irradiation with a concomitant boost. we found that there were no significant differences between the 2 regimens in any of the examined outcomes. thus, a shortened whole - breast hypofractionated irradiation schedule with a concomitant boost is as effective as standard radiation and may be a reasonable alternative following breast conservation surgery an earlier study by whelan et al. showed that a significant portion of early - stage breast cancer patients could benefit from accelerated, hypofractionated whole - breast irradiation (ah - wbi). they found that ah - wbi (42.5 gy in 16 fractions over 22 days) was not inferior to standard radiation (50 gy in 25 fractions over 35 days) in terms of acute skin reactions, local recurrence within 10 years, or cosmetic outcome. however, subgroup analysis showed that, compared with standard radiation treatment, ah - wbi had a lower efficiency in high - histological grade patients; the locoregional recurrence over 10 years in patients undergoing ah - wbi compared to those receiving standard radiation treatment was 15.6% and 4.7%, respectively (p=0.01). in 2012, the british columbia cancer center observed a total of 1335 early breast cancer patients with grade 3 disease (t1t2, n0, m0) and compared the local relapse rates between hypofractionated radiotherapy and conventionally fractionated schedules; 252 patients underwent conventional fractionation of 4550 gy in 25 fractions, and 1083 patients received a hypofractionated schedule of 42.544 gy in 16 fractions. the 10-year cumulative incidence of local relapse was 6.9% in the hypofractionated group and 6.2% in the conventionally fractionated group (p=0.99). the data show that the hypofractionated schedule was no worse than conventional fractionation, even for histologic grade 3 breast tumors. importantly, these are not consistent with the findings of the whelan study, previously described. another group compared the 5-year local relapse rates between hypofractionated radiotherapy and conventionally fractionated schedules. from this study showed that hypofractionated radiotherapy did not increase the local relapse rate. overall, both treatment regimens showed good efficacy and aesthetic outcomes. additionally, adverse reactions were acceptable, and the cost of treatment was significantly reduced. taken together, these studies show that hypofractionated radiotherapy following breast - conserving surgery is a feasible and worthwhile option. did not include a concomitant boost to the tumor bed, which may prevent direct comparison to our study. there was a statistically significant difference (p<.0001) in local recurrence at 10-year follow - up (10.2% versus 6.2%). there was also a significant increase in the amount of fibrosis detected in the boost group. in contrast to these 2 outcomes, there was no difference in overall survival between the 2 groups. another study reported that patients receiving a concomitant boost of 14gy to the tumor bed had an increased risk of breast hardening and telangiectasia. however, there were no reported differences in breast appearance, aesthetic outcomes, breast contractures, deformation, edema, or swelling between the 2 groups. recently, raza et al. compared accelerated intensity - modulated radiation therapy (imrt) with a concomitant boost to the tumor bed delivered over 3 or 5 weeks against standard 6-week accelerated radiotherapy with a sequential electron boost. acute complications such as breast pain, fatigue, and dermatitis were significantly less in the 3-week regimen compared to the other treatment schedules (p<0.05). from studies such as these have made whole - breast radiotherapy with a concomitant boost the standard of treatment in china. while our study supports a shorter radiation schedule, it has certain limitations, including the short follow - up time and the limited number of patients enrolled. thus, additional studies with larger sample sizes must be performed to understand the long - term effects of hypofractionated radiation. in , our show that abbreviated hypofractionated radiation is well - tolerated and comparable to longer standard treatment regimens. thus, shortened whole - breast hypofractionated irradiation schedule with a concomitant boost is as effective as standard radiation and may be a reasonable alternative following breast conservation surgery.

adjuvant radiation therapy is commonly administered to breast cancer patients who received breast - conserving surgery. however, lengthy treatment times of standard radiotherapy pose certain challenges. here , we performed a prospective controlled study comparing standard radiation to hypofractionated radiotherapy in terms of efficacy and outcome.material/methodseighty breast cancer patients (tumor stage pt1 - 2n0 - 1m0) who had undergone breast - conservation surgery were randomly divided into 2 groups (40 patients / group). the experimental group received 43.2 gy to the whole breast in 18 fractions for 24 days with a concomitant boost (50.4 gy) to the tumor bed. the control group received 45 gy to the whole breast in 25 fractions for 44 days with a boost to the tumor bed of 59 gy. survival, locoregional recurrence, adverse effects, and aesthetic were all considered for analysis.the following criteria were included as part of study follow - up: local control, survival, adverse skin reactions, cosmetic outcome, and hematological toxicity. at a median follow - up of 27 months (follow - up rate 100%), there were no statistical differences in any of the categories between the 2 groups. the 2-year survival rate of both groups was 100% without any locoregional recurrence. although there was some skin toxicity, these instances were not severe and they cleared on their own within 6 weeks. the most common problems encountered by patients were breast fibrosis and altered pigmentation.a shortened whole - breast hypofractionated irradiation schedule with a concomitant boost is as effective as standard radiation and may be a reasonable alternative following breast conservation surgery.

nearly a century has passed since insulin codiscoverers frederick banting and charles best observed the first patient to receive insulin therapy in 1922, instantly transforming the prognosis of type 1 diabetes (t1d) from a death sentence to a manageable disease. however, the root cause of t1d is still not known, but the processes that lead to the destruction of insulin - producing pancreatic beta - cells have been fairly well elucidated. t1d is now considered an autoimmune disease that causes the gradual, systematic destruction of pancreatic beta - cells within the islets of langerhans. proinflammatory cytokines play a prominent role in the pathophysiology of t1d , but increasing evidence also suggests a significant role for cytokines in islet dysfunction in t2d as well. in this review, i address the substantial differences in the inflammatory environment of the pancreatic islet in t1d versus t2d and then consider alternative models of cytokine exposure that may more accurately reflect the pancreatic environment in t2d, with particular emphasis on the islet. there are notable similarities and differences in the action of cytokines in the development of t1d versus t2d , as shown in figure 1 and described below. in t1d, beta - cells are the direct target of an autoimmune invasion beginning with peri - insulitis and ending in beta - cell death. in t2d , metabolic stress is thought to activate the innate immune system, ing in a chronic inflammatory state marked by increased cytokines, increased islet - associated macrophages, and beta - cell apoptosis. in t1d, autoimmunity is the prime effector of beta - cell destruction. in t2d, a number of metabolic stress factors related to excess nutrients are thought to contribute to beta - cell decline and destruction including glucotoxicity , lipotoxicity , oxidative stress , endoplasmic reticulum (er) stress , amyloid deposition, and inflammation. some evidence suggests that all of these factors may even be connected through inflammasome activation. to bring this discussion full circle, emerging research suggests that some of the putative factors causing beta - cell dysfunction in t2d, most notably er stress and/or oxidative stress, may play a role in antigen presentation to trigger the autoimmune response in t1d. the key proinflammatory cytokines in t1d are interleukin- (il-) 1beta, tumor necrosis factor alpha (tnf - alpha), and interferon - gamma (ifn - gamma). il-1beta is produced by monocytes (leukocytes), macrophages, and other immune cells. tnf - alpha is produced by macrophages, lymphoid, stromal cells, and many other cell types and can exist in membrane - bound as well as in soluble forms. ifn - gamma is primarily produced by natural killer cells and certain types of t cells, although macrophages can also produce ifn - gamma under certain conditions, such as exposure to il-12 and il-18. infiltrating immune cells are thought to secrete these three cytokines in close proximity to the beta - cell at high concentrations in t1d, and these cytokines interact synergistically to inflict cytotoxic effects. although rodent islets may succumb to high concentrations of any of these cytokines, classic studies demonstrated that combinations of these cytokines produced more consistent cytotoxic effects on human islets. in t2d, the metabolic stress of obesity is thought to elevate cytokine production in adipose tissue and other organs to a lesser extent, ing in an overall chronic low - grade inflammatory state. in the next section , key parameters will be examined to define a model of islet exposure to cytokines that may resemble the pancreatic environment more closely during the development of t2d. a number of proinflammatory cytokines are elevated in the general circulation of obese individuals compared to lean individuals, and these increased levels are associated with an increased risk of developing t2d. this low - grade systemic inflammation may also play a direct role in triggering beta - cell dysfunction, particularly in t2d. we have previously reported that cytokines can directly affect aspects of islet function in rodent islets at circulating concentrations in vitro. however, these effects, which are primarily related to intracellular calcium handling, do not appear to impact cell death rate or insulin secretion in normal islets. as shown in figure 2, the t2d - like cytokines, in the range of circulating levels in the blood, do not increase cell death or decrease insulin secretion significantly. the t1d - like concentrations of cytokines necessary to induce cell death are at least 50-fold higher than what is observed in low - grade systemic inflammation in t2d (see also similar published data in). the 5 ng / ml concentration of il-1beta commonly found in the literature is 10 times greater than the t1d - like concentration and 500 times greater than the t2d - like concentration shown in figure 2. this suggests that the use of high concentrations of death - inducing cytokines is not necessarily appropriate for recreating an islet environment related to t2d. to build an appropriate in vitro model of chronic low - grade inflammation that can reasonably mimic the islet environment in t2d in vivo, several questions must be addressed: what cytokines and chemokines are elevated in the systemic circulation in obesity and t2d that can negatively impact islet function?what immune cells are found in or near islets that can secrete cytokines in close proximity?what cytokines are secreted by islet cells themselves in response to damage or stress?what concentration and duration of exposure mimic chronic low - grade inflammation?each of these considerations is discussed in sections 3.13.4. what cytokines and chemokines are elevated in the systemic circulation in obesity and t2d that can negatively impact islet function? what immune cells are found in or near islets that can secrete cytokines in close proximity? circulating levels of several cytokines and chemokines have been identified as potential risk factors for developing t2d, including tnf - alpha, c - reactive protein (crp), monocyte chemoattractant protein-1 (mcp-1), il-1beta, and il-6. il-1beta at high enough concentrations can destroy beta - cells. antagonizing the actions of il-1beta has been shown to have clinical value in partially mitigating the symptoms of t2d , suggesting that il-1beta plays a role in the pathophysiology of the disease. numerous studies have also associated increased levels of il-6 with the development of type 2 diabetes. however, il-6 has also been associated with beneficial effects of exercise (reviewed in) and with improving islet function , ing in a complex and evolving view over the role(s) of il-6 in t2d. a prospective study focused primarily on links between nutrition and cancer generated an intriguing report that identified the combination of both il-1beta and il-6 as key cocontributors to the development of t2d. we reported that serum levels of il-1beta and il-6 in diabetes - prone mice at an age before hyperglycemia developed were 2 - 3 times higher than for age - matched heterozygous control mice, suggesting that low - grade systemic inflammation develops early in the disease process. in addition, many other cytokines and chemokines are increased in obesity including leptin, resistin, il-7, il-8, retinol binding protein- (rbp-) 4, plasminogen activator inhibitor- (pai-) 1, chemokine- (c - x - c motif-) ligand 5 (cxcl5), visfatin, chemerin, and vaspin. other cytokines are decreased with increased obesity including adiponectin, il-10, and omentin. we conducted a 32-plex cytokine detection array of mouse blood serum from leptin - receptor - deficient male bks.cg - dock7m+/+ leprdb / j (db / db) mice and heterozygous controls at different ages to identify additional cytokines and chemokines. many of the 32 cytokines tested were above or below the sensitivity for appropriate detection or were highly variable. however, cxcl1 and cxcl5 were increased significantly in serum at the onset of obesity and t2d. we further showed that exposure to circulating concentrations of these chemokines synergistically produced mild inhibition of islet function and that expression of cxcl1 and cxcl5 increased markedly in islets in response to low - grade inflammation. regarding the classic trio of t1d cytokines, il-1beta appears to also play a role in t2d, but tnf - alpha and ifn - gamma may or may not be as prominent. in our studies, we did not observe elevated serum levels of tnf - alpha in prediabetic mice (ifn - gamma was not examined), and tnf - alpha does not appear to have the same degree of impact as il-1beta and il-6 on the development of t2d in humans. ifn - gamma appears to be elevated in obesity and may play a role in islet dysfunction in t2d. however, at present there is not sufficient evidence that circulating levels of ifn - gamma impact islet function or that ifn - producing immune cells are localized within islets in models of t2d. additional studies are needed to determine the full milieu of obesity - associated cytokines and the extent to which this milieu of circulating cytokines can impact islet function. in addition to the chronic inflammatory state marked by increased circulating cytokines, signs of increased inflammation in t2d have also been observed within the pancreas itself. specifically, increased numbers of islet - associated macrophages have been reported in rodent models of t2d and in humans with t2d. in the db / db mouse model of diabetes, a 4-fold increase in m1 type macrophages was reported as early as 8 weeks of age. macrophages are involved in adaptation and cellular repair as well as in the discrete removal of dead and dying cells. thus, increased islet presence of macrophages could merely be an indicator of more dying cells that must be cleared away. however, macrophages could also be active instigators of destruction by secreting various proinflammatory cytokines in close proximity to islet cells. the potential of macrophage instigation of islet dysfunction has been confirmed by studies showing that palmitate causes macrophage accumulation, increased cytokine / chemokine production within islets, and islet dysfunction, but islet dysfunction is prevented when macrophage accumulation is blocked. these findings suggest that macrophages play an important role in inflammation caused by metabolic stress. in addition to macrophage involvement, recent work also suggests that clusters of differentiation- (cd-) 45-positive leukocytes are also increased in islets from t2d donors, suggesting a possible role of the adaptive immune system in t2d. chronic high levels of glucose, for example, can stimulate il-1beta production in beta - cells. for example, treatment with ifn - gamma + tnf - alpha increases islet expression of il-15, interferon - gamma - induced protein- (ip-) 10, cxcl9, cxcl11, and chemokine (c - c motif) ligand- (ccl-) 20. exposure to free fatty acids, palmitate in particular, can induce islet expression of numerous cytokines and chemokines, including il-1beta, tnf - alpha, il-6, il-8, cxcl1, and mcp-1. the importance of intraislet cytokine production is that cytokine concentrations within islets may be orders of magnitude higher when cytokines are produced by islet cells and/or resident immune cells within the islet in comparison to circulating levels. it should be noted that the increased cytokine expression in many of these studies could not be attributed to any specific cell type(s) within the islet. thus, it is possible that immune cells within the islet could be responsible for most or all of the increased expression of these cytokines, rather than insulin - producing beta - cells, glucagon - producing alpha cell, or other endocrine cell types in the islet. thus, intraislet secretion of cytokines, regardless of the source, may markedly increase the potential concentration of cytokine exposure in islet cells. the net effect of cytokine exposure on tissue depends markedly on factors such as cytokine concentration, duration of cytokine exposure, and the combination of cytokines involved (also called the cytokine milieu). il-1beta, for example, causes islet dysfunction or cell death in a number of studies of chronic exposure , and blockade of the il-1 receptor mitigates many of these effects. however, il-1beta can also enhance insulin secretion , and recent work even suggests il-1beta may play a key role in islet compensation for nutrient overload in the early stages of metabolic disorders. in one early series of studies, both positive and negative impacts of il-1beta were described based on differences and dose and duration of exposure. in the context of t2d, the duration of low - grade inflammation may be a key factor, as cytokines could have compensatory / protective effects initially that become deleterious under chronic conditions. thus, the dose and duration of cytokine exposure , species being tested , and milieu of interacting cytokines are all important factors that contribute to both positive or negative effects on the beta - cell. in addition, synergistic activity among multiple cytokines can alter or amplify signaling pathways , adding an additional layer of complexity to cytokine action. our recent work has focused on elucidating possible differences in response to chronic low - grade inflammation between islets from diabetes - prone and normal (nondiabetes - prone) environments. male db / db mice and heterozygous controls were used at 4 - 5 weeks of age, an age at which the capacity for glucose - stimulated insulin secretion and intracellular calcium responses are similar for both mouse strains. the combination of il-1beta and il-6 significantly impaired islet function in ways that either alone could not. further, islets isolated from prediabetic db / db mice and exposed in vitro to il-1beta + il-6 overnight at approximately circulating levels (in mice) caused a significant decrease in glucose - stimulated insulin secretion, a significant increase in er stress markers (nitric oxide synthase 2 ( nos2), 78-kda glucose - regulated protein (grp78), activating transcription factor 4 (atf4), and dna damage inducible transcript 3 (ddit3), also known as c / ebp homologous protein (chop) ), and increased cell death; these cytokines produced no such effect in heterozygous control islets. further, when nondiabetic control mice were implanted with subcutaneous osmotic mini - pumps containing il-1beta + il-6 to mimic the serum increases found in prediabetic db / db mice, we were able to produce 2 - 3-fold increases in circulating cytokines, thus reproducing cytokine levels observed in low - grade inflammation in obesity. the increased circulating levels of il-1beta + il-6 were insufficient to impact physiology in these normal mice. when compared with saline - implanted controls, however, isolated islets from cytokine - pump mice showed deficiencies in calcium handling and insulin secretion that were similar to cytokine effects on islets in vitro. these in mice suggest that mild increases in circulating cytokines may be sufficient to trigger islet dysfunction leading to islet failure in genetically susceptible individuals. the cytokine signaling pathways may also differ in conditions of low - grade inflammation when compared to classic cytokine cocktails associated with t1d. a microarray study of islet gene expression in response to low concentrations 10 pg / ml il-1beta + 20 pg / ml il-6 produced a large number of gene hits that were apparently not associated with canonical signaling pathways for il-1beta and il-6. several highly cytokine - induced genes related to proteins involved with iron regulation, including steap4 (six - transmembrane epithelial antigen of prostate 4), lipocalin-2 (lcn-2), and hepcidin antimicrobial peptide (hamp). moreover, seven cytokine - sensitive genes were identified for single nucleotide polymorphisms related to the acute insulin response to glucose (airg, a test of islet function) in a genome - wide association scan of a population with a high prevalence for t2d. the seven genes were arap3 (arfgap with rhogap domain, ankyrin repeat, and ph domain 3), f13a1 (coagulation factor xiii, a1 polypeptide), klhl6 (kelch - like protein 6), nid1 (nidogen 1), pamr1 (peptidase domain - containing protein associated with muscle regeneration 1), ripk2 (receptor - interacting protein kinase 2), and steap4. the microenvironment of the pancreatic islet during the development of t2d has yet to be fully elucidated. with regard to inflammatory - mediated processes, several sources of proinflammatory cytokines have been suggested throughout this review: immune - cell - derived cytokines from macrophages and lymphocytes, cytokines derived from inflamed fat tissues or other distal sources that increase cytokine concentration in the general circulation, and cytokines / chemokines derived from peptide - producing islet cells such as alpha - cells or beta - cells (see figure 3). creating accurate in vitro models of the concentration, duration, and combination of key cytokines involved with each of these sources will contribute greatly toward a better understanding of how islets and other organs such as the liver, muscle, fat, and kidney respond to low - grade inflammation. we have made a first attempt at a model of the circulating levels of proinflammatory cytokines utilizing il-1beta and il-6. although the inflammatory environment is dynamic, we focused on cytokines involved prior to the onset of hyperglycemia (the prediabetic stage). several observations served as our rationale for developing this model of circulating cytokines in t2d. first, the combination of these two cytokines is sufficient to significantly increase the risk of developing t2d in humans. second, increased levels of il-1beta and il-6 were observed in db / db mice prior to hyperglycemia or substantial differences in weight (< 10%) compared to control mice. third, exposure to combinations of these cytokines at circulating levels affected islets in unique ways that treatment with individual cytokines could not replicate. these studies collectively point to the combination of 510 pg / ml il-1beta and 1020 pg / ml il-6 as being sufficient to promote islet dysfunction in t2d. to produce these levels of low - grade systemic inflammation in vivo osmotic mini - pumps are ideally designed to produce the small and precise changes in the circulating levels of cytokines that define chronic inflammation. we induced low - grade inflammation in vivo in normal healthy mice loading alzet osmotic mini - pumps (model 1007d, rate 0.5 l / h for 7 days) with 32 g / ml for il-1b plus 4 g / ml for il-6 in saline or saline only controls. we inserted each pump subcutaneously into an incision at the nape of the neck to cause low - grade inflammation in mice without significantly impacting blood glucose or insulin levels. elisa assays confirmed that serum levels of il-1b and il-6 were approximately doubled in mice treated with cytokine mini - pumps versus saline control pumps. islets isolated from cytokine - pump - treated mice showed impaired function compared to saline - pump controls. these findings are detailed in and provide a method for elevating specific circulating cytokines related to chronic low - grade inflammation in t2d, independent of obesity. differences in susceptibility to cytokine - induced damage between diabetes - prone and nondiabetes - prone islets suggest that certain genetic predispositions may render some individuals much more susceptible to the negative impact of proinflammatory cytokines than others. the relatively small increases in circulating cytokines caused by obesity or other chronic inflammatory conditions could conceivably contribute to a plethora of conditions including asthma , rheumatoid arthritis, heart disease, various cancers , polycystic ovarian syndrome , and even mood disorders. thus, the combination of the genetic predisposition of the individual and the dose, duration, and milieu of cytokine exposure may significantly contribute to numerous chronic diseases.

proinflammatory cytokines have been implicated in the pathophysiology of both type 1 diabetes (t1d) and type 2 diabetes (t2d). t1d is an autoimmune disease involving the adaptive immune system responding to pancreatic beta - cells as antigen - presenting cells. this attracts immune cells that surround pancreatic islets (insulitis) and secrete cytokines, such as il-1beta, ifn - gamma, and tnf - alpha, in close proximity to pancreatic beta - cells. in contrast, there is little evidence for such a focused autoimmune response in t2d. instead, the innate immune system, which responds to cellular damage and pathogens, appears to play a key role. there are three major sources of proinflammatory cytokines that may impact islet / beta - cell function in t2d: from islet cells, from increased numbers of intraislet macrophages / immune cells, and from increased circulating levels of proinflammatory cytokines due to obesity, presumably coming from inflamed adipose tissue. these differences between t1d and t2d are reflected by significant differences in the cytokine concentration, duration, and milieu. this review focuses on chronic versus acute cytokine action, cytokine concentrations, and cytokine milieu from the perspective of the pancreatic islet in t2d. we conclude that new cytokine models may be needed to reflect the pathophysiology of t2d more effectively than what are currently employed.

we evaluated 25 fnac specimens from 25 patients with well - documented cases of pl according to serum parathyroid hormone (pth) level, immunohistochemical staining, ultrasonography, and pathology at the korea cancer center hospital from january 2006 to may 2011. cases that presented as a single mass were included, and those that were not confirmed by operation or immunohistochemistry were excluded. clinicoradiologic information, such as signs or symptoms, serum pth level, ultrasonography, and operation records, were obtained from patients' medical records. the aspirated material was smeared onto four glass slides that were immediately fixed in 95% alcohol and subjected to papanicolaou staining. thyroid transcription factor-1 (ttf-1), pth, and chromogranin a were selectively used for immunohistochemistry on cell blocks. diagnostic terminology of " parathyroid lesion " (pl) was used if the cytological findings or immunohistochemistry indicated a parathyroid origin. twenty patients underwent surgery, and five patients did not. in those patients who underwent surgery, all sections of the resection specimens the five patients who did not receive surgery, pl was confirmed by immunohistochemical staining of cell blocks. all the slides were reviewed with a multi - headed microscope by two separate pathologists. the cytological features were categorized as follows: architectural features (scattered naked nuclei, loose clusters, a papillary pattern with a fibrovascular core, tight clusters, and a follicular pattern), nuclear features (anisokaryosis, stippled chromatin, prominent nucleoli, intranuclear pseudoinclusion, and eccentric nuclei) and other features (a well - defined cell border, oxyphilic cytoplasm, colloid - like material, and macrophages). the patient population included six males and 19 females with an age range of 39 to 71 years (mean age, 51 years) (table 1). of the 25 patients, 22 were evaluated for serum pth before the cytological examination. the pl was found at the posterior surface of the thyroid gland in nine patients (36%), inferior to the thyroid gland in seven patients (28%), in the thyroid parenchyma in six patients (24%), and in the paratracheal area in three patients (12%). the clinicoradiologic impression consisted of 11 incidentalomas (44%), eight phpts (32%), four pls (16%), and two metastatic papillary thyroid carcinomas (ptc) (8%). eighteen of the 25 cases (72%) were cytologically diagnosed as pl; the remaining seven patients were diagnosed as follows: two with benign follicular cells (8%), two with adenomatous goiter (8%), two with atypical cells (8%), and one with metastatic carcinoma (4%). when the clinicoradiologic impression suggested pl, the sensitivity of the cytological diagnosis was 86.7% (13/15). in all other cases, a minimally invasive parathyroidectomy was performed in 17 of the 20 patients who underwent surgery, and the remaining three patients received thyroid surgery or neck dissection. eighteen of the 20 cases who received surgery were diagnosed as parathyroid adenoma, and the remaining two were determined to have parathyroid carcinoma. in the aspirates from the five patients who did not receive surgery, pth was expressed in two, and the remaining three aspirates demonstrated positivity for chromogranin a. ttf-1 was negative in all four cases who were examined (fig . the most impressive feature of the parathyroid aspirates was the diversity of the architectural features . most of the smears showed scattered naked nuclei ( 25/25, 100%) and loose clusters (22/25, 88%) in varying degrees. other architectural features included a papillary pattern with a fibrovascular core (17/25, 68%), tight clusters (14/25, 56%), and a follicular pattern (11/25, 44%) (table 2, fig . each aspirate showed a combination of foregoing architectures in varying degrees . in three aspirates, the entire specimen consisted of scattered naked nuclei and closely resembled thyroiditis ( 3/25, 12%). almost all nuclei were centrally located, round to oval and similar in size to red blood cells with a regular nuclear membrane. a small proportion of parathyroid cells showed mild to moderate anisokaryosis in each aspirate (24/25, 96%). prominent nucleoli and intranuclear pseudoinclusions were only detected in one aspirate (1/25, 4%) (table 2, fig . 3). many parathyroid cells had finely granular cytoplasm, and a small portion of cells revealed a well - defined cell border (9/25, 36%) and oxyphilic cytoplasm (9/25, 36%). fragments of hyaline colloid - like material were noted in three aspirates (12%). another two cases (patients nos . 3 and 4) were diagnosed as adenomatous goiter; these patients underwent minimally invasive parathyroidectomy because pl was diagnosed using an intraoperative frozen biopsy. clinical impression and cytological diagnosis were metastatic carcinoma, so neck dissection was performed. the dominant cytological architecture in this case consisted of tight clusters, although other cytological features of pl were present in small proportions. a papillary pattern with a fibrovascular core, anisokaryosis and stippled chromatin were also found. although pl was diagnosed using intraoperative frozen biopsy, the remaining lobe was removed by lobectomy. in patient no. 7, who had simultaneous ptc, the paratracheal mass was clinically thought to be metastatic ptc. the dominant architecture was loose clusters, and other cytological findings were a papillary pattern with scattered naked nuclei, tight clusters, a follicular pattern, anisokaryosis, and stippled chromatin. the patient population included six males and 19 females with an age range of 39 to 71 years (mean age, 51 years) (table 1). of the 25 patients, 22 were evaluated for serum pth before the cytological examination. the pl was found at the posterior surface of the thyroid gland in nine patients (36%), inferior to the thyroid gland in seven patients (28%), in the thyroid parenchyma in six patients (24%), and in the paratracheal area in three patients (12%). the clinicoradiologic impression consisted of 11 incidentalomas (44%), eight phpts (32%), four pls (16%), and two metastatic papillary thyroid carcinomas (ptc) (8%). eighteen of the 25 cases (72%) were cytologically diagnosed as pl; the remaining seven patients were diagnosed as follows: two with benign follicular cells (8%), two with adenomatous goiter (8%), two with atypical cells (8%), and one with metastatic carcinoma (4%). when the clinicoradiologic impression suggested pl, the sensitivity of the cytological diagnosis was 86.7% (13/15). in all other cases, a minimally invasive parathyroidectomy was performed in 17 of the 20 patients who underwent surgery, and the remaining three patients received thyroid surgery or neck dissection. eighteen of the 20 cases who received surgery were diagnosed as parathyroid adenoma, and the remaining two were determined to have parathyroid carcinoma. in the aspirates from the five patients who did not receive surgery, pth was expressed in two, and the remaining three aspirates demonstrated positivity for chromogranin a. ttf-1 was negative in all four cases who were examined (fig . the most impressive feature of the parathyroid aspirates was the diversity of the architectural features . most of the smears showed scattered naked nuclei ( 25/25, 100%) and loose clusters (22/25, 88%) in varying degrees. other architectural features included a papillary pattern with a fibrovascular core (17/25, 68%), tight clusters (14/25, 56%), and a follicular pattern (11/25, 44%) (table 2, fig . each aspirate showed a combination of foregoing architectures in varying degrees . in three aspirates, the entire specimen consisted of scattered naked nuclei and closely resembled thyroiditis ( 3/25, 12%). almost all nuclei were centrally located, round to oval and similar in size to red blood cells with a regular nuclear membrane. a small proportion of parathyroid cells showed mild to moderate anisokaryosis in each aspirate (24/25, 96%). prominent nucleoli and intranuclear pseudoinclusions were only detected in one aspirate (1/25, 4%) (table 2, fig . many parathyroid cells had finely granular cytoplasm, and a small portion of cells revealed a well - defined cell border ( 9/25, 36%) and oxyphilic cytoplasm (9/25, 36%). fragments of hyaline colloid - like material were noted in three aspirates (12%). another two cases (patients nos . 3 and 4) were diagnosed as adenomatous goiter; these patients underwent minimally invasive parathyroidectomy because pl was diagnosed using an intraoperative frozen biopsy. clinical impression and cytological diagnosis were metastatic carcinoma, so neck dissection was performed. the dominant cytological architecture in this case consisted of tight clusters, a papillary pattern with a fibrovascular core, anisokaryosis and stippled chromatin were also found. although pl was diagnosed using intraoperative frozen biopsy, the remaining lobe was removed by lobectomy. in patient no. 7, who had simultaneous ptc, the paratracheal mass was clinically thought to be metastatic ptc. the dominant architecture was loose clusters, and other cytological findings were a papillary pattern with scattered naked nuclei, tight clusters, a follicular pattern, anisokaryosis, and stippled chromatin. the ability to distinguish between pl and thyroid lesions using fnac has important clinicoradiologic significance for the treatment and quality of life of patients.6 in thyroid neoplasm, a more extensive operation, such as a lobectomy, total thyroidectomy, or neck dissection, is required, whereas pl can be selectively removed with minimal incisions.4 therefore, pathologists need to note the cytomorphologic features of pl to ensure accurate diagnoses. in our study, the variable architectural and cytological features of pl were well presented as follows: scattered naked nuclei, loose clusters, a papillary pattern with a fibrovascular core, tight clusters, a follicular pattern, anisokaryosis, stippled chromatin, a well - defined cell border, and oxyphilic cytoplasm. although a few cases showed absolute predominance of one type of architecture, most cases had variable cytological features, each being mixed in varying degrees. absher et al.2 reported that the presence of a pl may be suspected when recognizing a combination of diverse cytological features rather than a single distinct factor. a few attempts were made to distinguish between pl and thyroid lesions by using fnac. absher et al.2 recommended determining certain characteristic features of pl, including cohesive cellular fragments admixed with numerous single naked nuclei, absent or inconspicuous nucleoli, round to oval uniform nuclei, smaller nuclei than thyroid nuclei, anisokaryosis, and hyperchromatic chromatin rather than finely granular chromatin. dimashkieh and krishnamurthy6 documented that smaller nuclei than thyroid follicular cells, bare nuclei, stippled chromatin, and a prominent vascular network with epithelial cells indicated parathyroid origin. the papillary pattern with a fibrovascular core presented in our study corresponded well with the prominent vascular network and the attached epithelial cells as well as the specific " spot of fire " hypervascularity pattern of the parathyroid gland observed on the color doppler sonogram.4 past studies have stated that colloid - like substances and macrophage accumulation are distinguishing cytological features of thyroid lesions from pl.1,7,8 however, recent studies have revealed that they also can be seen in pl.2,3,6 similarly, colloid - like substances were observed in three of our cases (12%), even though macrophages were not detected. there was one case with intranuclear pseudoinclusion, which postoperatively proved to be a parathyroid carcinoma. however, intranuclear pseudoinclusion has also been described in parathyroid tumors,9,10 and it was noted in just a few cells in this case. that particular patient had a history of total thyroidectomy due to ptc, and there was no evidence of ptc recurrence. clinicoradiologic features and ancillary tests, such as immunohistochemical testing, can contribute to the correct diagnosis of pl.2,3,6 the sensitivity of cytological diagnosis varied substantially depending upon available clinicoradiologic information, such as serum pth, ultrasonography, and location. an unusual location and other radiologic findings of pl can increase diagnostic difficulty. in our series, six of 25 pls (24%) this number is higher than the previously reported incidence of 53 of 4,868 (1%) parathyroid adenomas.11 in general, there are no distinguishing cytological features between parathyroid adenoma and hyperplasia.2,6 therefore, it is recommended that pl is used in the routine cytological diagnosis if an aspirate suggests a parathyroid origin.2 however, a few studies have indicated that there might be distinguishing cytological features between parathyroid adenoma and hyperplasia.1,12,13 in , fnac is a very useful method for the diagnosis of pl, although a few reports have mentioned the limitations of parathyroid fnac, such as its low sensitivity, smear inadequacy, and contamination with thyroid tissue.3,8,13 on the basis of the findings from previous reports as well as our study, common cytological architectures of pl are scattered naked nuclei, loose clusters, prominent vasculature with epithelial cells resembling a papillary pattern, tight clusters, and a follicular pattern. common cellular features included anisokaryosis, stippled chromatin, a well - defined cell border, oxyphilic cytoplasm, and smaller nuclei than thyroid follicular cells. minor features include intranuclear pseudoinclusions, colloid - like material, prominent nuclei, and eccentric nuclei. fnac of the parathyroid can easily be confused with that of the thyroid because of their overlap in cytological features. although no single cytomorphologic characteristic can be used as a diagnostic tool,2 a combination of cytological parameters and clinicoradiologic findings should be used to improve the diagnostic sensitivity of pl.

there has been an increase in the use of fine needle aspiration cytology (fnac) for the diagnosis of parathyroid lesions (pls). differentiation between a thyroid lesion and a pl is not easy because of their similar features. we reviewed parathyroid aspirates in our institution and aimed to uncover trends in diagnostic criteria.methodswe selected 25 parathyroid aspirates (from 6 men and 19 women) confirmed surgically or immunohistochemically from 2006 to 2011.major architectural findings of pls include scattered naked nuclei, loose clusters, a papillary pattern with a fibrovascular core, tight clusters, and a follicular pattern. these architectures were commonly admixed with one another. cytological features included anisokaryosis, stippled chromatin, a well - defined cell border, and oxyphilic cytoplasm. eighteen of the 25 patients were diagnosed with pl using fnac. seven patients had been misdiagnosed with atypical cells (n=2), benign follicular cells (n=2), adenomatous goiter (n=2) and metastatic carcinoma (n=1) in fnac. using clinicoradiologic data, the sensitivity of the cytological diagnosis was 86.7%. the cytological sensitivity decreased to 50% without this information.fnac of pl is easily confused with thyroid lesions. a combination of cytological parameters and clinical data will be required to improve the diagnostic sensitivity of pls.

keratoconus (kc) is the most common primary human degenerative corneal disease with a prevalence of around 1 in 2000 worldwide. it is bilateral, asymmetric, and progressive, leading to corneal thinning and irregularity. onset primarily occurs in the 2nd decade of life and is associated with significant decreasing visual function and morbidity. kc is the main indication recorded for corneal grafts in australia, and currently its progression can only be halted through surgical interventions including collagen cross - linking that stiffens the cornea using riboflavin and uva. more recently a surgical procedure was developed transplanting isolated bowman's layer from donor corneas to kc eyes as a further late - stage intervention. the histopathology of kc is well described and includes epithelial and stromal thinning within the apical cone region, breaks in the bowman's layer, focal fibrosis, and anterior stromal keratocyte apoptosis. recent evidence indicates a role for inflammation in the disease, with increased recruitment of inflammatory cells (e.g., macrophages, lymphocytes, and antigen presenting cells) and inflammatory markers such as interleukin-1 (il-1) and transforming growth factor - beta (tgf-) observed in kc corneal tissue sections. increased expression of inflammatory markers such as interleukin-6 (il-6) , tumour necrosis factor alpha (tnf-), and matrix metalloproteinase 9 (mmp-9) has also been found in tears collected from kc patients compared to controls. furthermore, a recent review examining the biochemical changes in kc proposed a two - hit hypothesis with a genetic predisposition to the corneal disease and a second hit that may induce abnormalities of inflammatory components. single nucleotide polymorphism (snp) refers to a change in a single nucleotide within a dna sequence and is the most common type of genetic variation observed in the human genome. two parallel genome - wide association studies identifying potential snps associated with kc, using independent sample cohorts, reported a significant association between kc and the hepatocyte growth factor (hgf) gene, identifying two single nucleotide polymorphisms (snps ; rs3735520 and rs17501108) in the promoter region. also examined hgf protein abundance in the serum of controls correlating to the rs3735520 genotype and found a significant increase in hgf serum protein associated with the minor allele t. hgf is a pleiotropic growth factor that activates the hgf / c - met pathway after binding to its receptor, mesenchymal - epithelial transition factor (c - met / met). once activated, downstream pathways such as mitogen - activated protein kinase (mapk) cascades, pi3k - akt axis or janus kinase / signal transducers, and activators of transcription (jak / stat) pathways may be activated. for example, together with mmp-1, hgf is reported to initiate human corneal epithelial cell migration in vitro, and exogenous hgf has been found to promote the proliferation of both corneal epithelial and endothelial cells. in injured rabbit corneas, wilson et al. reported an obvious increase of hgf mrna expression in keratocytes and c - met mrna expression in epithelial cells compared to unwounded corneas, suggesting that the hgf / c - met pathway plays a role in corneal wound healing. studying bovine corneal wound healing in organ culture models, carrington and boulton showed that hgf delayed epithelial layer formation, together with increased differentiation of keratocytes to myofibroblasts, compared with untreated and keratinocyte growth factor (kgf) treated corneas. the expression of hgf and c - met proteins in human kc corneas has not been investigated to date. one study has reported increased serum hgf expression for at least the minor allele of hgf snp rs3735520, associated with increased potential for developing kc. as a first step in assessing the role of hgf protein and its receptor (c - met) in kc, we used corneal buttons from patients with severe kc and control human corneas to compare and examine the distribution and expression of these proteins. kc corneal tissue buttons (vision eye institute, chatswood, nsw australia) and donor corneas (lions new south wales ( nsw) eye bank ) were obtained with consent and approval from the sydney eye hospital human research ethics committee (hrec 2013/1041). normal donor corneas were obtained from the lions nsw eye bank following appropriate consent and hrec approval. ten corneal buttons were collected from kc patients (age range from 18 to 32 years) undergoing corneal transplantation at vision eye institute. all kc patients were diagnosed on the basis of clinical signs and corneal topography and were classified as kc grade 4 (most severe stage) (table 1). six normal donor corneas (age range 53 to 83 years) were obtained from the lions nsw eye bank (table 2). kc corneal buttons (~8 mm diameter), with the cone apex location marked, were fixed in 10% neutral buffered formalin (nbf). all specimens were paraffin embedded and cut at 6 m. sections were collected on super - frost plus slides (menzel - glaser, saarbruckener, germany) and dried before use. , sections were incubated in 0.01 m citrate buffer (ph 6) at 85c for 10 minutes, cooled to 40c, and rinsed in tris - buffered saline (tbs, ph 7.4) with 0.1% tween-20 (tbst). sections were incubated at room temperature (rt) in 5% bovine serum albumin (bsa) in tbst for 30 minutes, followed by incubation overnight at 4c in hgf or c - met primary antibodies, or appropriate negative controls (mouse or rabbit igg) (table 3). after overnight incubation , sections were washed in tbst and incubated in either goat anti - mouse alexa fluor 488 or donkey anti - rabbit alexa fluor 488. for co - immunolabelling experiments, a separate series of slides were prepared and hgf visualised with alexa fluor 488 and c - met visualised with alexa fluor 594, combined with nuclear hoechst stain (table 3). immunolabelling was repeated at least twice per specimen for each antibody, and appropriate ig controls were included for each experiment. all slides were mounted in 20% glycerol / pbs, coverslipped, sealed with nail varnish, and viewed using a zeiss lsm700 scanning laser confocal microscope and image software (zen 2011, carl zeiss microimaging ghbh, jena, germany). where more than one colour was to be detected, multichannel excitation bleed - through was minimized using fluorochromes with separated peak excitations. emission bleed - through was minimized by multitracking, where signal crosstalk between neighbouring channels was corrected by performing a sequential image capture routine. semiquantitative grading of single - immunolabelled sections was used to assess the intensity and distribution of hgf and c - met immunoreactivity in the corneal epithelium, stroma, and endothelium. grading for kc buttons was made in the region adjacent to the cone (adj) and for control corneas in a similar central corneal region. immunolabelling of the thinned cone area of kc buttons was examined in each specimen but was not graded for comparison with controls because of the obviously altered morphology (only 1 - 2 epithelial layers present). the grading scale used was based on the intensity of the immunofluorescence (0 = no staining, 0.5 = very weak, 1 = weak, 2 = moderate, and 3 = strong) and the percentage (%) area immunolabelled (0 = 0%, 1 = 1% to 10%, 2 = 11% to 50%, and 3 > 50%) as described previously. a final grade of 0 to 6 (intensity + % area immunolabelled) was then given for each specimen and this data is used to generate frequency histograms for hgf and c - met immunostaining in kc and control specimens, respectively. all kc corneas showed a thickened epithelium adjacent to the more central cone region (figures 1(a) and 2(a) ), compared to similar regions in the control corneas (figures 1(c) and 2(c) ), as we previously reported. only one to two layers of flattened epithelium were observed within the cone region (figures 1(b) and 2(b) ). weak and more evenly distributed cytoplasmic immunostaining for hgf and c - met was detected in the epithelium of control corneas (figures 1(c) and 2(c) ). in kc corneas, moderate - to - strong cytoplasmic immunostaining was seen within the basal and wing cell epithelial layers adjacent to the cone region (green fluorescence, figures 1(a) and 2(a) ). only weak - to - moderate immunostaining of hgf and c - met was detected in the kc cone region (figures 1(b) and 2(b) ). co - immunolabelling for hgf and c - met showed that c - met (red) was primarily localised in the epithelium of both control and kc corneas (figure 3). hgf (green) was primarily localised within the stroma (both keratocytes and extracellular matrix) of control and kc corneas (figure 3). weak and relatively uniform immunostaining was observed in similar regions from control corneas for hgf and c - met (figure 3(a) ). however, for kc corneas, increased hgf staining (green) was detected in the basal epithelium adjacent to the cone region and colocalised with increased c - met staining (red) (figure 3(d) ). limbal areas of control corneas showed slightly stronger stromal immunostaining of hgf compared to the central region of control corneas (figure 3(b) ). semiquantitative grading of hgf and c - met immunostaining in kc samples was increased overall compared to the control corneas for similar regions, as indicated by the skewed distribution of the frequency histograms (figures 4(a) and 4(b) ). overall, a greater proportion of kc corneas showed > grade 3 immunostaining for both hgf and c - met (kc : 70% and 90%, resp . , versus control : 16% and 66% resp .) (figures 4(a) and 4(b) ). despite the importance of the hgf / c - met pathway in regulating a number of key cellular activities, little is known about its potential role in normal or kc human corneas. as a first step to understand the possible role(s) of this pathway, we examined patterns of hgf and c - met protein expression in both control and kc corneas. higher levels of hgf and c - met immunostaining were detected in the basal epithelium adjacent to the kc cone, compared to the weaker and more uniform epithelial staining pattern seen in control corneas. in control corneas, we found hgf expression was more intense in the stroma compared to the epithelium. this is consistent with previous studies showing low level hgf mrna expression in the human corneal epithelium compared to keratocytes and endothelium. the surface of normal rabbit corneal epithelium was reported to show only low level hgf protein. our immunostaining experiments detected stronger c - met (hgf receptor) staining in epithelium compared to keratocytes in control corneas. this is consistent with c - met mrna findings, which showed that human corneal epithelium, keratocytes, and endothelium all expressed c - met, but with highest mrna expression in epithelium. these observations suggest that secreted keratocyte hgf may preferentially bind to the epithelium that expresses higher levels of c - met (hgf receptor), compared to keratocytes that express low levels of c - met, thus potentially regulating key epithelial cellular activities such as cell proliferation. hgf is reported to be involved in two major processes, cell proliferation and migration, and inflammatory - related signalling cascades. hgf is a potent enhancer for corneal epithelial cell proliferation and migration in vivo, and increased hgf and c - met mrna expressions have been detected during corneal wound healing. increased hgf protein expression has been detected in the whole corneal epithelium, keratocytes, and tears of wounded compared to unwounded rabbit corneas. hgf mrna expression was also upregulated in the lacrimal gland when studying injured compared to unwounded rabbit corneas. no difference in c - met immunostaining was detected between wounded and unwounded rabbit corneas. in secondary this was suggested to be most likely due to normal tissue repair pathways already being damaged or compromised, implying involvement of hgf in tissue regeneration. these studies also suggested that lacrimal gland and keratocyte - derived hgf are closely associated with corneal tissue repair. in kc corneal epithelium , we observed increased hgf in the region adjacent to the cone, as well as increased c - met; however it remains to be determined whether the increased expressions of hgf and c - met observed are directly involved in kc pathogenesis or are secondary responses. poorly regulated and overexpressed hgf may be detrimental to tissues, related to the involvement of hgf in inflammation. for example, delayed formation of the epithelium layer and increased formation of stromal myofibroblasts have been detected during rabbit corneal wound healing, for corneas treated with recombinant hgf. applying recombinant hgf to corneas of mice with pseudomonas aeruginosa keratitis also worsened the disease progression, with a significantly higher grade of corneal opacity and thinning compared to pbs - treated corneas. in vitro treatment of corneal stromal keratocytes with proinflammatory interleukin- (il-) elevated corneal hgf expression also enhanced proinflammatory cytokines and decreased anti - inflammatory cytokines in pseudomonas aeruginosa keratitis in mice, most likely under the control of inflammatory cytokines and cell growth kinases. , emerging evidence clearly indicates that inflammation - related processes within the epithelium and stroma are involved in the pathogenesis of kc. significant increases in proinflammatory molecules such as il-6, il-4, il-5, il-8, and il-12 , mmp-1, mmp-3, mmp-7, and mmp-13, and chemokine c - c motif ligand 5 (ccl5) and significantly decreased levels of lactoferrin, serum albumin, and secreted iga (siga) have been reported in kc compared to control tears. keratocytes in kc are reported to express more il-1 receptors than controls, and one group has proposed that keratocyte apoptosis observed in kc may be induced by the binding of il-1 receptors secondary to increased levels of il-1 associated with epithelial trauma, for example, due to eye - rubbing. most recently, increased mrna expression of mmp-9 and its inducer proteins il-6 and tnf- were detected in kc corneal epithelium in an indian cohort (> 100 patients) compared to healthy controls (n = 20), and tear protein levels of mmp-9 and il-6 were also increased in kc. treatment with topical cyclosporine a (an immunosuppressant) in a small group of kc patients (n = 20) reduced the tear levels of mmp-9 and appeared to halt the progression of kc, consistent with the involvement of inflammation in kc pathogenesis. the link between hgf variants and kc susceptibility was first reported by burdon et al., and then confirmed in an independent european cohort which replicated the association of snp rs3735520 and detected a new hgf snp rs2956540. in addition, an independent study of an australian population of european descent (n = 830) focused on the hgf locus and detected a different snp (rs4954218) significantly associated with kc. however, a study of rs3735520 implicated a possible regulatory effect of this snp for hgf protein expression in serum from kc patients. as the snps identified to date are all located in the noncoding region of the gene (rs3735520, rs17501108, and rs1014091 in the upstream of hgf and rs2286194 in the intron between exon 8 and exon 9), it is likely that they will affect gene expression through mechanisms such as rna splicing, transcription factor binding, and mirna regulation. our provide evidence of increased hgf protein in kc epithelium compared to control corneal epithelium; however the role of the reported hgf snps in the increased protein expression is unclear and will be further investigated. previous studies showed an independent, repeatable association between certain snps and hgf in kc. the snp variations detected were located in the noncoding region of hgf consistent with a role for these snps in the regulating hgf expression, and increased serum hgf expression associated with the minor snp allele has been reported. in the current study, we showed increased hgf protein expression within kc corneal epithelium. taken together previous snp studies, these observations indicate that the hgf / c - met pathway may be involved in the pathogenesis of kc. further studies investigating how the hgf / c - met pathway may be altered in kc, including the associations between snps and protein expression and the role of inflammation requires further investigation. studies on the downstream signalling pathways regulated by hgf / c - met, such as mapk cascades, the pi3k - akt axis, and the jak / stat pathway may help to identify the potential role of this pathway in kc and in normal corneal homeostasis.

keratoconus (kc) is a progressive degenerative inflammatory - related disease of the human cornea leading to decreased visual function. the pathogenesis of kc remains to be understood. recent genetic studies indicate that gene variants of an inflammation - related molecule, hepatocyte growth factor (hgf), are associated with an increased susceptibility for developing kc. however hgf protein expression in kc has not been explored. in this initial study, we investigated late - stage kc and control corneas for the expression of hgf and its receptor mesenchymal - epithelial transition factor (c - met / met). kc buttons (~8 mm diameter) (n = 10) and whole control corneas (n = 6) were fixed in 10% formalin or 2% paraformaldehyde, paraffin embedded and sectioned. sections were immunolabelled with hgf and c - met antibodies, visualised using immunofluorescence, and examined with scanning laser confocal microscopy. semiquantitative grading was used to compare hgf and c - met immunostaining in kc and control corneas. overall, kc corneas showed increased hgf and c - met immunostaining compared to controls. kc corneal epithelium displayed heterogeneous moderate - to - strong immunoreactivity for hgf and c - met, particularly in the basal epithelium adjacent to the cone area. taken together with the recent genetic studies, our further support a possible role for hgf / c - met in the pathogenesis of kc.

smoking generates oxidative stress in the lungs and is the principal risk factor for development of lung cancer and chronic obstructive pulmonary disease (copd). detoxification and elimination processes of noxious substances present in cigarette smoke are important for both disease prevention and progression, yet little is known on these processes in the lung so far. proteins of the atp - binding cassette (abc) superfamily such as the multidrug resistance - associated protein 1 (mrp1) may play a role, since they protect against oxidative stress and other xenobiotics (cole et al 1992). substrates for mrp1 are glutathione, glucuronate, and sulfate conjugates and unconjugated compounds in presence of glutathione, eg, tobacco - specific nitrosamines (leslie et al 2001). interestingly, the lung and trachea and other tissues with a barrier function highly express several abc transporters (langmann et al 2003). especially mrp1 is expressed at high levels in human lung tissue (van der deen et al 2005), mainly at the basolateral side of bronchial epithelium (brechot et al 1998 ; scheffer et al 2002). we observed that mrp1 expression is diminished in bronchial epithelial cells of copd patients (van der deen et al 2006) supporting the hypothesis that lower functional mrp1 activity is related to copd development. so far, copd is recognized as a relentlessly progressive disease in which only smoking cessation reduces the accelerated lung function decline. there is no cure for copd, yet recently it has been shown that some drugs are beneficial in the disease management. inhaled corticosteroids and long - acting beta - agonists such as budesonide and formoterol reduce the number of exacerbations in copd (alsaeedi et al 2002 ; sutherland et al 2003) and their combination has been shown to be very effective (calverley et al 2003). treatment of copd patients with the anticholinergic drug ipratropium bromide in a small improvement of lung function, yet does not influence the long - term decline in mild copd (anthonisen et al 1994). studies on oral use of the anti - mucolytic drug n - acetylcysteine (nac) have provided contradictory (stey et al 2000 ; decramer et al 2005). in contrast to the extensive knowledge on chemotherapeutic drugs as substrates for mrp1, limited data are available on the effect of pulmonary drugs on the functional expression of mrp1 (hamilton et al 2002). nac induces higher cellular glutathione levels and in this way can protect against oxidative stress that may indirectly affect mrp1 function (akan et al 2005) but no information is available about budesonide, formoterol, and ipratropium bromide in this respect. in the present study, we questioned whether medications commonly prescribed to copd patients affect mrp1-mediated transport. therefore, we analyzed the direct in vitro effects of budesonide, formoterol, ipratropium bromide, and nac on mrp1 by means of functional flow cytometry in immortalized human bronchial epithelial cells. bovine serum albumin (bsa, fraction v), minimal essential medium (mem, supplemented with earle s salts and l - glutamine) and rpmi 1640 medium (supplemented with 25 mm hepes and l - glutamine) were purchased from invitrogen life technologies (breda, the netherlands). carboxyfluorescein diacetate (cfda), ipratropium bromide, nac and propidium iodide (pi) were obtained from sigma - aldrich bv (zwijndrecht, the netherlands). budesonide (pulmicort) was obtained from astrazeneca bv (zoetermeer, the netherlands), formoterol fumarate dihydrate from astra draco ab (lund, sweden), ethylenedinitrilo tetraacetic acid disodiumsalt dihydrate (edta) from merck (darmstadt, germany), fetal calf serum (fcs) from bodinco bv (alkmaar, the netherlands), vitrogen from nutacon (leimuiden, the netherlands), and mk571 from omnilabo (breda, the netherlands). the human bronchial epithelial cell line 16hbe14o, immortalized with psvori plasmid transfection, dc gruenert (california pacific medical center research institute ; san francisco, ca) (cozens et al 1994). , cells were washed twice with phosphate buffered saline (pbs : 6.4 mm na2hpo4, 1.5 mm kh2po4, 0.14 mm nacl, 2.7 mm kcl, ph = 7.4) with 0.5 mm edta. 16hbe14o cells were grown on tissue culture plastics coated with vitrogen (30 g / ml) and bsa (10 g / ml). to determine mrp1-mediated transport, cells were incubated with 0.1 m cfda as described previously (van der kolk et al 1998) with slight modifications. cfda is intracellularly converted to carboxyfluorescein (cf) which is a fluorescent mrp1 substrate. to establish the effect of copd drugs on mrp1-mediated activity, 1 10 cells were incubated in 0.5 ml rpmi 1640 medium without fcs (37 c, 5% co2) with cfda and with or without the addition of drugs for 1 hour. mk571 (20 m) was used as a positive control for inhibition of mrp1 activity (van der kolk et al 1998) and always showed strong inhibition (more than 10-fold) of mrp1-mediated efflux of cf. budesonide and formoterol were added in concentrations of 10 m to 10 m. these drugs were dissolved in 96% ethanol and dimethyl sulfoxide (dmso), respectively. in part of the experiments , cells were simultaneously incubated with budesonide and formoterol. when budesonide and formoterol are combined in one inhaler (eg, symbicort, astrazeneca), the budesonide concentration is ~18- to 35-fold higher than the concentration of formoterol; therefore we added concentrations of either drug with a comparable proportion. ipratropium bromide was added in concentrations from 10 m to 2 10 m and nac from 10 m to 1.6 10 m and these drugs were dissolved in pbs and water respectively. cells were pelleted for 15 seconds at 12,000 g and resuspended in ice - cold rpmi medium without fcs. drug or mk571 was added a second time in appropriate concentrations for 1 hour, this time without substrate cfda (37 c, 5% co2). after pelleting, cells were put on ice to stop efflux of substrate and were resuspended in 350 l rpmi medium with 0.1 g / ml pi to distinguish dead from living cells. fluorescence of cf was analyzed with a facscalibur flow cytometer (becton dickinson medical systems ; franklin lakes, ny, usa).; topsham, me, usa ) was used to calculate mean fluorescence intensity (mfi) values. all measurements were corrected for the negative control (medium alone) and for incubation with the solvents 96% ethanol and dmso if appropriate. the percentage of dead cells (pi positive population) did not increase with all tested drug concentrations. none of the tested drugs caused autofluorescence (fluorescence without cfda and pi incubation). data are expressed as the mean standard error of the mean (sem). the paired student s t - test (two - tailed) was used to calculate modulating effects of the drug under study compared to control. bovine serum albumin (bsa, fraction v), minimal essential medium (mem, supplemented with earle s salts and l - glutamine) and rpmi 1640 medium (supplemented with 25 mm hepes and l - glutamine) were purchased from invitrogen life technologies (breda, the netherlands). carboxyfluorescein diacetate (cfda), ipratropium bromide, nac and propidium iodide (pi) were obtained from sigma - aldrich bv (zwijndrecht, the netherlands). budesonide (pulmicort) was obtained from astrazeneca bv (zoetermeer, the netherlands), formoterol fumarate dihydrate from astra draco ab (lund, sweden), ethylenedinitrilo tetraacetic acid disodiumsalt dihydrate (edta) from merck (darmstadt, germany), fetal calf serum (fcs) from bodinco bv (alkmaar, the netherlands), vitrogen from nutacon (leimuiden, the netherlands), and mk571 from omnilabo (breda, the netherlands). the human bronchial epithelial cell line 16hbe14o, immortalized with psvori plasmid transfection, was kindly provided by dr. dc gruenert (california pacific medical center research institute ; san francisco, ca) (cozens et al 1994). cells were cultured in mem supplemented with 10% heat inactivated fcs. before trypsinization, cells were washed twice with phosphate buffered saline (pbs : 6.4 mm na2hpo4, 1.5 mm kh2po4, 0.14 mm nacl, 2.7 mm kcl, ph = 7.4) with 0.5 mm edta. 16hbe14o cells were grown on tissue culture plastics coated with vitrogen (30 g / ml) and bsa (10 g / ml). to determine mrp1-mediated transport, cells were incubated with 0.1 m cfda as described previously (van der kolk et al 1998) with slight modifications. cfda is intracellularly converted to carboxyfluorescein (cf) which is a fluorescent mrp1 substrate. to establish the effect of copd drugs on mrp1-mediated activity, 1 10 cells were incubated in 0.5 ml rpmi 1640 medium without fcs (37 c, 5% co2) with cfda and with or without the addition of drugs for 1 hour. mk571 (20 m) was used as a positive control for inhibition of mrp1 activity (van der kolk et al 1998) and always showed strong inhibition (more than 10-fold) of mrp1-mediated efflux of cf. budesonide and formoterol were added in concentrations of 10 m to 10 m. these drugs were dissolved in 96% ethanol and dimethyl sulfoxide (dmso), respectively. in part of the experiments , cells were simultaneously incubated with budesonide and formoterol. when budesonide and formoterol are combined in one inhaler (eg, symbicort, astrazeneca), the budesonide concentration is ~18- to 35-fold higher than the concentration of formoterol; therefore we added concentrations of either drug with a comparable proportion. ipratropium bromide was added in concentrations from 10 m to 2 10 m and nac from 10 m to 1.6 10 m and these drugs were dissolved in pbs and water respectively. cells were pelleted for 15 seconds at 12,000 g and resuspended in ice - cold rpmi medium without fcs. drug or mk571 was added a second time in appropriate concentrations for 1 hour, this time without substrate cfda (37 c, 5% co2). after pelleting, cells were put on ice to stop efflux of substrate and were resuspended in 350 l rpmi medium with 0.1 g / ml pi to distinguish dead from living cells. fluorescence of cf was analyzed with a facscalibur flow cytometer (becton dickinson medical systems ; franklin lakes, ny, usa). the winlist 5.1 program (verity software house inc . ; topsham, me, usa) was used to calculate mean fluorescence intensity (mfi) values. all measurements were corrected for the negative control (medium alone) and for incubation with the solvents 96% ethanol and dmso if appropriate. the percentage of dead cells (pi positive population) did not increase with all tested drug concentrations. none of the tested drugs caused autofluorescence (fluorescence without cfda and pi incubation). data are expressed as the mean standard error of the mean (sem). the paired student s t - test (two - tailed) was used to calculate modulating effects of the drug under study compared to control. budesonide increased the accumulation of cf at concentrations of 10 m and 10 m with 26% and 97%, respectively. formoterol had a small yet significant effect on the accumulation of cf at 10 m, but at higher concentrations this effect was not significant (figure 1b). copd patients are often treated with a combined therapy of budesonide and formoterol. to address the question whether the effect of budesonide is altered by addition of formoterol , we incubated the highest concentration of budesonide (10 m) with increasing concentrations of formoterol. with this approach, the budesonide - induced cf accumulation decreased in a dose - dependent manner; with 10 m, 10 m, and 10 m formoterol, respectively, 198%, 124%, and 58% compared with the control (figure 2). ipratropium bromide increased cf accumulation at relatively low concentrations (10 m to 10 m) with a maximum of 24% at 10 m (figure 3a). increasing the concentration of ipratropium bromide to 2 10 m potentiated the cf efflux (56% compared with control), suggesting that mrp1-mediated activity is stimulated rather than inhibited with ipratropium bromide. similar were obtained with nac, ie, cf accumulation was reduced in a concentration - dependent manner (57% at 8 10 m) (figure 3b). increasing the nac concentrations to 1.6 budesonide increased the accumulation of cf at concentrations of 10 m and 10 m with 26% and 97%, respectively. formoterol had a small yet significant effect on the accumulation of cf at 10 m, but at higher concentrations this effect was not significant (figure 1b). copd patients are often treated with a combined therapy of budesonide and formoterol. to address the question whether the effect of budesonide is altered by addition of formoterol , we incubated the highest concentration of budesonide (10 m) with increasing concentrations of formoterol. with this approach, the budesonide - induced cf accumulation decreased in a dose - dependent manner; with 10 m, 10 m, and 10 m formoterol, respectively, 198%, 124%, and 58% compared with the control (figure 2). ipratropium bromide increased cf accumulation at relatively low concentrations (10 m to 10 m) with a maximum of 24% at 10 m (figure 3a). increasing the concentration of ipratropium bromide to 2 10 m potentiated the cf efflux (56% compared with control), suggesting that mrp1-mediated activity is stimulated rather than inhibited with ipratropium bromide. similar were obtained with nac, ie, cf accumulation was reduced in a concentration - dependent manner (57% at 8 10 m) (figure 3b). increasing the nac concentrations to 1.6 the membrane protein mrp1 is highly expressed in human lung tissue and may protect the airways against damage induced by cigarette smoking (scheffer et al 2002). this study shows for the first time the direct influence of pulmonary drugs on mrp1 functional activity, drugs that have already proven their effectiveness in the clinical management of copd. since mrp1 function is most likely cytoprotective in lung cells, we argued that modulation of its function with pulmonary drugs might induce either a positive or negative effect in copd with long - term treatment. this implies either that budesonide is an mrp1 substrate or that it acts on mrp1 function in another way. budesonide is intracellularly esterified to a fatty acid which is thought to be the mechanism of its prolonged activity after a single dose (oconnell 2003). mrp1 has been shown to be capable of rather slow outward transport of phospholipids, phophatidylcholine and sphingomyelin, and phospholipid analogues in the presence of oxidized glutathione and atp in human erythrocytes and epithelial cells (dekkers et al 2000). it is tempting to speculate that esterified budesonide could behave like a phospholipid analogue with a polar head and two fatty acid tails and is then outwards transported by mrp1. as such, budesonide can compete with other mrp1 (physiological) substrates and in that way affect the functional activity of mrp1. in addition, budesonide is also able to affect the transcriptional regulation of mrp1, as shown by diminished expression of mrp1 in the lung epithelial cell line calu-1 after long - term incubation with budesonide (10 m) (bandi and kompella 2002). taken together, the data suggest that treatment with budesonide could potentially have a negative effect in copd, given the cell protective properties of mrp1. our observation does not distract from the well known beneficial effects of budesonide in diseases like asthma, since steroids suppress virtually every step of the inflammatory cascade. however, our findings may contribute an alternative hypothesis why inhaled steroids are not capable of altering the long - term course of copd, particularly in smokers with copd (alsaeedi et al 2002). in combination with formoterol, the direct inhibitory effect of budesonide on cf accumulation of epithelial cells was reduced, whereas formoterol as such had only minor effects on mrp1-mediated transport. this indicates that budesonide or its esterified derivatives is less available as an mrp1 substrate / inhibitor when combined with formoterol or that formoterol interferes with the interaction between budesonide (esters) and mrp1. although formoterol is moderately lipophilic, it has a high affinity for cellular membrane lipid bilayers (anderson et al 1994). high concentrations of formoterol might interfere with the function of membrane proteins as this depends on the lipid environment of the protein. the effect with formoterol may also indirectly run via adenosine 3,5-cyclic monophosphate (camp) since formoterol stimulates cells via the beta-adrenergic receptor (johnson and rennard 2001) ing in higher intracellular levels of camp. however, it is not clear why this occurs in the presence of budesonide while formoterol alone had no effect. ipratropium bromide incubation ed in lower cf retentions with increasing concentrations in a concentration - dependent manner. ipratropium bromide competitively inhibits acetylcholine binding to the muscarinic receptor ing in decreased guanosine 3,5-cyclic monophosphate (cgmp) levels which lowers broncho - constriction. there are no reports in the literature on its mechanism of action with respect to mrp1, but it can be speculated that cgmp might play a role in this process since cgmp is a substrate of other members of the mrp family, mrp4 and mrp5 (wielinga et al 2003). effects of nac were similar to those observed with ipratropium bromide, ie, mrp1-mediated transport was stimulated with increasing concentrations of nac. glutathione and many glutathione conjugates are substrates for mrp1, and therefore, intracellular glutathione levels are of major importance for mrp1 function in antioxidant defense. it is known that transport of anionic species such as cf can be stimulated with glutathione (akan et al 2005 ; bagrij et al 2001). it was observed that the compounds sulfinpyrazone and indomethacin stimulate glutathione transport by mrp2 at low concentrations, whereas relatively high concentrations inhibit gsh transport into the medium (evers et al 2000). our with nac and ipratropium bromide might be explained by a similar co - transport of these substances with cf. it may occur that certain (at present undefined) toxic inhaled substances can be transported by mrp1 more effectively with increasing glutathione levels as well. for example, the tobacco - derived nnal - o - glucuronide was identified as an mrp1 substrate that requires glutathione for transport (leslie et al 2001). the pulmonary drugs that we tested are, apart from nac, clinically administered by inhalation. these drugs act directly on epithelial cells or pass the epithelial layer and act on eg smooth muscle cells or inflammatory cells. the intracellular levels that can be reached with treatment highly depend on the biochemical features of the substance, action of drug metabolism proteins (phase ii conjugation enzymes such as cytochrome p450 isoforms), drug efflux pumps (phase iii systems), as well as the route of administration. we have chosen the ranges of the applied drug concentrations based on published data (gustafsson and persson 1991 ; anderson et al 1994 ; gillissen et al 1997 ; nagy et al 1997 ; bandi and kompella 2002 ; borchard et al 2002) and theoretical models, ie, ranges that will be clinically effective. mrp1 is located in bronchial epithelium, the first cells that drugs have to encounter to reach the underlying tissue to act on eg smooth muscle cells, fibroblasts and inflammatory cells. thus, local high cellular concentrations of pulmonary drugs are very likely to affect mrp1 activity in bronchial epithelium which was indeed confirmed in vitro in the present study. in , our studies show that drugs that are clinically used in copd affect mrp1 function, a transporter that protects against oxidative stress and toxic compounds. budesonide inhibits this mrp1-mediated transport and addition of formoterol on its turn reduces this inhibitory effect whereas ipratropium bromide and nac stimulate mrp1 activity in a concentration - dependent manner. further studies are required to investigate whether stimulation of mrp1 activity is beneficial for long - term treatment of copd with regard to the protective properties of mrp1.

smoking is the principle risk factor for development of chronic obstructive pulmonary disease (copd). multidrug resistance - associated protein 1 (mrp1) is known to protect against toxic compounds and oxidative stress, and might play a role in protection against smoke - induced disease progression. we questioned whether mrp1-mediated transport is influenced by pulmonary drugs that are commonly prescribed in copd.methodsthe immortalized human bronchial epithelial cell line 16hbe14o was used to analyze direct in vitro effects of budesonide, formoterol, ipratropium bromide and n - acetylcysteine (nac) on mrp1-mediated transport. carboxyfluorescein (cf) was used as a model mrp1 substrate and was measured with functional flow cytometry.formoterol had a minor effect, whereas budesonide concentration - dependently decreased cf transport by mrp1. remarkably, addition of formoterol to the highest concentration of budesonide increased cf transport. ipratropium bromide inhibited cf transport at low concentrations and tended to increase cf transport at higher levels. nac increased cf transport by mrp1 in a concentration - dependent manner.our data suggest that, besides their positive effects on respiratory symptoms, budesonide, formoterol, ipratropium bromide, and nac modulate mrp1 activity in bronchial epithelial cells. further studies are required to assess whether stimulation of mrp1 activity is beneficial for long - term treatment of copd.

intrinsic, or ab initio, gene finders make no explicit use of information about dnas or proteins outside the sequence being studied. extrinsic gene finders utilize sequence similarity search methods to identify the locations of protein - coding regions. it is common for gene finders of both types to be used in concert in a gene finding project, owing to their complementary nature. such programs are the only means to identify genes with no homologues in current databases. as these genes make up a sizeable percentage of the whole gene complement for particular species, the importance of ab initio programs will not diminish in the foreseeable future. the genemark web software includes two major programs, called genemark and genemark.hmm. both programs employ inhomogeneous (three - periodic) markov chain models describing protein - coding dna and homogeneous markov chain models describing non - coding dna. genemark uses a bayesian formalism to calculate the a posteriori probability of the presence of the genetic code (in at least one of six possible frames) in a short dna sequence fragment, thus being a local approach. the genemark.hmm program uses a hidden markov model (hmm) framework and the generalized viterbi algorithm to determine the most likely sequence of hidden states (which are actually labels designating the coding or non - coding function) based on the whole observed dna sequence. additional details about the genemark and genemark.hmm algorithms can be found in refs. the architecture of the hmm itself can be altered to fit the organization of a particular type of genome under study in a better way. for example, the prokaryotic version of genemark.hmm contains hidden states for the characteristic features of genes in prokaryotes, including ribosomal binding sites (rbs), uninterrupted genes and gene overlaps. the eukaryotic version utilizes an extended hmm architecture, including states for splice sites, translation initiation (kozak) sites and interrupted genes (exons and introns). the web software programs have been extensively used currently > 21 000 nucleotide sequence and 329 000 protein sequence records in genbank contain references to the genemark programs. as many of the model parameters are species - specific, the accuracy of an ab initio gene finder is highly dependent on the selection of adequate training data as well as on the use of sound methods to create the models. the models available at the genemark website were constructed using our recently developed self - training methods and were tested locally before being released. both genemark and genemark.hmm can be used via the genemark website for the analysis of prokaryotic dna, with 175 pre - computed species - specific statistical models available. analysis of dna from any prokaryotic species is supported by (i) a special version of genemark.hmm using a heuristic model calculated from the nucleotide frequencies of an input sequence at least 400 nt long and (ii) a self - training program, genemarks, which can be used for longer sequences on the order of 1 mb in length. thus, the dna of any prokaryote can be analysed, via either a pre - computed species - specific model or a model created on the fly. as many of the programs at the genemark website share similar interfaces, we use here the prokaryotic genemark.hmm program as an exemplar and discuss program - specific differences below, where appropriate. the genemark.hmm web interface accepts as input a single dna sequence as an uploaded file or as text pasted into a textbox. if a fasta description line begins the sequence, all text on the line following the greater than symbol (>) is used as the title. in the remainder of the submission , digits and white space characters are ignored and letters other than t, c, a and g (assumed to appear rarely) are converted to n. the interface requires selection of the species name. selection of a model for the rbs (in the form of a position - specific weight matrix and a spacer length distribution) is optional. in certain cases, such as the crenarchaeote pyrobaculum aerophilum , the rbs model is replaced by a promoter model, which is the dominant regulatory motif located upstream to gene starts in this species. the interface also includes the option of using other types of genetic codes such as the mycoplasma genetic code. genemark.hmm reports all predicted genes in a format that includes the strand the gene resides on, its boundaries, length in nucleotides and gene class (table 1). class indicates which of the two markov chain models used in genemark.hmm, typical or atypical gene model, provided the higher likelihood for the gene sequence. genes of the typical class exhibit codon usage patterns specific to the majority of genes in the given species, while atypical class genes may not follow such patterns and frequently contain significant numbers of laterally transferred genes. the nucleotide sequences of predicted genes and translated protein sequences are available as an output to facilitate further analysis, such as blast searching. an option to generate genemark predictions in parallel with the genemark.hmm analysis provides important additional information. in this case , genemark is set up to use models derived from the same training data as models for the current run of genemark.hmm. it is worth noting that the genemark.hmm and genemark algorithms are complementary to each other in the same way as the viterbi algorithm and the posterior decoding algorithm are. therefore, though the two algorithms are distinct, they are supposed to generate predictions largely corroborating and validating each other. differences frequently indicate sequence errors and deviations in gene organization, very short genes, gene fragments, gene overlaps, etc. graphical output of the analysis is available in pdf or postscript format. a fragment of this output, illustrating the predictions of both genemark and genemark.hmm, is shown in figure 1. the graphical output clearly depicts the advantage of using multiple markov chain models representing different classes of genes. here , the coding potential graph obtained using the typical gene model, derived by genemarks, is denoted by a solid black line, and the coding potential graph obtained using the atypical gene model (derived by a heuristic approach) is denoted by a dotted line. whereas the first and last genes in figure 1 could be detected using either of the two models, as both of them produced high enough coding potentials, the gene located in positions from 846 to 1112 was detected only by the atypical model. further, figure 1 demonstrates the ability of the genemark programs to detect genes of both the typical and atypical gene classes. the genemark graph also includes indications of frameshift positions (also listed in the text report), which are often sequencing errors but in rare cases are natural and biologically very interesting. for the genemark program, the window size and step size parameters (96 nt and 12 nt, respectively, by default) define the size of the sliding window and how far this window is moved along the sequence in one step. the threshold parameter determines the minimal average coding potential for an open reading frame (orf) to be predicted as a gene. there are several options which allow fine - tuning of the genemark graphical output. in addition, there are options supporting the analysis of eukaryotic dna sequences by genemark including the ability to provide lists of putative splice sites and protein translations of predicted exons. as might be expected, genemark (the posterior decoding algorithm) does not produce high enough resolution for the precise prediction of exon intron borders. thus, genemark.hmm (the generalized viterbi algorithm) in its eukaryotic version is the major tool for the identification of exon intron structures in eukaryotic dna sequences. the output of the genemark program consists of a list of orfs predicted as genes, i.e. those with average coding potential above the selected threshold. although each predicted gene can have more than one potential start, additional data is provided to help the researcher annotate one of the alternatives as the true one. the start probability (abbreviated start prob) is derived from the sequences in the windows immediately upstream and downstream of each potential start. rbs information is provided in the form of a probability score along with the position and sequence of the potential rbs (abbreviated rbs prob, in addition to the list of predicted genes, genemark provides a list of regions of interest, spans of significant length between in - frame stop codons where spikes of coding potential are wide enough and may warrant further analysis even if no genes are predicted therein based on automatic comparison with the threshold . analysis of prokaryotic dna sequences for which there is no pre - computed species - specific model can be carried out using a program version which heuristically derives a model for any input sequence > 400 nt. this approach has also proven useful for the analysis of inhomogeneous genomes, particularly regions too divergent from the bulk of the genome, such as pathogenicity islands. if models (including rbs models) have to be computed de novo for an anonymous dna sequence with length of the order of 1 mb or longer, the genemarks program can be used. this program needs significantly more computational resources; thus, its output is provided via email. a modified version of genemarks tuned for the analysis of viruses of eukaryotic hosts creates a model for the kozak consensus sequence instead of a two - component rbs model. the eukaryotic version of genemark.hmm is currently available for the analysis of 11 eukaryotic genomes: homo sapiens, arabidopsis thaliana, caenorhabditis elegans, chlamydomonas reinhardtii, drosophila melanogaster, gallus gallus, hordeum vulgare, mus musculus, oryza sativa, triticum aestivum and zea mays. from the prediction accuracy tables given at the website , it follows that the latest versions of genemark.hmm produce remarkably accurate gene predictions for plant genomes such as rice and arabidopsis. this fact has not escaped the attention of plant genome sequencing consortiums, which have used the program intensively. the analysis of cdna and est sequences from eukaryotes, which typically contain no introns, is facilitated by a special version of genemark called genemark.spl. interestingly, eukaryotic genomes with rare introns present difficulty in terms of collecting enough statistics for the intron and internal exon related models, the important components of a full - fledged eukaryotic gene finder. for this reason, currently, this interface employs versions of prokaryotic genemark and genemark.hmm augmented with kozak start site models instead of the prokaryotic rbs model. the eukaryotic species - specific models are represented by several variants built for distinct g + c% ranges covering the whole scale of g + c inhomogeneity observed in a particular genome. genemark.hmm automatically selects the model variant which fits the g + c% of the input sequence. note that, in the eukaryotic case, the repeatmasker program (a. m. a. smit, r. hubley, and p. green,), which is frequently used for pre - processing, can introduce a significant number of these characters do not influence the selection of the markov chain model used in prediction. a sample text output produced by the eukaryotic version of genemark.hmm is shown in table 2. in the graphical output of the eukaryotic version of genemark.hmm, the thick horizontal bars (which represent whole genes in the prokaryotic case) vertical ticks on these bars show the starts and ends of predicted initial and terminal exons, respectively. for the analysis of virus and phage dna, the heuristic (for short genomes) and genemarks (for long genomes) options, mentioned above, are recommended. in addition, a database called violin containing pre - computed reannotations of > 1000 virus genomes is available. future directions for genemark web software development include detection of several genomic elements currently not predicted by either genemark or genemark.hmm, such as rrna and trna genes (which can be mis - predicted as protein - coding genes in low g+c% species) and improving the detection of gene 5 ends. currently, the server supports the analysis of sequences masked by trnascan or similar programs. n characters ); thus, the predictions will be compatible with this extrinsic information. the detection of exact gene starts remains a challenging problem in gene finding, as many genes have relatively weak patterns indicating sites of translation and transcription initiation. this problem is made especially difficult by the lack of available data sets containing verified gene start locations to be used for training and evaluation. refinements in the rbs and kozak models and the potential inclusion of hidden states representing upstream promoter sequences are currently being explored to address this issue. graphical output from the combination of genemark and genemark.hmm for a fragment of the escherichia coli k12 genome. the solid black and dashed traces indicate the coding potential calculated by the genemark program using the typical and atypical markov chain models of coding dna, respectively. only the three reading frames in the direct strand are shown as there are no genes (either predicted or annotated) on the reverse strand in this section of the genome. the thick grey horizontal bars indicate regions of interest provided by the genemark program. the thin black horizontal lines indicate (longest) orfs observed in each reading frame; ticks extending above and below this line indicate potential start and stop codons, respectively. gene predictions made by the prokaryotic version of genemark.hmm for a fragment of the escherichia coli k12 genome in the class column, 1 and 2 indicate typical and atypical, respectively. direct and reverse complement strands are indicated by + and , respectively. prediction of a single gene (with seven exons) made by the eukaryotic version of genemark.hmm for a fragment of the arabidopsis thaliana genome the gene that an exon belongs to and its strand are necessarily the same for all exons in a gene. the start frame and end frame indicate the position of the codon (first, second or third) that the exon begins and ends with, respectively. notably, all complete gene structures begin in codon position 1 and end in codon position 3.

the task of gene identification frequently confronting researchers working with both novel and well studied genomes can be conveniently and reliably solved with the help of the genemark web software . the website provides interfaces to the genemark family of programs designed and tuned for gene prediction in prokaryotic, eukaryotic and viral genomic sequences. currently, the server allows the analysis of nearly 200 prokaryotic and > 10 eukaryotic genomes using species - specific versions of the software and pre - computed gene models. in addition, genes in prokaryotic sequences from novel genomes can be identified using models derived on the spot upon sequence submission, either by a relatively simple heuristic approach or by the full - fledged self - training program genemarks. a database of reannotations of > 1000 viral genomes by the genemarks program is also available from the web site. the genemark website is frequently updated to provide the latest versions of the software and gene models.

v - erb - b2 avian erythroblastic leukemia viral oncogene homolog 2 erbb2 interacting protein human epidermal growth factor receptor 2 heat shock protein 90 mouse mammary tumor virus - neu psd95/discs large / zo-1 promyelocytic leukemia - retinoic acid receptor a polyomavirus middle t antigen the mode of action of approved targeted therapies for cancer treatment mostly consists of inhibiting the function of the oncogenic target. although effective, these drugs are not totally efficient, in part due to remaining active oncoprotein in the cancer cell. the perfect drug would ideally degrade the bad guy, definitively eliminating the roots of the disease. such a drug, arsenic trixoxyde, has been very successfully developed to treat acute promyelocytic leukemia by eradicating the promyelocytic leukemia retinoic acid receptor a (pml - rara) fusion product through sumo - dependent proteasomal degradation. it is thus anticipated that a better knowledge of the mechanisms of oncoprotein degradation would benefit the development of innovative and efficient drugs. v - erb - b2 avian erythroblastic leukemia viral oncogene homolog 2 (erbb2 ; also named human epidermal growth factor receptor 2 or her2) is a tyrosine kinase receptor whose gene amplification and protein overexpression leads to a poor prognosis in breast cancer. this genetic lesion is retrieved in 1520% of cases of breast cancer and is associated with metastatic development. for almost 10 years, so - called erbb2-positive patients have been treated with potent monoclonal antibodies (trastuzumab, pertuzumab), and more recently with tyrosine kinase inhibitors (lapatinib). although there is no doubt that these drugs have improved patient care, relapse is frequent. erbb2 induces a strong mitogenic pathway in cancer cells by activating classic tyrosine kinase downstream molecules such as ras, mapk, and pi3k. erbb2 protein levels are controlled by heat shock protein 90 (hsp90), a chaperon protein that binds the erbb2 intracellular domain and protects the protein from proteosomal degradation. hsp90 inhibitors such as geldanamycin that induce erbb2 degradation have been developed and have entered into clinical trials. more than a decade ago, our laboratories identified erbb2 interacting protein (erbb2ip, best known as erbin), which has a psd95/discs large / zo-1 (pdz) domain that interacts with erbb2. erbb2 interaction is highly specific as erbin has no affinity for other members of the erbb2/her family (egfr, erbb3/her3, and erbb4/her4). erbin stabilizes erbb2 at the plasma membrane through its pdz interaction, and is necessary for the function of the receptor in the myelination of axons in the peripheral nervous system. erbin knock - out mice (erbin) are viable and fertile, despite their neurological defects. erbin mutation leads to decreased amounts of erbb2 at the plasma membrane of mammary epithelial cells. these data obtained in a physiological context enabled investigation into the role of erbin in tumor growth promoted by erbb2. transgenic mouse mammary tumor virus (mmtv)-neu (neu) mice express oncogenic neu, the rat homolog of erbb2, in the mammary gland and form tumors similar to erbb2-positive breast cancer. remarkably, erbinneu mice exhibit a very significant delayed appearance of mammary tumor development compared to animals bearing oncogenic neu in a wild - type erbin . tumor growth was diminished in the absence of erbin and correlated with lower amounts of total and active erbb2. no effect was observed on protein levels of other erbin interactors such as integrin-4, smad2, and smad3. moreover, a lower rate of cell proliferation was detected in neu - driven tumors and in 2d and 3d cultures of human cancer cells deficient for erbin expression. this led to the that erbin stabilizes erbb2 and is a component of the oncogenic program of this tyrosine kinase receptor. complementary were obtained with 2 other mouse models developed to answer the following important questions. the answer is clearly no, as erbin deficiency had no effect on the development of mammary tumors induced by mmtv - pyvt (polyomavirus middle t antigen is another strong oncoprotein). the answer to this is yes, as deletion of the erbin pdz domain in the mouse genome led to a similar decrease in neu - driven tumoral development as complete erbin deficiency. so how does erbin function to stabilize erbb2? obviously not by transcriptional regulation, as erbin deficiency did not affect erbb2 mrna levels. in fact, the answer to this question came from the discovery that erbin promotes the formation of a complex containing itself, erbb2, and hsp90, another stabilizing molecule for the receptor. disruption of this complex by a competitor peptide for the erbin pdz - erbb2 interaction or by geldanamycin condemned erbb2 to proteasomal degradation. this mechanism highlighted the importance of the very c - terminal sequence of erbb2 in protein stability, as previously shown and the role of erbin in maintaining receptor levels in collaboration with a chaperon (fig . 1). it remains to be determined how erbin participates in this stabilizing process at the molecular level, and whether other parts of the protein outside of the pdz domain can perform the job, either alone or with associated partners. of interest, erbin was found to be overexpressed in erbb2-positive breast cancer, suggesting that it may represent a suitable target for drug development. even though development of protein protein interaction inhibitors mimicking the erbb2 pdz binding site is no easy task, these data form the basis for strategies aiming to target the erbin pdz erbb2 interface with the objective of promoting degradation of the oncoprotein. on the left, erbb2 forms a complex with erbin and hsp90 at the plasma membrane. this interaction stabilizes erbb2 and allows a potent mitogenic signal. on the right, disruption of the erbb2erbin complex with a peptide mimicking the c - terminal sequence of erbb2 erbb2, v - erb - b2 avian erythroblastic leukemia viral oncogene homolog 2; erbin, erbb2 interacting protein; hsp90, heat shock protein 90: pdz, psd95/discs large / zo-1. disruption of the erbb2erbin complex decreases erbb2 levels and limits oncogenicity. on the left, erbb2 forms a complex with erbin and hsp90 at the plasma membrane. this interaction stabilizes erbb2 and allows a potent mitogenic signal. on the right, disruption of the erbb2erbin complex with a peptide mimicking the c - terminal sequence of erbb2 induces degradation of erbb2 leading to lower amounts of erbb2 and decreased mitogenicity. erbb2, v - erb - b2 avian erythroblastic leukemia viral oncogene homolog 2; erbin, erbb2 interacting protein; hsp90, heat shock protein 90: pdz, psd95/discs large / zo-1. the laboratory of jpb is supported in part by grants from la ligue nationale contre le cancer (label ligue 2013), institut paoli - calmettes, cancrople paca and by siric (inca - dgos - inserm 6038).

erbb2 (v - erb - b2 avian erythroblastic leukemia viral oncogene homolog 2) is an oncogenic tyrosine kinase receptor that is overexpressed in breast cancer. antibodies and inhibitors targeting erbb2 are currently available, although therapeutic failures remain frequent. we discuss here recent data showing that the scaffold protein erbb2ip (erbb2 interacting protein, best known as erbin) regulates erbb2 stability and may represent a future therapeutic target.

a marine cyanobacterial assemblage comprising a small filament leptolyngbya species, based on morphological analysis, was collected in 2004 by hand using scuba from a reef pinnacle in coiba national park, panama. the alcohol - preserved tissue was extracted with ch2cl2meoh, and the extract fractionated by normal - phase silica gel vacuum liquid chromatography (np - vlc). in preliminary biological activity profiling, the 100% etoac fraction was cytotoxic to nci - h460 human lung tumor cells with an ic50 of 300 ng / ml and also showed preliminary activity in the icbg panel of antiparasitic assays: malaria (ic50 6 g / ml), american trypanosomiasis (ic50 35 g / ml), and leishmaniasis (ic50 > 50 g / ml). rp18 solid - phase extraction (spe), hplc, and further cytotoxicity testing of this fraction ed in the discovery of the potent antiproliferative metabolite coibamide a, to which was attributed the preliminary antiparasitic activities observed for the parent fraction. however, h nmr analysis of the rp18-spe subfractions indicated the presence of additional, but unrelated, peptidic metabolites in the 70% meoh hplc purification of this more polar spe fraction has afforded two new cyclic depsipeptides, companeramides a and b, subsequently found to be responsible for the observed antiparasitic activity of the parent fraction. laboratory culture of the 2010 field - collected cyanobacterial assemblage yielded predominantly companeramides a and b, with a relatively low yield of coibamide a during the first six months of culture. microscopic examination of these cultures also revealed that a small - filament cyanobacterium was the dominant organism in culture, over its companion large - filament cyanobacterium. beyond six months in laboratory culture, this assemblage no longer produced coibamide a, but continued to produce the companeramides, and microscopic examination revealed the presence of almost exclusively small filaments, with only sporadic large filaments in the cultured material (figure s21, supporting information). subsequently a monoculture of this small - filament cyanobacterium was achieved that could be associated with companeramide production (pac-10 - 3 csf1, genbank km882611). phylogenetically, this organism is most closely related to a taxonomically unassigned filamentous cyanobacterium associated with black band disease (flk9, genbank eu196364, see supporting information figures s19 and s20). companeramide a yielded a prominent ion by hrtofms for a molecular formula of c57h97n9o11, which was supported by nmr spectroscopic analysis. the h nmr spectrum for 1 exhibited signals typical for a relatively hydrophobic cyanobacterial peptide metabolite, including four n - methyl singlets (h 3.14, 3.06, 2.92, 2.69), four nh doublets (h 8.86, 6.92, 6.91, 6.75), nine -h multiplets (h 4.585.26), and numerous overlapped methyl doublets (h 0.771.32, table 1). the c nmr spectrum for 1 displayed nine distinguishable signals for ester / amide - type carbonyls (c 169.4174.4), one signal for an oxygenated sp - hybridized carbon (c 75.4), and two signals at c 83.8 and 68.6, diagnostic of a terminal acetylenic moiety (table 1). the hmbc spectrum displayed prominent three - bond hmbc correlations from the four n - methyl singlets, permitting n - methyls h3 - 15, h3 - 25, h3 - 42, and h3 - 52 to be associated with corresponding -carbons c-11, c-20, c-38, and c-50, respectively. the side chain spin systems of these four n - methylated amino acid residues were delineated by multiplicity - edited hsqc and hsqc - tocsy experiments as two n - methyl valine (n - me - val), one n - methyl leucine (n - me - leu), and one n - methyl alanine (n - me - ala) residue. further analysis of the cosy, hsqc, hsqc - tocsy, and hmbc experiments identified the regular amino acids alanine (ala), proline (pro), and two isoleucines (ile), as well as hydroxyisovaleric acid (hiva) (table s1, supporting information). cosy and tocsy nmr experiments were used to identify a spin system in which a methine doublet of quartets (h 2.51, h-2) was correlated to both a methyl doublet (h 1.31, h3 - 9) and a second downfield methine multiplet (h 3.97, h-3). this h-3 multiplet was in turn relay - coupled to the signals for three contiguous methylenes (h2 - 4 to h2 - 6) and also to an nh doublet at h 6.75 (nh-3 ; figure 1). hmbc correlations from the distal methylene h signal (h2 - 6, h 2.17) to nonprotonated sp - hybridized c-7 (c 83.8) and methine c-8 (c 68.6) signals completed the hydrocarbon chain with a terminal acetylene. finally, three - bond hmbc correlations from the h3 - 9 doublet to methine c-3 (c 51.2) and the c-1 carbonyl signal (c 174.3) aided in the establishment of this -methyl--amino acid as 3-amino-2-methyl-7-octynoic acid (amoya). key 2d nmr correlations for (a) companeramide a and (b) companeramide b. despite several closely overlapping -proton signals (h-17, h-32, h-38, h-44) correlated to overlapped carbonyl signals (c-10, c-31, and c-49) in the hmbc spectrum for companeramide a, all nine amino and one hydroxy acid subunits were sequenced from hmbc and roesy data collected at 700 mhz. two obvious fragments consisting of n - me - val-1ala n - me - leu pro (fragment 1) and ile-1n - me - val-2ile-2n - me - ala hiva (fragment 2) were assembled by hmbc correlations between n - methyl, amide, and/or -h and the carbonyl c signals for neighboring residues (figure 1 and table s1, supporting information). at the n - terminus of fragment 2, the ester linkage between hiva and the amoya residue was defined by hmbc correlations from the hiva -h (h-54, h 4.80), amoya -h (h-2, h 2.51), and amoya methyl (h3 - 9, h 1.31) to the amoya carbonyl c-1 signal (c 174.3). roesy correlations between the pro h2 - 30 multiplets (h 3.84 and 3.76) and the ile-1 -h-32 doublet of doublets (h 4.74) corroborated an hmbc correlation from the pro -h-27 doublet of doublets (h 4.92) to the overlapped ile carbonyl c-31 signal (c 170.0). although hmbc experiments with varying delay times failed to show correlations from the amoya nh-3 (h 6.75) or -h to any carbonyl c nmr signals, a roesy correlation between the nh-3 signal and h 4.58 (-h-11) was used to connect the amoya and n - me - val-1 residues to complete the planar macrocyclic structure of companeramide a (figure 1a). ms / ms data for 1 were consistent with the proposed amino acid sequence (figure s15, supporting information). companeramide b gave a prominent hresims peak at m / z 1056.7013, which was 28 mass units less than the major ion for companeramide a and indicated a molecular formula of c55h93n9o11 for 2. the h nmr spectrum for 2 displayed four n - methyl amide singlets (h 3.23, 3.06, 2.89, 2.74), four amide proton doublets (h 8.84, 7.48, 7.44, 6.80), and midfield multiplets integrating to 10 -h (h 3.815.40). in the c nmr spectrum for 2 there were two signals indicative of a terminal acetylene (c 83.4 and 69.4), as for 1, and eight distinct signals for ester / amide carbonyls, one of which was noticeably broad and of relatively higher intensity. analysis of the 2d nmr data for companeramide b, including the cosy, hsqc, hsqc - tocsy, and hmbc spectra, revealed a similar structural composition to 1, with the presence of amoya, two n - me - val, pro, ile, and hiva residues (tables 1 and s2, supporting information). the hsqc - tocsy spectrum for 2 additionally defined two val spin systems, each comprising an amide (nh-17 or nh-43), two coupled methines, and two methyl h nmr signals. two remaining sets of coupled -methine and methyl doublet h nmr signals could be attributed to two n - me - ala residues on the basis of hmbc correlations from the n - methyl singlets at h 2.74 (h3 - 24) and 2.89 (h3 - 50) to their respective coupled -methine signals (h 4.96, h-22 ; 5.40, h-48). as in the case of 1, the sequential assembly of amino and hydroxy acids in 2 was complicated due to a number of closely overlapping -h chemical shifts (h-11 and h-31, h-11 and h-43, h-31 and h-52) correlated to closely overlapped carbonyl signals (c-10, c-30, and c-47) in the hmbc spectrum. in this case, hmbc and roesy correlations supported a fragment 1 sequence of n - me - val-1val-1n - me - ala-1pro (figure 1b). although the order of val-1 and n - me - ala-1 in 2 was originally in question, when compared to the corresponding second and third residues (ala and n - me - leu, respectively) in 1, key hmbc correlations were present from the n - ch3 - 15 singlet (h 3.23, n - me - val-1) to the c-16 carbonyl signal (c 173.4, val-1), from the -h-17 triplet (h 4.58, val-1) to carbonyl c-21 (c 169.8 n - me - ala-1), and from the n - ch3 - 24 singlet (h 2.74, n - me - ala-1) to the c-25 carbonyl signal (c 171.8, pro). in addition, ms / ms fragments of m / z 183.11 (b2), 282.18 (b3), and 395.26 (b4) (figure s16, supporting information) supported the assigned fragment 1 sequence for 2. this consistent pattern of n - methylation for 1 and 2 is biosynthetically logical. hmbc and roesy correlations supported an extended fragment 2 for 2, in which the ile present in 1 was substituted for a val, providing a sequence of ile-1n - me - val-2val-2n - me - ala hiva. again, the two fragments could be connected by a roesy correlation between the pro methylene (h2 - 29) signal and ile -h-31 signal in combination with a hmbc correlation from the pro -h-26 signal (h 4.83) to the overlapped ile carbonyl c-30 signal (c 170.2). finally, the macrocycle could be closed by a roesy correlation between the signals at h 7.44 (amoya nh-3) and 4.72 (-h-11) together with an hmbc correlation from the nh-3 doublet to the c-10 carbonyl signal (c 170.2) of n - me - val-1. the ing planar structure of companeramide b (2, figure 1b) was again supported by ms / ms data (figure s16, supporting information). the absolute configurations of the amino and hydroxy acid units in companeramides a and b were determined by both marfey s methodology and direct chiral - phase hplc analyses of acid hydrolysates (0.5 mg, 6 n hcl, 110 c, overnight). for each natural product, a portion (ca . 0.25 mg) of the acid hydrolysate was derivatized with n--(2,4-dinitro-5-fluorophenyl)-l - alaninamide (l - fdaa, marfey s reagent) and analyzed by comparative rp18 hplc with fdaa - derivatized d- and l - amino acid standards. the additional 0.25 mg of each natural product acid hydrolysate was reconstituted with h2o, analyzed by chiral - phase hplc, and compared with the retention times of authentic r- and s - hiva standards. for companeramide a, an l - configuration for ala, n - me - ala, pro, and both ile, n - me - leu, and n - me - val residues, as well as s - hiva, was established. the hydrolysate from companeramide b contained l - pro, two l - n - me - val, two l - val, l - ile, and both d- and l - n - me - ala, as well as s - hiva. in order to correctly assign the relative position of the d- and l - n - me - ala residues in companeramide b, a partial hydrolysis and purification of ing fragments containing n - me - ala was attempted, for subsequent complete hydrolysis and marfey s analyses. to this end, 2 (4 mg) was partially hydrolyzed with 3 n hcl, and the product mixture analyzed by lc - ms to identify fragments containing a single n - me - ala residue. a tetrapeptide fragment (m / z 413.9) identified as n - me - val val n - me - ala pro from ms / ms data (figure s17, supporting information) was purified from the product mixture and subjected to complete hydrolysis and derivatization with n--(2,4-dinitro-5-fluorophenyl)-l - leucinamide (l - fdla, advanced marfey s reagent) for analysis. the retention times for the derivatized amino acids from the latter (tetrapeptide fragment 1 of 2) corresponded to fdla - derivatized standards for n - me - l - val, l - val, n - me - d - ala, and l - pro. no other partial hydrolysis fragments could be isolated in reasonable amount for marfey s analysis; however, assignment of the n - me - d - ala in the northern hemisphere (fragment 1) of 2 led n - me - l - ala to be assigned in the southern hemisphere (fragment 2) of 2. the -amino acid amoya present in companeramides a and b was first identified in the molluscan metabolite onchidin, but has since been reported as a component of several marine cyanobacterial metabolites including ulongapeptin, guineamide c, and malevamide c. while the absolute configurations of the amoya residues in guineamide c and malevamide c have not been determined, those in ulongapeptin were assigned as 2s, 3s by marfey s analysis using comparison with a synthetic mixture of c-2 diastereomers (2s, 3r and 2r, 3r) derivatized with both d- and l - fdla enantiomers. the (2s,3s)-configuration was also assigned to the amoya unit in onchidin based on noe studies and h nmr coupling constant analysis; however, the total synthesis of onchidin suggested that a revision of the structure was necessary. therefore, it was planned to use marfey s analysis for assignment of the amoya unit in companeramides a and b, facilitated by the availability of the synthetic 3(r)-amino-2(r, s)-methyloctanoate (amo) standards. two portions of the synthetic material were derivatized separately with d- or l - fdla to provide hplc retention times for all four possible amo diastereomers, with retention times of l - fdla-(2r,3s)-amo and l - fdla-(2s,3s)-amo being inferred from the retention times of the enantiomeric d - fdla-(2s,3r)-amo and d - fdla-(2r,3r)-amo standards, respectively. hydrogenation of each companeramide to reduce the terminal alkyne was followed by acid hydrolysis to release amo for separate derivatization with d- and l - fdla. reported retention times for the four amo diastereomers using similar hplc conditions were used to assign the order of elution of the pairs of standards generated in our protocol. for each companeramide, the d - fdla - amo product coeluted with the d - fdla - derivatized (2s,3r)-amo standard, and similarly the l - fdla - amo product coeluted with the l - fdla - derivatized (2s,3r)-amo, supporting a 2s,3r - amoya unit in companeramides a and b. companeramides a and b showed no significant cytotoxicity at 1 m against four human cancer cell lines (nci - h460 non - small - cell lung carcinoma, mda - mb-231 breast adenocarcinoma, sf-295 glioblastoma, and sk - ov3 ovarian carcinoma cells). instead preliminary antiparasitic activity of the parent fractions led to testing of the two pure compounds against three strains of the malaria parasite plasmodium falciparum in a fluorescence - based assay. neither compound was as active as the chloroquine control against the chloroquine - sensitive d6 or chloroquine - insensitive dd2 and 7g8 strains (table 2, figure s18, supporting information). however, the differential activity between the two companeramides across all strains is noteworthy considering their structural similarity. the chloroquine - sensitive d6 strain was approximately twice as sensitive to companeramide a as the chloroquine - resistant dd2 and 7g8 strains. in contrast, companeramide b showed comparable activity against the chloroquine - sensitive d6 and chloroquine - resistant strain dd2, but was about three times less active against the chloroquine - resistant 7g8 strain. the companeramides add to the growing repertoire of cyanobacterial depsipeptides that show some degree of antiplasmodial activity, including dolastatin 10, the venturamides, carmabins and dragonamides, and symplostatin 4 (first reported as gallinamide). while some of these compounds (e.g., dolastatin 10) are potently toxic to mammalian cells as well as to the malarial parasite, others such as the venturamides and carmabin a show relatively little toxicity to mammalian cells. at the concentrations tested, it is not possible to distinguish any selective antiplasmodial activity for companeramide a, although the for companeramide b suggest potential antiplasmodial selectivity. the requisite testing of 2 for mammalian cell toxicity at higher micromolar concentrations was not pursued in the interest of saving material for other assays in which the target activity may be more pronounced than the moderate antiplasmodial activity presented here. it is interesting to speculate that related large cyclic alkynoic depsipeptides that are reported to be nontoxic, such as the malevamides, may possess antiparasitic activity. noteworthy also is that despite the structural relationship between the companeramides and ulongapeptin, only the heptameric ulongapeptin displays nanomolar cytotoxicity. uv and ir data were obtained on a hitachi u-2000 spectrophotometer and a nicolet ir100 ft - ir instrument, respectively. nmr spectra were acquired on bruker avance 700 mhz and bruker avance drx 600 mhz spectrometers with the residual chcl3 solvent used as an internal standard (c 77.23, h 7.26). lr fabms and hrtofms (es) mass spectra were recorded on absciex 3200 qtrap and waters micromass mass spectrometers. the isolation of compounds 1 and 2 was performed using a waters hplc system consisting of two waters 515 pumps, a rheodyne 7725i injector, and a waters 996 photodiode array detector. marfey s and chiral - phase hplc analyses were conducted on a shimadzu hplc system equipped with two lc-20ad pumps and an spd - m20a photodiode array detector. the marine cyanobacterial assemblage (mcphail laboratory voucher number pac-6/25/04 - 2) was first collected in june 2004 by hand using scuba from a reef pinnacle off the west coast of coiba national park. the material collected for chemical extraction (1 l) was stored in 50% etoh for transport and then stored at 20 c until extraction. for phylogenetic analyses, subsamples of the field collection (0.5 ml of cyanobacteria in 5 ml of rna later solution) were stored at room temperature (rt) and then 4 c before dna extraction, and live cultures were also initiated. additional field collections were made subsequently in 2010 (voucher number pac-10 - 03) and 2012 (voucher number pac-7/10/12 - 1), for chemical and dna extraction, as well as laboratory culture. dna extraction of the 2010 rna later samples and comparison of the genomic 16s rdna (rdna) sequence with the ncbi blast database led to the identification of the cf. symploca strain, which will be assigned to a new genus, hyalidium. monoculture of the small - filament cyanobacterium (pac-10 - 3 csf1) associated with companeramide production in the 2010 collection provided material for genomic dna isolation (genbank accession number km882611), and this organism was found to be 99% identical to that of filamentous cyanobacteria associated with black band disease (see supporting information, s19 and s20). approximately 95.6 g dry weight of the 2004 cyanobacterial collection was extracted repeatedly with ch2cl2meoh (2:1) to produce 5.75 g of an organic extract. the extract was fractionated by normal - phase vlc on si gel with a stepwise gradient solvent system from hexanes to etoac to meoh, yielding nine fractions. the fraction eluting with 100% etoac was cytotoxic (ic50 300 ng / ml) to nci - h460 lung cancer cells and further chromatographed by rp18 spe using a stepwise solvent gradient (meoh h2o, 6:4, 7:3, 8:2, 9:1, 100% meoh and ch2cl2). the noncytotoxic spe fraction eluting with 70% meoh possessed interesting peptide - like h nmr spectroscopic parameters and was purified further using rp18 hplc (85:15 meoh h2o, phenomenex synergi fusion 4 m, 10 250 mm, 2 ml / min, uv detection at 216 nm) to yield companeramide a (1, 2.3 mg, tr 30.5 min) and companeramide b (2, 5.3 mg, tr 21.4 min). colorless oil; d 140 (c 0.6, meoh); uv (meoh) max (log) 215 (3.45) nm; ir max (neat) 3311, 2963, 2930, 1720, 1628, 1517, 1462, 1231, 1098 cm; h and c nmr data, see tables 1 and s1 (supporting information); hrtofms (es+) m / z 1084.7395 (calcd for c57h98n9o11, 1084.7386). colorless oil; d 78 (c 0.5, meoh); uv (meoh) max (log) 225 (4.06) nm; ir max (neat) 3311, 2963, 2930, 1720, 1628, 1517, 1462, 1231, 1098 cm; h and c nmr data, see tables 1 and s2 (supporting information); hrtofms (es+) m / z 1056.7115 (calcd for c55h94n9o11, 1056.7073), m / z 1078.6865 (calcd for c55h93n9o11na, 1078.6892). the absolute configurations of the amino acid residues in companeramides a and b were determined by marfey s methodology. approximately 0.5 mg of each of companeramides a and b were separately hydrolyzed (6 n hcl at 110 c for 16 h), evaporated to dryness, and reconstituted in h2o (50 l). fdaa solutions in acetone (0.1%, 100 l) and 1 n nahco3 (50 l) were added to each hydrolysate and heated to 37 c for 1 h. the solutions were allowed to cool to rt, neutralized with 2 n hcl (25 l), and evaporated to dryness. the residues were resuspended in dmso h2o (1:1, 100 l) and analyzed by rp18 hplc (waters symmetry shield c18 column, 3.9 150 mm, 1 ml / min, uv detection at 340 nm) using a linear gradient of 9:1 40 mm ammonium acetate buffer (ph 5.2)ch3cn to 1:1 40 mm ammonium acetate buffer the absolute configurations of the amino acids in the hydrolysates of 1 and 2 were determined by direct comparison with the retention times (tr, min) for marfey s derivatives of authentic standards. the retention times (min) of the fdaa - derivatized -amino acids in the hydrolysate of 1 matched those of l - pro (12.8 ; d - pro, 13.5), n - me - l - val (23.2 ; d - n - me - val, 27.5), l - allo - ile (21.4 ; l - ile, 21.2, d - ile, 28.3, d - allo - ile, 28.8), l - ala (12.3 ; d - ala, 16.9), n - me - l - ala (14.3 ; n - me - d - ala, 16.6), and n - me - l - leu (26.6 ; n - me - d - leu, 30.3). the retention times (min) of the derivatized amino acids in the hydrolysate of 2 matched l - pro (12.8 ; d - pro, 13.5), n - me - l - val (23.2 ; n - me - d - val, 27.5), l - val (17.3 ; d - val, 24.0), l - allo - ile (21.4 ; l - ile, 21.2, d - ile, 28.3 and d - allo - ile, 28.8), and both n - me - l - ala (14.3) and n - me - d - ala (16.6). chiral - phase hplc analysis was used to determine the absolute configurations of the hiva residues in the two depsipeptides. a portion of the acid hydrolysate of 1 (0.25 mg) and 2 (0.25 mg) was reconstituted in h2o prior to chiral - phase hplc analysis (85:15 2 mm cuso4ch3cn ; column phenomenex chirex 3126 ( d), 4.6 250 mm, flow 1.0 ml / min, uv detection at 254 nm ). the hiva residue in the hydrolysates of 1 and 2 eluted with the same retention time (tr, min) as the s - hiva standard (40.0) but not that of r - hiva (61.7). to assign the position of n - me - l - ala versus n - me - d - ala in companeramide b, 4 mg of 2 was partially hydrolyzed in 3 n hcl (2 ml, constant stirring, 100 c); the reaction was monitored by lc - ms at 30 min intervals and was halted after 3 h by cooling to 20 c, after which the acid was removed under high vacuum. h2o (100 l) for lc - ms / ms analysis (es+) to identify fragments containing single n - me - ala residues. tetrapeptide n - me - val - val - n - me - ala - pro (m / z 413.9, 0.1 mg) was purified from the total hydrolysate by rp18 hlc using a linear gradient of 5100% ch3cn tfa over 60 min (phenomenex synergi hydro column, 10 250 mm, 2.5 ml / min, 340 nm). subsequent complete hydrolysis of the tetrapeptide (6 n hcl, microwave irradiation, 1000 w, 50 s) was followed by marfey s derivatization of the ing hydrolysate (with l - fdla) and commercial amino acid standards (with l - fdla and d - fdla). in each case , 1 m nahco3 (20 l) was added to a 50 mm amino acid solution in h2o, followed by fdla (1% w / v in acetone, 44 l), and the reaction mixture heated to 40 c (1 h). the reactions were quenched by cooling to rt and acidification (20 l of 1 n hcl), before drying under high vacuum and resuspension in 100 l of dmso for lc - ms analysis (thermo aquasil c18 column, 3 150 mm, 2555% linear gradient of ch3cn tfa over 45 min, 1 ml / min, 340 nm, es+). retention times (m / z , tr min) for derivatized residues of the tetrapeptide corresponded to standards for n - me - l - val (426.2, 23.55 ; n - me - d - val, 28.00), l - val (412.2, 19.06 ; d - val, 28.40), n - me - d - ala (398.2, 18.31 ; n - me - l - ala, 17.84), and l - pro (ion not identified, 16.34, d - pro, 19.50). assignment of the amoya residues relied on hydrogenation of the amoya unit in 1 and 2 to 3-amino-2-methyloctanoic acid (amo), peptide hydrolysis, marfey s derivatization with fdla, and comparison with fdla - derivatized synthetic amo standards. in each case, approximately 0.5 mg of cyclic depsipeptide was dissolved in dry meoh and stirred over 5% pd / c under an atmosphere of hydrogen (rt, 18 h). after filtration over celite, washing with etoh, and concentration in vacuo, the products (m / z 1088, tetrahydro-1, and 1060, tetrahydro-2) were resuspended in 6 n hcl and stirred overnight at 100 c. the acid hydrolysate of tetrahydro-1 was derivatized with l - fdla, as described above, while that of tetrahydro-2 was divided into two portions and derivatized with d- and l - fdla. h2o ) was derivatized with each of d- and l - fdla for rp18 hplc - ms analysis (50% ch3cn h2o ; phenomenex synergi hydro column, 4.6 250 mm, flow 0.2 ml / min, uv detection at 340 nm). observed elution times of the standards were 9.4 min (d - fdla-2s,3r - amo = l - fdla-2r,3s - amo), 10.0 min (d - fdla-2r,3r - amo = l - fdla-2s,3s - amo), 17.4 min (l - fdla-2r,3r - amo), and 20.9 min (l - fdla-2s,3r - amo), as assigned by comparison to reported elution times. the l - fdla - hydrolysate of tetrahydro-1 provided a small peak at 20.9 min for l - fdla-2s,3r - amo, which was supported by retention times of 20.9 min (l - fdla-2s,3r - amo) and 9.4 min (d - fdla-2s,3r - amo) for the two derivatized portions of tetrahydro-2. plasmodium falciparum strains d6, dd2, and 7g8 were cultured in human erythrocytes at 2% hematocrit in rpmi 1640 containing 0.5% albumax, 45 g / l hypoxanthine, and 50 g / l gentamicin, as previously described. in vitro antimalarial activity was determined by the malaria sybr green i - based fluorescence (msf) assay described previously with slight modification. stock solutions of each test sample were prepared in sterile distilled h2o at a concentration of 10 mm. the sample solutions were serially diluted with culture medium and distributed to asynchronous parasite cultures on 96-well plates in quadruplicate in a total volume of 100 l to achieve 0.2% parasitemia with a 2% hematocrit. automated pipetting and dilution were carried out with a programmable precision 2000 robotic station (bio - tek). after incubation, 100 l of lysis buffer with 0.2 l / ml sybr green i was added to each well. the plates were incubated at rt for 1 h in the dark and then placed in a 96-well fluorescence plate reader (spectramax gemini - em ; molecular diagnostics) with excitation and emission wavelengths at 497 and 520 nm, respectively, for measurement of fluorescence. the 50% inhibitory concentration (ic50) was determined by nonlinear regression analysis of logistic dose cell viability was assessed with a standard mtt assay as described previously with the viability of vehicle - treated cells defined as 100% and coibamide a (30 nm) included as a control cancer cell toxin. all compounds were reconstituted in 100% dmso and stored at 20 c until the day of treatment; final concentrations of dmso never exceeded 0.1%. human h460 lung and sf-295 glioblastoma cells were from the national cancer institute cell line repository, and mda - mb-231 breast and sk - ov3 ovarian carcinoma cells were from the american type culture collection (atcc). all cells were maintained under standard growth conditions and seeded into 96-well plates at a density of 3000 cells per well the day before treatment.

two new cyclic depsipeptides, companeramides a and b, have been isolated from the phylogenetically characterized cyanobacterial collection that yielded the previously reported cancer cell toxin coibamide a (collected from coiba island, panama). the planar structures of the companeramides, which contain 3-amino-2-methyl-7-octynoic acid (amoya), hydroxy isovaleric acid (hiva), and eight -amino acid units, were established by nmr spectroscopy and mass spectrometry. the absolute configuration of each companeramide was assigned using a combination of marfey s methodology and chiral - phase hplc analysis of complete and partial hydrolysis products compared to commercial and synthesized standards. companeramides a and b showed high nanomolar in vitro antiplasmodial activity but were not overtly cytotoxic to four human cancer cell lines at the doses tested.

the literature concerning this rare syndrome alternatively knows it as maladie des tics, which was mainly descriptive since it was first mentioned by itard and later by gilles de la tourette's syndrome in 1885. the prevalence of tourette's disorder was 4.9 per 10,000 males and 3.1 per 10,000 females. the most common co - occurring disorders with tourette's disorder are attention deficit hyperkinetic disorder (adhd) 50 - 60%; obsessive compulsive disorder (ocd) 30 - 70%, but there is no report regarding prevalence of impulse control disorder (icd).however, icd is described as a rare one. the children with tourette's disorder are prone to rage attacks due to underlying brain dysfunction. hence, the clinician should be aware of the possibility of co - morbidity of tourette's disease (td) with icd. in tourette's disorder , there is triad of multiple motor tics, coprolalia 2 - 6% and childhood / adolescent onset. shapiro and shapiro described impulsion, a manifestation of rage, which is interchangeable with intermittent explosive disorder. confirmed a relationship between levels of 5-hydroxyindoleacetic acid (5-hiaa) in cerebrospinal fluid (csf) and impulsive or aggressive behavior. there is a defect in the serotonergic system, which acts as an inhibitor of motor activity (staner, 1998) and positron emission tomography (pet) scan shows decrease in glucose metabolism in the prefrontal and frontal cortex of patients with impulsive acts (raine et al 1994). to our knowledge , this is the first case report from india which delineates the co - morbidity of td with icd. x, a 17-year - old, unmarried male and a 11th class student, reported in the outpatient clinic / department of psychiatry, government medical college, patiala. history dates back to 1 year when the patient had symptoms of sudden irregular involuntary body movements of head turn and shoulder shrug with isolated episodes of vocalization of obscene phrase bloody kill him. the symptoms were of waxing and waning in nature, and there was no period of remission for more than 3 months during the consecutive 12 months of illness. for the last 3 months, the patient complained of irresistible urge to throw stones without any provocation or any other motive upon his neighbors and destroyed their property. he derived pleasure after acting out and did not try to escape from the scene or resisted his arrest. when he tried to control his rage of throwing stones, there was mounting of tension and increased restlessness within him. anatomically, he felt intense discomforts on his palms, shoulders, midline abdomen and throat. the body movements started from the face, eye blinking, frowning and grimacing of the mouth, further progressing down to involve neck with shoulder shrug and wringing of hands. these movements were exaggerated by excitement, emotional stress or anxiety and attenuated by focused attention or relaxation. for the last 2 months, the patient had an irresistible urge to stab his brother with a knife. realizing his urge getting out of control, he asked for help. though he did not complete or act upon the urge, yet found it irresistible, followed by ejaculatory words of coprolalia (bloody kill him ! !). neither was it a persistent idea, thought or an image nor he tried to substitute it with any other subsidiary thought or engaged in any ritual of undoing it. in the family history, his father was aged 45 years and illiterate. he persistently nagged the patient and was strict, rigid and demanding with obsessive traits. his mother was aged 40 years, illiterate, housewife with no abnormal pre - morbid personality traits. in personal history, the patient was full - term, wanted child, delivered through cesarean section and third in birth order. he was breast fed, had mild physiological jaundice on 7th day and passed his developmental milestones normally. he was fully immunized and his schooling started at the age of 5 years, and had a normal peer group relationship. he was educated in government school till the age of 15 years and left it on financial grounds. after leaving the school, as assessed on wechsler adult intelligent scale (wais), his iq was 95. he described his relationship with his brother as loving and caring and denied any conflict with him. there was no pre - morbid personality traits of borderline, antisocial, histrionic, narcissistic and anankastic (obsessive) personality disorder as assessed on international personality disorders examination (icd-10 : ipde). there was no history of mood disorder, anxiety disorder, adhd, ocd, conduct disorder or learning disorder. there was no history of substance abuse or medical history of head injury, seizure, athetosis, restless leg syndrome, huntington's chorea, syndehams chorea, dystonia, meig's syndrome, blepharospasm or family history of involuntary movement disorders. the patient was assessed using dsm - iv - tr diagnostic criteria and diagnosed as a case of tourette's disorder with icd, specifier: intermittent explosive disorder. the global functional impairment as assessed on global assessment of functioning scale score (gaf score) was 61. he was treated with sertraline 50 mg / day and gradually titrated upward within 4 weeks to the target level of 150 mg / day in divided doses. low dose of haloperidol was also given (less than 4.5 mg / day) and the dose of clonidine ranged from 0.1 to 0.3 mg / day in divided doses. as assessed on yale global tic severity scale (ygtss), there was 50% improvement. the cardinal features of tourette's disorder and other tic disorders are motor and vocal tics and childhood onset.in simple motor tics, there is a brief movement of individual muscle group like eye blinking, head shaking and shoulder shrugging, whereas in complex motor tics there is involvement of multiple muscle groups. in our case study, the onset of motor tics was at the age of 15 years, which confirms the findings of sandor et al. , i.e., the onset of disease between the age of 1 and 21 years. it started as simple motor tics involving eye blinking and frowning, which gradually became complex with head turn, shoulder shrug, neck cracking and trunk bending. simple vocal tics include sniffing, grunting, throat clearing, snorting, and sucking, puffing, squeaking, barking and other meaningless sounds. complex vocal tics may present as stereotyped words or phrases, palilalia (repeating one 's own words), echolalia or coprolalia. though coprolalia is often incorrectly considered essential for the diagnosis of tourette's disorder, it is an uncommon symptom as it is seen in only 2 - 6% of the cases. in our case , the development of vocal tics (coprolalia) follows the usual pattern of development, i.e., motor tics. coprolalia has, indeed, been regarded as hostile reaction toward the authority figures, especially within the family (tobin and reinhart, 1961 as cited in enoch and ball). the patient does not have sufficient outlets for his hostility; being afraid of punishment, his pent up feelings of hostility toward his father build up to the point where it is released in crescendo of explosive tics and obscene utterances. this view is supported by the fact that the father of the patient was strict, rigid and demanding with obsessive traits. tics are exacerbated by excitement or emotional stress and can be attenuated during periods of focused productive activity, attention and sleep. tics are involuntary; yet because they are briefly suppressible and can be triggered by environmental stimuli, they may appear as volitional acts. in our case, it was triggered by emotional stress. other movement disorders such as chorea and dystonia are continuous, whereas tics are intermittent. mannerisms are often not impairing; stereotypy tends to occur exclusively in children and adults with developmental disabilities and mental retardation. hence, it supports the diagnosis of tourette's disorder as assessed using dsm - iv - tr diagnostic criteria, i.e., both multiple motor and vocal tics; tics occurring many times a day for more than 12 months with no period of remission for more than 3 months; significant functional impairment as assessed on global assessment of functioning scale (gaf score 61); onset before 18 years of age and absence of substance abuse or medical / neurological cause of disease. the complex motor tics of tourette's disorder may be difficult to distinguish from the ocd compulsions. both tics and compulsion are preceded by an intrusive urge followed by feeling of relief. however, ocd compulsion is preceded by fear and obsessional concern with the mental urge to do something repeatedly until it is felt just right , whereas in tourette's disorder there is a physical urge to perform a motor tic and not preceded by fear. an impulsion is an action performed until a sense of rightness is achieved, rather than compulsion which is designed to reduce anxiety brought by obsession. there is inability to resist the impulse, rather than the rapid transduction of thought to action as in ocd. there is considerable evidence that there is a broad range of co - occurring clinical problems like mood disorder, ocd, impulse control, anxiety, adhd and learning / conduct disorder. up to 50% of clinically ascertained children and adolescents with tourette's disorder may be affected with problems of attention, concentration activity level or impulse control.in some individuals, tics are preceded or provoked by a thought or physical sensation referred to as premonitory urges. budman suggested that the clinical phenomenon of rage attack in children with tourette's disorder resembles intermittent explosive disorder which may reflect specific neurobiological disturbances. these rage attacks in tourette's disorder may be related to the presence of co - morbid disorder. the lifetime prevalence of intermittent explosive disorder is 6.3%; the age of onset is in teens and the incidence is more in males as compared to females. in our case, the patient described the aggressive episodes of throwing stones or an urge to stab his brother as spell or attack. it was preceded by the sense of tension or arousal and was followed immediately by a sense of relief or release of tension. the obsession in tourette's disorder has multiple concerns such as symmetry, violent, sexual images, urges, worries or loss of control, whereas in intermittent explosive disorder, the behavior has no gratifying element. in addition, fear is the underlying drive in ocd that leads to compulsion which in turn decreases anxiety. in icd, the aggressive acts of throwing stones toward neighbors and destruction of their property and an urge to kill his brother were without any premeditation. these impulsive acts were performed in response to instinctive, or involuntary, irresistible impulses. there was failure to resist the aggressive impulse and the aggressive acts were not accounted by any another mental illness, personality disorder, psychotic, mood, conduct, adhd and are not due to direct physiological effect of a substance or general medical condition (head trauma, seizure, alzheimer 's disease). the essential feature of intermittent explosive disorder was the occurrence of urge for destruction of property. therefore, more epidemiological research is necessary to extend these intriguing findings to provide estimates for a range of presentations that reflect the clinical reality of the tourette's disorder. it is important to know how often people with tourette's disorder have tics specific impairment or co - morbidity and are in need of medication.

we report a case of tourette's disease (td) with impulse control disorder which is rare;these type of patients are prone to rage attack and explosive outbursts in the childhood and adolescence which can be detrimental. hence, a case is reported to understand the phenomenology of its co - morbidity in td.

the rapid development of genome - sequencing techniques has led to the generation of complete genome sequences for > 600 species. most of these sequences belong to model organisms, covering only small portions of the tree of life. the generation of massive numbers of expressed sequence tag (est) libraries that can now be sequenced inexpensively by third - generation sequencing is a cheap alternative to whole genome sequencing, and has also provided a wealth of phylogenetically relevant data. several recent phylogenomic studies have used est sequences to generate large data matrices. these studies generated and assembled est sequences, which were screened for orthologous sequence regions to build useful orthologous gene alignments. orthologous sequences from a speciation event, and are likely to have a conserved function, whereas paralogous sequences evolve through a gene duplication event within a species, and are less likely to maintain their original function, due to processes such as neo-/or sub - functionalization. orthologous and paralogous together are called homologues. since the prime goal of building reliable phylogenetic trees is to decipher the evolutionary relationships among organisms based on their shared common ancestry a reliable protocol is needed to build sets of orthologous sequences from est libraries for successive phylogenomic analyses. we recently proposed such a protocol, which we called orthoselect (schreiber et al . the main idea is to keep user interaction simple, by simultaneously using state - of - the - art methods for orthology assignment, est translation and elimination of paralogues, as well as construction and automated refinement of multiple sequence alignments . orthoselect has been extensively tested and proven to be a useful tool for managing this complex task . figure 1.the main workflow of orthoselect : each est ( a) is assigned to a pre - defined orthologous group (og) by the kog database, and translated (b). after all ests have been assigned to ogs, a subset of the ogs can be selected which will be further processed to exclude all redundant sequences, compute a sequence alignment and refine it in the last step (c). the main workflow of orthoselect: each est (a) is assigned to a pre - defined orthologous group (og) by the kog database, and translated (b). after all ests have been assigned to ogs, a subset of the ogs can be selected which will be further processed to exclude all redundant sequences, compute a sequence alignment and refine it in the last step (c). here, we present a web interface to orthoselect, the first web - based est analysis pipeline for constructing orthologous gene alignments from est libraries. the user simply uploads est libraries and chooses parameters (or uses default settings) to conduct the analysis. orthoselect then provides the user with a dataset useful for subsequent phylogenetic analysis, as well as numerous helpful data and statistics, such as annotations, a data matrix showing est assignments to the orthologous groups (ogs) and visualizations of the orthologous gene alignments. the main purpose of our web server is the construction of orthologous gene alignments from assembled est libraries or other nucleotide sequences. after the user uploads pools of est sequences, ests are assigned to ogs and the sequences most likely to be orthologous in case there were multiple sequences per species are used to compute an alignment that is post - processed in a final step. the workflow of the pipeline is depicted in figure 1, and is described in more detail in the next section. using the ogs defined by the eukaryotic orthologous groups (kog) database, each est is assigned to the closest og. the assignment is done using a reimplementation of blasto that clusters hits from a similarity search of the est against the kog database. the similarity between a query sequence and an og is defined as the mean e - value between the query and the sequences from the og. we then translate the ests using the tools estscan and genewise, to account for frame shift errors. considering the best blast hit of the est as a reference sequence, our program selects the translated sequence that is most similar to the reference sequence. only ogs with at least three taxa are further considered. at this stage of the analysis, it is possible to preselect individual or groups of taxa. the set of ogs is then further reduced to contain only ogs containing all preselected taxa. redundant (e.g. paralogous) sequences are removed from each og. this is done by considering only the sequence from each species that maximizes a global alignment score as being most likely orthologous. all sequences from each orthologous group are then aligned using either muscle, t - coffee or dialign - tx. these alignments are used to build hidden markov models that will be used to search the est libraries for additional hits. est sequences may only partially cover genes, there is an option to exclude sequences from the alignment that are too short. this procedure outputs gene alignments whose member sequences are the ones most likely to be orthologous, given the dataset. our web server allows the use of orthoselect with default or adapted parameter values, e.g. the e - value for similarity searches using blast or methods for computing multiple sequence alignments. orthoselect accepts nucleotide sequences in fasta format. in the absence of a standard format for sequence identifiers in fasta headers, sequence identifiers have to be adapted at some stage of a phylogenetic analysis to allow viewing taxa. orthoselect requires the fasta header to be in a certain format (the first word up to the first whitespace is taken as an accession number), and uploaded files have to match that format, or can be adapted using a converter supplied on the web page. several syntax checks for the uploaded est sequences have been implemented to ensure optimal performance from orthoselect. our web interface offers the possibility to set up sequence identifiers (e.g. abbreviated taxon names) that will be used throughout the analysis. our web server will then return a list of those ogs to which the submitted ests have been assigned, as well as a subset of those ogs containing the preselected taxa. the maximum number of input sequences is 30 000, and the maximum number of est libraries is 10. an email address has to be supplied, since notification about the will be sent via email. having generated est libraries for the species under study, one of the main questions that arise is what genes those est libraries have in common. these set of common genes can be used as a base for subsequent phylogenetic analysis. our web server outputs those genes present in all est libraries, but also provides additional information that will help the user to interpret the data and to decide which data are useful as input for phylogeny programs. the web interface offers a wide range of diagrams, charts, tables, etc. to supply the user with useful information (figure 2). the most important part is the graphical representation of individual ogs with all assigned and translated est sequences, and an overview of its taxonomical composition. single sequences can be viewed along with their translation, as well as the computed multiple sequence alignment prior and subsequent to the final post - processing step in which the program gblocks or aliscore are used. the web server outputs an overview of the ests functional classifications and og assignments as a data matrix with presence / absence information for each gene and species in the study, and annotations for each species. the data matrix shows how many sequences from which taxa have been assigned to an og. this way, the user can easily select ogs with all or a certain percentage of taxa present. figure 2.the output of the orthoselect web server. besides a general overview page of the (a), our web server reports information about functional annotations (b), a gene / taxa presence / absence matrix (c, d), annotations for each taxon (e), as well as an overview of the orthologous groups (f). additionally, for each orthologous group the ing alignments are visualized using the jalview applet (g). the output of the orthoselect web server. besides a general overview page of the (a), our web server reports information about functional annotations (b), a gene / taxa presence / absence matrix (c, d), annotations for each taxon (e), as well as an overview of the orthologous groups (f). additionally, for each orthologous group the ing alignments are visualized using the jalview applet (g). besides an overview of all ogs with sequences assigned (all orthologous groups), orthoselect automatically builds a subset of ogs (best orthologous groups) that have either at least three different taxa or the pre - defined taxa present. the all orthologous groups contain all orthologous groups to which sequences have been assigned, whereas the best orthologous groups only contain one sequence per taxon (see methods section). the page is intended to give the user an elaborate overview and useful information, but also provides all to be downloaded for further examination and use in phylogenetic studies. the orthoselect server consists of a web interface, a mysql database management system (dbms), and the core program orthoselect. the web interface for orthoselect has been constructed using grails, which is a web application framework that uses the groovy scripting language on the java platform to help standardize the development of web interfaces (http://www.grails.org). grails follows the idea of keeping data and web pages separate with a controller functioning as a mediator between them. all jobs are split into equal chunks to be computed in parallel on our computer cluster, and all data is stored in the dbms. german research foundation (dfg, project wo896/6 - 1,2) within dfg priority programme spp 1174 deep metazoan phylogeny. funding for open access charge: deptartment of bioinformatics, georg - august - universitt gttingen. conflict of interest statement.

in the absence of whole genome sequences for many organisms, the use of expressed sequence tags (est) offers an affordable approach for researchers conducting phylogenetic analyses to gain insight about the evolutionary history of organisms. reliable alignments for phylogenomic analyses are based on orthologous gene sequences from different taxa. so far, researchers have not sufficiently tackled the problem of the completely automated construction of such datasets. existing software tools are either semi - automated, covering only part of the necessary data processing, or implemented as a pipeline, requiring the installation and configuration of a cascade of external tools, which may be time - consuming and hard to manage. to simplify data set construction for phylogenomic studies, we set up a web server that uses our recently developed orthoselect approach. to the best of our knowledge, our web server is the first web - based est analysis pipeline that allows the detection of orthologous gene sequences in est libraries and outputs orthologous gene alignments. additionally, orthoselect provides the user with an extensive section that lists and visualizes all important , such as annotations, data matrices for each gene / taxon and orthologous gene alignments. the web server is available at http://orthoselect.gobics.de.

the philadelphia chromosome (ph+) is derived from t(9;22)(q34 ; q11) or variants and carries the chimeric bcr / abl1 fusion gene which is classically associated with chronic myeloid leukemia (cml) 1. chimeric bcr / abl1 fusion transcript e13a2(b2a2)/e14a2(b3a2) encodes for a 210kda protein (p210), e1a2 encodes for a 190kda protein (p190), and less commonly e19a2 encodes for a 230kda protein (p230) ing in constitutively active tyrosine kinase that leads to oncogenic transformation of cell to leukemia 2. ph+ is found in 1530% of adults and 25% of children with acute lymphoblastic leukemia (all), and on rare occasions, < 3%, in acute myeloid leukemia (aml) 3. ph+ lymphoma and myeloma have been described in the literature 4, 5. among mixed phenotype leukemia with abnormal cytogenetics, acute leukemia presenting with ph+ blasts can mean cml in myeloid blast crisis (cml / mbc), ph+ all, ph+ aml, or ph+ mixed phenotype acute leukemia. certain features such as the presence of basophilia, often seen in cml / mbc, or the absence of basophilia commonly seen in ph+ aml can help distinguish one from the other 7. however, it can be very challenging to delineate these separate entities even in expert hands. efforts are being made to characterize the genetic differences between the malignancies to better manage treatment as these patients typically have poor prognosis 3. mixed phenotype acute leukemia patients with t(9;22) or 11q23 cytogenetic abnormalities have very adverse prognosis 8. central nerves system (cns) involving leukemia add to the disease complexity as extramedullary aml with marrow involvement is considered independent adverse prognostic factor with 5year survival rate ranging from 20% to 30% 9. here, we present a challenging case of ph+ aml with cns involvement treated with intensive chemotherapy, tyrosine kinase inhibitor (tki), cns directed therapy, and achieved lasting remission after allogeneic hematopoietic stem cell transplantation (allohsct). patient is in complete remission for over 18 months despite its poor prognosis and remains on maintenance tki for relapse prevention. a previously healthy 61yearold man presented with easy bruising, slow wound healing sustained during routine household chores, and general malaise for the past 3 months. physical examination revealed petechiae on lower extremity, mild splenomegaly, and axillary adenopathy. complete blood count (cbc) showed significant leukocytosis with white blood count of 150,100/l, normocytic anemia with hemoglobin of 6.7 g / dl, and thrombocytopenia with platelet count of 37,000/l. initial laboratory workup bone marrow biopsy (bmb) showed diffuse infiltration by numerous blast cells, which effaced the normal marrow architecture (fig . 1a). cytogenetics and fluorescence in situ hybridization (fish) analysis revealed the loss of chromosome 7 and the presence of philadelphia chromosome: 45,xy,7, t(9;22)(q34;q11.2)/46,xy. morphologically these cells were atypical with immature expression of cd34 and coexpression of cd13 and hladr favoring an early myeloid differentiation rather than lymphoid origin. flow cytometry on the marrow aspirate showed an abnormal population of cells expressing dim cd45, moderate cd19, dim cd13, bright cd34, hladr, with a subset expressing dim cd117, dim mpo, and dim tdt is identified, comprising 72% of total events. these blasts were negative for surface kappa and surface lambda light chain immunoglobulins, as well as cd10, cd11b, cd14, cd16, cd64, cd56. molecular studies showed bcr / abl p190 mrna pcr of 33.8% on international scale and negative p210. (a) core biopsy of the marrow showed diffuse infiltration by numerous blast cells, which effaced the normal marrow architecture (h&e, x400). (b) a large number of blast cells are also evident in the marrow aspirate (wirght giemsa, x1000). on admission, patient was started on hydroxyurea for cytoreduction along with routine tumor lysis prophylaxis with hydration and allopurinol. dasatinib was started after 2 days once initial reports became available showing positive philadelphia chromosome. patient was then started on hypercvad a few days later after extensive discussions about therapy choice. five days after admission, patient developed headache, blurry vision, and floaters bilaterally. a computerized tomography (ct) of head was performed, and it did not show any acute intracranial bleed, mass, or midline shift. ophthalmology was consulted, and they noted the following on fundus examination: right eye has preretinal hemorrhage near fovea and left eye with intraretinal and preretinal hemorrhage. their final impression was preretinal hemorrhages on both eyes and intraretinal hemorrhages on left eye secondary to thrombocytopenia. he underwent lumbar puncture (lp), and eight blasts were found in cerebral spinal fluid (csf) on smear consistent with cns involvement with leukemia. patient was placed on hypercvad (cyclophosphamide, vincristine, doxorubicin, dexamethasone) with dasatinib 100 mg daily. biweekly intrathecal chemotherapy including intrathecal cytarabine (arac) was initiated with subsequent lp showing clearance of blast cells in csf. dasatinib was continued throughout his treatment course, and no significant adverse effect was noted. bmb performed 3 months after diagnosis showed mildly hypocellular bone marrow without morphologic or immunophenotypic evidence of aml. patient achieved complete cytogenetic response (ccyr) and major molecular response (mmr) at this point. due to the high risk of relapse in ph+ aml, patient subsequently received allohsct. he had 10/10 hlamatched haploidentical allohsct from his brother (a total of 4.31 10e6 cd34 + cells were transfused) with conditioning regimen consists of cyclophosphamide 60 mg / kg and total body irradiation (tbi) 1200 centigray (cgy). his course was complicated by chronic graftversushost disease (gvhd), but overall patient had been doing well for more than 18 months. patient continued on dasatinib with plan to stop after 2 years from the date of allohsct. fish study for t(9;22) was negative, and monthly bcr / abl p190 mrna pcr level remained negative. by all tests, patient is in complete remission and minimal residual disease (mrd) negative. philadelphia chromosomepositive de novo aml is a rare condition with estimated incidence rate of 0.53% 1, 6, 10. the 2016 revision of the world health organization (who) classification has recognized mixed phenotype aml with t(9;22) as a separate entity 11. distinction between unrecognized cml / mbc and de novo aml is possible on many grounds. compare to cml / mbc, patients with ph+ aml lack a history of cml or chronic phase / accelerated phase cml after aml induction chemotherapy. coexistence of normal metaphases along with ph+ metaphases, absence of basophilia, and lack of moderate / massive splenomegaly at the time of diagnosis favors ph+ aml over cml / mbc 3, 12. normal karyotype after induction therapy is suggestive of ph+ acute leukemia, and in cml / mbc, cytogenetic abnormality often persist 13. in addition to the t(9;22), cml / mbc is associated with trisomy 8, trisomy 19, and isochromosome 17q, whereas monosomy is associated with ph+ aml 1, 3, 14. the p190 bcrabl fusion protein is common in ph+ aml but only rarely present in cml / mbc 1. on the molecular level, konoplev et al. found 22% of its patients with ph+ aml had npm1 mutation and none in patients with cml / mbc 2. while none of the aforementioned features alone is diagnostic of ph+ aml, the summation provides a good indicator of the correct diagnosis, which is important because of its potential treatment implications. outcome of ph+ aml is generally poor with some studies showed median survival of 918 months 1, 14. the mainstay of treatment includes imatinib and allohsct, but the optimal treatment is not clear because of the limited number of cases and short followup reported in literature. with the presence of ph chromosome, complete response is thought to be unlikely with imatinib alone 1. current recommendation according to the national comprehensive cancer network (nccn) clinical practice guidelines in oncology for aml is to treat ph+ aml as cml / mbc with tki (alone or with induction chemotherapy) followed by allohsct 15. a recent study by chantepie et al. showed a promising 68% 2year overall survival in patients with ph+ aml after receiving allohsct 10. there are also reports that showed benefits with using imatinib as interim therapy prior to allohsct and even imatinib or later generations of tki alone 16, 17, 18. reboursiere et al. reported 21 cases of patients with ph+ aml in the literature treated with tki therapy 1. eight of the patients received allohsct and three of them continued tki as maintenance therapy afterward. it appears tki after allohsct provides survival benefits, but it is far from conclusive at this point given the limited number of cases reported. more studies will be needed to elucidate the best treatment in this rare subtype of aml. any type of aml with cns involvement is uncommon with incidence rate range from 3.1% to 3.6% 19. incidence rate is even lower at 0.37% when cns involvement is found at diagnosis according to a study conducted by shihadeh et al. these patients tend to be younger, have a higher white blood cell (wbc) count, lactate dehydrogenase (ldh), and peripheral blood blasts at diagnosis 19, 20. one study found 80% of patients with cns involvement had ldh levels > 700 iu / l 19. other predictive factors include flt3itd, african american ethnicity, and cytogenetic abnormalities of chromosome 11, inv, and trisomy 20. unlike acute all where cns involvement is more common in which routine intrathecal prophylaxis chemotherapy at the time of diagnosis has shown benefits, such prophylactic measure is not recommended for aml due to the lack of supporting data. different treatment options include intrathecal chemotherapy, systemic chemotherapy, and brain / craniospinal radiation 20. it is important to note that aml with cns involvement confers a worse diseasefree and overall survival (os) compared to those without cns involvement, so any clinical suspicion should prompt timely diagnostic workup such as lumbar puncture and/or head imaging 19. our patient presented with features that are suggestive of primary ph+ aml including no history of chronic phase, a lack of basophilia, monosomy, and positive p190. his course was complicated by cns involvement at diagnosis, and he had a number of predictive factors such as high wbc, ldh, blasts, and chromosome 11 abnormalities. patients with biphenotypic acute leukemia treated with allbased induction regimen have reported 87% complete remission (cr) while only 20% of patients treated with an amlbased regimen achieved cr 21. based on strongly favorable data of intrathecal component to treat cns disease he was successfully treated and later received allohsct who is now in complete remission for more than 18 months. philadelphia chromosomepositive mixed phenotype de novo aml is rare and can be challenging to distinguish from cml / mbc. features associated with ph+ aml include lack of chronic phase, absence of basophilia or splenomegaly, monosomy, npm1 mutation, and presence of p190. nonamltype intensive induction therapy followed by postremission therapy by allohsct in suitable patients can potentially overcome adverse prognostic factors, achieve complete remission with longterm survival, and be cured. the authors declare that there is no conflict of interest regarding the publication of this article.

key clinical messagemixed phenotype acute leukemia with t(9;22) is a rare disease with poor prognosis, and information on optimal treatment is limited. we describe a case where our patient experienced positive outcome after nonacute myeloid leukemiatype intensive induction therapy followed by postremission therapy with stem cell transplant.

tor proteins are ser / thr kinases with highly conserved homologues that are found in all eukaryotes (schmelzle and hall, 2000). in mammals, the two best - characterized targets of mtor are the ribosomal s6 kinases (s6k1 and s6k2) and the eukaryotic initiation factor 4e (eif4e)binding protein 1 (4e - bp1). mtor activity leads to s6k1/2 phosphorylation and activation and to 4e - bp1 phosphorylation and release from the cap - dependent translation initiation factor eif4e. these two events, likely combined with other mtor targets, lead to an increase in ribosomal biogenesis and the selective translation of specific mrna populations. this ability to increase the protein synthesis capacity of the cell is responsible, at least in part, for the ability of tor proteins to drive cell growth and proliferation (for review see fingar and blenis, 2004). full activation of the mtor pathway requires signals from both nutrients (e.g., amino acids and glucose) and growth factors. although the mechanism by which amino acids are sensed by tor proteins is unknown, their presence is essential for basal activation of mtor signaling, as well as for further stimulation by growth factors (hara et al ., 1998). interestingly, amino acids have been shown to modulate insulin signaling through mtor - dependent effects on irs-1 (takano et al . it has been demonstrated that, via mtor regulation, excess amino acids attenuate and amino acid starvation stimulates akt activation and subsequent glucose uptake in response to insulin . these effects are concomitant with changes in the state of irs-1 phosphorylation, localization, and/or degradation . therefore, the levels of circulating amino acids and other nutrients may have a profound effect on the insulin sensitivity of peripheral tissues, such as fat and skeletal muscle, through mtor - mediated regulation of irs-1 function . the primary pathway by which most growth factors and cytokines activate mtor and its downstream targets appears to be the pi3k akt pathway ( for review see fingar and blenis, 2004). as with amino acids , several research groups have found that pi3k akt - mediated activation of mtor also leads to attenuation of insulin signaling. their studies have demonstrated that activation of the pi3k akt mtor pathway in various cell lines by pdgf (li et al ., 1999), tnf- (ozes et al ., 2001), or insulin itself (haruta et al ., 2000 carlson et al ., 2004) leads to irs-1 and/or irs-2 serine phosphorylation and down - regulation. although all of these studies found that irs protein phosphorylation is sensitive to the mtor - specific inhibitor rapamycin, the serine residues that are proposed to be involved vary. pdgf - mediated inhibition of irs-1 required the proline - directed sites s632, s662, and s731 (li et al ., 1999), tnf- treatment correlated with s632 and s635 phosphorylation (ozes et al ., 2001), , 2001; greene et al., 2003; carlson et al., 2004; all numbering is for mouse irs-1 ). however, s307 (s312 in humans) is the only site demonstrated to be required for insulin - stimulated irs-1 down - regulation (greene et al ., 2003). although this site was independently found to be phosphorylated by jnk in response to tnf- (aguirre et al . 2002), jnk does not appear to be responsible for s307 phosphorylation in response to insulin (rui et al . , 2001 ; greene et al ., 2003), and the identity of the mtor - dependent kinase that directly phosphorylates this site remains unknown. tuberous sclerosis complex (tsc) is a syndrome characterized by a pleiotropic array of benign tumors, collectively referred to as hamartomas, often containing abnormally large cells (for a recent review see kwiatkowski, 2003). although these tumors rarely become malignant, their presence in various tissues gives rise to severe clinical manifestations, including neurological disorders, skin lesions, cardiac dysfunction, and kidney and lung failure. the disease has been mapped to mutations in either of the two tumor suppressor genes tsc1, which encodes the hamartin protein, and tsc2, which encodes the tuberin protein. the hamartin and tuberin proteins form a complex that has recently been found to act within the pathway leading from pi3k akt activation to mtor signaling (for review see manning and cantley, 2003). hamartin complex potently inhibits tor - dependent signaling in both flies and mammals (gao et al ., 2002 ; , 2002 ; tee et al ., 2002), and this inhibition is relieved by akt - directed phosphorylation of tuberin (inoki et al ., 2002 ; manning et al ., 2002 ; potter et al ., 2002) therefore, in response to growth factors such as insulin, the pi3k akt pathway activates mtor signaling through phosphorylation and inhibition of tuberin (fig . interestingly, the responsiveness of tor proteins to other signals, such as nutrient and energy availability, also appears to be dependent on the tsc genes ( gao et al . , 2002 ; inoki et al ., 2003, shaw et al ., 2004). therefore, homozygous loss of tsc1 or tsc2 leads to high constitutive activation of mtor signaling, as detected in mouse embryonic fibroblasts (mefs ; jaeschke et al ., 2002 ; kwiatkowski et al ., 2002 ; zhang et al ., 2003), in the tumors of rodent models of tsc (kenerson et al ., 2002 ; el - hashemite et al ., 2003a), and in human tsc cells and tumors (goncharova et al model of the pi3k akt tsc tor pathway and feedback regulation of insulin / igf - i signaling . ( a) under normal conditions, insulin / igf - i engagement of the insulin or igf - i receptor (ir) leads to tyr phosphorylation of irs proteins and subsequent recruitment and activation of pi3k. pi3k activity leads to activation of akt, which phosphorylates and inhibits many downstream substrates, including bad, foxo transcription factors, gsk3, and tuberin (tsc2). through these and other targets , akt activity stimulates glucose uptake and cell growth and proliferation, and inhibits apoptosis. akt - directed phosphorylation of tuberin relieves its inhibition of tor, via activation of the small g protein rheb (not depicted). tor activation by this pathway requires the presence of nutrients and in activation of s6k and inhibition of 4e - bp1. s6k phosphorylates the ribosomal s6 protein (not depicted) and can feedback and inhibit irs proteins (see s6k1 : closing the loop section for details). (b) under atypical conditions of constitutive tor activation, arising from chronic insulin / igf - i exposure, excess nutrients (e.g., amino acids and free fatty acids), inflammatory cytokines, or genetic loss of specific tumor suppressor genes, such as tsc2 (encoding tuberin), aberrantly high s6k activity shuts down insulin / igf - i signaling. therefore, although this unregulated tor activity leads to constitutive eif4e activation, it leads to insulin / igf - i resistance through down - regulation of irs protein function. this feedback inhibition prevents akt - mediated glucose uptake and regulation of its downstream substrates. the study of tsc1 and tsc2 mefs has uncovered a striking inability of serum, pdgf, and, especially, insulin and igf - i to activate the pi3k akt pathway in these cells (jaeschke et al . , 2002 ; kwiatkowski et al ., 2002 ; zhang et al ., 2003). this strong attenuation of akt activation is also seen in drosophila melanogaster tsc1 cells and larvae (radimerski et al ., 2002). the insensitivity of tsc - deficient mefs to pdgf is due to a loss of expression of both pdgf receptor and pdgf receptor (zhang et al ., 2003). however, two groups have now characterized the mechanism of insulin / igf - i resistance in these mefs (harrington et al ., 2004 ; they found that the resistance is a consequence of constitutive down - regulation of irs-1 and irs-2 that is due to sustained and unregulated mtor signaling . found that in the absence of tsc2, both irs-1 and irs-2 shift to a slower mobility during sds - page, which is generally indicative of an increase in ser / thr phosphorylation, and this shift is blocked by short (1 h) treatments with rapamycin. this group found that the mrna and protein levels of irs-1 alone are decreased in tsc - deficient mefs, whereas shah et al. found that the levels and stability of both the irs-1 and irs-2 proteins are decreased in these cells. importantly, both studies found that long - term (24 h) treatment of tsc - deficient mefs with rapamycin completely restores irs-1 protein levels and the insulin / igf - i responsiveness of the pi3k akt pathway. together, these studies demonstrate that high levels of constitutive mtor activity strongly down - regulate insulin / igf - i signaling through combined effects on irs-1 expression and irs-1 and irs-2 serine phosphorylation and protein stability. it is now well established that mtor signaling is intimately involved in many pathways that lead to insulin resistance through irs-1/2 down - regulation. the finding in drosophila that larvae doubly mutant for dtsc1 and ds6k have normal levels of akt activity, relative to the attenuated state of dtsc1 larvae, was the first suggestion that this feedback mechanism downstream of mtor is likely to be mediated by s6k (radimerski et al ., 2002). have now found that small interfering rna knockdown of s6k1 or s6k2 can restore the expression of irs-1 mrna in tsc2 mefs. 2004 ) found that a kinase - dead version of s6k1 can block this negative feedback loop and activate akt. although the mechanism of transcriptional regulation of irs-1 by s6k1/2 remains unknown, it appears that the effect of s6k1 on irs-1 serine phosphorylation is direct. in in vitro kinase assays, s6k1 directly phosphorylates s302 of mouse irs-1 (harrington et al ., 2004), a site matching its preferred substrate recognition motif (alessi et al ., 1996). furthermore, s302 is constitutively phosphorylated in tsc2 mefs, and this phosphorylation is inhibited by treatment with either rapamycin or s6k1 small interfering rnas. the from in vitro binding assays suggest that phosphorylation of this site by s6k hinders the ability of irs-1 to associate with the insulin receptor (harrington et al ., 2004). a recent independent study also found that insulin and igf - i stimulate mtor - dependent s302 phosphorylation in various cell lines, as well as in skeletal muscle (giraud et al ., 2004). however, the of overexpression experiments led this group to conclude that s302 phosphorylation enhances, rather than inhibits, insulin signaling. although additional experiments are necessary to resolve this discrepancy, two other research groups have recently provided further evidence that s302 phosphorylation inhibits signaling from irs-1 (greene et al . a critical in vivo role for s6k1 in desensitizing tissues to insulin was demonstrated in a recent study on s6k1-deficient mice ( um et al ., 2004). the researchers found that these mice are hypersensitive to insulin and impervious to the obesity - induced insulin resistance detected in wild - type mice. in the adipose tissue of wild - type mice, a dramatic increase in s6k1 activation is observed in response to a high fat diet or in genetic models of obesity, and this is accompanied by an increase in irs-1 phosphorylation on s307, s632, and s635 and by attenuation of insulin - induced akt activation. however, under these same conditions in s6k1 mice, these sites on irs-1 are not phosphorylated, and akt remains responsive to insulin. although this study did not examine s302, an increase in phosphorylation of this site has recently been demonstrated in mouse models of obesity and insulin resistance (werner et al ., 2004). these demonstrate that s6k1 is essential to multiple pathways that render cells unresponsive to insulin, including those originating from chronic exposure to insulin, ffas, and perhaps tnf-. interestingly, mice lacking either s6k1 or jnk1 are similarly protected from obesity - induced insulin resistance and display loss of irs-1 phosphorylation on s307 (hirosumi et al ., 2002 ; um et al ., 2004) , a site found to be a direct target of jnk (aguirre et al ., 2000). it appears that phosphorylation of s307 and other serines on irs-1 might, therefore, exhibit cooperation with one another in response to some stimuli. because mtor signaling appears to be required for the effects of several pathways on irs-1, it seems likely that there is an initial s6k1-mediated phosphorylation event, perhaps on s302, that is required for other serine kinases to subsequently phosphorylate irs-1. if this model is correct, this mechanism would prevent any pathway from blocking insulin signaling when mtor activity is low, such as during glucose or amino acid starvation. this would allow cells, and mtor, to remain maximally sensitive to the insulin pi3k akt pathway, which is involved in nutrient uptake. as a downstream target, s6k is poised to sense the degree of activation of mtor, which is stimulated by both nutrients and growth factors (fig . 1 a). as a critical component of the negative feedback loop, s6k then has the ability to modify accordingly the level of input from some growth factors, such as insulin and igf - i. however, under nutritional conditions in which mtor is chronically activated, such as in the presence of excess amino acids or during high fat diets, s6k will constitutively shut down the responsiveness of certain cells to insulin (fig . this suggests that rapamycin ( or, more specifically, an inhibitor of s6k) might be an effective treatment for certain metabolic disorders, such as type 2 diabetes, characterized by insulin resistance (um et al ., 2004). chronic activation of mtor signaling can also from genetic loss of certain tumor suppressor genes, including pten, tsc1/2, and lkb1 (neshat et al ., 2001 ; kwiatkowski, 2003 ; shaw et al ., 2004). interestingly, mutations in these genes are associated with the specific tumor syndromes cowden's disease (pten), tsc (tsc1/2), and peutz - jegher's syndrome (lkb1), which are all characterized by the development of benign tumors classified as hamartomas. although aberrant mtor activation appears to be a common biochemical link between cells lacking these tumor suppressors, the activation state of the pi3k akt pathway varies. loss of pten (phosphatase and tensin homologue deleted on chromosome 10), which counters the activity of pi3k, leads to constitutive activation of akt (for review see cantley and neel, 1999). however, as discussed above for tsc1/2, mefs lacking lkb1 also exhibit some attenuation of akt activation (shaw et al ., 2004). this discrepancy might have profound effects on the nature of the tumors that arise in patients with these different syndromes. the overall development of a hamartoma might be the of constitutive mtor activity, but the severity and malignancy potential of the tumor might depend on the ability to overcome the feedback inhibition of akt. the attenuation of akt is strongest in the absence of the tsc genes, and the inability of akt to phosphorylate and inhibit its downstream substrates other than tuberin, such as specific members of the foxo family of transcription factors, gsk3, and bad, could limit the proliferation and survival capacity of tsc hamartomas (fig . consistent with this idea, malignant tumors are very rare in tsc patients ( kwiatkowski, 2003), whereas in cowden's disease, where loss of pten activates akt independently of growth factor signaling, the development of malignant tumors is much more common (cantley and neel, 1999). because rapamycin relieves this feedback inhibition of akt (harrington et al ., 2004 ; shah et al ., 2004), a difficult question in the field is whether this mtor inhibitor would be beneficial or detrimental to patients with hamartomas exhibiting aberrant mtor activation. consistent with mtor activation driving initial tumor development in these related syndromes, rapamycin has been proven to be effective at blocking the formation of tumors in rodent models of tsc (kenerson et al ., 2002). however, it is less clear whether rapamycin would be a useful treatment once the tumor has already been established, as is the case in patients diagnosed with tsc. in fact, studies of tsc1/2- and lkb1-deficient mefs have demonstrated that under some growth conditions rapamycin actually enhances survival of cells devoid of these tumor suppressors (inoki et al ., 2003 ; , 2004 ; shaw et al ., 2004), and this is likely due to the restoration of critical survival pathways controlled by akt. however, these studies are done on a short - term basis and under very specific conditions. it seems likely that, although rapamycin's inhibition of s6k activation would eliminate the negative feedback mechanism, it would also shut down the aberrant activation of eif4e and cap - dependent translation. based on many studies suggesting that eif4e is a potent oncogene (e.g., for review see mamane et al ., 2004 ; ruggero et al ., 2004 ; wendel et al ., 2004), it would appear that the mtor - dependent activation of eif4e, through 4e - bp1 phosphorylation, may be at the heart of tumorigenesis in syndromes exhibiting constitutive mtor signaling. if this is true, then long - term treatment with rapamycin would block the primary cellular process driving tumor growth, irrespective of akt activity. furthermore, it is currently unknown whether the down - regulation of pdgf receptors observed in tsc - deficient mefs is sensitive to rapamycin (zhang et al ., 2003). it remains to be established whether the state of this mtor - dependent feedback mechanism is what delineates the severity of some tumors and metabolic diseases. however, studies on this pathway, and the diseases to which it contributes, underscore the importance of elucidating the complex wiring of seemingly basic signal transduction pathways.

proper regulation of the phosphoinositide 3-kinase akt pathway is critical for the prevention of both insulin resistance and tumorigenesis. many recent studies have characterized a negative feedback loop in which components of one downstream branch of this pathway, composed of the mammalian target of rapamycin and ribosomal s6 kinase, block further activation of the pathway through inhibition of insulin receptor substrate function. these findings form a novel basis for improved understanding of the pathophysiology of metabolic diseases (e.g., diabetes and obesity), tumor syndromes (e.g., tuberous sclerosis complex and peutz - jegher 's syndrome), and human cancers.

asians and pacific islanders (apis) constitute 5% of the united states (us) population and 13% of the new york city (nyc) population. from 2000 through 2010, the api population experienced the fastest rate of growth than any other racial / ethnic groups both nationally (43% increase) and in nyc (32% increase). the city's api population is the largest among us cities, 1.1 million persons, according to the 2010 census. while cardiovascular disease is the leading cause of mortality among non - hispanic whites, non - hispanic blacks, and hispanics, cancer is the leading cause of death among apis, both nationally in 2007 (106.7 per 100,000) and in nyc in 2010 (105.9 per 100,000). apis experience lower death rates for certain common cancers (i.e., lung, colorectal, breast, and prostate) than other racial / ethnic groups in the united states. however, apis experience the highest death rates from some less common cancers, particularly those associated with infectious agents, such as nasopharynx, liver, and stomach cancer. published analyses on cancer mortality among apis have largely focused on california and new york city , except one study utilizing data from selected sites throughout the us, but all these studies are based on data that is more than a decade old. in california, mccracken et al. examined cancer mortality among asian americans overall from 2000 to 2002, and kwong et al. described cancer mortality by asian subgroups from 1997 through 2001. in nyc, freeman et al. examined cancer mortality by overall racial / ethnic group between 1990 and 2000. miller et al. examined cancer incidence and mortality by asian subgroups for 19982002 from selected state and regional cancer registries covering 54% of the us api population. cancer mortality among api subgroups has not been studied in nyc, aside from a 20-year - old nyc study (1988 through 1992) examining site - specific cancer mortality rates among the chinese only. thus, to our knowledge, no recent analyses of api or api subgroup cancer mortality rates within a us population have been conducted. we aim to characterize site - specific cancer mortality rates among apis in nyc, comparing them to other racial / ethnic groups for the 10-year period from 2001 through 2010. we will also examine site - specific cancer rates among different api subgroups (i.e., chinese, korean, asian indian, and filipino) to explore heterogeneity within the diverse api group. this analysis was based on new york city annual population estimates obtained from the us census bureau, the american community survey public use microdata samples (2005 through 2009), and mortality data for the 10-year period (2001 through 2010) from the new york city department of health and mental hygiene's (nyc dohmh) bureau of vital statistics. variables included sex, age, race / ethnicity, ancestry, birthplace, education, marital status, and underlying cause of death. underlying causes of death were coded according to the 10th revision of the international statistical classification of diseases and related health problems (icd-10). common site - specific cancers across all racial / ethnic groups (i.e., lung, colon and rectum, esophagus, prostate, breast, ovary, and cervix) and those specifically common in apis (i.e., nasopharynx, liver, and stomach) were selected for study. api subgroups were determined by the races reported on death certificates, which is provided by funeral directors in nyc. we did not differentiate between foreign - born versus us - born apis because the proportion of us - born was only 2.1% of deaths which is too small to merit analysis. this is likely because of the relatively young age of new york city's us - born asian population; 81% are less than 45 years of age based on us census bureau's 20052009 american community survey microdata sample. cancer mortality rates and 95% confidence intervals were calculated for major racial / ethnic groups using nyc dohmh mortality data and us census population estimates. cancer mortality rates and 95% confidence intervals for chinese, korean, asian indian, and filipino subgroups were calculated using nyc dohmh mortality records and the 20052009 american community survey public use microdata sample. the 2010 us census reports information on 6 asian subgroups: chinese, korean, asian indian, filipino, japanese, and vietnamese. new york city's japanese and vietnamese populations were too small to allow for inclusion in this analysis. in 2010, 13,333 (25.4%) new york city decedents died from a malignant neoplasm. among api decedents, 943 (29.9%) died from a malignant neoplasm. apis have the overall lowest cancer mortality rates across all racial / ethnic groups. however, between 2001 and 2010, cancer mortality rates declined in all racial / ethnic groups in nyc, except apis. the api cancer mortality rate has remained stable from 2001 through 2010 (see figure 1). from 2001 through 2010, a total of 82,608 decedents died in new york city from the 10 selected cancers (table 4); 5,548 (6.7%) of these decedents were apis (3,200 males and 2,348 females) (table 1). compared with non - hispanic whites, api decedents were more likely to be male, married, and having less than a high school education. on average, api decedents died 5 years earlier from cancer than did non - hispanic whites. the age - adjusted nasopharyngeal cancer mortality rate among api men (2.6 per 100,000) was the highest across all racial / ethnic groups and nearly 10 times higher than the rate for non - hispanic white men (0.29 per 100,000 ; see table 2). the mortality rates of liver and stomach cancer were twice as high in api men as in non - hispanic white men (liver cancer, 15.9 versus 7.9 per 100,000 and stomach cancer, 11.8 versus 6.0 per 1000,000, resp .). the mortality rate for nasopharyngeal cancer among api women (0.7 per 100,000) was the highest of all racial / ethnic groups, 7 times as high as in non - hispanic white women (0.1 per 100,000). the liver cancer mortality rate was almost 2 times higher in api women (5.4 per 100,000) than in non - hispanic white women (3.0 per 100,000), and api women had the highest stomach cancer mortality rate (5.6 per 100,000) of all racial / ethnic groups. among both men and women, apis have the lowest colorectal cancer mortality rates of all racial / ethnic groups. api men and women have age - adjusted lung cancer mortality rates (38.2 and 16.7 per 100,000, resp .) that surpass those of hispanic men and women (33.5 and 14.7 per 100,000, resp .) but not non - hispanic whites or blacks. for all studied sites, cancer death rates were higher in api men than api women. from 2001 through 2010, api males experienced higher age - adjusted liver cancer mortality rates than non - hispanic white males (15.6 to 14.0 per 100,000 versus 8.3 to 10.3 per 100,000, resp . ; , api females experienced a slight increase in age - adjusted liver cancer mortality rates, while this rate was stable among non - hispanic white females ( 5.3 to 6.1 per 100,000 versus 3.1 to 3.0 per 100,000, resp . , api males did not experience the dramatic decline in age - adjusted lung cancer mortality rates that non - hispanic white males did ( 38.6 to 38.2 per 100,000 versus 62.2 to 47.0 per 100,000, resp . ; see figure 4). age - adjusted lung cancer mortality rates among api females increased during 2001 through 2010, while non - hispanic white females experienced a decrease (17.4 to 19.7 per 100,000 versus 39.8 to 34.5 per 100,000, resp . ; see figure 5). overall, 5,548 cancer deaths occurred among apis in new york city from 2001 through 2010 (see table 3). chinese comprise the majority (66.7%) of api cancer decedents. compared to other api subgroups, chinese decedents were more likely to be male, have less than a high school education, and die at an older age on average. compared to other api subgroups, asian indian cancer decedents were more likely to be married and die at a younger age. filipino decedents were more likely to be female and have more than a high school education. chinese men and women had the highest lung cancer mortality rates (47.1 and 20.0 per 100,000, resp .) as compared to other api subgroups. lung cancer mortality rates in the chinese have increased from 2001 to 2010 (40.0 to 51.5 per 100,000 in men and 16.2 to 25.8 per 100,000 in women), while they have decreased in non - hispanic whites (62.2 to 47.0 per 100,000 in men and 39.8 to 34.5 per 100,000 in women). among apis, chinese women had the highest colon cancer mortality rate (10.6 per 100,000) among women and chinese men had the second highest colon cancer mortality rate (16.1 per 100,000) among men. chinese men and women had the highest nasopharyngeal cancer mortality rates (4.5 and 1.2 per 100,000, resp .) across all racial / ethnic groups, and these rates were statistically significantly higher than in the other racial / ethnic groups. korean men had the highest liver and colon cancer rates (25.3 and 17.6 per 100,000, resp .) compared to other api subgroups. korean women had the highest liver cancer mortality rate, which was statistically significantly higher than other api subgroups (7.5 per 100,000 versus 6.0 per 100,000 for chinese women, 2.9 per 100,000 for asian indian women and 2.6 per 100,000 for filipino women). korean men and women had the highest stomach cancer rates, which were statistically significantly higher than other api subgroups (27.7 and 11.0 per 100,000, resp .). korean men and women had the second highest age - adjusted lung cancer mortality rates (38.9 and 12.9 per 100,000, resp .). among api subgroups, filipino men had the highest prostate mortality rate (9.5 per 100,000). filipino women had the highest breast and ovarian cancer mortality rates (13.4 and 6.4 per 100,000, resp .). we investigated site - specific cancer mortality rates among apis and api subgroups, given that cancer is the leading cause of death among apis. while apis in nyc have the lowest overall cancer mortality rate of the 4 major racial / ethnic groups, certain site - specific cancer rates are much higher among apis than other racial / ethnic groups and among api subgroups. examining rates for apis as a group masks the true heterogeneity of their cancer burden. varying environmental and behavioral influences, including tobacco use, exposure to infectious agents, and diet, are likely driving these observed patterns of api cancer mortality. these findings warrant consideration of targeted cancer mortality prevention efforts for affected subgroups, including hepatitis vaccination, screening, and treatment; smoking cessation; and cancer screening. lung cancer death rates in chinese new yorkers are high and have increased from 2001 through 2010 for both men and women. it is known that tobacco smoking, primarily cigarettes, is the major cause of lung cancer. in nyc, the 2010 community health survey, a nyc dohmh population - based telephone survey, found a smoking prevalence of 20% among chinese men and less than 1% among chinese women. the increasing lung cancer death rate among chinese women in the setting of low prevalence of smoking is worrisome, and more research is needed to determine factors contributing to the increased lung cancer death rates in chinese women. second - hand smoke exposure at home and work and exposure to cooking oil from high - temperature frying may contribute to the increased risk of lung cancer in chinese women. in addition, the smoking prevalence among the chinese population overall has remained the same over 8 years (9.2% in 2002 and 10.2% in 2010), whereas the overall smoking prevalence in nyc dropped significantly from 21.5% in 2002 to 14% in 2010. given that nyc mounted an aggressive tobacco control program during this period, the increasing lung cancer death rates and the stable smoking prevalence rates among the chinese suggest that we may not be reaching this subgroup. global data suggest that lung cancer mortality rates in china are lower than in nyc. it is not known why this is true, but two possibilities would be increased immigration to nyc from areas of china with higher rates of lung cancer and decreased accuracy of cause of death information. in 2008, according to the international agency for research on cancer, lung cancer mortality rates were 39.6 per 100,000 in chinese men and 18.3 per 100,000 in chinese women versus 47.1 and 20.0 per 100,000 for chinese men and women in nyc, respectively, per our analyses. one reason that could explain these persistently high rates of lung cancer mortality among the chinese in nyc is the culturally accepted nature of tobacco smoking in certain asian countries, particularly among men. according to the global adult tobacco survey, 63% of men in china currently smoked in 2010. in addition, giving cigarettes as gifts during special occasions is a cultural norm, and immigrants have access to low - cost cigarettes from taiwan and china. since 2005, the nyc mayor's office and nyc dohmh have launched several culturally relevant media campaigns focusing on the chinese, including translated posters and print advertisements to promote tobacco cessation. the nyc dohmh has a long - standing nicotine patch and gum give - away program, and in 2010, online registration was made available in chinese along with translated nicotine patch and gum kit contents. dohmh also has a 311 hotline with chinese language capability to help interested callers quit smoking. in 2012, a dedicated chinese hotline was created to attract more participants to the nicotine patch and gum give - away program. despite these efforts, more research is needed to determine the barriers to quitting smoking in the asian population. closely following the chinese, koreans have the second highest lung cancer mortality rates (38.9 per 100,000 for men and 12.9 per 100,000 for women) among apis. in south korea from 2008 to 2010, 42.3% of korean men have been reported to smoke. in nyc, for 20062008, korean's self - reported rate of smoking was 16.9% based on the nyc community health survey, although small numbers limit the precision of that estimate. nyc has not focused on special tobacco cessation efforts in this group due to its small population size (only 1.0% of nyc population). nasopharyngeal cancer death rates in nyc are higher among apis than other racial / ethnic groups, and this cancer disproportionately affects the chinese. the nasopharyngeal cancer mortality rate in chinese men (4.5 per 100,000) was 15 times that of non - hispanic whites (0.3 per 100,000). in chinese women, the nasopharyngeal cancer mortality rate (1.2 per 100,000) was 12 times as high as in non - hispanic white women (0.1 per 100,000). according to the world health organization, nasopharyngeal cancer mortality rates in 2008 in china for males and females were 1.9 and 0.8 per 100,000, respectively, based on data from the international agency for research on cancer. most immigrants from china to the us come from guangdong province, where the incidence of nasopharyngeal cancer is very high (25 cases per 100,000). several epidemiologic studies have proposed that the epstein - barr virus (ebv), smoked fish products, and genetic predisposition are associated with high nasopharyngeal cancer rates among the chinese. in certain parts of southern china, particularly guangdong province where nasopharyngeal cancer is common, at - risk individuals are being screened by ebv testing, followed by periodic examination of the nasopharynx and neck if positive. earlier detection of nasopharyngeal carcinoma allows for treatment with improved 5-year survival. in the united states, no nasopharyngeal cancer screening guidelines exist. in cities like nyc where a large proportion of chinese immigrants are from southern china, a high index of clinical suspicion for this type of cancer should be maintained by clinicians caring for these immigrants. further research is indicated to evaluate whether the screening approach used in southern china is effective at preventing mortality and to determine whether screening is appropriate for chinese new yorkers. in nyc , the liver cancer death rate is higher among apis, particularly koreans and chinese, than in other racial / ethnic groups. hepatitis b virus (hbv) infection is highly prevalent in asian countries, including south korea and china, and is the main risk factor for developing liver cancer in countries endemic with hbv. other contributing risk factors include cirrhosis (associated with infection, obesity, and alcohol) and aflatoxin exposure. nyc dohmh surveillance for chronic hbv infection from june 2008 to november 2009 identified 156 cases of infection, of whom 87 (56%) were in chinese born persons and few (<3) were in persons originating from korea. however, a 2008 prevalence study found that koreans represented 4.6% of nyc's chronic hbv infections, while they constituted only 1.0% of the nyc population, confirming their higher risk for chronic infection compared to other new yorkers. in one 2005 nyc study of a clinic - based screening program, chinese had the highest prevalence (21%) of chronic hbv infection among the patients screened, but koreans also had a high prevalence (5%) of infection. given elevated rates of liver cancer among the chinese and koreans, nyc clinicians should be aware that the centers for disease control and prevention and dohmh currently recommend screening individuals at risk for hbv infection, including persons from hbv - endemic countries such as korea and china, treatment of chronic hbv infection, and periodic liver cancer screening in those with chronic hbv infection, which may help to prevent progression and death from liver cancer. given that current evidence suggests that koreans have a lower chronic hbv infection rate compared with chinese, other factors may be playing a role in elevating their liver cancer death rate above that of the chinese. when liver cancer was listed as the underlying cause of death, the use of alcohol as a contributing cause was mentioned in more korean decedents (1.8%) than chinese decedents (0.2%), although its mention was infrequent for both. in nyc's community health survey for the period 2002 to 2008, individuals of korean descent reported binge drinking almost 4 times more frequently than individuals of chinese descent (23.5% versus 5.8%, resp .) and heavy drinking was reported more frequently as well (3.4% versus 1.3%), although small numbers limit the precision of these later estimates. synergism between alcohol consumption and chronic hbv infection may possibly increase the risk of cirrhosis and thus liver cancer deaths in koreans in nyc. further research is needed to determine the contribution of alcohol use to liver cancer deaths among koreans. in addition, stomach cancer has also been associated with eating preserved food and foods rich in nitrites and/or nitrates. no routine stomach cancer screening is recommended in the us, but screening programs have been implemented in japan. the risk of stomach cancer deaths in the japanese living in japan (20.5 per 100,000 in men) is comparable to mortality rates in nyc chinese (12.9 per 100,000 in men) and koreans (27.7 per 100,000 in men). an improving trend in the stomach cancer survival rate has been observed in japan and can possibly be attributed to early cancer detection. a formal evaluation of the risk and benefits of using the japanese screening approach in high - risk groups should be considered for nyc and other urban areas with a sizeable api population. breast cancer is the leading cause of cancer death in nyc asian indian and filipina women. multiple factors contribute to breast cancer mortality, including genetic predisposition and socioeconomic and cultural circumstances, but late - stage diagnosis is strongly associated with higher mortality rates. in the nyc community health survey, women of asian descent aged 40 years and older reported a lower rate of mammography screening (67.5%) than nyc women overall (75.3%), and specifically, asian indian women in nyc had a substantially lower rate of screening (58.9%). the rate for filipina women was a few percent below the citywide rate (72.1%) based on their self - reported responses. however, a hawaiian study found that filipina women are less compliant with mammography guidelines and are diagnosed at a more advanced stage of cancer at time of diagnosis. efforts to target specific racial / ethnic groups to increase breast cancer screening may be needed. more research is also needed in nyc to determine the reasons for lower rates of mammography screening in asian indian and possibly filipina women, which may contribute to higher breast cancer mortality rates. the limitations of this study include potential underascertainment of api population size by the 2010 census or american community survey, which would in an overestimation of mortality rates in the population. in particular, undocumented immigrants may not be captured in the census because of their reluctance to be counted, contributing to underestimation of the population. underestimation of deaths among apis may also occur if persons who spend most of their working life here in the us return to their home country for retirement and death. asians are the fastest growing racial / ethnic group in the us, and they have a unique cancer burden that requires special attention. the leading cause of death in apis nationally and in nyc is malignant neoplasms. apis are disproportionately affected by some less common cancers, including nasopharyngeal, liver, and stomach cancers. hepatitis b screening has been recommended by cdc for high - risk groups, including persons from china and korea. culturally tailored tobacco control policies and programs have been implemented over the last decade, and further evaluation is needed to determine their impact. in those populations that have reduced smoking rates, the impact may become more apparent in the next decade as fewer people develop smoking - related cancer, given that there is a long lag time between smoking exposure and cancer development. potential population - based screening approaches to detect and treat early nasopharyngeal and stomach cancers in apis require more evaluation before implementation but should be considered. the substantial variation in cancer - specific mortality rates across asian racial subgroups indicates that api subgroup analyses such as these are necessary to plan public health interventions.

asians and pacific islanders' (apis) leading cause of death is cancer. we compared apis' age - adjusted cancer mortality rates to other racial / ethnic groups and by api subgroup (i.e., chinese, koreans, asian indians, and filipinos) using new york city (nyc) mortality data and census bureau population estimates for 20012010. while other racial / ethnic groups' overall cancer mortality rates declined in nyc during the last decade, apis remained stable. apis overall had the lowest mortality rates for more common cancer types (i.e., lung, colorectal, breast, and prostate), but the highest mortality rates for certain less common cancers (i.e., nasopharyngeal, stomach, and liver). chinese new yorkers' lung cancer death rates were very high compared to other apis and comparable to non - hispanic whites (47.1/100,000 versus 49.5/100,000, resp .). chinese men had much higher nasopharyngeal cancer mortality rates (4.5/100,000 versus 0.3/100,000 for non - hispanic whites). korean men had the highest liver and stomach cancer mortality rates (25.3/100,000 and 27.7/100,000, resp ., versus 7.9/100,000 and 6.0/100,000 for non - hispanic whites). analysis of cancer rates by api subgroup provides the detailed information needed to plan cancer prevention efforts. these findings warrant consideration of targeted cancer mortality prevention efforts for affected subgroups, including hepatitis vaccination, screening, and treatment; smoking cessation; and cancer screening.

periodontitis is a chronic infectious disease of the supporting tissues of the teeth, with multiple related factors. in recent years , interest has grown regarding the relationship with other systemic conditions to identify some new aspects to improve diagnostic tools and treatment outcomes. metabolic syndrome (mes) consists of a combination of impaired glucose regulation, abdominal obesity, dyslipidemia, and high blood pressure. it is estimated that approximately one - fourth of the world's adult population is affected by mes. it is generally accepted that the origin is a pro - inflammatory state derived from excessive calorie intake and over nutrition and other chronic inflammatory diseases. oxidative stress has been proposed as a potential common link to explain relationship among each component of mes and periodontitis. a cross - sectional study conducted by shimazaki et al. , in 2007, showed that individuals exhibiting more components of mes had a higher odds ratio (or) for a greater periodontal probing depth (pd) and clinical attachment level (cal). in addition, other cross - sectional studies revealed that individuals with mes had a higher risk for poor periodontal status, and those individuals with poor periodontal status had a higher risk for mes. elevated blood levels of inflammatory markers, such as c - reactive protein (crp) and interleukin (il)-6, were reported in patients with periodontal disease, suggesting that periodontal disease is a mild chronic inflammatory disease affecting the systemic conditions. aggravation of glucose tolerance in people with deep periodontal pockets was also shown epidemiologically, suggesting that infection with periodontal disease pathogens enhance tumor necrosis factor - alpha (tnf-) production, induces the prediabetic condition, and leads to abnormal glucose tolerance. furthermore, negative influences by lipopolysaccharide and cytokines produced by inflammation, such as tnf- and il-1 on lipid metabolism, were reported, suggesting that gram - negative anaerobe - induced periodontal disease has some influence on lipid metabolism. considering these findings, it is possible that periodontal disease increases the risk of developing mes as a gram - negative anaerobe - induced mild chronic inflammatory disease. prevalence of mes is high among asians including indians and is rising, particularly with the adoption of modernized lifestyle. however, little information is available on the possible association between periodontitis and mes as a sole clinical entity in indian population; this study was carried out to assess the association between mes and chronic periodontitis. the total percentage of rural population in himachal pradesh 90.20% of the total population residing in 17,495 inhabited villages. the rural population has limited access to health - care facility as compared to the urban population. himachal pradesh has the highest percentage of rural population among all the states of the country with poor socioeconomic status as compared to those residing in urban areas. the cases and controls were screened on the basis of cal according to the american academy of periodontology classification system. subjects meeting the inclusion / exclusion criteria were invited to take part in the study and were provided with the subject information sheet and consent form. three hundred and seventy - eight subjects were enrolled for the study of duration 22 months. among all the consecutive patients population referred for care to the outpatient department of periodontology and services of h.p. government dental college and hospital, shimla, india, 119 (out of 378 subjects) patients did not complete the study. therefore, among the 259 subjects who completed the study, 130 individuals diagnosed with moderate / severe periodontitis were enrolled in the periodontal clinic, whereas 129 patients without periodontitis were invited to participate while attending the oral surgery or restorative clinics and served as nonaffected controls. thus, patients with moderate / severe chronic periodontitis formed the study population and those without periodontitis on clinical evaluation formed the control group. however, screening of cases, i.e., individuals with moderate / severe periodontitis, was based on mean clinical attachment loss measured at four sites per tooth (mesiobuccal, buccal, distobuccal, and lingual) of the entire dentition except for third molars. inclusion criteria for the cases were diagnosis of moderate / severe periodontitis based on the 1999 consensus classification of periodontal disease by armitage and thomas f. flemmig. inclusion criteria for cases with moderate / severe chronic periodontitis: patients with at least 20 teeth and more than 30% of sites exhibiting 34 mm of mean clinical attachment loss for moderate chronic periodontitis and more than 30% of sites 5 mm of mean clinical attachment loss for severe chronic periodontitis, minimum age of 20 years, and patients consenting to participate. inclusion criteria for controls: the absence of the clinical and radiographic manifestations of periodontal disease, at least, 20 teeth present in the oral cavity and no history of periodontal treatment in the past, minimum age of 20 years, and patients consenting to participate. exclusion criteria: patients not willing to participate in the study, pregnant patients, patients of rheumatic heart disease, congenital heart disease and/or with prosthetic valves who were at high risk for endocarditis, and all patients medically unfit to participate in the study. mes was diagnosed based on the presence of three of the five following components using modified adult treatment panel iii criteria: abdominal / central obesity (as indicated by increased waist circumference or markedly increased body mass index and defined as waist circumference with ethnicity - specific values): waist circumference 90 cm in men and 80 cm in women (based on a chinese, malay, and asian indian population), hypertriglyceridemia: 150 mg / dl (1.69 mmol / l), low- and high - density lipoprotein cholesterol: < 40 mg / dl (1.04 mmol / l) in men; < 50 mg / dl (1.29 mmol / l) in women, high blood pressure: 130/85 mmhg, and high fasting glucose: 100 mg / dl (6.1 mmol / l). spss package version 16 (spss inc ., chicago, il, usa) was used. the significance of difference in mean values of continuous variables between study and control group was estimated by student's t - test. the significance of the difference in the distribution of discrete variables between the groups was estimated with chi - square test. association between chronic periodontitis and mes was estimated by calculating or with 95% confidence interval (ci). the cases and controls were screened on the basis of cal according to the american academy of periodontology classification system. subjects meeting the inclusion / exclusion criteria were invited to take part in the study and were provided with the subject information sheet and consent form. three hundred and seventy - eight subjects were enrolled for the study of duration 22 months. among all the consecutive patients population referred for care to the outpatient department of periodontology and services of h.p. government dental college and hospital, shimla, india, 119 (out of 378 subjects) patients did not complete the study. therefore, among the 259 subjects who completed the study, 130 individuals diagnosed with moderate / severe periodontitis were enrolled in the periodontal clinic, whereas 129 patients without periodontitis were invited to participate while attending the oral surgery or restorative clinics and served as nonaffected controls. thus, patients with moderate / severe chronic periodontitis formed the study population and those without periodontitis on clinical evaluation formed the control group. however, screening of cases, i.e., individuals with moderate / severe periodontitis, was based on mean clinical attachment loss measured at four sites per tooth (mesiobuccal, buccal, distobuccal, and lingual) of the entire dentition except for third molars. inclusion criteria for the cases were diagnosis of moderate / severe periodontitis based on the 1999 consensus classification of periodontal disease by armitage and thomas f. flemmig. inclusion criteria for cases with moderate / severe chronic periodontitis: patients with at least 20 teeth and more than 30% of sites exhibiting 34 mm of mean clinical attachment loss for moderate chronic periodontitis and more than 30% of sites 5 mm of mean clinical attachment loss for severe chronic periodontitis, minimum age of 20 years, and patients consenting to participate. inclusion criteria for controls: the absence of the clinical and radiographic manifestations of periodontal disease, at least, 20 teeth present in the oral cavity and no history of periodontal treatment in the past, minimum age of 20 years, and patients consenting to participate. exclusion criteria: patients not willing to participate in the study, pregnant patients, patients of rheumatic heart disease, congenital heart disease and/or with prosthetic valves who were at high risk for endocarditis, and all patients medically unfit to participate in the study. mes was diagnosed based on the presence of three of the five following components using modified adult treatment panel iii criteria: abdominal / central obesity (as indicated by increased waist circumference or markedly increased body mass index and defined as waist circumference with ethnicity - specific values): waist circumference 90 cm in men and 80 cm in women (based on a chinese, malay, and asian indian population), hypertriglyceridemia: 150 mg / dl (1.69 mmol / l), low- and high - density lipoprotein cholesterol: < 40 mg / dl (1.04 mmol / l) in men; < 50 mg / dl (1.29 mmol / l) in women, high blood pressure: 130/85 mmhg, and high fasting glucose: 100 mg / dl (6.1 spss package version 16 ( spss inc ., chicago, il, usa) was used. the significance of difference in mean values of continuous variables between study and control group was estimated by student's t - test. the significance of the difference in the distribution of discrete variables between the groups was estimated with chi - square test. association between chronic periodontitis and mes was estimated by calculating or with 95% confidence interval (ci). the study is attempted to evaluate the association between mes and chronic periodontitis in case control study design. the cases and controls were well matched with insignificant difference for age (mean age : 39.06 vs. 37.9, p < 0.475), sex (male : 46.9% vs. 58.9%, p < 0.070), residential (rural : 60.8% vs. 55.8%, p < 0.494), educational status (illiterate : 6.2% vs. 3.9%, p < 0.655), and tobacco consumption status (current tobacco consumers : 14.6% vs. 11.6%, p < 0.597). the prevalence of diabetes (3.1% vs. 0.8%, p < 0.37), hypertension (15.4% vs. 13.2%, p < 0.741), and coronary artery disease / myocardial infarction (0.8% vs. 1.6%, p < . clinical characteristics of the study population clinical characteristics of the study population risk factors and atherovascular disease distribution among the study population however, the association of mes and chronic periodontitis was strong and significant with or : 2.64, 95% ci : 1.365.18, and p < 0.003 . comparison of mean values of components of mes and lipid parameters between cases and controls reveals that the mean waist circumference ( mean difference : 4.8 , p < 0.002), mean total cholesterol level (mean difference : 9.43 , p < 0.047), mean triglycerides level (mean difference : 25.75 , p < 0.032), and mean ldl level (mean difference : 7.6 , p there was no significant difference in the mean systolic blood pressure ( 2.9 , p < 0.126), diastolic blood pressure (1.69 , p < 0.127), and hdls levels (0.05% , p < 0.963) in the two groups. although mean systolic blood pressure, diastolic blood pressure, and fasting blood sugar level were higher in cases (125.77, 82.99, and 86.38, respectively) compared with control (122.81, 81.3, and 83.68, respectively), it was statistically insignificant. the observed strong association between chronic periodontitis and mes is unlikely to be influenced by the tobacco consumption pattern in the study and control groups as comparison of the distribution of tobacco consumption index in two groups was statistically insignificant. frequency and distribution of biological variables among the study population frequency and distribution of tobacco consumption index of current tobacco consumers among the subjects with or without metabolic syndrome the of this study suggest that there is a strong association of mes and chronic periodontitis was strong and significant with or: 2.64, 95% ci: 1.365.18, and p < 0.003. the association was independent of the various potential confounding risk factors affecting the chronic periodontitis such as age, gender, residential , and tobacco consumption. chronic periodontitis has been associated with systemic alteration such as low - grade inflammation, dyslipidemia, a procoagulant state, glucose intolerance, and endothelial dysfunction. it leads to the state of the chronic systemic inflammatory state through the release of various inflammatory cytokines such as il-6, il-1, tnf-, and crp, which may worsen the insulin resistance. further, the production of oxidative stress - enhancing reactive oxygen species in affected periodontal tissues is a potential factor which could increase insulin resistance that may in mes. mes is characterized by state of constellation of metabolic and biological risk factors presumed to be the of insulin resistance which is thought to be the outcome of the complex interaction of acquired environmental factors related to lifestyle and genetic predisposition. the studies also suggested the association between mes and chronic periodontitis but the exact mechanism is not known. mes could also be a predisposing factor for chronic periodontitis and it can influence the periodontal disease process. mes is known to affect normal vascular function and it may increase the vulnerability for infection because of its predisposition to glucodysregulation. individual risk factors of mes such as obesity can affect the levels of plasminogen activator inhibitor-1, alterations in t - cells and monocytes for macrophage and increased cytokine production, abnormally elevated cytokines il-6 and -1 and tnf-, all of which contribute to periodontitis. adipose tissue can produce cytokines and prostaglandins, which play an important role in tissue destruction during periodontal disease. in obese, hypertensive rats, hyperplasia and hypertrophy of the walls of blood vessels supplying the periodontium was observed. thus, all the individual risk factors for mes either affect the normal periodontium or the periodontal disease process. conversely, as an infectious process with a prominent inflammatory component, periodontal disease can adversely affect mes. the underlying cause - and - effect relationship needs to be explored in future interventional studies that would have important implications in management and preventive strategies of both these conditions more effectively. the finding from this study is in agreement with the findings by shimazaki et al., which reported that mes among 584 japanese women was associated with an increased risk for periodontitis. showed that the severity of periodontal disease, as measured by average pd and average cal, and the extent of periodontal disease, as measured by the percentage of sites with cal 3 mm and the percentage of sites with pd 3 mm, were significantly greater among patients with mes compared to those without mes. it is generally accepted that the origin of metabolic disorders is in pro - inflammatory state derived from excessive caloric intake, over nutrition, and other chronic inflammatory conditions. the pro - inflammatory status also leads to an increase in oxidative stress, which has potential to impair several crucial biological mechanisms. a study has demonstrated an increase in the products of oxidative damage in peripheral blood from persons with periodontitis, which emphasized the influence of periodontitis on serum and/or plasma oxidative markers. reduced antioxidant capacity pro / anti - oxidant capacity between periodontitis and mes could add the evidence for elucidating the mechanism of the link between periodontitis and mes. by removing all confounding potential risk factors for periodontitis and mes, the strong association between the two diseases in our study was not by chance, thus disproving the null hypothesis which was also substantiated by the other previous studies. the design of the study is such that the causal relationship between periodontal diseases and mes can not be identified. attrition of 119 subjects from the study can lead to important loss of the information of the study. multiple logistic regression and multivariate analysis were not done which would have helped in finding out the correlation of the different variables in the study. other confounding variables, for example, in between snacking, lack of exercise, genetics, and hereditary were not included in the study. as the study is conducted in the individuals with more than 20 teeth, the relation between periodontal disease and mes with < 20 teeth and among edentulous remains unknown. in future, the design of the study is such that the causal relationship between periodontal diseases and mes can not be identified. attrition of 119 subjects from the study can lead to important loss of the information of the study. multiple logistic regression and multivariate analysis were not done which would have helped in finding out the correlation of the different variables in the study. other confounding variables, for example, in between snacking, lack of exercise, genetics, and hereditary were not included in the study. as the study is conducted in the individuals with more than 20 teeth , the relation between periodontal disease and mes with < 20 teeth and among edentulous remains unknown. in future, the association was independent of the various potential confounding risk factors affecting the chronic periodontitis such as age, gender, residential , and tobacco consumption. keeping in mind this association, dental professionals in general and periodontists in particular should take mes into consideration when evaluating periodontitis.

: prevalence of metabolic syndrome (mes) is high among asians, including indians and is rising, particularly with the adoption of modernized lifestyle. various studies have reported a significant relationship between periodontal status and mes. the objective of this study is to investigate the association between periodontitis and mes.materials and methods: the study included 259 subjects (130 cases with chronic periodontitis, 129 controls without chronic periodontitis) who underwent medical and periodontal checkup. five components (obesity, high blood pressure, low- and high - density lipoproteins, cholesterol, hypertriglyceridemia, and high plasma glucose) of mes were evaluated, and individuals with 3 positive components were defined as having mes. the periodontal parameter was clinical attachment level (cal) on the basis of which cases were selected with moderate (cal loss 34 mm) and severe (cal loss 5 mm) generalized chronic periodontitis. the association between chronic periodontitis and mes components was investigated using odds ratios (ors) and 95% confidence intervals (cis).: the association of mes and chronic periodontitis was strong and significant with or: 2.64, 95% ci: 1.365.18, and p < 0.003. comparison of mean values of components of mes between cases and controls reveals that the mean waist circumference (mean difference : 4.8 , p < 0.002) and mean triglycerides level (mean difference : 25.75 , p < 0.032) were significantly higher in cases than in control groups. although mean systolic blood pressure, diastolic blood pressure, and fasting blood sugar level were higher in cases (125.77, 82.99 and 86.38, respectively) compared with control (122.81, 81.3 and 83.68, respectively), it was statistically insignificant.:the of this study suggest that there is a strong association between chronic periodontitis and mes. the association was independent of the various potential confounding risk factors affecting the chronic periodontitis such as age, sex, residential , and tobacco consumption.

classifying and managing dcis has always been a thorny issue, often dividing various groups of pathologists around the world. amongst dcis features, the architectural pattern, its prognostic value, and role in grading dcis the current literature on the subject accepts the existence of 3 major architectural patterns of dcis, namely, the solid, cribriform, and micropapillary patterns. it has been variably considered as an early micropapillary dcis or even a subvariant of the atypical ductal hyperplasia. other special types of dcis, such as the apocrine, the endocrine (argyrophilic), and signet ring dcis, are all defined on histological criteria, rather than architectural pattern and they actually belong to the solid pattern of growth. with respect to grading, it is universally accepted that the nuclear grade is the essential feature, recurring in all classification systems previously proposed and currently in use. an association, albeit inconsistent, exists between the nuclear grade and the architectural growth pattern. it is generally accepted that most micropapillary and cribriform in situ carcinomas are of low nuclear grade and relatively indolent. however, in a recent publication by fisher and colleagues, micropapillary dcis was found to be associated with both ipsilateral and contralateral recurrence of malignancy in a statistically significant number of cases. as this appears somewhat puzzling in the light of our present knowledge and understanding of dcis , we decided to have a new, fresh look at all cases of dcis reported during the past approximately 10 years at the department of pathology of the royal darwin hospital, nt, australia and report our findings. all cases of dcis reported at the royal darwin hospital, northern territory, australia between january 2001 and september 2010, representing 60% of all breast cancers in the nt were retrospectively reviewed. in order to capture all the cases, including those that may have been incorrectly coded, we verified all breast tissue reports and then selected for active review all cases of dcis. the architectural and cytological aspects of dcis were assessed. the presence or absence of necrosis the 3 main types of dcis, according to the architectural growth pattern (micropapillary, cribriform, and solid) were assessed. 90% of the in situ tumour displayed one architectural pattern, and mixed when the dominant pattern constituted < 90% of the in situ carcinoma. nuclear grading is based on the size of malignant cells nuclei in comparison to normal ductal epithelial cells. grade 1 is applied when the nuclei of the malignant cell are between 1.5 and 2 times that of normal ductal epithelial cell. grade 2 is applied when the nuclei of the malignant cell are between 2 and 2.5 times that of normal ductal epithelial cell. grade 3 is applied when the nuclei of the malignant cell are greater than 2.5 times that of normal ductal epithelial cell. the term comedonecrosis, which is poorly defined in the literature, was applied when significant necrosis, creating an appearance similar the comedos, seen in cutaneous acne, was noted in ducts with dcis. a total of 289 breast carcinomas had been received at the royal darwin hospital during the period of the study. these consisted of 265 invasive and 24 pure in situ cancers. these cancers consisted of 231 infiltrating duct carcinomas of no special type, 24 infiltrating lobular carcinomas and 10 carcinomas of special type. of these special breast cancers, 5 were pure mucinous carcinomas, 2 were invasive papillary carcinomas, 2 were tubular carcinomas, and 1 was medullary carcinoma. the proportion of invasive ductal carcinomas containing dcis varied from year to year, ranging from 16/26 (62%) in 2002 to 8/26 (31%) in 2007. table 1 also shows the frequency of pure dcis and dcis associated with invasive cancer. of the 157 cases of dcis (pure dcis and invasive cancers with dcis), 91 (58%) displayed a single growth pattern and 66 (42%) showed a mixed growth pattern (table 1). of the 91 cases of dcis with a single growth pattern, 4 (5%) were micropapillary, 23 (25%) were cribriform, 61 (67%) were solid, and 3 (3%) were encysted papillary (table 1). of the 66 mixed type dcis cases, all 3 growth patterns (micropapillary, cribriform, and solid) were noted in 11 (17%) of the cases. micropapillary and cribriform was present in 14 (21%), micropapillary and solid was seen in 11 (17%), and cribriform and solid was identified in 30 (45%) of the cases (table 1). most of the pure in situ carcinomas were of the mixed type and there were no cases of single pattern micropapillary dcis (table 2). overall, a micropapillary pattern was seen in 40/157 (25%), a cribriform component in 78/157 (50%), a solid component in 113/157 (72%), and macropapillary (encysted papillary carcinoma) in 3 (1.9%) of the dcis cases (table 2). comedonecrosis was present in 18/40 (45%) of the micropapillary, 67/113 (59%) of the solid, and 20/78 (26%) of the cribriform cases of dcis. these findings show that comedonecrosis is more likely to be seen in micropapillary and solid types dcis than in the cribriform type. this association is statistically significant with p values of 0.033 and 0.00004, respectively. at the same time, no statistically significant difference in comedonecrosis occurrence was noted between the micropapillary and solid dcis (p = 0.117). of the 157 cases of dcis high nuclear grade was recognised in 70 (45%), intermediate nuclear grade in 65 (41%), and low nuclear grade in 22 (14%) of the cases (table 3). of the 40 cases of micropapillary dcis, 2 (5%) were low grade, 18 (45%) were intermediate grade, and 20 (50%) were high grade (table 3). of the 78 cases of cribriform dcis, 17 (22%) were low grade, 39 (50%) were intermediate grade, and 22 (28%) were high grade (table 3). of the 113 cases of solid dcis, 8 (7%) were low grade, 47 (42%) were intermediate grade, and 58 (51%) were high grade (table 3). these show a statistically significant difference between the presence of high nuclear grade (grade 3) in the solid and micropapillary types of dcis compared to the cribriform type (p = 0.0008, and p = 0.019 resp .). on the other hand, no statistically significant nuclear grade differences were noted between the micropapillary and solid types (p = 0.884). all three cases of macropapillary (encysted papillary carcinoma) were low grade (table 3). overall, we identified 40 cases with a micropapillary dcis component (table 2). thirty - six of these were mixed with other growth patterns and 4 had only micropapillary growth pattern. in the cases of dcis with no invasive cancer, there was a micropapillary component in 9 cases; all mixed with other growth patterns. of these, 4 had only a micropapillary growth and in 27 the micropapillary pattern was mixed with other growth patterns. of all the 91 cases of dcis with a single growth pattern, 4 (4%) had only micropapillary growth (table 1). of the 24 cases of pure dcis, none was only micropapillary but 9 had a micropapillary component, mixed with other architectural patterns. of these, 6 cases were high grade, 2 cases were intermediate grade, and 1 case was low grade (table 3). the age of patients who had a micropapillary component ranged from 38 years to 88 years with a median of 50 years. the age range for the low grade was 52 years to 63 years, for the intermediate grade was 38 years to 88 years, and for the high grade was 40 years to 80 years. the invasive component of cases that included a micropapillary type dcis was grade 3 in 22.6% (7/31), grade 2 in 38.7% (12/31), and grade 1 in 38.7% (12/31) of the cases. of the 4 cases with micropapillary dcis not associated with any other type of dcis (pure micropapillary type), 3 were intermediate grade and 1 was low grade. the micropapillary dcis represented only a minor component (< 25%) in 9/36 of the cases in which it appeared in combination with the other types of dcis. the cases had been reported by 6 pathologists, of which three were responsible for reporting 94% of cases. the overall concordance for all cases of dcis, in respect to nuclear grade, regardless of architectural pattern, was very good, with a kappa score of 0.87. in cases with micropapillary component there was a good concordance between our evaluation and the initial report with a kappa score of 0.61. the nonconcordant cases, which differed by 1 grade, were all between low to intermediate grade; none involved a high grade. the concordance rate for cases not including a micropapillary pattern was also very good with a kappa score of 0.88. it is beyond the scope of this study to chronologically recapitulate all aspects of this complex histopathological entity. regarding the micropapillary dcis, our are surprising, but may explain partly the increased correlation with breast carcinoma recurrence identified by fisher et al.. fisher and colleagues reviewing dcis from 1456 patients enrolled in the national surgical adjuvant breast and bowel project (nsabp) protocol b-24 to determine predictors for ipsilateral breast tumour recurrences and contralateral breast cancers, after a median follow - up time of 10.5 years, found ductal comedonecrosis, micropapillary histological tumour type to be independent high risk factors for ipsilateral breast tumour recurrence and for contralateral breast cancers. in a recent article, castellano et al. have shown the nuclear grade to be crucial in determining the biology of micropapillary dcis. they also showed that high nuclear grade micropapillary dcis more frequently overexpressed her2, showed a higher proliferation index, and displayed necrosis and microinvasion. logistic regression analysis confirmed high nuclear grade (odds ratio, 6.86 ; confidence interval, 1.4033.57) as the only parameter associated with elevated risk of local recurrence after breast - conserving surgery. however, the recurrence rate of 19 micropapillary dcis, which were part of a cohort of 338 consecutive dcis, was significantly higher (log - rank test, p = 0.019) than that of nonmicropapillary, independent of nuclear grade. the authors concluded that although nuclear grade may significantly influence the biological behaviour of micropapillary ductal carcinoma in situ, micropapillary growth pattern represents a risk factor for local recurrence after breast - conserving surgery. while the number of cases in which the micropapillary growth pattern constitutes the only pattern was very small in our study, it may not be a coincidence that none of these cases was high nuclear grade and none was associated with necrosis. this finding would be consistent with the dogma defining micropapillary dcis as a predominantly low grade dcis. the frequent association between micropapillary dcis and necrosis may explain why authors, considering comedonecrosis a separate pattern of dcis, have been discarding all those cases also displaying comedonecrosis from the micropapillary group. another notion on which all publications seem to agree is the fact that the solid variant tends to be associated with high grade dcis, whereas the micropapillary and cribriform variants most often form a low grade dcis. we believe comedocarcinoma is not a separate type of dcis and should not be used as such. in other words , each dcis should be given an architectural pattern label and the presence or absence of necrosis should be described separately. in all the cases of dcis with a pure micropapillary growth pattern, the nuclear grade was low or intermediate. this may mean that those cases of low grade dcis that may have a certain genetic makeup will maintain an indolent course, whereas the more aggressive ones will undergo the changes described above and assume a mixed growth pattern with comedonecrosis. because of this supposed evolution, the micropapillary component may represent a minor proportion of the entire dcis at the time the tissue is removed for histological evaluation. if comedocarcinoma is considered a separate type of dcis in which the architectural pattern is neglected and not reported, then our , with only 4 cases of micropapillary dcis, are all in keeping with the old dogma that micropapillary dcis is a low or intermediate grade with no necrosis. we agree with fisher and colleagues in considering comedocarcinoma a separate feature, rather than a histological type. pinder and o'malley hit the nail on the head and explain that comedonecrosis may be seen in association with any dcis architectural type, and that it is not a type itself, as it is neither a grade nor an architectur. so they clearly recommend that the term comedo - type dcis should not be used as a characterisation of growth pattern. also in keeping with our view is the fact that the rare, special type dcis, namely, the signet ring, endocrine, and other types, also have a growth pattern that is, the solid one, even if the cells show specific our show that over 40% (66/157) of the dcis cases are not of pure architectural type, but in fact they are mixed. approximately half of the solid (51%) and micropapillary (50%) cases in our series are high grade. these values are statistically significant when compared to the occurrence of high grade cribriform dcis, of which only 28% are high grade. just under half (45%) of the micropapillary and nearly two - thirds (67%) of the solid cases of dcis are associated with comedonecrosis, while only a quarter (26%) of the cribriform cases of dcis are associated with comedonecrosis. the micropapillary type is the rarest one, occurring seldom as a pure form with less than 3% of all cases of dcis and less than 5% of dcis cases with a single pattern being pure micropapillary ones. slightly more than half (54%) of the solid and almost a third (30%) of the cribriform cases occur on their own, whereas in only 10% of the cases low grade micropapillary and solid dcis were very rare in our series (5% and 7%, resp .). our can be translated into the newly accepted din system by replacing the nuclear grade value with the equivalent din. after carefully observing and analysing our data, we draw the that in many cases the pattern of growth may be a continuum, starting as micropapillary, with papillae then either joining one another to form arches resembling the cribriform pattern or even continuing to proliferate until a solid sheet of cells fills the entire lumen. as necrosis occurs toward the tips or on the sides of the micropapillary structures, the lesion becomes intermediate grade. with progression, nuclear pleomorphism and a comedo - type necrosis appear, and the lesion then qualifies as a high grade dcis. we therefore believe the study of the determinants of growth pattern in dcis would be the key to unravelling the diverse, often nonconcordant evidence one encounters in the literature.

mammary ductal carcinoma in - situ (dcis), a malignant appearing lesion on cytological and histological grounds, is in fact a non - obligate precancer. dcis is difficult to manage and is sometimes treated more aggressively than invasive carcinoma. although most dcis classifications take into account the architectural growth pattern, when it comes to architecture, the literature is full of contradictory information. we examined 289 breast cancers and found dcis in 265 of the cases. the majority of the dcis cases were seen in the setting of invasive cancer and only 9% of the cases represented pure dcis with no invasive cancer. the dcis commonly displayed a mixed pattern with micropapillary, cribriform and solid components with the micropapillary type being the rarest, occurring seldom on its own. a continuum of growth with a micropapillary pattern evolving into a cribriform type could be seen in some of the cases. this may explain some of the conflicting information, in the literature, regarding the different architectural types of dcis. the comedo - pattern of necrosis could be seen in all types of dcis. we therefore conclude that the study of the determinants of growth pattern in dcis would be the key to unravelling the diverse, often non - concordant evidence one encounters in the literature.

several recent meta - analyses have questioned the usefulness of beta - blockers as the primary tools to treat hypertension. this has led to hesitation in the proper use of beta - blockers in the management of hypertension and chronic kidney disease (ckd). this review describes the opinion base promoting the use of beta - blockers for the treatment of hypertension and ckd. prichard classified beta - blockers into three types according to their beta1-selectivity and vasodilatory potential. an additional classification is lipophilic or hydrophilic beta - blockers. atenolol is a beta1-selective agent, and it has been widely used as the control drug in large randomized prospective controlled trials of newer antihypertensive agents such as calcium channel blockers and renin - angiotensin (ra) system blockers. table 1 summarizes the plausible reasons why beta - blockers are considered to be relatively ineffective for the prevention of cardiovascular events. in the anglo - scandinavian cardiac outcomes trial - blood pressure lowering arm (ascot - bpla) study, blood pressure values were lower in those allocated to the calcium channel blocker - based regimen as compared to those allocated to the beta - blocker - based regimen throughout the trial period. recently, webb et al. reported a meta - analysis in which they described visit - to - visit blood pressure instability in patients receiving beta - blocker treatment, and also that this instability was associated with an increased risk of stroke. atenolol was used in the ascot - bpla study, and not only the analysis conducted by webb et al. but some studies demonstrated that once - daily atenolol does not provide adequate blood pressure control during the night - time and early morning periods because of its pharmacokinetic profile and half - life. these drug profiles of atenolol may be the cause for its relatively weak blood pressure - lowering effect and the blood pressure instability. on the other hand, metoprolol or bisoprolol have been shown to be more effective in sustaining 24-hour and early morning bp reductions as compared with atenolol. in the arterial tree, the arteries branch and taper as they reach peripheral sites, associated with the increase of the arterial resistance. reflected pulse wave (from the periphery to the heart) occurs at sites of abrupt increase of the arterial resistance, such as at sites of arterial branching. interaction between the incident pulse wave (from the heart to the periphery) and reflected pulse wave (from the periphery to the central region) is observed in the arterial tree (figure 1); therefore, the blood pressure values differ between central and peripheral sites of the arterial tree. central (aortic and carotid) blood pressure is pathophysiologically more relevant than the peripheral pressure in the pathogenesis of cardiovascular disease. augmentation index (ai), a marker of the interaction of incident pressure wave and reflected pressure wave, was significantly and inversely related to heart rate due to an alteration in the relative timing of the reflected pressure wave. beta - blockers reduce the heart rate and decrease ai, which reduces their efficacy in reducing the central blood pressure as compared to other antihypertensive agents. in their meta - analysis, fagard et al. reported that beta - blockers exert a relatively weak effect in causing regression of the left ventricular mass. in fagard et al.'s review, atenolol was used in about 70% of the study subjects prescribed beta - blockers, and no study involving the use of vasodilatory beta - blockers was included. recently, the advantages of nebivolol, a vasodilatory beta - blocker, over conventional -blockers in reducing the central blood pressure and inducing regression of the left ventricular mass have been reported. compared with atenolol, nebivolol exerts a more favorable effect on 24-hour blood pressure profile. furthermore, nebivolol and telmisartan, an angiotensin ii receptor blocker, decreased the left ventricular mass to a similar degree. reported that angiotensin - converting enzyme inhibitors improve endothelial function and are superior antihypertensive agents as compared to calcium channel blockers and beta - blockers. however, in all of the studies cited in their meta - analysis, atenolol had been used as the beta - blocker. in contrast to atenolol, carvedilol and nebivolol also improve the endothelial function. while the meta - analysis conducted by messerli et al. reported the unfavorable effects of beta - blockers on the metabolic profiles, this analysis did not include studies in which vasodilatory beta - blockers had been used. more recent studies have reported the relatively less harmful effects of vasodilatory beta - blockers on the metabolic profiles and also on weight gain. as described above, new generations of beta - blockers, such as the long - acting and/or vasodilatory beta - blockers may overcome the relatively weak effect of beta - blockers in preventing cardiovascular events. , which was a representative analysis to evaluate the usefulness of beta - blockers in the management of hypertension, suggested that first - line beta - blocker use was not as good as other classes of antihypertensive drugs to decrease the mortality or morbidity. in his review, 6070% of the subjects were receiving atenolol. as mentioned above, there is some doubt about the suitability of atenolol as a first - line antihypertensive drug, because of its low lipophilic profile and relatively weak effect on cardiovascular protection. the maphy study demonstrated the significantly lower risk for coronary events in patients on metoprolol, a lipophilic beta - blocker, as compared to those on diuretics. the usefulness of lipophilic beta - blockers for the prevention of cardiovascular events is still under debate. (total number of analyzed subjects, 91561) reported the higher risk for cardiovascular events in patients on beta - blockers as compared to those on diuretics. however, the number of study subjects prescribed metoprolol included in their meta - analysis was 7663 (8.4%). on the other hand, the meta - analysis conducted by turnbull et al. (total number of study subjects for the comparison of the outcomes of major cardiovascular events ( angiotensin converting enzyme inhibitor or calcium antagonist versus beta - blocker) was 14583, which was calculated from figure 4 in ) demonstrated no evidence of any difference in the effect between beta - blockers and other classes of antihypertensive agents in preventing major cardiovascular events. this meta - analysis included two studies in which metoprolol alone was used in the beta - blocker arm and two other studies in which metoprolol was used as one of the beta - blockers in the beta - blocker arm. the number of study subjects prescribed metoprolol included in this meta - analysis was 10062 (13.5%). thus, the meta - analysis conducted by turnbull et al. might have included a lower number of subjects prescribed atenolol and higher number of study subjects prescribed metoprolol, as compared to the meta - analysis conducted by wiysonge et al.. then, recently, turnbull et al. suggested that lipophilic beta - blockers may be preferable to hydrophilic beta - blockers for reducing the mortality in patients with coronary artery disease, though lipophilic -blockers are associated with an increased risk of depressive symptoms. reported that the differential effects between nonatenolol beta - blockers and other antihypertensive drugs on the risk of major cardiovascular events could not be fully evaluated because of the small number of studies including subjects prescribed nonatenolol beta - blockers. anyhow, atenolol is one of the most widely used beta - blockers, and more than 50% of the data in previous meta - analyses were derived from subjects prescribed atenolol. a meta - analysis to examine the effects of lipophilic and/or vasodilatory beta - blockers on the risk of major cardiovascular events the meta - analysis conducted by bangalore et al. demonstrated an inverse relationship between beta - blocker - induced heart rate lowering and the reduction in the risk of future cardiovascular events in patients with hypertension. this heart rate lowering causes a pseudoantihypertensive effect; that is, the central aortic pressure becomes less than the brachial pressure. this phenomenon is thought to be one of mechanisms underlying the lower cardiovascular - protective effects of beta - blockers. on the other hand, the risk seems to increase as the heart rate begins to exceed 70 bpm. the aforementioned meta - analysis conducted by bangalore et al. demonstrated an inverse relationship between heart rate and cardiovascular events in subjects with heart rates under 70 bpm, and no study until the current date has examined the relationship between the heart rate and the risk for cardiovascular events in cases receiving beta - blocker treatment with heart rates over 70 bpm. furthermore, atenolol was used in more than 80% of the study subjects of the meta - analysis conducted by bangalore et al.. thus, no study has examined the association between the reduction in heart rate and the increased risk of cardiovascular events by the treatment with beta - blockers other than atenolol. our previous prospective study identified high heart rate as an independent risk factor for vascular damage (increase in arterial stiffening). therefore, it has not been concluded that beta - blocker - induced heart rate lowering increases the risk of cardiovascular events in cases with heart rate over 70 bpm. in the case of patients with hypertension and a high heart rate, we have sometimes experienced good efficacy of beta - blockers for the control of both the blood pressure and the heart rate (figure 2). in addition to the prevention of cardiovascular events, renal protection is crucial in the management of ckd. recent guidelines recommended ra system blockers as the agents of first choice for the management of hypertension in patients with ckd, because of the significant renal - protective effects of this class of drugs. on the other hand, the sympathetic nervous system is activated in ckd , which acts as a key player in the progression of renal dysfunction and may also contribute to the onset / progression of cardiovascular disease. however, reduction of the cardiac output and the consequent impairment of renal perfusion caused by beta - blockers are thought to be harmful in patients with ckd. actually, recent studies have demonstrated that only 2030% of patients with ckd are prescribed beta - blockers. the kidney disease outcomes quality initiative (k / doqi) guideline recommend that beta - blockers be used as the third - line antihypertensive agents in patients with proteinuria. however, as mentioned above, while meta - analyses have suggested the demerits of atenolol in the management of hypertension , controversial of the renal - protective effects of atenolol as compared to those of other antihypertensive agents were reported in subjects with hypertension accompanying ckd. concerning other beta - blockers, the african american study of kidney disease and hypertension study (aask trials) demonstrated the absence of any significant differences in the clinical composite outcomes (renal function decline, onset of end - stage renal disease, and/or death) between patients treated with metoprolol and those treated with amlodipine. two multicenter studies reported the renal protective effects of carvedilol, a vasodilatatory beta - blocker. thus, it has not yet been concluded whether beta - blockers (especially vasodilatory beta - blockers) may simply represent third - line of antihypertensive agents for the management of hypertension in patients with ckd. further study is proposed to clarify whether calcium channel blockers (especially nondihydropyridines, which reduce proteinuria) or vasodilatory beta - blockers may be more suitable for renal protection and controlling blood pressure in patients with ckd. traditional risk factors such as hypertension, diabetes mellitus, and dyslipidemia, and nontraditional risk factors such as inflammation, oxidative stress, abnormal mineral metabolisms, hyperparathyroidism, homocysteinemia, and anemia are known to be associated with cardiovascular events in patients with ckd. the reported rate of sudden cardiac death in patients with end - stage renal disease is 50-fold higher than that in the general population. in addition to the prevalence of coronary artery disease and/or heart failure, left ventricular hypertrophy, electrolyte abnormalities such as hyperkalemia, and vascular calcification might also be associated with sudden cardiac death in patients with ckd.'s meta - analysis demonstrated that beta - blockers reduce the risk of all - cause and cardiovascular mortality in patients with ckd and systolic heart failure. basically, in their review, studies involving the use of beta - blockers other than atenolol were included, and ace inhibitors were concomitantly prescribed for most of the patients in these studies. however, their meta - analysis did not evaluate the effect of beta - blockers on sudden cardiac death. the hemodialysis (hemo) study suggested a trend towards the benefit of beta - blockers for the prevention of sudden cardiac death in patients with ckd and coronary heart disease, but not in ckd patients without coronary artery disease (this study did not describe the details about the kind of beta - blockers used, e.g., atenolol). this review does not support the use of beta - blockers as the primary tool to treat hypertension. however, it does propose proper use of beta - blockers, especially in cases with high heart rate and/or resistant hypertension, considering their long - acting, vasodilatory, and/or lipophilic profiles. beta - blockers are recommended as second - line agents after ra system blockers for controlling hypertension in patients with ckd and systolic heart failure. as compared to other antihypertensive agents, except ra system blockers , it has been confirmed that there are no demerits to using beta - blockers for renal protection. in addition, vasodilatory beta - blockers may also have beneficial renal - protective effects. even in patients with ckd, control of blood pressure is crucial for the prevention of cardiovascular events. while the combination of ra system blockers with diuretics is effective for reducing the blood pressure, in ckd often three or more different antihypertensive drugs are required to control blood pressure level. then, there is no evidence that beta - blockers, especially vasodilatory beta blockers, are inferior to diuretics or calcium channel blockers as second- or third - line agents for renal protection and control of blood pressure in patients with ckd.

this minireview provides current summaries of beta - blocker use in the management of hypertension and/or chronic kidney disease. accumulated evidence suggests that atenolol is not sufficiently effective as a primary tool to treat hypertension. the less - than - adequate effect of beta - blockers in lowering the blood pressure and on vascular protection, and the unfavorable effects of these drugs, as compared to other antihypertensive agents, on the metabolic profile have been pointed out. on the other hand, in patients with chronic kidney disease, renin - angiotensin system blockers are the drugs of first choice for achieving the goal of renal protection. recent studies have reported that vasodilatory beta - blockers have adequate antihypertensive efficacy and less harmful effects on the metabolic profile, and also exert beneficial effects on endothelial function and renal protection. however, there is still not sufficient evidence on the beneficial effects of the new beta - blockers.

in addition to transmitting genetic information from dna to proteins, rna molecules participate actively in a variety of cellular processes. examples are translation (rrna, trna, and tmrna), editing of mrna, intracellular protein targeting, nuclear splicing of pre - mrna, and x - chromosome inactivation. the rna molecules involved in these processes do not code for proteins but act themselves as functional products. in addition, some rna molecules prepared in vitro can bind to specific molecules such as atp. in all these cases, the information encoded in the sequence of nucleotide bases of each rna molecule determines its functional tertiary structure. the forces which stabilize the secondary structure of rna are stronger than interactions responsible for the tertiary structure and hence these two structures are characterized by two different energy scales. according to one of the currently accepted concepts of rna folding the secondary structure elements, such as helices and loops hairpins, are formed first and then stack together to form a three - dimensional tertiary structure. however, some experiments show that the folding rate of large rna is lower than that predicted by the hierarchical mechanism. the landscape of the energy function of large rna is extremely rugged and contains multiple deep minima which act as kinetic traps in the folding pathways. the molecule can remain trapped in the states distinct from the native structure for time periods even longer than the average lifetime of rna in a living cell. it should be noted that excellent models for describing the rna secondary structure formation have been developed as well. what remains relatively poorly understood is the full path of formation of the tertiary structure and, in particular, the mutual interplay between the secondary and tertiary structures. it has been shown that the characteristic pattern of the secondary structure of rna is a tree - like structure, formed by relatively short double - stranded helices. the hierarchical folding scenario has been studied, for example, in, where the folding of rna with fixed secondary structure is described by the model of tree - like polymer with quenched random branching. in the concept of annealed randomly branched polymer has been applied to study the equilibrium characteristics of rna. the completely annealed branching patterns describe the ensemble of secondary structures, wherein the tertiary structure is being formed as a of substantial rearrangements of secondary structure elements. this scenario is typical for large rnas, to which the hierarchical folding mechanism is most probably not applicable. while the model of a polymer with randomly annealed branching can be applied successfully for studying the equilibrium features of rna folding, it can not describe the folding kinetics efficiently. at the same time , the kinetic effects in the folding process are viewed to be of great importance due to the existence of long - living intermediates, as mentioned above. in the present study we focus on the thermodynamic behaviour of the rna molecule in the steady nonequilibrium state that occurs in case of well - defined separation between the relaxation timescales of secondary and tertiary structures. we introduce a reasonable coarse - grained model of rna and study its behavior through analytical equations. the obtained provide evidence for the existence of a nonequilibrium phase transition of the second order between the glassy phase and the ensemble of freely fluctuating spatial structures. the ssrna molecule is dissolved initially in the solvent at temperature t which satisfies inequalities < t < tm, where tm is the melting temperature and is the flory temperature. under these conditions next, transfer a very small amount of our rna containing solution into the same kind of solvent but with the temperature t, such that t <. in the beginning, the secondary and spatial structures in this state still correspond to the temperature t but they start to relax to the new temperature t. in the end of the process, the rna will arrive at a compact globular state with some secondary structure pattern. the tertiary structure of rna is stabilized by interactions between different elements of secondary structure: helical stems, hairpins, internal loops, mismatches, and so forth (figure 1). interactions between helical stems can be considered as homogeneous since the nitrogen bases are located inside the double helix. at the same time, interactions between single - stranded regions are heterogeneous because of the interactions between nitrogen bases of different types. to describe the conformation of rna in a coarse - grained approximation , n. the center of mass of monomer i is placed at the point with coordinates xi. secondary structure is described in terms of the randomly branched polymer (figure 2) as the matrix b^=bij, where bij = 1 if the ith and jth monomers are linked by helical stems and bij = 0, otherwise.. then we introduce the following hamiltonian of the model:h = hii+i < jvijxixj+vconf, where the term hiixi, b^=dt/22i < jbijxi - xj2 mimics the helical spring elasticity between the ith and jth effective monomers and is the equilibrium distance between neighbouring monomers which here coincides with the mean length of the helical stem. thus, the helical spring elasticity constant is assumed to be equal to dt. here vij is the second virial coefficient of interaction between the ith and jth effective monomers, which refer to the tertiary contacts between nonpaired regions (loops). the interactions between nonpaired regions (loops) i and j are governed by their size and nucleotide sequences (figure 3). since many nucleotides contribute to the interaction of these effective monomers i and j, it is reasonable to consider coefficients vij as statistically independent random gaussian variables with distributionpvijexpvij222,where is the variance of virial coefficients vij. collapse of the ssrna molecule is driven mostly by electrostatic interactions and has been investigated experimentally and theoretically. their impact is present implicitly in the confinement potential vconfxi = tv0i < jxi - xj+g0i < j < kxi - xjxk - xj, which ensures existence of globular state of the rna molecule. here v0 < 0 and g0 > 0 are the second and third virial coefficients of interactions between helical stems, correspondingly. two types of conformational rearrangements in rna are possible: rearrangement of the secondary structure with characteristic time scale 2 and tertiary structure fluctuations with characteristic time scale 3. thus as shown in the collapse of 400-nucleotide - long rna takes about 3 - 4 ms while the two - order shorter 21-nucleotide sequence of rna folds into a hairpin in about 10 ms. the reason for this is not only the higher stability of base pairs as compared with the tertiary contacts. formation of the base pairs requires twisting of two single - stranded subchains into a double helix, which is kinetically hampered to unwind. on the timescale , such that 3 2, secondary structure and spatial arrangement of the effective monomers (nonpaired regions) are not in thermal equilibrium, and this stationary nonequilibrium steady state can be described in terms of the effective partition functionz = zb^,v^nb^v^,where zb^,v^ is the partition function of a branched molecule with the given branching pattern b^ and interaction matrix v^=vij. b^ means the average over all possible branching patterns, v^ is the average over intermonomer interactions, and n = t / t. t and t are the effective temperatures of coarse - grained spatial and secondary structures correspondingly. the effective partition function is calculated by using the replica technique developed for systems with quenched disorder (see, e.g.,). in our case the limit n 0 corresponds to the quenched disorder, n = 1 describes the completely annealed disorder, and 0 < n < 1 for the partially annealed disorder. following the method described in we rewrite the partition function in the formz=def,where =1/t, x=i=1na=1nxia - xa, x=x1, ,xn, and f{} = e{} ts{} is the n - replica free energy withe=avconfca24abdx dyqab2x,ysl42dxx2x. here cax=dxxxa - x is the one - replica density of monomers, qabx,y=dxxxa - xxb - y is the two - replica overlapping parameter, and is the laplace operator in the nd - dimensional space. further in all equations we set kb = 1. the energy term in is obtained by averaging the nth power of the partition function over variables vij, and the entropy term includes averaging over all possible branching patterns corresponding to the rooted tree with coordination number equal to three. for function the confinement potential is written as a virial expansion vconf(c) = nt((1/2)v0c + (1/3!)g0c ). in compactly packed chain the density of monomers c0 and vconf(c0) nt(v0/4). like in we parameterize the offdiagonal entries of the matrix k by function k(u), where u , and the diagonal entries as kaa = k. the inverse matrix m^=k^-1 is parameterized by m(u), u , and maa = m~=2n2/d. by introducing notations (u) = uk(u) ndvk(v) and k = n(du / u)(u), the free energy functional takes a formf = nvconfc0+44nn2d22dn1dum~2mu2d/2tl42dnnk2+n1duu2ku2k~2. variation of the free energy over k~ and (u) as independent variables gives that the free energy per monomer has two branches:f = f0nn = tv04ld+f<,nu0f>,n > u0,where the disordered free energy isf<=f<0na1tl4dz02,where n u0, = (3d + 4)/(4 d), and a1(d, , ,) . f < = f<(t, d, ,) is the free energy of ssrna with frozen secondary structure at n u0. at the same time, for n u0 the disordered free energy can be written asf>=f>0na1u01+tl4dz02u0+11n2,where f > = f>(t, d, ,). here the following notations are introduced: z0(d, , ,) , (d) = (4 d)/8, (d) = (d + 4)/(4 d), and u0(d) = (3/2)((3(d/2 + 1)+(d + 1)(d/2 1))/(d + 2) ) 0.6 if d = 3. thus, at n < u0 a glassy phase with replica - symmetry breaking is observed while at values n > u0 a replica symmetric phase is obtained. the second derivatives of the free energy branches and with respect to variable n at the point of transition n = u0 satisfy equations2f<n2n = u0=1u02a1tl4dz022f>n2n = u0=2u02tl4dz02while the first derivatives are as follows: (f</n)|n = u0 = (f>/n)|n = u0 = u0(a1 tdz0). n < 1 the model exhibits nonequilibrium phase transition of the second order. the temperature of transition istc=tu0. in the framework of the proposed model the value of the parameter 1 n serves a measure of the distance from the equilibrium state, corresponding to the n = 1. if rna molecule is far enough from the equilibrium (n < u0), then the glassy phase is realized which is dominated by a few long - lived intermediates, which were observed experimentally in. the interplay between the secondary structure formation and fast collapse of the single - stranded rna is addressed in terms of the model with interaction between heterogeneous nonpaired and homogeneous double - stranded regions. thus, the nucleotide sequence heterogeneity is approximately described in terms of statistical - mechanical model with disorder. the memory effects in rna compact structure formation are governed by slow rearrangements of the secondary structure with subsequent fast relaxation of the spatial degrees of freedom. under these conditions, monomers rapidly attain their equilibrium at the temperature t and thus, because of the wide timescale gap, their equilibrium free energy acts as a driving force pushing the slow dynamics of the elements of secondary structure to reach a nonequilibrium stationary state at long times. this scheme is known generally as the adiabatic elimination of fast variables. the observed experimentally long - lived intermediates are obtained if rna molecule is far enough away from the state of thermodynamic equilibrium.

the mutual influence of the slow rearrangements of secondary structure and fast collapse of the long single - stranded rna (ssrna) in approximation of coarse - grained model is studied with analytic calculations. it is assumed that the characteristic time of the secondary structure rearrangement is much longer than that for the formation of the tertiary structure. a nonequilibrium phase transition of the 2nd order has been observed.

visual information embedded in histologic specimens carries prognostic value and reflects the underlying molecular traits of disease. human evaluation of histology is a time - honored practice and serves as the basis of modern pathology, yet is highly subjective and known for its inter- and intra - observer variations. human observers are also limited by scale and the need to reduce information into summary categorical descriptions. diagnostic evaluation of histologic specimens is often performed over a prescribed number of high - power fields, and reasonable reporting can not possibly capture detailed descriptions of the tissue heterogeneities observed in many diseases. the digitization of pathologic specimens has advanced with improvements in charge - coupled device (ccd) sensors, storage and network performance. early versions of slide scanning hardware suffered from slow image acquisition, and their practical use was limited by the expense of storage and network limitations that made file transfer and remote viewing difficult. contemporary slide scanning devices are now capable of digitizing a single slide at 40x objective magnification in two minutes or less, and can produce hundreds of whole - slide images (wsis) in a single day. with each image occupying hundreds of megabytes to several gigabytes, the recent precipitous decline in storage costs in the past decade has made generation and analysis of large wsi archives more practical. faster networks and improved software have enabled users to fluidly view and interact with large wsi archives at their desktop by streaming imaging data directly from remote servers. currently, no universal standards exists for file format or image compression within a wsi, despite some work by the dicom working - group 26, creating significant challenges in the interoperability of various hardware and software platforms from different wsi vendors. improvements in computing performance and image analysis algorithms enable large wsi archives to be mined to extract quantitative and objective imaging features that describe the visual characteristics of tissue architecture and microanatomy. advances in the theory of image analysis algorithms make it possible to reliably delineate objects across biological scales from cell nuclei and membranes (where stained) to complex multicellular structures and tissue interfaces. with these objects delineated, a set of descriptive features can be calculated to describe their appearance including shape, texture, and spatial relation to one another. new computing hardware like multi - core processors and graphics cards enable these techniques to be scaled to wsi archives that can contain billions of such objects. a collection of algorithms has even emerged to mitigate technical effects introduced by the physical processing of tissues, allowing the automatic detection of artifacts, and the correction of color differences caused by variations in section thickness and staining. these procedures improve the robustness of image segmentation processes and in uniform features that reflect biological properties, while reducing noise introduced by technical artifacts. the size in bytes of features extracted from an image can rival that of the image itself, and the management and standardization of image features and their provenance is not trivial. image analysis algorithms that precisely describe microscopic features within pathologic specimens provide tremendous opportunities for integration with genomic analyses and a new platform for advancing genotype - phenotype comparisons. contemporary genomic platforms have generated a new view of the genetic, transcriptional and epigenetic events that are embedded within tissue samples. deep molecular characterizations of biospecimens are increasingly available and gaining clinical relevance and the complementary nature of genomic and quantitative imaging descriptions creates new opportunities for their integrated analysis. genomics provide extremely high molecular resolution but poor spatial resolution, and the genomic signature of a specimen therefore represents an aggregate measure of heterogeneous molecular profiles within distinct components of the tissue analyzed. laser capture microdissection provides a way to increase the purity of genomic measurements, but is labor - intensive and difficult to carry out on large cohorts, although image analysis has been used to reduce this burden. an alternative approach is the integration of genomic and imaging features through computational means to deconvolve distinct profiles from the aggregate profile, with the goal of recovering information that is lost when tissue is homogenized for genomic analysis. histology is also a manifestation of underlying molecular profiles within tissues, so quantitative imaging features can be expected to contain predictive power as biomarkers of genetic alterations and gene expression patterns. by integrating imaging and genomic features into risk models, the availability of large de - identified data - sets from the cancer genome atlas (tcga) has greatly facilitated integrated analyses that use imaging, genomic and clinical data. this well - characterized and comprehensive data set would be difficult to duplicate at a single institution due to prohibitive cost, privacy concerns, and patient volumes. tcga is a large public resource that provides comprehensive molecular characterizations of more than 22 cancers types. although intended primarily as a genomic resource, tcga contains over 22,000 whole - slide images from more than 10,000 tumors, in addition to detailed clinical descriptions, and serves as an open platform to perform studies that integrate quantitative histology with molecular and clinical data. the use of these existing resources to conduct in silico scientific investigations has enabled researchers in this area to focus effort on developing analysis methods rather than data production, and to scale studies to a number of samples that would be otherwise difficult to achieve (fig 1). while tcga is an exceptional resource at this point in time, such multifaceted descriptions of tissues will likely become more commonplace within academic research institutions with increasing clinical adoption of genomics and digital pathology, and as the information management systems that manage these data improve. in this paper we present a review of developments in the area of quantitative histology, using examples from glioblastoma to illustrate how imaging features can be integrated with genomic and clinical data to improve understanding. the first example explores issues of tumor microenvironment (tme), and how imaging features can illuminate the impact of the tme on the genomic signatures and molecular classifications. in the second example we present a pipeline for the morphometric characterization of nuclei that is capable of extracting quantitative descriptions of billions of cell nuclei in digital wsi archives. we show how this pipeline can be used along with statistical and statistical learning techniques to define imaging biomarkers of genetic alterations and epigenetic and transcriptional patterns, as well as clinical outcomes. we finish by describing near - term potential opportunities for quantitative imaging and integrated studies, and discuss the limitations and challenges associated with these approaches. the cancer genome atlas (tcga) was established in 2005 to improve understanding of the molecular basis of human cancers through large - scale genomic analysis. with a goal of accruing 500 tumors for each cancer selected for study , tcga has expanded beyond initial pilot projects in glioblastoma, lung and ovarian carcinoma to now span more than 22 tumor types. this effort relies on a pipeline of participating institutions that submit frozen tissues and clinical data to a central repository, a set of de - centralized genomic analysis centers that produce messenger rna, micro rna, dna exome sequencing, dna copy number, dna methylation, and protein expression profiles, and an electronic clearinghouse that then makes this data available to the public (https://tcga-data.nci.nih.gov). an important, yet underappreciated aspect of acquiring clinical data from tissue source sites includes the collection of digitized whole - slide images of submitted tumors. frozen sections are produced from the top and bottom of tissue samples that are submitted for genomic analysis, and are used for quality control to evaluate the percentage of tumor, the presence of necrosis and other factors that will influence the quality of genomic . these images are a valuable resource since they are immediately adjacent to tissues used for genomics, and provide the most faithful representation of genomic - annotated tissues. the higher quality of these images and lack of freezing artifacts makes them more suitable for algorithmic analysis, particularly at high magnification. expert pathology committees that are selected by disease area review these permanent sections to ensure correct diagnosis and to evaluate the presence of important pathologic criteria. examples from the gbm project include the categorical scoring (0, 1 +, 2 +) of qualities like microvascular proliferation, pseudopalisading necrosis and lymphocytic infiltration. all permanent and frozen sections are digitized at 20x or 40x objective magnification and made publicly available for download. one of the main outcomes of the tcga analyses has been the development of genomic sub - classifications of many cancers. using clustering analysis of gene expression and other molecular platforms, the goal of these analyses is to define cohesive sub - classes of tumors with distinct molecular signatures that may benefit from class - specific targeted therapies. in glioblastoma, two studies using tcga data have identified tumor sub - classifications based on gene expression and dna methylation. the initial tcga analysis of gbms identified four gene expression classes (gess): proneural, neural, classic and mesenchymal. these classes exhibit clear and distinct patterns of gene expression, and are highly correlated with genetic alterations in egfr, idh1, nf1, pdgfra, and tp53. a subsequent analysis of dna methylation data revealed that pro - neural gbms are further subdivided into two groups - those with idh mutations that have significant hypermethylation of cpg islands (gcimp) and are typically secondary gbms afflicting younger patients, and idh wildtype tumors that do not exhibit dna hypermethylation patterns. one of the first goals of our in silico research was to investigate the relationships between gene expression classifications and the tumor microenvironment in gbms. most tissue - based transcriptional classification studies of tumors are subjective in that neoplasms are highly heterogeneous, and gene expression measurements can vary significantly among different samples from the same tumor. glioblastomas are no exception, being spatially complex tumors that harbor a variety of non - neoplastic cell types and microenvironmental elements that can significantly impact gene expression measurements. pseudopalisading necrosis and micro - vascular proliferation are perhaps the most notable elements, being part of the diagnostic criteria that distinguish glioblastomas from lower - grade gliomas, and indicators of poor prognosis. the development of necrosis and microvascular proliferation is can be focal at first, but then expands, and signals severe underlying hypoxia with ant profound transcriptional changes. having access to both frozen sections and comprehensive molecular profiles from adjacent tissues from tcga, we sought to measure the impact of necrosis and angiogenesis on gene expression patterns used to classify gbms. we hypothesized the extent of necrosis and angiogenesis in a histologic section are tightly associated with the presence of hypoxia, which could play an important role in establishing ges signatures by activation of hypoxia - inducible transcription factors. with the degree of hypoxia varying spatially throughout a tumor , multiple ges classes could possibly co - exist within the same tumor, and so classification by gene expression could be subject to random effects in tissue sampling. intra - tumoral variations in ges classification would have significant implications in using these classes as platforms for the development of targeted therapies. using a human - computer interface, we annotated 177 digitized frozen section images to define the boundaries of necrosis and angiogenesis for 99 tumors. the sections in these images are immediately adjacent to those used for genomic analysis, and these annotations therefore provide the most faithful representation of the microenvironmental conditions in genomically analyzed tissues (fig 2a). the extent of necrosis and angiogenesis was calculated as a percentage of the total tissue area, and these quantitative features were linked to gene expression and other genomic measurements from the same tissue (fig 2b). we first examined the abundance of necrosis and angiogenesis in tumors organized by tcga transcriptional class. tumors with a mesenchymal ges were clearly enriched with higher amounts of necrosis (one - way anova p = 8.7e-4, see fig 2c), suggesting a strong association between the mesenchymal gene - expression signatures, necrosis, and hypoxia. there were only 4 outliers with much higher levels of angiogensis and 3 were from the mesenchymal ges and 1 was from the proneural ges. while we would expect the presence of angiogenesis to influence gene expression, the ability to detect this feature may in part be limited by the relatively small contribution these regions make to the total amount of dna / rna extracted for analysis. next we performed a genome - wide analysis of transcriptional data to discover the impact of necrosis on gene expression. a normalized linear regression coefficient was calculated for each transcript to measure the strength of relationship between extent of necrosis and gene expression for more than 22,000 transcripts measured with affymetrix arrays. significance analysis of microarrays (sam) correction was applied to obtain multiple - test corrected p - values for each gene. this analysis identified 2422 genes that are significantly correlated with extent of necrosis at 5% false - discovery rate or below, suggesting that necrosis has tremendous influence on gene expression in gbms. among the genes most significantly correlated with necrosis were a set of transcription factors known as mesenchymal master regulators: cebpb, fosl2, cebpd, stat3, bhlhe40 (ranked 4, 10, 60, 213 and 221 respectively). these transcription factors have been shown to form a small module regulating a much broader gene expression network that is responsible for mesenchymal tumor phenotype in glioblastomas. at the top of this regulatory module are the transcription factors ce - bpb / cebpd and stat3, whose coexpression is necessary and sufficient for activating the mesenchymal expression network. to explore the expression of these regulators in tissues, we performed immunohistochemistry on archived surgically resected glioblastomas from our own institution. we observed that cebpb / cebpd expression was strongly and specifically expressed in the hypoxic pseudopalisading cells surrounding areas of necrosis (fig 1d). cebpb was strongly expressed in nuclei of the first 25 cell layers immediately surrounding necrosis, and cebpd expression was found in both nuclear and cytoplasmic regions of perinecrotic cells but extending slightly farther beyond cebpd. in regions between foci of necrosis, gene expression classifications are made by measuring the distances in gene expression space between a tumor s expression profile and a set of points or centroids that represent each class. the tumor is assigned to the class with the nearest centroid, which can be measured using a variety of metrics including simple euclidean distance. while a given tissue could potentially contain individual cells / regions with diverging gene expression profiles, these classifications force a selection of a single gene expression class that best defines the entire sample. to explore how the formation of necrosis influences the expression patterns of non - mesenchymal gbms , we examined the relationship between extent of necrosis and distance to the mesenchymal expression centroid in this cohort. we observed a clear trend the more necrosis that a sample contains, the more its expression profile resembles the mesenchymal centroid (fig 1e). this finding further suggests that mesenchymal gene expression is strongly impacted by hypoxia and that expression signatures are strongly impacted by regionally varying elements of the microenvironment. the morphologic characteristics of cell nuclei convey important clinical information in many types of neoplasms. besides determining histologic classification and subtype, nuclear qualities including shape, texture and spatial arrangement can be indicative of more specific molecular alterations and patient prognosis. gains, losses and rearrangements of dna along with epigenetic modifications affecting chromatin structure can manifest in observable changes within nuclei of neoplastic cells. in the diffuse gliomas, nuclear features are of particular importance, as their classification of oligodendroglioma or astocytoma is based in large part on nuclear morphology. however, histopathologic classification based on human review is subjective and prone to substantial interobserver variation. understanding the relationships between nuclear morphology, tumor genetics and clinical outcomes will provide a better understanding of tumor biology and further improve the precision of clinical predictions. our studies of tumor microenvironment used human markups and annotations to generate quantitative features from whole - slide images. the limitations of human annotations are apparent when dealing with nuclear morphology - nuclei can number in the hundreds of millions in even a modestly sized set of images, and qualities of interest like nuclear texture are difficult to accurately characterize objectively by human observers. to address these challenges we have developed a computational system for the study of nuclear morphometry in large archives of whole - slide images (fig 3a). this system uses image analysis algorithms to delineate individual cell nuclei, and to calculate a set of objective nuclear features for each nucleus to describe its shape and texture. high performance and parallel computing approaches are used to scale this approach to hundreds of millions of cells. this system presents opportunities to define quantitative morphologic biomarkers of molecular and clinical endpoints by enabling the extraction of objective, repeatable measurements from wsi archives. our initial morphometric study focused on the quantitative characterization of oligodendroglial differentiation in glioblastomas. although gbm is defined as a grade iv astrocytoma, an important subset exhibits varying degrees of oligodendroglial differentiation in addition to the dominant astrocytic component. neoplasms with pure oligodendroglial differentiation typically have slower growth and better survivals when compared with astrocytomas of the same grades. the morphologic characteristics of oligodendrogliomas distinguish them from astrocytomas: oligodendroglial nuclei tend to be smaller, round and hyperchromatic with a lack of detailed texture, in contrast with astrocytoma nuclei that are larger, irregularly shaped, typically elongated and unevenly textured. in most instances, gbms contain a heterogeneous mixture of neoplastic cells with wide variations in nuclear characteristics, many of which are not clearly astrocytic or oligodendroglial. the volume and heterogeneity of cells present in gbms combined with subtle differences in morphologic diversity make them an ideal candidate for computational morphometric approaches. using our computational pipeline, we analyzed 200 million nuclei from digitized images of diagnostic slides corresponding to 117 tcga gbms. twenty - three quantitative features from four categories (shape, intensity, texture and gradient) were calculated to describe each nucleus. to represent the differentiation of each nucleus along the oligodendroglial / astrocytic spectrum , we built a regression model that uses the nuclear feature values to calculate a score for each nucleus representing its degree of oligodendroglial appearance (fig 3b). combining the 200 million scores obtained from our pipeline with gene expression, copy number, dna sequence and methylation data from the same tcga tumors, we were able to clearly separate a set of tumor enriched with oligodendroglial - like cells that had strong associations with pdgfra amplification, proneural transcriptional class, and expression of the oligodendrocyte signature genes mbp, hoxd1, plp1, mobp and pdgfra. these provide molecular validation that the quantitative features extracted by our software pipeline can capture the morphologic variations of nuclei encountered in gliomas. our differentiation study used a supervised approach to build a quantitative model of the oligodendroglial / astrocytic spectrum in gliomas. model - based approaches are a powerful way to incorporate prior knowledge into morphologic analyses, and to use quantitative measures of recognized morphologic patterns to explore their molecular correlates. because model - based approaches are built on prior knowledge, their ability to reveal previously unrecognized or unknown morphologic patterns is limited. to address this limitation, we have developed several unsupervised or model - free approaches that do not impose established constructs in the morphological analysis of wsi data. instead, these approaches let data speak for itself, using clustering analysis and other statistical learning techniques to reveal natural structure within the feature data in a bottom - up fashion. our first study with unsupervised methods investigated patient clustering of gbms into morphologically defined subtypes. using nuclear features , we sought to determine if there are clear and distinct groups of tumors that emerge from clustering analysis, similar to gene expression studies where transcriptional profiles are clustered to reveal molecular tumor subtypes. taking the nuclear features from the tcga cohort, a morphologic signature was calculated for each tumor to represent the morphologic properties of its average nuclei. these signatures were analyzed using a consensus - clustering algorithm to find natural groups within the data and to measure their robustness. three clear clusters emerged from this analysis and we named them for themes observed in their molecular correlates: cell cycle (cc), protein biosynthesis (pb) and chromatin modification (cm). we observed that these clusters had significant differences in patient survival (logrank p=1.4e-3), with the pb cluster containing patients with relatively better outcomes and the cm cluster relatively worse. these clusters were also observed in an independent dataset of 84 gbms where the relative differences in outcomes between the clusters were also confirmed. to explore the meaning of these clusters we used the various genomic platforms made available by tcga including gene expression, dna methylation, copy number and dna sequencing. a pathway analysis found that the clusters varied in the extent of tp53 wnt, and nfkb signaling, and had variations in the extent of total dna methylation. an analysis of the pathologic features using categorical human annotations (0,1+,2 +) found that tumors in the cm cluster had enriched presence of lymphocytes, and that pb cluster tumors exhibited a conspicuous lack of inflammation. to further explore model - free associations of nuclear morphometry in gbm and clinical and genomic endpoints , we took a more direct approach of correlating raw nuclear features with genomic and clinical endpoints. for each patient , we calculated the mean and standard deviation of each feature as metrics and correlating them directly with patient survival using cox proportional hazards analysis using sam. notably, the mean circularity was significantly associated with longer patient survival, an observation consistent with prolonged clinical outcomes in gliomas with oligodendroglial differentiation. other features that were significantly associated with outcome include major axis length, with longer nuclei associated with a shorter survival, and min nuclear pixel intensity, with higher values associated with longer survival. the fact that these features emerged from a more data - driven approach provides some level of confidence in our analysis workflow. to correlate these features with genomic measurements we performed a one - way anova for each feature metric across transcriptional classifications, somatic mutations and dna copy number alterations. features distinguishing transcriptional classes include nuclear eccentricity (p = 3.81e-4), minor axis length (p = 8.87e-3) and nuclear extent (p = 3.2e-2). those hypermethylated (gcimp) tumors within the proneural group had greater variation of pixel intensities within their nuclei (nuclear energy, p = 2.28e-5), and greater variation in nuclear size. genetic events having significant differences in nuclear morphometry included pten and tp53 mutations, and pdg - fra amplification. pten and tp53 mutant tumors were both associated with less circular nuclei (p = 9.68e-3, 3.77e-2 respectively). pdgfra amplified tumors were associated with greater circularity (p = 2.31e-2), consistent with pdgfra amplifications being associated with oligodendroglial differentiation. other genetic alterations with significant associations included egfr amplification, which was associated with greater nuclear eccentricity and canny, and mdm2 amplifications, which were associated with greater minor axis length, area and circularity. the cancer genome atlas (tcga) was established in 2005 to improve understanding of the molecular basis of human cancers through large - scale genomic analysis. with a goal of accruing 500 tumors for each cancer selected for study , tcga has expanded beyond initial pilot projects in glioblastoma, lung and ovarian carcinoma to now span more than 22 tumor types. this effort relies on a pipeline of participating institutions that submit frozen tissues and clinical data to a central repository, a set of de - centralized genomic analysis centers that produce messenger rna, micro rna, dna exome sequencing, dna copy number, dna methylation, and protein expression profiles, and an electronic clearinghouse that then makes this data available to the public (https://tcga-data.nci.nih.gov). an important, yet underappreciated aspect of acquiring clinical data from tissue source sites includes the collection of digitized whole - slide images of submitted tumors. frozen sections are produced from the top and bottom of tissue samples that are submitted for genomic analysis, and are used for quality control to evaluate the percentage of tumor, the presence of necrosis and other factors that will influence the quality of genomic . these images are a valuable resource since they are immediately adjacent to tissues used for genomics, and provide the most faithful representation of genomic - annotated tissues. the higher quality of these images and lack of freezing artifacts makes them more suitable for algorithmic analysis, particularly at high magnification. expert pathology committees that are selected by disease area review these permanent sections to ensure correct diagnosis and to evaluate the presence of important pathologic criteria. examples from the gbm project include the categorical scoring (0, 1 +, 2 +) of qualities like microvascular proliferation, pseudopalisading necrosis and lymphocytic infiltration. all permanent and frozen sections are digitized at 20x or 40x objective magnification and made publicly available for download. one of the main outcomes of the tcga analyses has been the development of genomic sub - classifications of many cancers. using clustering analysis of gene expression and other molecular platforms, the goal of these analyses is to define cohesive sub - classes of tumors with distinct molecular signatures that may benefit from class - specific targeted therapies. in glioblastoma, two studies using tcga data have identified tumor sub - classifications based on gene expression and dna methylation. the initial tcga analysis of gbms identified four gene expression classes (gess): proneural, neural, classic and mesenchymal. these classes exhibit clear and distinct patterns of gene expression, and are highly correlated with genetic alterations in egfr, idh1, nf1, pdgfra, and tp53. a subsequent analysis of dna methylation data revealed that pro - neural gbms are further subdivided into two groups - those with idh mutations that have significant hypermethylation of cpg islands (gcimp) and are typically secondary gbms afflicting younger patients, and idh wildtype tumors that do not exhibit dna hypermethylation patterns. one of the first goals of our in silico research was to investigate the relationships between gene expression classifications and the tumor microenvironment in gbms. most tissue - based transcriptional classification studies of tumors are subjective in that neoplasms are highly heterogeneous, and gene expression measurements can vary significantly among different samples from the same tumor. glioblastomas are no exception, being spatially complex tumors that harbor a variety of non - neoplastic cell types and microenvironmental elements that can significantly impact gene expression measurements. pseudopalisading necrosis and micro - vascular proliferation are perhaps the most notable elements, being part of the diagnostic criteria that distinguish glioblastomas from lower - grade gliomas, and indicators of poor prognosis. the development of necrosis and microvascular proliferation is can be focal at first, but then expands, and signals severe underlying hypoxia with ant profound transcriptional changes. having access to both frozen sections and comprehensive molecular profiles from adjacent tissues from tcga, we sought to measure the impact of necrosis and angiogenesis on gene expression patterns used to classify gbms. we hypothesized the extent of necrosis and angiogenesis in a histologic section are tightly associated with the presence of hypoxia, which could play an important role in establishing ges signatures by activation of hypoxia - inducible transcription factors. with the degree of hypoxia varying spatially throughout a tumor, multiple ges classes could possibly co - exist within the same tumor, and so classification by gene expression could be subject to random effects in tissue sampling. intra - tumoral variations in ges classification would have significant implications in using these classes as platforms for the development of targeted therapies. using a human - computer interface, we annotated 177 digitized frozen section images to define the boundaries of necrosis and angiogenesis for 99 tumors. the sections in these images are immediately adjacent to those used for genomic analysis, and these annotations therefore provide the most faithful representation of the microenvironmental conditions in genomically analyzed tissues (fig 2a). the extent of necrosis and angiogenesis was calculated as a percentage of the total tissue area, and these quantitative features were linked to gene expression and other genomic measurements from the same tissue (fig 2b). we first examined the abundance of necrosis and angiogenesis in tumors organized by tcga transcriptional class. tumors with a mesenchymal ges were clearly enriched with higher amounts of necrosis (one - way anova p = 8.7e-4, see fig 2c), suggesting a strong association between the mesenchymal gene - expression signatures, necrosis, and hypoxia. there were only 4 outliers with much higher levels of angiogensis and 3 were from the mesenchymal ges and 1 was from the proneural ges. while we would expect the presence of angiogenesis to influence gene expression, the ability to detect this feature may in part be limited by the relatively small contribution these regions make to the total amount of dna / rna extracted for analysis. next we performed a genome - wide analysis of transcriptional data to discover the impact of necrosis on gene expression. a normalized linear regression coefficient was calculated for each transcript to measure the strength of relationship between extent of necrosis and gene expression for more than 22,000 transcripts measured with affymetrix arrays. significance analysis of microarrays (sam) correction was applied to obtain multiple - test corrected p - values for each gene. this analysis identified 2422 genes that are significantly correlated with extent of necrosis at 5% false - discovery rate or below, suggesting that necrosis has tremendous influence on gene expression in gbms. among the genes most significantly correlated with necrosis were a set of transcription factors known as mesenchymal master regulators: cebpb, fosl2, cebpd, stat3, bhlhe40 (ranked 4, 10, 60, 213 and 221 respectively). these transcription factors have been shown to form a small module regulating a much broader gene expression network that is responsible for mesenchymal tumor phenotype in glioblastomas. at the top of this regulatory module are the transcription factors ce - bpb / cebpd and stat3, whose coexpression is necessary and sufficient for activating the mesenchymal expression network. to explore the expression of these regulators in tissues, we performed immunohistochemistry on archived surgically resected glioblastomas from our own institution. we observed that cebpb / cebpd expression was strongly and specifically expressed in the hypoxic pseudopalisading cells surrounding areas of necrosis (fig 1d). cebpb was strongly expressed in nuclei of the first 25 cell layers immediately surrounding necrosis, and cebpd expression was found in both nuclear and cytoplasmic regions of perinecrotic cells but extending slightly farther beyond cebpd. in regions between foci of necrosis, gene expression classifications are made by measuring the distances in gene expression space between a tumor s expression profile and a set of points or centroids that represent each class. the tumor is assigned to the class with the nearest centroid, which can be measured using a variety of metrics including simple euclidean distance. while a given tissue could potentially contain individual cells / regions with diverging gene expression profiles, these classifications force a selection of a single gene expression class that best defines the entire sample. to explore how the formation of necrosis influences the expression patterns of non - mesenchymal gbms , we examined the relationship between extent of necrosis and distance to the mesenchymal expression centroid in this cohort. we observed a clear trend the more necrosis that a sample contains, the more its expression profile resembles the mesenchymal centroid (fig 1e). this finding further suggests that mesenchymal gene expression is strongly impacted by hypoxia and that expression signatures are strongly impacted by regionally varying elements of the microenvironment. the morphologic characteristics of cell nuclei convey important clinical information in many types of neoplasms. besides determining histologic classification and subtype , nuclear qualities including shape, texture and spatial arrangement can be indicative of more specific molecular alterations and patient prognosis. gains, losses and rearrangements of dna along with epigenetic modifications affecting chromatin structure can manifest in observable changes within nuclei of neoplastic cells. in the diffuse gliomas, nuclear features are of particular importance, as their classification of oligodendroglioma or astocytoma is based in large part on nuclear morphology. however, histopathologic classification based on human review is subjective and prone to substantial interobserver variation. understanding the relationships between nuclear morphology, tumor genetics and clinical outcomes will provide a better understanding of tumor biology and further improve the precision of clinical predictions. our studies of tumor microenvironment used human markups and annotations to generate quantitative features from whole - slide images. the limitations of human annotations are apparent when dealing with nuclear morphology - nuclei can number in the hundreds of millions in even a modestly sized set of images, and qualities of interest like nuclear texture are difficult to accurately characterize objectively by human observers. to address these challenges we have developed a computational system for the study of nuclear morphometry in large archives of whole - slide images (fig 3a). this system uses image analysis algorithms to delineate individual cell nuclei, and to calculate a set of objective nuclear features for each nucleus to describe its shape and texture. high performance and parallel computing approaches are used to scale this approach to hundreds of millions of cells. this system presents opportunities to define quantitative morphologic biomarkers of molecular and clinical endpoints by enabling the extraction of objective, repeatable measurements from wsi archives. our initial morphometric study focused on the quantitative characterization of oligodendroglial differentiation in glioblastomas. although gbm is defined as a grade iv astrocytoma, an important subset exhibits varying degrees of oligodendroglial differentiation in addition to the dominant astrocytic component. neoplasms with pure oligodendroglial differentiation typically have slower growth and better survivals when compared with astrocytomas of the same grades. the morphologic characteristics of oligodendrogliomas distinguish them from astrocytomas: oligodendroglial nuclei tend to be smaller, round and hyperchromatic with a lack of detailed texture, in contrast with astrocytoma nuclei that are larger, irregularly shaped, typically elongated and unevenly textured. in most instances, gbms contain a heterogeneous mixture of neoplastic cells with wide variations in nuclear characteristics, many of which are not clearly astrocytic or oligodendroglial. the volume and heterogeneity of cells present in gbms combined with subtle differences in morphologic diversity make them an ideal candidate for computational morphometric approaches. using our computational pipeline , we analyzed 200 million nuclei from digitized images of diagnostic slides corresponding to 117 tcga gbms. twenty - three quantitative features from four categories (shape, intensity, texture and gradient) were calculated to describe each nucleus. to represent the differentiation of each nucleus along the oligodendroglial / astrocytic spectrum , we built a regression model that uses the nuclear feature values to calculate a score for each nucleus representing its degree of oligodendroglial appearance (fig 3b). combining the 200 million scores obtained from our pipeline with gene expression, copy number, dna sequence and methylation data from the same tcga tumors , we were able to clearly separate a set of tumor enriched with oligodendroglial - like cells that had strong associations with pdgfra amplification, proneural transcriptional class, and expression of the oligodendrocyte signature genes mbp, hoxd1, plp1, mobp and pdgfra. these provide molecular validation that the quantitative features extracted by our software pipeline can capture the morphologic variations of nuclei encountered in gliomas. our differentiation study used a supervised approach to build a quantitative model of the oligodendroglial / astrocytic spectrum in gliomas. model - based approaches are a powerful way to incorporate prior knowledge into morphologic analyses, and to use quantitative measures of recognized morphologic patterns to explore their molecular correlates. because model - based approaches are built on prior knowledge, their ability to reveal previously unrecognized or unknown morphologic patterns is limited. to address this limitation, we have developed several unsupervised or model - free approaches that do not impose established constructs in the morphological analysis of wsi data. instead, these approaches let data speak for itself, using clustering analysis and other statistical learning techniques to reveal natural structure within the feature data in a bottom - up fashion. our first study with unsupervised methods investigated patient clustering of gbms into morphologically defined subtypes. using nuclear features , we sought to determine if there are clear and distinct groups of tumors that emerge from clustering analysis, similar to gene expression studies where transcriptional profiles are clustered to reveal molecular tumor subtypes. taking the nuclear features from the tcga cohort, a morphologic signature was calculated for each tumor to represent the morphologic properties of its average nuclei. these signatures were analyzed using a consensus - clustering algorithm to find natural groups within the data and to measure their robustness. three clear clusters emerged from this analysis and we named them for themes observed in their molecular correlates: cell cycle (cc), protein biosynthesis (pb) and chromatin modification (cm). we observed that these clusters had significant differences in patient survival (logrank p=1.4e-3), with the pb cluster containing patients with relatively better outcomes and the cm cluster relatively worse. these clusters were also observed in an independent dataset of 84 gbms where the relative differences in outcomes between the clusters were also confirmed. to explore the meaning of these clusters we used the various genomic platforms made available by tcga including gene expression, dna methylation, copy number and dna sequencing. a pathway analysis found that the clusters varied in the extent of tp53 wnt, and nfkb signaling, and had variations in the extent of total dna methylation. an analysis of the pathologic features using categorical human annotations (0,1+,2 +) found that tumors in the cm cluster had enriched presence of lymphocytes, and that pb cluster tumors exhibited a conspicuous lack of inflammation. to further explore model - free associations of nuclear morphometry in gbm and clinical and genomic endpoints , we took a more direct approach of correlating raw nuclear features with genomic and clinical endpoints. for each patient, we calculated the mean and standard deviation of each feature as metrics and correlating them directly with patient survival using cox proportional hazards analysis using sam. notably, the mean circularity was significantly associated with longer patient survival, an observation consistent with prolonged clinical outcomes in gliomas with oligodendroglial differentiation. other features that were significantly associated with outcome include major axis length, with longer nuclei associated with a shorter survival, and min nuclear pixel intensity, with higher values associated with longer survival. the fact that these features emerged from a more data - driven approach provides some level of confidence in our analysis workflow. to correlate these features with genomic measurements we performed a one - way anova for each feature metric across transcriptional classifications, somatic mutations and dna copy number alterations. features distinguishing transcriptional classes include nuclear eccentricity (p = 3.81e-4), minor axis length (p = 8.87e-3) and nuclear extent (p = 3.2e-2). those hypermethylated (gcimp) tumors within the proneural group had greater variation of pixel intensities within their nuclei (nuclear energy, p = 2.28e-5), and greater variation in nuclear size. genetic events having significant differences in nuclear morphometry included pten and tp53 mutations, and pdg - fra amplification. pten and tp53 mutant tumors were both associated with less circular nuclei (p = 9.68e-3, 3.77e-2 respectively). pdgfra amplified tumors were associated with greater circularity (p = 2.31e-2), consistent with pdgfra amplifications being associated with oligodendroglial differentiation. other genetic alterations with significant associations included egfr amplification, which was associated with greater nuclear eccentricity and canny, and mdm2 amplifications, which were associated with greater minor axis length, area and circularity. advances in whole - slide imaging and computing hardware have made it possible to approach increasingly difficult image analysis problems in pathology. at the same time, the increasing availability of rich genomic data have made pathology image analysis studies more interesting by allowing linkage of histologic features with comprehensive molecular measurements. within the last decade, the goals of pathology image analysis have shifted from attempts to implement computer - aided diagnostic procedures, to more creative analyses that explore complex genotype - phenotype associations and define novel prognostication methods. the emerging goal is to go beyond computational replication of pathologists and to develop novel techniques to unmask latent content within image sets that has as yet unrecognized clinical and scientific value. this convergence of image analysis and bioinformatics has produced some exciting in several different areas. in glioblastoma , morphometry - driven tumor subtypes were identified on the basis of nuclear morphometry and cellularity and found to be predictive of clinical outcomes and pathway activation. in breast cancer, morphologic features describing stromal / tumor interface were found to be predictive of overall survival independent of other clinical, pathological and molecular features in two independent cohorts. features of cellularity derived from images were found to improve the estimation of copy number variations, and a prognostic model that combines image measurements with gene expression features was developed and validated in independent cohorts. focusing specifically on triple - negative breast cancer, a prognostic model based on morphologic features was also developed and validated in an independent dataset of triple negative breast cancers containing histology images. a gene - expression signature derived from this prognostic model was then developed and used to further validate the prognostic value of this model in gene expression datasets where histology images were not available. one barrier to progress in this area is the dissemination of algorithms and image features beyond image analysis experts to the broader research community. making software and feature data publicly accessible will facilitate advances in this field by more fully engaging the pathology community and providing opportunities for comparative studies. establishing the computational resources needed to execute image analysis algorithms on the primary images is difficult, and the sharing of derived feature data is limited by a lack of standardization. to begin to address these issues, we have developed web - based interfaces and data standards to support the visualization, federation and analysis of pathology image data. the cancer digital slide archive (cdsa, http://cancer.digitalslidearchive.net/) is a web - based resource that was originally developed to facilitate the visualization and analysis of pathology imaging and clinical data from tcga. the cdsa currently hosts over 22,000 images and associated clinical data from over 22 different cancers represented in tcga. this interface provides access to pathology and clinical data through a simple web - browser interface. although currently focused on serving primary images for visualization, the cdsa and other similar resources could naturally serve as clearinghouses that allow a broader set of users to interact with image analysis algorithms and feature sets. cloud - based services could be established that enable primary image data, derived features and computational tools to reside in a common computing environment, avoiding the need for costly transfer of massive amounts of image and feature data. users could then perform end - to - end integrated analyses of pathology imaging online without the need to establish local computing resources or shepherding primary image or feature data between systems across the internet. in cases where feature data and algorithms are exchanged, we have also developed the pathology analytic imaging standards (pais) to support the standardization of image analysis algorithms and image features. pais provides data standards that enable users to capture software and algorithm parameter provenance, and a common file format that enables to be stored in a database for search and exchange. this issue is particularly important in studies identifying image biomarkers of clinical outcomes or genomic features. one of the risks in using image features to predict outcome or genomic measurements is overfitting are we are learning meaningful relationships that will generalize to new unseen datasets, or simply generating predictions that are specific to the noise and artifacts of the dataset that were analyzed? validating findings in external datasets, when available, or by proper cross - validation of a single dataset is important for distinguishing true findings from artifacts. sometimes the morphologic features that are predictive can be visualized, while other times their predictive power is clearly measurable but not apparent to the human eye. some features are calculated (e.g. standard deviation of canny) are difficult to correlate with concrete histologic findings that can be visualized. in the cases where visualization is not possible or does not produce any obvious visible distinction, it is difficult to interpret the meaning of predictive features or to link them to existing knowledge about the histology of that disease. greater availability of benchmark datasets containing whole - slide images, and genomic and clinical data could help to establish reproducibility and improve confidence in the relationships defined through computational analysis. another area for growth is the integration of radiology imaging with pathology, genomics and clinical data. image analysis methods for radiology data are more mature than for pathology, and are able to extract meaningful features from mr, pet, ct and other medical imaging modalities. the global perspective of tumor provided by medical imaging is complementary to the tissue and molecular scale measurements provided by pathology and genomics, and a number of studies have already explored the relationships between quantitative radiology imaging features, genomic profiles and clinical outcomes. integrating complementary features across biological scales into prognostic models is a promising avenue to improve the precision of clinical predictions and risk stratification of patients. advances in whole - slide imaging and computing hardware have made it possible to approach increasingly difficult image analysis problems in pathology. at the same time, the increasing availability of rich genomic data have made pathology image analysis studies more interesting by allowing linkage of histologic features with comprehensive molecular measurements. within the last decade, the goals of pathology image analysis have shifted from attempts to implement computer - aided diagnostic procedures, to more creative analyses that explore complex genotype - phenotype associations and define novel prognostication methods. the emerging goal is to go beyond computational replication of pathologists and to develop novel techniques to unmask latent content within image sets that has as yet unrecognized clinical and scientific value. this convergence of image analysis and bioinformatics has produced some exciting in several different areas. in glioblastoma , morphometry - driven tumor subtypes were identified on the basis of nuclear morphometry and cellularity and found to be predictive of clinical outcomes and pathway activation. in breast cancer, morphologic features describing stromal / tumor interface were found to be predictive of overall survival independent of other clinical, pathological and molecular features in two independent cohorts. features of cellularity derived from images were found to improve the estimation of copy number variations, and a prognostic model that combines image measurements with gene expression features was developed and validated in independent cohorts. focusing specifically on triple - negative breast cancer, a prognostic model based on morphologic features was also developed and validated in an independent dataset of triple negative breast cancers containing histology images. a gene - expression signature derived from this prognostic model was then developed and used to further validate the prognostic value of this model in gene expression datasets where histology images were not available. one barrier to progress in this area is the dissemination of algorithms and image features beyond image analysis experts to the broader research community. making software and feature data publicly accessible will facilitate advances in this field by more fully engaging the pathology community and providing opportunities for comparative studies. establishing the computational resources needed to execute image analysis algorithms on the primary images is difficult, and the sharing of derived feature data is limited by a lack of standardization. to begin to address these issues, we have developed web - based interfaces and data standards to support the visualization, federation and analysis of pathology image data. the cancer digital slide archive (cdsa, http://cancer.digitalslidearchive.net/) is a web - based resource that was originally developed to facilitate the visualization and analysis of pathology imaging and clinical data from tcga. the cdsa currently hosts over 22,000 images and associated clinical data from over 22 different cancers represented in tcga. this interface provides access to pathology and clinical data through a simple web - browser interface. although currently focused on serving primary images for visualization, the cdsa and other similar resources could naturally serve as clearinghouses that allow a broader set of users to interact with image analysis algorithms and feature sets. cloud - based services could be established that enable primary image data, derived features and computational tools to reside in a common computing environment, avoiding the need for costly transfer of massive amounts of image and feature data. users could then perform end - to - end integrated analyses of pathology imaging online without the need to establish local computing resources or shepherding primary image or feature data between systems across the internet. in cases where feature data and algorithms are exchanged, we have also developed the pathology analytic imaging standards (pais) to support the standardization of image analysis algorithms and image features. pais provides data standards that enable users to capture software and algorithm parameter provenance, and a common file format that enables to be stored in a database for search and exchange. this issue is particularly important in studies identifying image biomarkers of clinical outcomes or genomic features. one of the risks in using image features to predict outcome or genomic measurements is overfitting are we are learning meaningful relationships that will generalize to new unseen datasets, or simply generating predictions that are specific to the noise and artifacts of the dataset that were analyzed? validating findings in external datasets, when available, or by proper cross - validation of a single dataset is important for distinguishing true findings from artifacts. sometimes the morphologic features that are predictive can be visualized, while other times their predictive power is clearly measurable but not apparent to the human eye. some features are calculated (e.g. standard deviation of canny) are difficult to correlate with concrete histologic findings that can be visualized. in the cases where visualization is not possible or does not produce any obvious visible distinction, it is difficult to interpret the meaning of predictive features or to link them to existing knowledge about the histology of that disease. greater availability of benchmark datasets containing whole - slide images, and genomic and clinical data could help to establish reproducibility and improve confidence in the relationships defined through computational analysis. another area for growth is the integration of radiology imaging with pathology, genomics and clinical data. image analysis methods for radiology data are more mature than for pathology, and are able to extract meaningful features from mr, pet, ct and other medical imaging modalities. the global perspective of tumor provided by medical imaging is complementary to the tissue and molecular scale measurements provided by pathology and genomics, and a number of studies have already explored the relationships between quantitative radiology imaging features, genomic profiles and clinical outcomes. integrating complementary features across biological scales into prognostic models is a promising avenue to improve the precision of clinical predictions and risk stratification of patients.

technological advances in computing, imaging and genomics have created new opportunities for exploring relationships between histology, molecular events and clinical outcomes using quantitative methods. slide scanning devices are now capable of rapidly producing massive digital image archives that capture histological details in high - resolution. commensurate advances in computing and image analysis algorithms enable mining of archives to extract descriptions of histology, ranging from basic human annotations to automatic and precisely quantitative morphometric characterization of hundreds of millions of cells. these imaging capabilities represent a new dimension in tissue - based studies, and when combined with genomic and clinical endpoints, can be used to explore biologic characteristics of the tumor microenvironment and to discover new morphologic biomarkers of genetic alterations and patient outcomes. in this paper we review developments in quantitative imaging technology and illustrate how image features can be integrated with clinical and genomic data to investigate fundamental problems in cancer. using motivating examples from the study of glioblastomas (gbms), we demonstrate how public data from the cancer genome atlas (tcga) can serve as an open platform to conduct in silico tissue based studies that integrate existing data resources. we show how these approaches can be used to explore the relation of the tumor microenvironment to genomic alterations and gene expression patterns and to define nuclear morphometric features that are predictive of genetic alterations and clinical outcomes. challenges, limitations and emerging opportunities in the area of quantitative imaging and integrative analyses are also discussed.

this study was supported by a grant - in - aid for scientific research on priority areas (19039013 to t.m . and 20061016 to t.y .) and a grant - in - aid for the gcoe programs (systems biology) of the ministry of education, culture, sports, science, and technology of japan.

the two - component systems (tcs), or histidine - to - aspartate phosphorelays, are evolutionarily conserved common signal transduction mechanisms that are implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes including plants. among higher plants, legumes including lotus japonicus have a unique ability to engage in beneficial symbiosis with nitrogen - fixing bacteria. we previously presented a genome - wide compiled list of tcs - associated components of mesorhizobium loti, which is a symbiont specific to l. japonicus (hagiwara et al . 2004, dna res . , 11, 5765). to gain both general and specific insights into tcs of this currently attractive model legume, here we compiled tcs - associated components as many as possible from a genome - wide viewpoint by taking advantage that the efforts of whole genome sequencing of l. japonicus are almost at final stage. in the current database (http://www.kazusa.or.jp/lotus/index.html), it was found that l. japonicus has, at least, 14 genes each encoding a histidine kinase, 7 histidine - containing phosphotransmitter - related genes, 7 type - a response regulator (rr)-related genes, 11 type - b rr - related genes, and also 5 circadian clock - associated pseudo - rr genes. these suggested that most of the l. japonicus tcs - associated genes have already been uncovered in this genome - wide analysis, if not all. here, characteristics of these tcs - associated components of l. japonicus were inspected, one by one, in comparison with those of arabidopsis thaliana. in addition, some critical experiments were also done to gain further insights into the functions of l. japonicus tcs - associated genes with special reference to cytokinin - mediated signal transduction and circadian clock.

the design of the study has been described previously.9 in short, a prospective, observational study was performed among females undergoing hysterectomy for benign disease in 13 teaching and nonteaching hospitals in the netherlands. the study was approved by all local ethical committees, and written informed consent was obtained from all patients. we recruited consecutive females who had been offered hysterectomy between january 1999 and july 2000. exclusion criteria were known endometriosis and symptomatic descensus of the uterus as indication for hysterectomy. before hysterectomy, the gynecologist who had set the indication completed a standardized form to score age, obstetric history, history of abdominal surgery, indication for hysterectomy, duration of complaints, postmenopausal status, maximal diameter of the uterus as assessed by ultrasound, indication for vaginal or abdominal hysterectomy, and indication for removal or preservation of the cervix. after surgery, the gynecologist who had performed the hysterectomy completed a standardized form to score the following variables: duration of surgery in minutes, amount of blood loss in ml, and complications during surgery. at the day of discharge , this form was completed by documenting the duration of hospital stay and complications during hospital stay. all patients completed before, and at three years after, surgery the defecation distress inventory (ddi).10 the ddi is a dutch validated questionnaire that was developed identical to the urogenital distress inventory11,12 by our research group and is used to assess the presence and experienced discomfort of defecation symptoms. the 15 questions were developed after studying the literature and international definitions, interviewing patients who presented with constipation or fecal incontinence, and by interviewing three experts in the field from the department of surgery and department of obstetrics and gynecology from the university medical centre utrecht, the netherlands. eventually, a structured interview of the 15 selected items was held with 20 female patients. for this study, we used the response to two questions of the ddi: do you have less than three bowel movements per week? and the question: do you have to strain > 25 percent of the time to have a bowel movement? according to the definition of drosmann et al. , constipation was considered to be present if the patient responded positive to both of these questions.13 this statistical analysis was designed to calculate the risk of constipation after hysterectomy and to identify which patient characteristics are prognostic factors for the development or persistence of constipation. the prevalence of constipation that persisted or had developed after hysterectomy was compared for the presence or absence of different patient characteristics and tested for statistical significance by using fisher s exact test. the risk of constipation after hysterectomy was expressed by the relative risk (rr) and 95 percent confidence interval (ci). the rr expresses the risk of a patient in whom a condition is present compared with a patient in whom this condition is not present. variables that were tested for their statistical significance were age, body mass index, parity, history of abdominal surgery, presence of comorbidity, indication for hysterectomy, presence of fibroma, maximal diameter of the uterus, vaginal or abdominal approach, and removal of the cervix. the statistical package spss 11.5 was used to perform our analysis (spss, inc ., this statistical analysis was designed to calculate the risk of constipation after hysterectomy and to identify which patient characteristics are prognostic factors for the development or persistence of constipation . the prevalence of constipation that persisted or had developed after hysterectomy was compared for the presence or absence of different patient characteristics and tested for statistical significance by using fisher s exact test . the risk of constipation after hysterectomy was expressed by the relative risk ( rr) and 95 percent confidence interval (ci). the rr expresses the risk of a patient in whom a condition is present compared with a patient in whom this condition is not present. variables that were tested for their statistical significance were age, body mass index, parity, history of abdominal surgery, presence of comorbidity, indication for hysterectomy, presence of fibroma, maximal diameter of the uterus, vaginal or abdominal approach, and removal of the cervix. the statistical package spss 11.5 was used to perform our analysis (spss, inc ., of the 413 included patients, 344 ( 83 percent) responded at three - year follow - up. if abdominal hysterectomy was performed, the cervix was preserved in one of three patients. table 1patient characteristics (n = 334)age (yr)44parous (n)281body mass index (kg / m)25history of abdominal surgery (n)126comorbidity (n)235duration of symptoms (mo)34indication for hysterectomy (n) menorrhagia231 metrorrhagia104 abdominal pain144 dysmenorroe76fibroma present on ultrasound (n)228maximal diameter of uterus (cm)10.3(3.5)descensus of uterus (cm)-5.4(2.9)values are means with standard deviations in parentheses or numbers with percentages in parentheses.not mutually exclusive.measured under anesthesia by pulling down the cervix with a forceps.table 2surgical procedures and complicationsno. of patients 334surgical procedures total abdominal hysterectomy158 subtotal abdominal hysterectomy91 vaginal hysterectomy85surgery time (min)62blood loss (ml)275complications during surgery18 bleeding requiring transfusion14 bleeding requiring reoperation2 bladder lesion2complications during hospital admission14 vault abscess2 vault hematoma1 cystitis3 bladder retention4 fever of unknown origin2 pulmonary embolism2values are means with standard deviations in parentheses or numbers with percentages in parentheses.some patients had more than one complication. patient characteristics (n = 334) values are means with standard deviations in parentheses or numbers with percentages in parentheses. measured under anesthesia by pulling down the cervix with a forceps. surgical procedures and complications values constipation had developed in 7 of 309 patients (2 percent) without constipation before surgery. table 3 shows the risk on development of constipation according to the different patient characteristics and surgical parameters. it seemed that preservation of the cervix was associated with a higher risk to develop constipation after hysterectomy. we reviewed the medical files of the seven patients who developed constipation and found that in three of these patients total hysterectomy was planned; however, during surgery because of difficult surgical conditions, such as adhesions and/or fibroma extending into the cervix, it was decided to preserve the cervix. all patients who developed constipation had undergone abdominal hysterectomy, had fibroma on ultrasound, had not undergone previous abdominal surgery, and did not have metrorragia as indication for hysterectomy. table 3risk of development or persistence of constipation three years after hysterectomy for different patient characteristics and surgical parameterspatient characteristic / surgical parameterrisk of development of constipationrisk of persistence of constipationn(%)rr95 percent cip valuen(%)rr95 percent cip valueage (yr)<403/702.3(0.221.2)0.635/10**140503/1790.9(0.18.4)19/21**0.45>501/5311/11body mass index (kg / m)<220/51**0.335/100.9(0.42)0.8722252/990.6(0.13)0.71/50.4(0.12.3)0.32>255/14518/151parousyes4/252114/291no3/503.8(0.916.4)0.091/30.7(0.13.6)1history of abdominal surgeryyes0/11614/101no7/186**0.0611/221.3(0.53)0.71comorbidityyes3/8514/111no4/2080.5(0.12.4)0.4210/201.4(0.63.4)0.71menorrhagia**yes5/206110/251no2/960.9(0.24.3)15/71.8(0.93.5)0.21metrorrhagia**yes0/9515/91no7/207**0.110/230.8(0.41.7)0.7abdominal pain**yes4/13316/111no3/1690.6(0.12.6)0.79/210.8(0.41.6)0.71dysmenorroe**yes1/6715/91no6/2351.7(0.214)110/230.8(0.41.7)0.7fibroma present on ultrasoundyes7/20617/191no0/92**0.18/131.7(0.83.5)0.28maximal diameter of uterus (cm)<80/52**0.183/61(0.32.8)18102/1420.3(0.11.5)0.247/160.9(0.42)1>105/10815/101surgical approachvaginal0/7416/111abdominal7/228**0.29/210.8(0.41.6)0.71removal of the cervixtotal2/219111/231subtotal5/836.6(1.333.3)0.024/90.9(0.42.2)1rr = relative risk; ci = confidence interval.*cannot be calculated.**indication for hysterectomy.calculated by fisher s exact test. risk of development or persistence of constipation three years after hysterectomy for different patient characteristics and surgical parameters rr = relative risk; ci = confidence interval. constipation persisted in 16 of 35 patients (46 percent) with constipation before surgery. table 3 shows the risk on persistence of constipation according to the different patient characteristics and surgical parameters. at three years after surgery, constipation had developed in 7 of 309 patients (2 percent) without constipation before surgery. table 3 shows the risk on development of constipation according to the different patient characteristics and surgical parameters. it seemed that preservation of the cervix was associated with a higher risk to develop constipation after hysterectomy. we reviewed the medical files of the seven patients who developed constipation and found that in three of these patients total hysterectomy was planned; however, during surgery because of difficult surgical conditions, such as adhesions and/or fibroma extending into the cervix, it was decided to preserve the cervix. all patients who developed constipation had undergone abdominal hysterectomy, had fibroma on ultrasound, had not undergone previous abdominal surgery, and did not have metrorragia as indication for hysterectomy. table 3risk of development or persistence of constipation three years after hysterectomy for different patient characteristics and surgical parameterspatient characteristic / surgical parameterrisk of development of constipationrisk of persistence of constipationn(%)rr95 percent cip valuen(%)rr95 percent cip valueage (yr)<403/702.3(0.221.2)0.635/10**140503/1790.9(0.18.4)19/21**0.45>501/5311/11body mass index (kg / m)<220/51**0.335/100.9(0.42)0.8722252/990.6(0.13)0.71/50.4(0.12.3)0.32>255/14518/151parousyes4/252114/291no3/503.8(0.916.4)0.091/30.7(0.13.6)1history of abdominal surgeryyes0/11614/101no7/186**0.0611/221.3(0.53)0.71comorbidityyes3/8514/111no4/2080.5(0.12.4)0.4210/201.4(0.63.4)0.71menorrhagia**yes5/206110/251no2/960.9(0.24.3)15/71.8(0.93.5)0.21metrorrhagia**yes0/9515/91no7/207**0.110/230.8(0.41.7)0.7abdominal pain**yes4/13316/111no3/1690.6(0.12.6)0.79/210.8(0.41.6)0.71dysmenorroe**yes1/6715/91no6/2351.7(0.214)110/230.8(0.41.7)0.7fibroma present on ultrasoundyes7/20617/191no0/92**0.18/131.7(0.83.5)0.28maximal diameter of uterus (cm)<80/52**0.183/61(0.32.8)18102/1420.3(0.11.5)0.247/160.9(0.42)1>105/10815/101surgical approachvaginal0/7416/111abdominal7/228**0.29/210.8(0.41.6)0.71removal of the cervixtotal2/219111/231subtotal5/836.6(1.333.3)0.024/90.9(0.42.2)1rr = relative risk; ci = confidence interval.*cannot be calculated.**indication for hysterectomy.calculated by fisher s exact test. risk of development or persistence of constipation three years after hysterectomy for different patient characteristics and surgical parameters rr = relative risk; ci = confidence interval. constipation persisted in 16 of 35 patients (46 percent) with constipation before surgery. table 3 shows the risk on persistence of constipation according to the different patient characteristics and surgical parameters. the purpose of our study was to investigate whether hysterectomy causes constipation and to identify prognostic factors for the development or persistence of constipation. preservation of the cervix seemed to be associated with an increased occurrence of constipation, but the small numbers make the relevance of this finding questionable. in nearly half of the patients reporting constipation before hysterectomy, the present study was based on a multicenter cohort study of 413 females undergoing vaginal, subtotal abdominal, or total abdominal hysterectomy. data were prospectively collected, potential confounders were accurately documented, and a validated questionnaire was used to asses the occurrence of constipation. first, the number of patients in the stratum with constipation present before surgery was relatively small. second, the number of patients that developed constipation was so low that it is questionable whether one should attempt to identify risk factors for this occurrence. one of the strengths of our study is that we managed to assess complete follow - up of more than 80 percent of our cohort at three years after surgery. there is a widespread but poorly quantified belief that hysterectomy is associated with bowel problems, especially constipation. taylor and coauthors6 compared females with bowel symptoms after hysterectomy with age - matched healthy control subjects. posthysterectomy females reported more commonly infrequent bowel movement, use of laxatives, and consulted more often a doctor because of constipation. van dam et al.7 compared bowel function in 593 females who had undergone hysterectomy to a control group consisting of 100 women who had undergone laparoscopic cholecystectomy and found that bowel dysfunction was significantly more common after hysterectomy. heaton and coauthors5 also observed that constipation was more common after hysterectomy than after laparoscopic cholecystectomy. because of the retrospective design of these studies, it seems reasonable to assume that the operation precedes the onset of bowel dysfunction. furthermore, retrospective studies may be biased by recall bias, in specific, patients may have forgotten the exact timing of the onset of constipation. similar to our findings, one other study prospectively evaluating the effects of hysterectomy on constipation did not observe an increased incidence of constipation postoperatively.14 in a randomized trial comparing the effects of total and subtotal abdominal hysterectomy on pelvic floor function, the prevalence of constipation after surgery was lower than before surgery. the authors did not present the data stratified for presence or absence of constipation before hysterectomy. the reported prevalence of constipation in the community ranges from 2 to 28 percent and depends on the used definition.15 the definition for constipation we use has been described by drossman et al.13 and includes both frequency of defecation and the necessity to strain. in most studies, constipation is defined as a bowel frequency of less than three times per week.15 the use of our stricter definition may explain the low prevalence of constipation both before and after hysterectomy. however, it has been shown that adding the necessity to strain to the definition significantly increases the sensitivity of the symptoms to identify individuals with constipation.16 studies initiated to identify prognostic factors for the development or persistence of constipation have, as far as known, not been published. in this study, the small number of patients that developed constipation (7/309) and the small number with constipation before surgery (35/344) limited the identification of prognostic variables for the development and persistence of constipation. however, it was an interesting observation that preservation of the cervix seemed to be associated with an increased incidence of constipation after hysterectomy. in a randomized trial comparing the effects of total and subtotal abdominal hysterectomy on pelvic floor function, preservation of the cervix and prevalence of constipation after surgery were not related to each other.17 as mentioned in the section, review of the medical files of the seven patients who developed constipation showed that in three of these patients during surgery the cervix could not be removed because of difficult surgical conditions, such as adhesions and/or fibroma extending into the cervix. as a consequence, the prognostic value of the variable preservation of the cervix seems to be confounded by the technical difficulty of the procedure. even while preserving the cervix, it is likely that these surgical procedures have caused more autonomic nerve damage with the development of constipation as a . because the prevalence of constipation increases with age, the observed development in our study might reflect the natural course of this symptom.18 therefore, we conclude that hysterectomy does not affect the occurrence of constipation. preservation of the cervix was associated with an increased risk on the development of constipation after hysterectomy, but this was largely explained by more difficult and extensive surgery. if present before hysterectomy, constipation had disappeared at three years after surgery in almost half of the patients. to the editor hysterectomy is the most common major gynecologic operation and the procedure usually has a low risk for postoperative morbidity. sometimes patients report unwanted effects on bowel function after hysterectomy, but there are a limited number of studies 18 on this subject and most of them are retrospective in nature. roovers et al. should be commended for conducting a prospective large - scale study to determine the development of constipation after hysterectomy. the 83 percent response rate after three years is impressive. in a recent prospective study, 9 we found similar as roovers et al. we evaluated the influence of hysterectomy on bowel function in 120 consecutive patients, and we found a trend toward more anal incontinence symptoms but no deterioration of constipation. roovers et al. have defined constipation as the presence of less than three bowel movements per week and straining more than 25 percent of the time to have a bowel movement. we do not agree with this restrictive definition of constipation, and there is ample support from our standpoint in the literature. it is correct that koch et al.10 found that straining is a sensitive criterion for constipation. in their , however, they recommend physiologic testing in the assessment of constipated patients rather than using the definition used in the present study. the authors reference a study by drossman et al. 11 for their definition of constipation. this study is 15 years old and is primarily focused on describing the range of bowel patterns in the general population and not to define constipation. drossman is otherwise one of the authorities behind the rome criteria for functional bowel disorders, including constipation. 12 in the latest communication from the rome initiative, constipation is defined as persistently difficult, infrequent, or seemingly incomplete defecation, which do not meet ibs criteria. 13 the american college of gastroenterology states that constipation is characterized by unsatisfactory defecation that from infrequent stools, difficult stool passage, or both. 14 the restrictive definition of constipation in the present study limits the number of constipated patients, which may have influenced the and . it would be interesting to see whether the outcome would change with a more liberal, and more commonly used, definition of constipation. we are still lacking large prospective studies focused on evaluating the influence of hysterectomy on different aspects of bowel function. this is an important topic for future studies, given the high incidence of this operation in the female population. to the editor colleagues mellgren and altman do not agree with the restrictive definition of constipation. in our study, constipation was considered to be present if the patient responded both positive to the questions: do you have less than three bowel movements per week? and do you have to strain > 25 percent of the time to have a bowel movement? the ddi questionnaire does not only question whether a symptom is present but also the experienced amount of bother by that symptom. we reanalyzed our data and found that 2.4 percent of the asymptomatic patients developed infrequent bowel movement, whereas 17.6 percent of the asymptomatic patients developed the need to frequently strain. we also analyzed how bothered the patients were by these symptoms and found that three years after surgery 38 percent of the patients were severely bothered by infrequent bowel movements, whereas only 11 percent of the patients were severely bothered by the need to frequently strain. we concluded that frequency of bowel movement and the need to strain are represented in the definition of constipation according to the rome criteria and the american college of gastroenterology. based on our own data, we state that quality of life related to constipation is mainly determined by the frequency of bowel movement. in our study, 2.4 percent of the preoperative asymptomatic patients had developed this symptom at three years after surgery.

purposethis study was designed to evaluate the risk on development and persistence of constipation after hysterectomy.methodswe conducted a prospective, observational, multicenter study with three - year follow - up in 13 teaching and nonteaching hospitals in the netherlands. a total of 413 females who underwent hysterectomy for benign disease other than symptomatic uterine prolapse were included. all patients underwent vaginal hysterectomy, subtotal abdominal hysterectomy, or total abdominal hysterectomy. a validated disease - specific quality - of - life questionnaire was completed before and three years after surgery to assess the presence of constipation.of the 413 included patients, 344 (83 percent) responded at three - year follow - up. constipation had developed in 7 of 309 patients (2 percent) without constipation before surgery and persisted in 16 of 35 patients (46 percent) with constipation before surgery. preservation of the cervix seemed to be associated with an increased risk of the development of constipation (relative risk, 6.6 ; 95 percent confidence interval, 1.333.3 ; p = 0.02). statistically significant risk factors for the persistence of constipation could not be identified.hysterectomy does not seem to cause constipation. in nearly half of the patients reporting constipation before hysterectomy, this symptom will disappear.

the placement of fixed orthodontic appliances increases the difficulty in mechanical plaque removal and thus predisposes orthodontic patients to plaque accumulation and enamel demineralization.1 - 3 fixed orthodontic treatment is also associated with increased probing pocket depths, elevated bacterial count in plaque, and a shift in the healthy microbial composition of subgingival plaque to a periodontal pathogenic one.4 - 6 therefore, optimal oral home care and professional prophylactic programs are of paramount importance in patients undergoing fixed orthodontic treatment.7,8 during professional oral hygiene procedures, ultrasonic scalers are used around the bracket base, a critical site of plaque accumulation, and can affect the bracket - enamel interface.9,10 prolonged ultrasonic vibration at maximum power is used to facilitate the removal of posts, crowns, or bridges and could similarly debond orthodontic brackets.11 - 15 this action is attributable to the propagation of vibrations from the ultrasonic device to the object that is to be removed, as well as to the biophysical action of ultrasound within the coolant (i.e., cavitational activity and acoustic microstreaming).16 however, to date, no study of the effects of ultrasonic instrumentation for oral hygiene maintenance on the shear bond strength (sbs) of orthodontic brackets has been conducted. the aim of this study was to evaluate the effects of ultrasonic instrumentation with different scaler - tip angulations on the sbs and bond failure mode of metallic orthodontic brackets. seventy - two premolars extracted for orthodontic reasons from patients aged 12 - 18 years were collected after receiving adequate informed consent. the teeth did not have cracks visible under 4 magnification, hypoplasia, white spots, caries, or labial restorations.17 they were washed in water to remove contamination and stored in distilled water in a refrigerator (i.e., nominal 4) for 1 - 6 months, in accordance with the iso / ts 11405 standard.18 in strict accordance with the protocol described by alessandri bonetti et al.,19 the labial enamel surfaces were cleaned, polished, and etched. a thin uniform coat of primer (transbond xt ; 3 m unitek, monrovia, ca, usa) was applied by using a microbrush. adhesive pre - coated metallic brackets (apc ii adhesive coating on victory series brackets ; 3 m unitek) were then placed on the teeth, adjusted to their final positions, and pressed firmly. after removing excess resin from the periphery of the bracket base with a dental probe, the adhesive was cured by using a light - emitting diode light source (ortholux luminous curing light ; 3 m unitek) for 6 s (3 s mesially and 3 s distally), according to the manufacturer's instruction. after storage in distilled water at 37 for 24 h (iso / ts 1140518 ; test type 1 : short - term test), the teeth were embedded in autopolymerizing acrylic resin (orthocryl ; dentaurum, bologna, italy) in polyvinyl chloride molds (15-mm diameter and 20-mm height) so that the roots were fully embedded in acrylic resin and the bonding surface of the brackets remained perpendicular to the horizontal plane and parallel to the direction of the force to be applied, in an effort to minimize peel and maximize shear during testing. to ensure the fulfillment of these requirements, the roots of each tooth were initially inserted in a wax pedestal, which was molded until the correct position was achieved and verified with the aid of a parallelometer (paraline ; dentaurum). the teeth were randomly divided into 3 groups of 24 specimens each: control group, no treatment; 45-angulation group, ultrasonic instrumentation with a scaler - tip angulation of 45 (figure 1); 0-angulation group, ultrasonic instrumentation with a scaler - tip angulation of 0 (figure 2). to maintain the desired angulation in the 45-angulation group, an external support with a component forming an angle of 45 to the scaler tip was fastened to the ultrasonic device and the component was maintained parallel to the tooth surface, thus achieving an angle of 45 between the scaler tip and the tooth surface. a piezoelectric ultrasonic scaler (piezosteril 5 ; castellini, bologna, italy) was used with insert " c1 " (recommended by the manufacturer for supragingival scaling) at a power setting of 7.5 w. tap water, delivered directly from the dental unit, was used as the coolant. ultrasonic instrumentation was performed for 1 min, with 20 s each on the mesial, distal, and occlusal sides of the bracket base. the gingival side of the bracket base was omitted because the roots were embedded in acrylic resin up to the cementoenamel junction, preventing the access of the scaler tip to that surface. the bracket slot was not instrumented because the bracket wings did not allow the chosen scaler - tip angulations to be consistently achieved along that surface. after storage in distilled water at room temperature for 24 h, the specimens were tested according to the iso / ts 1140518 standard in a universal testing machine (instron, milan, italy). each acrylic block was placed in the machine so that the bracket base was parallel to the direction of the force to be applied and a chisel - shaped blade was placed on the occlusal side of the ligature groove between the bracket base and the wings. a shear debonding force was applied in the occlusogingival direction at a crosshead speed of 1 mm / min,18 and the amount of shear force required to debond each bracket was recorded in newtons. stress values were calculated as the ratio of the debonding load (in newtons) to the area of the bracket base (9.79 mm) and measured in megapascals (mpa).20 to evaluate the mode of bond failure, the specimens were examined at 20 magnification with an optical microscope.21 by using open - source image - analysis software (imagej ; national institutes of health, bethesda, md, usa), the amount of adhesive remaining on the tooth was expressed as a percentage of the total bonded area.19 the analysis was performed by an examiner trained to use the software and blinded to the groups. the evaluations were repeated by the same examiner 1 week later; if any discrepancies arose, a third assessment was performed to determine the final score. an adhesive remnant index (ari) score was assigned to each specimen: 0, no adhesive left on the tooth; 1, less than half of the adhesive remained on the tooth; 2, more than half of the adhesive remained on the tooth; 3, all the adhesive was left on the tooth, with a distinct impression of the bracket mesh.22,23 statistical analyses were performed by using statistical software (pasw statistics for windows, version 18.0 ; ibm co., armonk, ny, usa). the significance level was set at 0.05. a pilot study estimated the expected difference in the mean sbs values between the untreated and the treated specimens as 2.3 mpa and the within - group standard deviation as 2.4 mpa. given the level of 0.0167 (bonferroni adjustment for multiple comparisons) with a power of 80% the shapiro - wilk test showed that the sbs data were consistent with a gaussian distribution in the control and 0-angulation groups, but they were not normally distributed in the 45-angulation group. pairwise comparisons were performed by using the mann - whitney u - test with bonferroni correction (adjusted level = 0.0167). the kruskal - wallis test was also used to assess the significance of the differences in the ari scores among the groups. seventy - two premolars extracted for orthodontic reasons from patients aged 12 - 18 years were collected after receiving adequate informed consent. the teeth did not have cracks visible under 4 magnification, hypoplasia, white spots, caries, or labial restorations.17 they were washed in water to remove contamination and stored in distilled water in a refrigerator (i.e., nominal 4) for 1 - 6 months, in accordance with the iso / ts 11405 standard.18 in strict accordance with the protocol described by alessandri bonetti et al.,19 the labial enamel surfaces were cleaned, polished, and etched. a thin uniform coat of primer (transbond xt ; 3 m unitek, monrovia, ca, usa) was applied by using a microbrush. adhesive pre - coated metallic brackets (apc ii adhesive coating on victory series brackets ; 3 m unitek) were then placed on the teeth, adjusted to their final positions, and pressed firmly. after removing excess resin from the periphery of the bracket base with a dental probe, the adhesive was cured by using a light - emitting diode light source (ortholux luminous curing light ; 3 m unitek) for 6 s (3 s mesially and 3 s distally), according to the manufacturer's instruction. after storage in distilled water at 37 for 24 h (iso / ts 1140518 ; test type 1 : short - term test), the teeth were embedded in autopolymerizing acrylic resin (orthocryl ; dentaurum, bologna, italy) in polyvinyl chloride molds (15-mm diameter and 20-mm height) so that the roots were fully embedded in acrylic resin and the bonding surface of the brackets remained perpendicular to the horizontal plane and parallel to the direction of the force to be applied, in an effort to minimize peel and maximize shear during testing. to ensure the fulfillment of these requirements, the roots of each tooth were initially inserted in a wax pedestal, which was molded until the correct position was achieved and verified with the aid of a parallelometer (paraline ; dentaurum). the teeth were randomly divided into 3 groups of 24 specimens each: control group, no treatment; 45-angulation group, ultrasonic instrumentation with a scaler - tip angulation of 45 (figure 1); 0-angulation group, ultrasonic instrumentation with a scaler - tip angulation of 0 (figure 2). to maintain the desired angulation in the 45-angulation group, an external support with a component forming an angle of 45 to the scaler tip was fastened to the ultrasonic device and the component was maintained parallel to the tooth surface, thus achieving an angle of 45 between the scaler tip and the tooth surface. a piezoelectric ultrasonic scaler (piezosteril 5 ; castellini, bologna, italy) was used with insert " c1 " (recommended by the manufacturer for supragingival scaling) at a power setting of 7.5 w. tap water, delivered directly from the dental unit, was used as the coolant. ultrasonic instrumentation was performed for 1 min, with 20 s each on the mesial, distal, and occlusal sides of the bracket base. the gingival side of the bracket base was omitted because the roots were embedded in acrylic resin up to the cementoenamel junction, preventing the access of the scaler tip to that surface. the bracket slot was not instrumented because the bracket wings did not allow the chosen scaler - tip angulations to be consistently achieved along that surface. a single experienced and trained operator performed all these procedures. after storage in distilled water at room temperature for 24 h, the specimens were tested according to the iso / ts 1140518 standard in a universal testing machine (instron, milan, italy). each acrylic block was placed in the machine so that the bracket base was parallel to the direction of the force to be applied and a chisel - shaped blade was placed on the occlusal side of the ligature groove between the bracket base and the wings. a shear debonding force was applied in the occlusogingival direction at a crosshead speed of 1 mm / min,18 and the amount of shear force required to debond each bracket was recorded in newtons. stress values were calculated as the ratio of the debonding load (in newtons) to the area of the bracket base (9.79 mm) and measured in megapascals (mpa).20 to evaluate the mode of bond failure, the specimens were examined at 20 magnification with an optical microscope.21 by using open - source image - analysis software (imagej ; national institutes of health, bethesda, md, usa), the amount of adhesive remaining on the tooth was expressed as a percentage of the total bonded area.19 the analysis was performed by an examiner trained to use the software and blinded to the groups. the evaluations were repeated by the same examiner 1 week later; if any discrepancies arose, a third assessment was performed to determine the final score. an adhesive remnant index (ari) score was assigned to each specimen: 0, no adhesive left on the tooth; 1, less than half of the adhesive remained on the tooth; 2, more than half of the adhesive remained on the tooth; 3, all the adhesive was left on the tooth, with a distinct impression of the bracket mesh.22,23 statistical analyses were performed by using statistical software (pasw statistics for windows, version 18.0 ; ibm co., armonk, ny, usa). the significance level was set at 0.05. a pilot study estimated the expected difference in the mean sbs values between the untreated and the treated specimens as 2.3 mpa and the within - group standard deviation as 2.4 mpa. given the level of 0.0167 (bonferroni adjustment for multiple comparisons) with a power of 80% the shapiro - wilk test showed that the sbs data were consistent with a gaussian distribution in the control and 0-angulation groups, but they were not normally distributed in the 45-angulation group. pairwise comparisons were performed by using the mann - whitney u - test with bonferroni correction (adjusted level = 0.0167). the kruskal - wallis test was also used to assess the significance of the differences in the ari scores among the groups. three specimens of the 0-angulation group could not be tested because debonding of the brackets occurred during ultrasonic instrumentation. in these cases, an sbs value of 0 mpa was attributed because it was considered more representative of the clinical situation than a value obtained by interpolation or considering the loss of the specimen. a significant difference in the mean sbs values was noted among the groups (p = 0.004). the mean sbs value of the control group (9.92 mpa) was significantly higher than that of the 45-angulation (7.77 mpa ; p = 0.004) and 0-angulation (7.16 mpa ; p = 0.006) groups. the kruskal - wallis test showed no significant differences among the groups. in many specimens (62.5% of the 45-angulation group and 66.67% of the control and 0-angulation groups) , most of the adhesive remained on the enamel (ari scores of 2 or 3). proper professional oral hygiene maintenance is of paramount importance in patients undergoing fixed orthodontic treatment to reduce the risk of periodontal diseases and enamel demineralization.1 - 3 ultrasonic instrumentation is usually carried out around the bracket base, which is a critical site of plaque accumulation,9,10 but no studies have been performed yet to assess whether there is any influence on the sbs and mode of bond failure of metallic orthodontic brackets. to simulate extreme conditions and highlight the most detrimental effect of ultrasonic instrumentation on the sbs of metallic orthodontic brackets, the time of instrumentation was overrated when compared with a routine clinical procedure, in which the scaler tip probably contacts the bracket - enamel interface for only a short period. therefore, 1 minute was chosen as the total application time.24 further, because manufacturers generally recommend a power setting of medium to high for supragingival scaling and chapple et al.25 demonstrated that a half power setting is as effective as a full one, a power setting of 7.5 w (50% of the maximum power of the device) was selected in this study. finally, the experienced operator who performed all the ultrasonic procedures had been trained to maintain the force of scaler - tip application as low as possible while contacting the bracket in a consistent manner, similarly to the routine clinical setting. however, as this method is not a standardized way of force application, it may limit the reproducibility of the study. in this study, ultrasonic instrumentation around the bracket base decreased the sbs of the metallic orthodontic brackets compared with the control specimens. the mean sbs values of both the treated groups were still beyond 6 mpa, which is reportedly adequate for orthodontic purposes.26 however, such a reduction is undesirable because it increases the risk of bracket failure after professional oral hygiene procedures, consequently impeding treatment progress and prolonging treatment time.27 the angulation of the scaler tip has been investigated in relation to the potential damage to the tooth surface but not to the impairment of the sbs of metallic orthodontic brackets. the extent of damage to the tooth surface is reportedly the highest with a tip angulation of 45 when using piezoelectric ultrasonic scalers.28,29 in this study, no significant differences in the sbs were found between the 0-angulation and the 45-angulation groups, suggesting that the tested angulations did not influence to bracket bond failure after professional oral hygiene procedures. however, the scaler tip should preferably be parallel to the tooth surface (i.e., 0 angulation), or at an angle less than 15, to orient the pattern of ultrasonic vibration parallel to the tooth surface and decrease the risk of damage.28,29 interestingly, 3 brackets detached in the 0-angulation group before the sbs testing; the reason for their failure is not fully understood. finally, the ari scores were similar among the groups, indicating that ultrasonic instrumentation did not affect the mode of bracket bond failure. in all the groups, most of the adhesive remained on the enamel, indicating a low risk of enamel damage after debonding. further studies are required to evaluate the influence of other parameters (e.g., power setting, load, instrumentation time) pertaining to ultrasonic instrumentation for oral hygienic purposes on the sbs of orthodontic brackets. under the conditions of this in vitro study, 1 minute ultrasonic instrumentation around the bracket base at medium power reduced the sbs of metallic orthodontic brackets, indicating a higher risk of bracket bond failure after professional oral hygiene procedures. therefore, clinicians should avoid prolonged ultrasonic instrumentation around the bracket base during plaque removal. the scaler - tip angulation does not influence the sbs reduction and failure mode of metallic orthodontic brackets.

objectiveto evaluate the effects of ultrasonic instrumentation with different scaler - tip angulations on the shear bond strength (sbs) and bond failure mode of metallic orthodontic brackets.methodsadhesive pre - coated metallic brackets were bonded to 72 extracted human premolars embedded in autopolymerizing acrylic resin. the teeth were randomly divided into 3 groups (n = 24 each) to undergo no treatment (control group) or ultrasonic instrumentation with a scaler - tip angulation of 45 (45-angulation group) or 0 (0-angulation group). sbs was tested in a universal testing machine, and adhesive remnant index (ari) scores were recorded. the kruskal - wallis test and mann - whitney u - test were used for statistical analysis.the control group had a significantly higher mean sbs value than the treated groups, which showed no significant differences in their mean sbs values. the ari scores were not significantly different among the groups.ultrasonic instrumentation around the bracket base reduces the sbs of metallic orthodontic brackets, emphasizing the need for caution during professional oral hygiene procedures in orthodontic patients. the scaler - tip angulation does not influence the sbs reduction and bond failure mode of such brackets.

diabetes mellitus (dm) is a chronic metabolic disorder characterized by hyperglycemia, and ing from defects in insulin secretion, insulin action, or both.1 the chronic hyperglycemia occurring in dm is associated with long - term damage, as well as dysfunction and failure of different organs, especially in the eyes, kidneys, nerves, heart and blood vessels.1 the world health organization (who) estimates that about 347 million people worldwide are presently living with dm.2 of this figure, approximately 80% are from low and medium income countries.3 who also projects that by 2030, dm will be the seventh leading cause of death worldwide.4 there are two main types of dm: type 1 which usually develops in childhood and adolescence, and is insulin dependent; and type 2 which develops in adulthood and represents more than 90% of cases worldwide. risk factors for type 2 dm include sedentary lifestyle, obesity, and old age. diagnosis of dm can be made with a simple fasting plasma glucose test with values 126 mg / dl (7.0 mmol / l) being diagnostic of dm (fasting is defined as no caloric intake for at least 8 hours).5 in the presence of symptoms of hyperglycemia (polyuria, polydipsia, polyphagia, weight loss), a casual plasma glucose level of > 200 mg / dl (11.1 mmol / l) is diagnostic.5 recently, various researchers have proposed that type 2 dm is connected to a state of subclinical chronic inflammation.6,7 it may be that abnormal levels of chemokines released by the expanded adipose tissue in obesity activates monocytes, and increases the secretion of proinflammatory adipokines. such cytokines in turn enhance insulin resistance in adipose and other tissues, thereby increasing the risk of type 2 dm.8,9 the red blood cell distribution width (rdw) is a measure of variation in size of the circulating erythrocytes (anisocytosis)10 which is routinely obtained from a standard automated complete blood count. high rdw indicates the presence of anisocytosis which is related to impairment of erythropoiesis and degradation of erythrocytes,10 reflecting chronic inflammation and increased levels of oxidative stress, both of which are telltale signs in type 2 diabetics, and this may significantly contribute to development of atherosclerotic diseases.11,12 many recent studies have investigated changes in rdw in association with cardiac and noncardiac related deaths.1319 most of these studies report a positive correlation of rdw with the erythrocyte sedimentation rate (esr) and c - reactive protein (crp) levels an increase in the rdw during inflammation, similar to that seen in other inflammatory parameters. malandrino et al20 recently reported a positive correlation between a high rdw and increased incidence of both macro- and microvascular complications in dm patients without marked vascular complications. the aim of this study was to evaluate the relationship between the rdw and fasting blood sugar / blood pressure, and compare with nondiabetic controls. the research was approved by the ethics review committee of lagos state university teaching hospital (lasuth). this was a case - control study consisting of 100 type 2 dm patients receiving treatment, attending the diabetic clinic of the lasuth, and 100 nondiabetic controls consisting of medical students, nurses and doctors in the same institution. during the study period between june to september 2013 , all patients who gave informed consent and satisfied the study inclusion criteria were recruited into the study. they were asked to fill structured questionnaires including demographic information, height, weight, last fasting blood sugar, blood pressure, drug history, and family history of diabetes. all the diabetics were on oral hypoglycemic and antiplatelet drugs, like clopidogrel and vasoprin tablets, and some were on antihypertensive and lipid lowering drugs. information on family history of diabetes was also obtained from the controls and they were subjected to fasting blood sugar testing before enlistment. blood was withdrawn with minimal stasis from the antecubital vein using a dry sterile disposable syringe and needle. the edta samples were kept at room temperature until processed within 4 hours of collection. a full blood count was performed using the sysmexkx-21n (sysmex corporation, kobe, japan), a three - part auto analyzer able to run 19 parameters per sample, including hemoglobin concentration, packed cell volume, red blood cell concentration, mean corpuscular hemoglobin, mean cell volume, mean corpuscular hemoglobin concentration, white blood cell and platelet count, and mean platelet volume. standardization, calibration of the instrument, and processing of the samples were carried out according to the manufacturer s instructions. well - mixed blood samples were aspirated, by leaving the equipment sampling probe in the blood sample and then pressing the start button. the research was approved by the ethics review committee of lagos state university teaching hospital (lasuth). this was a case - control study consisting of 100 type 2 dm patients receiving treatment, attending the diabetic clinic of the lasuth, and 100 nondiabetic controls consisting of medical students, nurses and doctors in the same institution. during the study period between june to september 2013 , all patients who gave informed consent and satisfied the study inclusion criteria were recruited into the study. they were asked to fill structured questionnaires including demographic information, height, weight, last fasting blood sugar, blood pressure, drug history, and family history of diabetes. all the diabetics were on oral hypoglycemic and antiplatelet drugs, like clopidogrel and vasoprin tablets, and some were on antihypertensive and lipid lowering drugs. information on family history of diabetes was also obtained from the controls and they were subjected to fasting blood sugar testing before enlistment. blood was withdrawn with minimal stasis from the antecubital vein using a dry sterile disposable syringe and needle. the edta samples were kept at room temperature until processed within 4 hours of collection. a full blood count was performed using the sysmexkx-21n (sysmex corporation, kobe, japan), a three - part auto analyzer able to run 19 parameters per sample, including hemoglobin concentration, packed cell volume, red blood cell concentration, mean corpuscular hemoglobin, mean cell volume, mean corpuscular hemoglobin concentration, white blood cell and platelet count, and mean platelet volume. standardization, calibration of the instrument, and processing of the samples were carried out according to the manufacturer s instructions. well - mixed blood samples were aspirated, by leaving the equipment sampling probe in the blood sample and then pressing the start button. data were analyzed using spss version 16.0 (spss inc ., chicago, il, usa). a total of 200 participants were enrolled into the study consisting of 100 diabetics and 100 nondiabetic controls. the mean age of the control group was 32.386.44 years, with a minimum age of 17 years, and a maximum age of 70 years. the mean age of the type 2 dm group was 62.359.84 years, the minimum was 34 years old, and the maximum 90 years old. the overall female: male ratio was 68% to 32%. in the type 2 dm participants, 73% were female, and 27% were male. in the nondiabetic participants, 63% were female, and in the diabetic group, the mean body mass index was 32.104.85 kg / m, and the mean fasting blood sugar level was 147.8572.54 mg / dl. in the nondiabetic group, the mean body mass index was 255.23 kg / m, and the mean fasting blood sugar was 95.2030.10 mg / dl. the minimum blood pressure of the diabetics was 100/90 mmhg, maximum was 200/90 mmhg, and the mean was 138/90 mmhg. amongst the diabetics, a total of 45 out of 100 (45%) gave a positive family history of type 2 diabetes, while 55% had no family history of diabetes. the rdw - cv (rdw coefficient of variation) in the control group was 14.741.94, and 14.800.71 in the type 2 dm group. the rdw - sd (rdw standard deviation) was 46.444.64 in the control group, and 46.843.18 in the type 2 dm group (table 2). both the rdw - sd and the rdw - cv were not statistically significantly correlated with the fasting blood sugar level in the type 2 dm group; p - values were 0.10 and 0.55 respectively; spearman s rho values were 0.61 and 0.12 respectively. the rdw - sd was not statistically significantly correlated with blood pressure; p=0.99; spearman s rho of 0.11. a statistically significant correlation was achieved between the rdw - cv and the blood pressure of type 2 dm patients; p=0.02, spearman s rho of 0.02. rdw - sd and the rdw - cv values were not statistically significantly correlated with the duration of diagnosis of diabetes in the patients; p - values were 0.8 and 0.38 respectively; and spearman s rho of 0.096 and 0.035 respectively. tukey s post hoc analysis could not be performed between the rdw - cv and blood pressure because at least one group had fewer than two cases, the significant level of 0.29 was obtained with test of homogeneity of variance. dm affects a sizeable proportion of the working population, and has economic consequences for both the individual and the society as a whole. the rdw, a widely available and inexpensive test conducted as part of the complete blood cell count, measures the degree of anisocytosis. it is calculated as follows: rdw - cv=(standard deviation of red blood cell volumemean cell volume)100 the normal range for the rdw - cv is 11.5%14.5%, and higher values indicate greater variations in cell sizes.21 high rdw indicates a high degree of anisocytosis which is associated with distortion and degradation of erythropoiesis,21 reflecting chronic inflammation and an increased level of oxidative stress.22 dm increases vascular inflammation and oxidative stress while vascular inflammation affects erythropoiesis and deformability of red blood cells thus elevating rdw levels.23 rdw is strongly associated with chronic inflammation and is a strong indicator of risk of cardiovascular mortality in people with cardiovascular diseases, dm, as well as in the general population.13,14,18,24 increased rdw may also arise as a of anemia. thus, causes like iron deficiency, and megaloblastic anemia with associated micro- or macrocytosis are potential confounders because our participants were not screened for iron, vitamin 12, nor folic acid. however, the effect of such confounders is negligible as all the participants selected for the study were healthy individuals (control group) and diabetics without complications or obvious comorbidities as at the time of the study. the effect of other causes of chronic inflammation such as tuberculosis, cancers, and connective tissue disorders as confounders is similarly negligible. the mean age of diagnosis of type 2 dm among adults aged 1879 years in the us between 1997 and 2011 was 54 years,25 and is similar to the 62.359.84 years obtained in our study; however this value doubles the mean age of our controls of 32.386.44 years. all controls used for this study were relatively young men and women working in our institution including nurses, doctors, and medical students, which are not representative of the general population; this could impact on obtained and is a possible limitation of the study. however, the sex distribution in the type 2 dm group and the control group was similar, with approximately 2:1 female: male in both cases. our type 2 dm study population had relatively well controlled diabetes, hence rdw - cv and rdw - sd of the type 2 dm group (14.800.71 and 46.843.18) was almost the same with control values (14.741.94 and 46.444.64). also, the mean fasting blood sugar of 147.8572.54 mg / dl in diabetics compared well with the nondiabetic controls of 95.2030.10 mg / dl. these closely related in our study may be accounted for by the fact that the majority of diabetics enrolled had long been on treatment before this study, and their medications performed well. this finding is in contrast with the findings of studies by lee and partley,26 malandrino et al20 and heba et al27 who found significant associations between the rdw and macrovascular complications of dm, suggesting that rdw may be a predictor of the onset of diabetic macrovascular complications. also in contrast to the studies of malandrino et al20 and sherif et al,27 our showed a statistically insignificant negative correlation between rdw and fasting blood sugar levels in diabetics. while in keeping with many other studies,2831 a statistically significant positive correlation was achieved between rdw and blood pressure in our patients. elevated rdw had been reported as a prognostic marker reflecting an underlying inflammatory state.28 high rdw was strongly associated with poor clinical outcomes in patients with heart failure,28 coronary artery disease,29,30 pulmonary hypertension31 and peripheral arterial disease.32 increased rdw was also associated with increased mortality in diabetic patients with coronary artery disease treated with percutaneous coronary intervention.33 we could only establish a statistically significant correlation between rdw and blood pressure, not between rdw and fasting blood sugar. we appreciate the efforts of mr isa usman who assisted in blood collection from the participants, and mr phillip o oluwamuhuru who carried out the full blood count on the samples.

high red blood cell distribution width (rdw) is related to impairment of erythropoiesis, reflecting chronic inflammation and increased levels of oxidative stress, both of which are telltale signs of type 2 diabetics. the aim of this study was to evaluate the relationship between the rdw and fasting blood sugar / blood pressure, and compare the from diabetics with nondiabetic controls.methodsthis was an unmatched case - control study involving 200 participants consisting of 100 diabetics and 100 nondiabetic controls. blood (4.5 ml) was collected from all of the diabetics and nondiabetic controls, and placed into edta anticoagulant tubes. a full blood count was performed using the sysmex kx-21n, a three - part auto analyzer able to run 19 parameters per sample, including rdw. blood pressure was measured during sample collection and in a sitting position.the mean fasting blood sugar level was 95.2030.10 mg / dl in the controls, and 147.8572.54 mg / dl in the diabetics. the mean blood pressures for diabetics was 138/90 mmhg and for non - diabetics 120/80 mmhg. the mean rdw - sd (rdw standard deviation) was 46.444.64 fl in the controls, and 46.843.18 in the diabetics. the mean rdw - cv (rdw coefficient of variation) was 14.74%1.94% in controls, and 14.800.71 for diabetics. no statistically significant correlation was found between the rdw - sd and fasting blood sugar / blood pressure in the diabetics. a statistically significant positive correlation was found between the rdw - cv and blood pressure in the diabetics.a positive correlation between the rdw - cv and blood pressure was established in the diabetics in this study.

totally ten feet of eight patients with a congenital hallux varus deformity were surgically treated at our institute between 1993 and 2008 by the senior author. indications for surgery were to improve appearance and to enable the wearing of normal shoes.5 ) there were seven male and one female with a mean age at the time of surgery of 33 months (range, 7 to 103 months). mean duration of the follow - up was 5.9 years after index operation (range, 2.3 to 13.8 years). six of the patients had hallux varus as an isolated congenital anomaly, while two patients had other anomalies; one had a congenital anterolateral bowing of the tibia on the ipsilateral side, and the other patient had a congenital anterolateral bowing of the tibia and hypoplasia of the second finger on the ipsilateral side. a preoperative, simple radiograph was used to evaluate the angular deformity, the presence of supernumerary bones or toes and abnormal appearances of the first metatarsal or phalanges, including a leb. based on a preoperative radiograph and operative findings, pathological structures in patients reported to be associated with congenital hallux varus deformity by mcelvenny11 ) were described as follows: short, thick metatarsal, accessory bones or toes, varus deformity of one or more of the four metatarsals, and a firm fibrous band extending from the medial side of the great toe to the base of the first metatarsal. residual varus deformity or recurrence was evaluated by postoperative, simple radiographs and clinical photographs in all patients at follow - up. clinical outcomes were assessed according to the criteria of phelps and grogan.7 ) pain, calluses, residual deformities and scar cosmesis were examined in all patients and the final were graded as excellent, good or poor. the final was rated as the most unsatisfactory grade out of the four categories. furthermore, the patients were asked if they experienced any difficulty in wearing shoes and if they felt that the cosmetic were satisfactory. all of the deformities in our patients were associated with one or more of the four components described by mcelvenny11 ) (table 1). five feet of three patients had been operated to remove polydactyly approximately 12 months after birth at another institute (patients 5, 7, and 8). all remaining five patients had preaxial polydactyly of incompletely duplicated phalanges (type 4 according to venn - watson classification7) ), although most of the phalanges were poorly formed with little bony contact and arose from the medial border of the foot at the level of the metatarsophalangeal joint.5 ) these were removed at surgery. a firm fibrous band that extended from the medial side of the great toe to the base of the first metatarsal was also common, present in nine of the ten feet. a combined leb was identified in four feet and involved the first metatarsal in three feet (right foot of patient 5 and both feet of patient 7) and the proximal phalanx of the great toe in one foot (patient 8). interestingly, varus deformities were still present even though all of them had a previous history of surgical removal of polydactyly. surgical procedures varied according to the severity of the deformity and the anatomic configuration of the first metatarsal or proximal phalanx (table 1). decisions regarding surgical options were individualized, mainly depending on the pathology and severity of the deformity as well as on the age of the patient. a soft tissue procedure, as described by farmer1 ) or mcelvenny,11 ) was in isolation performed for three feet (patients 1, 3, and 4) and in conjunction with osteotomy of the first metatarsal or proximal phalanx in four feet. specifically, a medial open - wedge osteotomy of the first metatarsal was performed for the three feet with a combined leb of the first metatarsal (right foot of patient 5 and both feet of patient 7), followed by an interposition of an appropriately sized allogenous strut graft (fig . an osteotomy was performed at the proximal phalanx for the foot with hallux varus combined with a leb of the proximal phalanx . the farmer technique was initially performed for patient 6 with a severe deformity, but the varus alignment was not sufficiently corrected . a lateral closing - wedge osteotomy of the first metatarsal was performed for further correction because the first metatarsal was not shortened . after the farmer technique was performed, the varus deformity was still present in patient 2 with an interphalangeal joint and a severe varus preoperative angulation of 60. further correction with a medial open - wedge osteotomy at the proximal phalanx was necessary to achieve a neutral alignment ( fig . the clinical were excellent in two feet, good in six and poor in two feet ( table 1). gross appearances were satisfactory compared with preoperative appearances, although none of the feet could be described as cosmetically normal.5 ) an unsatisfactory rating was generally related to scar cosmesis. one patient with poor (patient 5) who had been bilaterally treated was not satisfied due to scar cosmesis of both feet, even though he did not experience pain or footwear problems. the mean varus angulation of 29.1 at the metatarsophalangeal joint was improved to a mean angle of 4.0 of valgus at follow - up (p < 0.05) (table 1). overcorrection was seen in one patient, who had 30 of valgus angulation at follow - up, although he did not complain of pain or difficulties wearing footwear. one patient (patient 1) with a previous surgery using the farmer technique complained of a painful bump on the medial aspect of the great toe and persistent widening of the first web space at weight - bearing at year 6 postoperative. soft tissue procedures were performed to resect the medial skin bump and to narrow the first web space. the patients' outcome was rated as good at the final follow - up. the other patient (patient 3) experienced footwear problems due to shortening of the first ray with mild, residual varus after the farmer procedure. this patient required a medial open - wedge osteotomy of the first metatarsal and insertion of a strut allograft for further correction 7.5 years postoperative. all of the deformities in our patients were associated with one or more of the four components described by mcelvenny11 ) (table 1). five feet of three patients had been operated to remove polydactyly approximately 12 months after birth at another institute (patients 5, 7, and 8). all remaining five patients had preaxial polydactyly of incompletely duplicated phalanges (type 4 according to venn - watson classification7) ), although most of the phalanges were poorly formed with little bony contact and arose from the medial border of the foot at the level of the metatarsophalangeal joint.5 ) these were removed at surgery. a firm fibrous band that extended from the medial side of the great toe to the base of the first metatarsal was also common, present in nine of the ten feet. a combined leb was identified in four feet and involved the first metatarsal in three feet (right foot of patient 5 and both feet of patient 7) and the proximal phalanx of the great toe in one foot (patient 8). interestingly, varus deformities were still present even though all of them had a previous history of surgical removal of polydactyly. surgical procedures varied according to the severity of the deformity and the anatomic configuration of the first metatarsal or proximal phalanx (table 1). decisions regarding surgical options were individualized, mainly depending on the pathology and severity of the deformity as well as on the age of the patient. a soft tissue procedure, as described by farmer1 ) or mcelvenny,11 ) was in isolation performed for three feet (patients 1, 3, and 4) and in conjunction with osteotomy of the first metatarsal or proximal phalanx in four feet. specifically, a medial open - wedge osteotomy of the first metatarsal was performed for the three feet with a combined leb of the first metatarsal (right foot of patient 5 and both feet of patient 7), followed by an interposition of an appropriately sized allogenous strut graft (fig . an osteotomy was performed at the proximal phalanx for the foot with hallux varus combined with a leb of the proximal phalanx . the farmer technique was initially performed for patient 6 with a severe deformity, but the varus alignment was not sufficiently corrected . a lateral closing - wedge osteotomy of the first metatarsal was performed for further correction because the first metatarsal was not shortened . after the farmer technique was performed, the varus deformity was still present in patient 2 with an interphalangeal joint and a severe varus preoperative angulation of 60. further correction with a medial open - wedge osteotomy at the proximal phalanx was necessary to achieve a neutral alignment ( fig . the clinical were excellent in two feet, good in six and poor in two feet ( table 1). gross appearances were satisfactory compared with preoperative appearances, although none of the feet could be described as cosmetically normal.5 ) an unsatisfactory rating was generally related to scar cosmesis. one patient with poor (patient 5) who had been bilaterally treated was not satisfied due to scar cosmesis of both feet, even though he did not experience pain or footwear problems. the mean varus angulation of 29.1 at the metatarsophalangeal joint was improved to a mean angle of 4.0 of valgus at follow - up (p < 0.05) (table 1). overcorrection was seen in one patient, who had 30 of valgus angulation at follow - up, although he did not complain of pain or difficulties wearing footwear. one patient (patient 1) with a previous surgery using the farmer technique complained of a painful bump on the medial aspect of the great toe and persistent widening of the first web space at weight - bearing at year 6 postoperative. soft tissue procedures were performed to resect the medial skin bump and to narrow the first web space. the patients' outcome was rated as good at the final follow - up. the other patient (patient 3) experienced footwear problems due to shortening of the first ray with mild, residual varus after the farmer procedure. this patient required a medial open - wedge osteotomy of the first metatarsal and insertion of a strut allograft for further correction 7.5 years postoperative. in the current study, congenital hallux varus deformities were treated with various surgical techniques on a case - to - case basis depending on the severity and anatomic characteristics of the deformity. overall, these techniques yielded favorable in terms of pain, ability to wear footwear and recurrence of the deformity. several studies have reported surgical outcomes for congenital hallux varus (table 2).1,4,5,8,10,12 ) these studies evaluated patients with mixed diseases or were based on only a few case reports. no uniform method was used to assess clinical outcome and also various surgical procedures were used. this heterogeneity in previous studies, as well as in ours, is most likely not only due to the rarity of congenital hallux varus, but also to the lack of an established, clear definition of the deformity, its underlying pathomechanisms and the surgical treatment of choice for correction. general guidelines recommend that the issues to be considered for surgery are the correction of a polydactyly if present; correction of the soft tissue tether on the medial side of the foot and the enlarged web space between the great and second toe; correction of a metatarsal - phalangeal incongruity and the correction of a metatarsal or bracket epiphyseal deformity.18 ) soft tissue procedure by mcelvenny11 ) or farmer1 ) is recommended if the first metatarsal is normal. we followed this guideline in our series and decisions regarding surgical options were based on the pathology and severity of deformity and were individualized for each patient. mills and menelaus5 ) reported surgical outcomes of 20 feet of 12 patients followed - up for an average of 12.7 years. surgical procedures varied, including mcelvenny technique for nine feet, farmer technique for four, metatarsal osteotomy for two and arthrodesis of the first metatarsophalangeal osteotomy for one foot. the of soft tissue procedures, such as mcelvenny or farmer technique, and those of arthrodesis were satisfactory, but the metatarsal osteotomy produced unsatisfactory . they reported that metatarsal osteotomy could cause footwear problems due to varus deformity of the second and third toes or shortening of the first ray. even amputations of digits were needed to improve the symptoms in their series.5 ) in the current study, an osteotomy of the first metatarsal or proximal phalanx was more frequently performed than in that of mills and menelaus5 ) and good to excellent were yielded in five of seven feet. specifically, a soft tissue procedure alone was performed in three feet, an osteotomy alone in three and a combined soft tissue procedure and osteotomy in four feet (table 1). as , two of three feet with soft tissue procedure alone had revision surgeries (patients 1 and 3) and two of three feet with osteotomy alone had poor clinical outcomes because of unsatisfactory cosmesis (bilateral in patient 5). on the other hand, all of four feet with combined procedures had excellent or good without any complication or subsequent surgery. our study demonstrated that an osteotomy in conjunction with adequate soft tissue procedure would be a reliable option for the correction of congenital hallux varus. in this series, a soft tissue procedure alone did not provide satisfactory correction and further correction was deemed necessary in case of a combined bony deformity of the metatarsal. our technique, consisting of farmer technique and opening wedge osteotomy, provided satisfactory correction and good clinical . we also postulated that an osteotomy plays some role in congenital hallux varus surgery, like described as follows: first, an osteotomy can be used to further correct any residual deformity after the soft tissue procedure. it is possible that varus angulation could remain at the first metatarsophalangeal joint or interphalangeal joint due to the severity and complexity of the deformity, and widening of the first web space could still be present after a soft tissue procedure alone. widening of the forefoot should be corrected because it can in difficulties wearing footwear or painful calluses. a lateral closing osteotomy or medial open wedge osteotomy of the first metatarsal can reduce the first and second intermetatarsal angles and cause relief of symptoms. an osteotomy can not only reduce varus angulation, but can also narrow the gap between the first and second toes. second, a short first metatarsal can be corrected by interposition of a bone graft after osteotomy. uncorrected shortening of the first metatarsal can cause footwear problems and residual deformities.5 ) graft positioning following a medial open - wedge osteotomy may be used for lengthening as well as to reduce varus angulation.8 ) leb involving the first metatarsal is a common cause of hallux varus.2,6,13,15,16 ) in the current study, it was clear that congenital hallux varus in four feet ed from a leb of the first metatarsal or proximal phalanx. all of these feet had a residual hallux varus even after previous removal of polydactyly approximately 12 months after birth.13,14,15,19 ) therefore a leb could not have been considered at the initial surgery until two years of age as it is the time point where the ossification of the proximal and distal ossification centers occurs and it can not be detected on radiographs before.13,14,15,19 ) a longitudinal growth of the digit is impossible because the longitudinal bony bracket can not elongate sufficiently if a leb is not treatedand the growth occurs in a c - shape along the shortened side of the bone. bracketing along the medial shaft of the bone probably led to varus deviation with growth in these patients. sobel et al.13 ) emphasized that a leb should not be overlooked when treating pediatric deformities, including congenital hallux varus. we agree that a bracket epiphysis should be suspected if a bone appears short or wide on radiographs in conjunction with one of the known associated anomalies or syndrome.13,19 ) diagnostic modalities, including magnetic resonance imaging or ultrasonography, have been shown to be effective in children less than two years of age before the occurrence of ossification of the bracket.14,19 ) numerous surgical procedures to treat a metatarsal leb have been described; these include bracket chondro - osteotomy accompanied by fat interposition, resection of the leb with silicone polymer or polymethylmethacrylate interposition, simple bracket excision before ossification, distraction osteogenesis and metatarsal osteotomy after complete closure of the leb.6,15,16,17,20 ) the overall goal of any of these procedure is to eliminate the tethering effect of the growth plate by removing the bar and therefore to promote growth in a lengthwise fashion.6,19 ) we performed a medial open - wedge osteotomy at the diaphysis of the first metatarsal with interposition of an allogenous strut graft to resect the abnormal longitudinal section of the epiphysis, to correct varus angulation and to lengthen the short first metatarsal. three of four feet with a leb were sufficiently corrected and showed favorable clinical outcomes using this method. however, in patient 5 (poor ), the varus angulation of the first metatarsal shaft still remained and the longitudinal growth of the first metatarsal was not sufficient at the final follow - up, although the hallux varus did not have a gross physical appearance. the radiographic of this patient were inferior to those reported by mubarak et al.21 ); these authors demonstrated successful longitudinal growth and a normally - shaped metatarsal after central physiolysis for metatarsal leb. although the small size of our series precludes a definitive on the optimal treatment strategy, based on our findings we only regard an osteotomy in conjunction with soft tissue reconstruction as being of importance. furthermore, patient's age is another factor that must be assessed when considering surgery. as shown in our series, a leb is one of the causes of the deformity and should be taken into consideration at the initial evaluation. resection of a bracket epiphysis should be performed as with other procedures to prevent recurrent deformity. we also recommend further evaluation with magnetic resonance imaging or ultrasonography before surgery if the presence of a leb is in doubt, especially for patients aged two years or less.

the purpose of this study was to report outcomes of congenital hallux varus deformity after surgical treatment.methodswe evaluated ten feet of eight patients with a congenital hallux varus deformity, including four feet combined with a longitudinal epiphyseal bracket (leb). there were seven male patients and one female patient with a mean age of 33 months (range, 7 to 103 months) at the time of surgery. two patients were bilaterally involved. the mean duration of follow - up was 5.9 years (range, 2.3 to 13.8 years). clinical outcomes were assessed according to the criteria of phelps and grogan. surgical procedures included the farmer procedure, the mcelvenny procedure or an osteotomy at the first metatarsal or proximal phalanx.the clinical were excellent in two feet, good in six and poor in two feet. the leb was associated with hallux varus in four feet and were treated by osteotomy alone or in conjunction with soft tissue procedure.congenital hallux varus was successfully corrected by surgery with overall favorable outcome. preoperatively, a leb should be considered as a possible cause of the deformity in order to prevent recurrent or residual varus after surgery.

over the last hundred years, neuroscience has approved a presumption that the process of neurogenesis is not present in the structures of the adult brain. beginning with the pioneer studies by santiago ramon y cajal and camillo golgi , it was commonly thought that neural cells forming mature cns are definitively postmitotic, do not arise de novo, and that plasticity adaptation mechanisms are based only on the origin of projections and synaptic connections between neurons formed in early brain development stages (de castro et al . mitotic divisions of ependymal cells located along lateral ventricles of mature rat brain were found for the first time by ezra allen already in 1912 . however, only after recognition of molecular mechanisms of dna synthesis, it was possible to unequivocally identify undifferentiated, proliferatively active neural stem cells ( nscs) in mature brain (allen 1912 ; balu and lucki 2009). it was achieved in 1965 by joseph altman group (altman and das 1965) using tritium labeled thymidine to detect replicating dna of precursor cells. this discovery opened the brand new, promising, and intriguing chapter of contemporary neurobiology. nevertheless, the significance of the adult neurogenesis is still far from being explained. a number of recent studies unequivocally reveal the role of this phenomenon in the processes of cns plasticity, learning, and memory (deng et al . the problem of relations between adult neurogenesis and pathogenesis or / and course of psychiatric diseases like bipolar disorder ( walton et al . 2012 ; braun and jessberger 2014), schizophrenia (reif et al . 2007 ; eisch et al . 2008 ; christian et al . 2010 ; decarolis and eisch 2010 ; de koning et al . 2012), and particularly depression (krishnan and nestler 2008 ; lucassen et al . 2010 ; mahar et al . experiments on animal models have proven that elevated levels of stress hormones, observed in depression, often correlated with neurogenesis process reduction . numerous antidepressants, commonly used in the therapy of patients suffering from depression, have strongly stimulating influence on new neurons formation in particular brain structures ( santarelli et al . 2003 ; boldrini et al . 2009). however, there are some doubts and assumptions stating that attenuation of neurogenesis is not a cause of depression, but it is rather one of its functional outcomes (airan et al . for quite a long - time postulated association between disturbances in formation, proliferation, and differentiation of nscs and pathogenesis of neurological diseases, alzheimer s ( veereraghavalu et al . 2009 ; rodriguez et al . 2008) and parkinson s (bertilsson et al . 2008 it is suggested that decrease of self - renewal rate of certain neural cells populations, e.g., in substantia nigra, hippocampus, or some neocortex areas ( bonfanti and peretto 2011), could constitute one of the causes of mentioned diseases. based on this assumption, intensive search for the substance selectively stimulating neurogenesis in cns is currently in progress. relatively promising and quite well - studied aminopropyl carbasole and its newly synthesized derivatives (pieper et al . 2013) might become potential, but still hypothetical drugs being able to improve clinical state of patients with neurodegenerative disorders. a decrease in proliferation rate and survival of mature brain neural progenitors is probably related to chronic stress (mirescu and gould 2006) and long - lasting insomnia (meerlo et al . on the other hand, some data suggest cautiously that among consequences of ischemic stroke and traumatic brain injury, there is also stimulation of adult neurogenesis as one of potential brain repair mechanisms ( kernie and parent 2010 ; bellenchi et al . up till now, two distinctive regions of active and constant neurogenesis process in adult mammalian cns have been identified : subgranular zone ( sgz) in dentate gyrus structure and subventricular zone (svz) located subependymally in the vicinity of brain lateral ventricles (fig . 1). sgz stem cells are the source of dentate gyrus granular cells, whereas svz forms progenitor cells migrating as rostral migratory stream (rms) to olfactory bulb, where they differentiate into interneurons suitable for this brain area. the neural precursor cells (npcs) must be located in neurogenic niche, a permissive milieu, which maintain their constant ability to divide and form mature neurons even in the adult brain. the hippocampal sgz niche is created mainly by astrocytes and blood vessel endothelium, numerously represented in this layer of dentate gyrus (rolando and taylor 2014). in sgz cytoarchitectonics three types of proliferatively active cells have been identified: radial glia - like stem cells, type i cells; non - ciliated cells with nestin expression, type ii cells also described as transiently activated progenitor cells, tap cells; and neuroblasts with doublecortin protein (dcx) and ki67 expression. on the other hand, in svz structure, four mitotically active and self - renewing cell populations have been distinguished: ependymocytes with cd133 expression; gfap - positive astrocytic stem cells, type b1 cells; transiently activated progenitor (tap) cells with mash1 expression, type c cells; and dcx protein expressing neuroblasts, type a cells (okano and sawamoto 2008).fig. 1the canonical sites of adult neurogenesis in the rat brain and the scheme of subventricular zone (svz) stem cell niche. a subgranular zone of dentate gyrus (sgz, dark blue), subventricular zone (svz, red) surrounding the lateral ventricle (lv, black), red arrow indicates direction of neuron migration from svz to olfactory bulb (rostral migratory stream, rms). b cellular composition of svz niche sectioned vertically to ependymal surface; e1 cells (yellow) and e2 cells (red) concentrically surround centrally located cilia of b1 cells (dark blue) forming the pinwheel structure. neuroblasts a cells (gray) and transiently activated stem cells c (green) are also presented. c horizontal view of the mentioned svz niche (color figure online) the canonical sites of adult neurogenesis in the rat brain and the scheme of subventricular zone (svz) stem cell niche. a subgranular zone of dentate gyrus (sgz, dark blue), subventricular zone (svz, red) surrounding the lateral ventricle (lv, black), red arrow indicates direction of neuron migration from svz to olfactory bulb (rostral migratory stream, rms). b cellular composition of svz niche sectioned vertically to ependymal surface; e1 cells (yellow) and e2 cells (red) concentrically surround centrally located cilia of b1 cells (dark blue) forming the pinwheel structure. neuroblasts a cells (gray) and transiently activated stem cells c (green) are also presented. c horizontal view of the mentioned svz niche (color figure online) ependymocytes play a fundamental role in svz niche structure formation. here , three cell subpopulations have been distinguished: previously mentioned cd133 cells functioning as nscs as well as e1 and e2 ependymal cells with cd24 expression. multiciliated e1 cells are dominating, whereas e2 ependymocytes make up less than 5 % of ependymal cell population and possess only two cilia with particularly big basal bodies. studies of rat brain svz niche cytoarchitecture have revealed unusually peculiar spatial pattern of stem cells and ependymocytes. central part of the niche is formed by gfap - positive b1 stem cells surrounded in their apical, ciliated part by several e1 and e2 ependymocytes forming a unique rosette or pinwheel - like structure (mirzadeh et al . on the other hand, central and basal part of b1 cells remain in contact with concentrically located c and a cells . b1 cells have long cytoplasmatic projections reaching the surface of blood capillaries ( fig . 1). extracellular matrix, in the form of three - dimensional spatial structures called fractones, plays also an important role in the niche functioning. in human and rodent brain, fractones are characterized by collagen iv, 1 and 1 laminin, perlecan and nidogen expression, and lacking laminin 2 (kerever et al . limited fractone subpopulation surrounding highly mitotically active cells additionally demonstrates immunoreactivity toward n - sulfate heparan sulfate proteoglycan ( n - sulfate hspg). fractones and subependymal capillaries are probably capable of binding fibroblast growth factor 2 (fgf-2), which substantially promotes nscs proliferation (kerever et al . matrix metaloproteinases mmp-1 and mmp9are also considered to play a significant role in internal niche environment modification and nscs fate determination ( tonti et al . the origin of neurons in the sgz and svz is probably strictly regulated by various growth factors ( duan et al . 2008 ; balu and lucki 2009 ; llorens - martn et al . 2010), classical neurotransmitters (petrus et al . 2009 ; okeeffe et al . 2009 ; yanpallewar et al . 2010), neuropeptides (howell et al . 2005 ; garza et al . 2008 ; moon et al . 2009 ; manda and reiter 2010), and cytoskeleton proteins (jiao et al . 2010). a large spectrum of commonly administered pharmaceuticals such as selective serotonin reuptake inhibitors (encinas et al . 2009), antipsychotics (wang et al . 2004 ; keilhoff et al . 2010 2013), hypnotic and normothymic drugs (yu et al . 2009 ; boku et al . 2010), lithium ions (wexler et al . 2008) and even steroid hormones (brummelte and galea 2010), and cyclooxygenase-2 inhibitors (goncalves et al . 2010) can also significantly affect the course of adult neurogenesis. there are numerous evidences, that new neural cells can also be formed, usually in limited number, outside sgz and svz, in potential niches located in distinct structures of mature brain. 2002; luzzati et al. 2003 ), striatum (emsley et al . 2005 ; bdard et al . multipotent neural precursors have been also isolated from certain neocortical areas ( dayer et al . currently, there is not enough evidence proving that these putative, non - classical neurogenic sites are stable in time and space, and that the continuous origin of new neurons is really taking place . therefore, the presence of established and permanently functional nscs niches in mentioned brain areas is still very controversial . however, hypothesis suggesting existence of a stable hypothalamic neurogenic site located in subependymal zone of the third ventricle ( hypothalamic ventricular zone, hvz) seems to be particularly intriguing and relatively well documented (kokoeva et al . 2005 ; more importantly, it is also suggested that neurogenesis process is significantly involved in various hypothalamic regulatory mechanisms, especially in the energy balance regulation ( pierce and xu 2010). the key role of ependymocytes lining lateral ventricles has become a source of presumptions that these cells could also take part in hypothalamic nscs niche formation, located in the vicinity of the third ventricle. studies held on rodents have revealed that proliferatively active cells are present not only in ependyma, but also in surrounding neuropilus (kokoeva et al . 2007 ; migaud et al . 2010). to date, two adult hypothalamic neurogenesis regions have been distinguished: located in lateral walls of the third ventricle at the level of paraventricular and arcuate nuclei (hypothalamic ventricular zone, hvz) and hypothalamic proliferating zone (hpz) formed by tanycytes located at the bottom of the third ventricle in median eminence region (fig . 2). tanycytes that form rat and human hvz are characterized by expression of proteins typical for neural precursor cells e.g., nestin (wei et al . 2002), vimentin (bolborea and dale 2013), and doublecortin - like protein (dcl) (saaltink et al . 2012). they are also enriched for neural stem and progenitor genes such as sox9, notch 1 and 2, he s 1 and 5, cd63, fzd5, dirc, ntrk-2t1, and thrsp (rodrguez et al . 2005 ; shimogori et al . recently, the expression of sox2, a selective marker of nscs, was also reported ( lee et al . 2012 ; li et al . 2012). four types of radial glia - like tanycytes have been identified, differing from each other in gene profile and location in the third ventricle wall. tanycytes 1 are present at the level of ventromedial nuclei, whereas 2 subpopulation in the vicinity of arcuate nuclei. both cell types send their long processes toward blood vessels, hypothalamic neurons, and glial cells. elongated 1 tanycytes form the lateral part of infundibular recess, while 2 cells line the floor of third ventricle inside the median eminence forming the hpz. the basal processes of 1 tanycytes are in contact with endothelial cells and terminals of gnrh - expressing neurons, whereas processes of 2 cells head toward blood vessels of hypothalamo - pituitary portal system and also pia mater (lee and blackshaw 2012) (fig . 2). noteworthy, the 2 tanycytes show particularly high level of aforementioned hes1 and hes2 marker expression (lee et al . some authors report the presence of the following zones of the third ventricle wall : ventrally located tanycytic zone composed of both and 2 tanycytes, transition zone with 1 tanycytes, and the most dorsal ependymal zone which contains only ependymal cells ( mathew 2008). most recently, two kinds of tanycytes have been found, dorsally located fgf10, blbp, gfap+, glast+, s100+ subtype and ventral fgf10 +, blbp+, gfap, glast subtype (haan et al . 2013). probably, the fgf10 + tanycytes are the neural stem / progenitor cells and can continuously proliferate and form parenchymal neurons and glial cells. importantly, tanycytes located on the median eminence are sensitive to the hormones and nutritional substances carried by the blood flowing in capillary vessels. in turn, the somata of tanycytes lining the third ventricle are able to receive the molecular signals e.g., growth factors from the cerebrospinal fluid.fig. 2schematic representation of the subependymal npcs niche in the animal hypothalamus. the neurogenic zones are located in the vicinity of the third ventricle at the level of paraventricular, ventromedial, and arcuate nuclei (hypothalamic ventricular zone, hvz), while tanycytes of the median eminence form the hypothalamic proliferating zone (hpz). ciliated and non - ciliated ependymocytes line the third ventricle as well as four types of tanycytes. various populations of glial cells (fibrous astrocytes, subependymal astrocytes, and oligodendrocyte precursor cells) and neural cells (peptidergic neurons, parenchymal proliferative cells, gnrh - releasing neurons, and doublecortin - expressing immature neurons) are located beneath the ependymal layer forming the npcs niche. neuropeptides and other regulatory factors are released from the capillary network schematic representation of the subependymal npcs niche in the animal hypothalamus. the neurogenic zones are located in the vicinity of the third ventricle at the level of paraventricular, ventromedial, and arcuate nuclei (hypothalamic ventricular zone, hvz), while tanycytes of the median eminence form the hypothalamic proliferating zone (hpz). ciliated and non - ciliated ependymocytes line the third ventricle as well as four types of tanycytes. various populations of glial cells (fibrous astrocytes, subependymal astrocytes, and oligodendrocyte precursor cells) and neural cells (peptidergic neurons, parenchymal proliferative cells, gnrh - releasing neurons, and doublecortin - expressing immature neurons) are located beneath the ependymal layer forming the npcs niche. neuropeptides and other regulatory factors are released from the capillary network the identity of hypothalamic neural progenitor cells (npcs) remains so far very controversial. some studies suggest that -tanycytes are the main proliferating cells in the hypothalamus of young adult mice (haan et al . they are the neural progenitors in the median eminence but not in other hypothalamic regions . in rats, the rate of neurogenesis within hvz and median eminence is significantly higher in the first weeks of postnatal life than in adult animals ( lee et al . 2012). on the other hand, a recent report identifies the lateral -tanycytes as local npcs and crucial cells in the mouse hypothalamic niche. the long - term study using a lineage tracing in vivo shows that -tanycytes form a self - renewing population that differentiate to new tanycytes, astrocytes, and neurons. moreover, this finding demonstrates clearly that only gfap - positive dorsal -tanycytes, but not -tanycytes or parenchymal cells may form neurospheres (robins et al . 2013). probably, the fibroblast growth factor (fgf) signaling is required to maintain -tanycyte proliferation, and increased fgf level enhances the rate of mitotic divisions. interestingly enough, the hypothalamic tanycytes are characterized by selective expression of transcription factor called retina and anterior pituitary neural fold homeobox (rax). this molecule shows a similar pattern of expression to retinoic acid receptor responder (rarres-2), a marker of third ventricle ependymal cells. the rax action is considered as required for tanycyte and ependymocyte differentiation at the level of hypothalamus (miranda - angulo et al . the regulatory mechanism by which rax determines the fate of tanycytes is still unknown ; however, some recent studies suggest the involvement of six6 and hmgb2, the factors selectively expressed in hypothalamic progenitor cells ( andreazzoli et al . acetylcholine, histamine, and atp sensitive tanycytes can activate intracellular calcium signaling regulatory pathway ( frayling et al . it should be noted that atp stimulates ca waves in the subventricular progenitor cells acting via p2y1 receptor ( weissman et al . tanycytes as the radial glial cells can release atp to control neural progenitor cell proliferation, and they also have the aforementioned type of purynergic receptors . moreover, tanycytes synthesize ectonucleoside triphosphate diphosphohydrolase 2 ( ntpdase 2) that inactivate the extracellular atp (braun et al . although tanycytes appear to be the key players in the npcs niche formation, they are not only nscs in the hypothalamus . a population of sox2 expressing cells scattered within the parenchymal regions also has a proliferative activity ( kokoeva et al . undoubtedly, advanced genetic tools such as using of inducible cre lines e.g., hgfapcreer, pdgfr-crert, or glast - creer should be useful to reveal whether these brdu - positive cells can form a quiescent hypothalamic neural progenitor pool ( ganat et al . the proliferating gfap - positive subependymal astrocytes have an apical process with single cilium and their periventricular basement membranes form a three - dimensional network that is typical for svz microachitecture ( prez - martin et al . numerous granular dcx - positive immature neuroblasts were identified close to the wall of the third ventricle ( fig . 3). on the other hand, the bipolar dcx cells found in the parenchyma of ventromedial hypothalamus have fusiform perikarya and sometimes elongated processes displaying the features of migrating cells that may undergo maturation within this region (batailler et al . , the neuroblasts located inside the subependymal niche can form a kind of migratory stream spreading to the hypothalamic nuclei ( haan et al . 2013).fig. 3hypothalamic neurogenic zones in the rat brain fluorescence (20-m - thick frozen sections) and classical immunostaining (7-m paraffin sections) of the adult (3-month - old) rat hypothalamus. primary antibodies dilutions: dcx 1:100 (santa cruz biotechnology), nestin 1:500 (millipore), and ki67 1:100 (abcam). brains were fixed with 4 % buffered formalin, dehydrated with ethanol or sucrose solutions, and then embedded on paraffin or mounted in oct medium. the photomicrograph shows the doublecortin (dcx) expressing cells in the parenchyma of paraventricular nucleus (a, b, a single cell in higher magnification nestin expressing cells : tanycytes at the level of ventral hypothalamus, arcuate nuclei and within the median eminence ( d, zone of tanycytes inset d1, 1 tanycytes, h), and ependymocytes in the dorsal hvz (e g). scale bars 50 m, excluding c, d, i (100 m), and a1, b1 (10 m). arc arcuate nucleus, me median eminence, pa paraventricular nucleus, vmh ventromedial hypothalamus, 3v third ventricle hypothalamic neurogenic zones in the rat brain fluorescence (20-m - thick frozen sections) and classical immunostaining (7-m paraffin sections) of the adult (3-month - old) rat hypothalamus. primary antibodies dilutions: dcx 1:100 (santa cruz biotechnology), nestin 1:500 (millipore), and ki67 1:100 (abcam). brains were fixed with 4 % buffered formalin, dehydrated with ethanol or sucrose solutions, and then embedded on paraffin or mounted in oct medium. the photomicrograph shows the doublecortin (dcx) expressing cells in the parenchyma of paraventricular nucleus (a, b, a single cell in higher magnification nestin expressing cells : tanycytes at the level of ventral hypothalamus, arcuate nuclei and within the median eminence ( d, zone of tanycytes inset d1, 1 tanycytes, h), and ependymocytes in the dorsal hvz (e g). scale bars 50 m, excluding c, d, i (100 m), and a1, b1 (10 m). arc arcuate nucleus, me median eminence, pa paraventricular nucleus, vmh ventromedial hypothalamus, 3v third ventricle a large spectrum of endogenous regulatory substances, especially growth factors, is able to regulate the course of adult hypothalamic neurogenesis. the brain - derived growth factor (bdnf), insulin - like growth factor 1 (igf-1), fibroblast growth factor (fgf), epidermal growth factor (egf), and ciliary neurotrophic factor (cntf) seem to play a key role as the activators of neuronal progenitor divisions. a recent report suggests that also gonadotropin - releasing hormone (gnrh) can increase the proliferating activity of hypothalamic npcs in aged mice (zhang et al . it should be emphasized that the aforementioned growth factors promote neurogenesis in different hypothalamic centers in a distinctly selective manner ( sousa - ferreira et al . thus, bdnf administration increases cell proliferation in the paraventricular nucleus, both fgf and cntf induce neurogenesis in the parenchymal zone of the arcuate nucleus, while igf-1 in the medial periventricular region ( pencea et al . injection of fgf and egf promotes cell proliferation in the ependymal layer, whereas extended subcutaneous injection increases neurogenesis in the arcuate nucleus ( xu et al . as hypothalamus is responsible for the constant control of many autonomous functions of the organism, it was considered that neurogenesis in this region may support some of these processes or act as a compensatory mechanism in response to different environmental signals . indeed, in the light of latest discoveries, it is possible that neurogenesis in hypothalamus is potentially involved in regulation of energy homeostasis via modulation of eating behavior . neuronal populations located within distinct regions of the hypothalamus express various neuropeptides modulating food intake . in the arcuate nucleus ( arc), there is an expression of orexigenic factors like agouti - related peptide (agrp), neuropeptide y (npy), as well as anorexigenic factors like pro - opiomelanocortin (pomc) and its derivative-melanocyte - stimulating hormone (-msh). on the other hand, , there is a dynamic balance between regulatory neuropeptides activities, but diet - related factors like obesity and high saturated fatty acid (sfa) intake cause hypothalamic neurogenesis inhibition and simultaneous defective energy balance. it suggests a link between eating behavior and intensity of hypothalamic neurogenesis (fig . the limited neuronal loss, homeostasis, and some growth factors promote the npcs proliferation, while severe cellular loss, obesity, and saturated fatty acids ( sfa) inhibit the neurogenesis. arc arcuate nucleus, pvn paraventricular nucleus, lha lateral hypothalamus, pomc pro - opiomelanocortin, npy neuropeptide y, agrp agouti - related protein, crh corticotrophin - releasing hormone, trh thyrotropin - releasing hormone, mch melanin - concentrating hormone, bdnf brain - derived neurotrophic factor, cntf ciliary neurotrophic factor, igf-1 insulin - like growth factor 1, fgf fibroblast growth factor, egf epidermal growth factor hypothalamic neurogenesis plays a role in the regulation of energy balance. the limited neuronal loss, homeostasis, and some growth factors promote the npcs proliferation, while severe cellular loss, obesity, and saturated fatty acids (sfa) inhibit the neurogenesis. arc arcuate nucleus, pvn paraventricular nucleus, lha lateral hypothalamus, pomc pro - opiomelanocortin, npy neuropeptide y, agrp agouti - related protein, crh corticotrophin - releasing hormone, trh thyrotropin - releasing hormone, mch melanin - concentrating hormone, bdnf brain - derived neurotrophic factor, cntf ciliary neurotrophic factor, igf-1 insulin - like growth factor 1, fgf fibroblast growth factor, egf epidermal growth factor it was also shown that newborn neural cells in the hypothalamus are functional they express orexigenic peptides (agrp, npy)and anorexigenic peptide (pomc) (kokoeva et al . 2005 ; pierce and xu 2010). simultaneously, some of them have ability to respond to leptin administration by inducing strong phospho - signaling transducer and activator of transcription 3 (pstat3) immunoreactivity (kokoeva et al . 2005). interesting data concerning significance of hypothalamic neurogenesis have been provided with the use of antimitotic agent arabinosylcytosine (arac) or focal irradiation procedure both applied in order to silence neurogenesis process. in wild - type mice, however, this effect is not observed in mutant mice with degenerated agrp - positive cells. as such genetic manipulation causes also the increase in cell proliferation rate in arcuate nucleus of the hypothalamus and because inhibition of this proliferation (by arac) in decreased feeding and weight loss, it is suggested that hypothalamic neurogenesis plays a compensatory role in preventing anorectic effects of neurodegeneration (pierce and xu 2010). in another study, mice were kept on high - fat diet (hfd) and infused with ctnf to induce neurogenesis within hypothalamic energy balance centers (kokoeva et al . ctnf treatment ed in weight loss, but the long - term effect was present only in mice with undistrupted neurogenesis process . if arac was administered, then mice gained weight 20 days after ctnf treatment, which indicates that neurogenesis in hypothalamus is crucial for maintaining ctnf - mediated metabolism regulation ( kokoeva et al . later, it was found that hfd alone enhances neurogenesis in the median eminence ( me) of mouse hypothalamus. blocking of this process by focal irradiation leads to attenuation of weight gain and increase in animal activity levels, which proves that neurogenesis in me may promote fat storage and body mass increase (lee et al . 2012 ; lee and blackshaw 2012). on the other hand, studies on rodent obesity models revealed the significant decrease in hypothalamic neurogenesis process rate in arcuate nucleus of obese mice. it further suggests that regulation of energetic homeostasis can be mediated by hypothalamic neurogenesis (mcnay et al . are considered as new neural cells precursors, detailed studies have been performed to characterize these cells and trace the fate of their progeny . experiments held on mice confirmed that tanycytes positive for fibroblast growth factor 10 ( fgf10) give rise to cells that are located mainly in arcuate nucleus. some of them expressed npy and were sensitive to fasting alone or followed by leptin administration, which was proven by c - fos and pstat immunostaining. moreover, newly formed neurons were able to significantly expand their axonal / dendritic arborization (haan et al . tanycytes are also known to respond to glucose by releasing atp ca waves, especially if it is applied selectively to cell bodies ( frayling et al . another interesting feature of tanycytes, suggesting their contribution to energy metabolism, is expression of orphan receptor gpr50member of melatonin receptor family ( sidibe et al . gpr50 is functionally linked to energy homeostasis, which was proven by observation of knock - out ( gpr50) mice having more stable body weight they were more resistant to obesity induced by high - energy diet and to fasting - induced weight loss (ivanova et al . 2007 ; bolborea and dale 2013). another intriguing point of view, from which adult hypothalamic neurogenesis can be analyzed, is the relationship between this process, obesity, and macronutrients dietary content. diet rich in saturated fatty acids (sfa) not only induced obesity and pre - diabetes in mouse model, but also caused reduction in the number of nscs and their proliferation potential, which was associated with inhibitor kappa/nuclear factor kappab (ikk/nfb) activation (li et al . 2012 ; yon et al . different effects were observed when rats were fed with sfa - rich diet during pregnancy . ing offspring showed higher proliferation rate of npcs together with the increase in blood circulating lipids and orexigenic peptides expression ( chang et al . 2008 ; yon et al . 2013). additionally, some researchers linked neurogenesis process with endocannabinoid system. treatment with the cannabinoid-1 receptor inverse agonist (am251) reduced food intake and modulated neurogenesis in rats, but the neurogenic change was present only in hfd - fed animals there was a reduction of neurogenesis levels in svz and hypothalamus, but induction in sgz. it suggests that hfd sensitizes the endocannabinoid system to modulate neurogenesis (rivera et al . apart from well - documented contribution of neurogenesis in hypothalamus to energy balance regulation, some other potential functions of newly formed neural cells have been reported ( yuan and arias - carrion 2011). it is supposed that social environment especially pheromones influences hypothalamic neurogenesis in adult female prairie voles. two - day - long male exposure increased the number of new formed cells in amygdala and hypothalamus in comparison to condition of female exposure and social isolation (fowler et al . 2002). moreover, immunohistochemical staining of female pig hypothalamus have revealed that new oxytocin - containing neuron formation rate in paraventricular nucleus (pvn) is greater in lactating sow and adult gilts in comparison to puberty ones (raymond et al . formation rate of new neurons expressing vasopressin in vasopressin and oxytocin - containing nucleus ( von) was positively correlated with the growth of this brain region more newly formed vasopressin positive neurons were shown in adult pigs, than in adolescent individuals (rankin et al . it may support the hypothesis that adult hypothalamic neurogenesis is involved in sexual maturation process and can be sensitive to environmental conditions . the pubertal increase in anteroventral periventricular nucleus ( avpv) volume seems to be connected with the hormone - dependent formation of new cells (juraska et al . , brdu was administered to rats that were at different stages of puberty, and the labeled cell was quantified 3 weeks later. a number of avpv cells that had been born during puberty had transformed into mature neurons. interestingly, more brdu cells were found in the female than in the male avpv. unexpectedly enough, brdu - labeled cells were also found in the sexually dimorphic nucleus of the preoptic area (sdn), with greater numbers of pubertally born cells in males than in females (ahmed et al . however, these brdu - positive cells coexpressed neither neural nor glial markers, so their phenotype remains unclear . possibly, they require more than 34 weeks to become the fully differentiated neurons or astrocytes, as suggested for cortical interneurons ( cameron and dayer 2008 ; juraska et al . recently, accumulating reports seem to confirm the real existence of adult hypothalamic neurogenesis in the animal brain and highlight its potential role in the energy balance regulation . the cellular architecture of hypothalamic subependymal neurogenic niche is quite well described ; however, many crucial questions remain so far unanswered . first of all, the presence of npcs in the human hypothalamus is not yet reliably proven . moreover, we also do not know whether hypothalamic neurogenesis can contribute to energy homeostasis in humans . both eating disorders and obesity pathogenesis are strictly related to the disturbances at the level of hypothalamic neuronal populations . the blockage of hypothalamic neurogenesis may alter the body weight in rodents supporting the hypothesis that local npcs play role in the control of energy expenditure . having in mind that hypothalamic neurogenesis may be regulated by various factors, an intriguing idea of potential pharmacomodulation of this process should not be excluded . it would be interesting to study the influence of some drugs modulating appetite ( as their main or side effect) on hypothalamic neurogenesis process in order to better understand their mechanism of action. in the future , it could have some practical implication allowing modification and improvement of existing therapies interfering with organism energetic homeostasis for example, treatment of eating disorders and schizophrenia. taking to the account recent from experiments on pigs and prairie voles, it is also justified to perform research on hormonal regulation of hypothalamic neurogenesis and its impact on various physiological processes connected with hormonal activity. finally, promising future direction of research concerns hypothalamus - derived nscs cultures. it would contribute to detailed characterization of neural precursors and ing application of hypothalamic regenerative potential.

the discovery of undifferentiated, actively proliferating neural stem cells (nscs) in the mature brain opened a brand new chapter in the contemporary neuroscience. adult neurogenesis appears to occur in specific brain regions (including hypothalamus) throughout vertebrates life, being considered an important player in the processes of memory, learning, and neural plasticity. in the adult mammalian brain, nscs are located mainly in the subgranular zone (sgz) of the hippocampal dentate gyrus and in the subventricular zone (svz) of the lateral ventricle ependymal wall. besides these classical regions , hypothalamic neurogenesis occurring mainly along and beneath the third ventricle wall seems to be especially well documented. neurogenic zones in sgz, svz, and in the hypothalamus share some particular common features like similar cellular cytoarchitecture, vascularization pattern, and extracellular matrix properties. hypothalamic neurogenic niche is formed mainly by four special types of radial glia - like tanycytes. they are characterized by distinct expression of some neural progenitor and stem cell markers. moreover, there are numerous suggestions that newborn hypothalamic neurons have a significant ability to integrate into the local neural pathways and to play important physiological roles, especially in the energy balance regulation. newly formed neurons in the hypothalamus can synthesize and release food intake regulating neuropeptides and they are sensitive to the leptin. on the other hand , high - fat diet positively influences hypothalamic neurogenesis in rodents. the nature of this intriguing new site of adult neurogenesis is still so far poorly studied and requires further investigations.

the term scrub means the type of vegetation (terrain between woods and clearings) that harbors the vector and typhus means scrub typhus is endemic in so called tsutsugamushi triangle such as japan, taiwan, china, and south korea on the north, india and nepal on the west, and australia and indonesia in the south. a billion people are at risk, and nearly a million cases are reported every year. o. tsutsugamushi is an obligate intracellular gram - negative bacterium; it grows freely in the cytoplasm of infected cells since it lacks vacuolar membrane. it has 5 major serotypes - boryon, gilliam, karp, kato, and kawazaki. scrub typhus is one of the differential diagnosis for fever with thrombocytopenia or hemorrhagic fever. scrub typhus can manifest with either nonspecific febrile illness or constitutional symptoms (fever, rash, myalgia, and headache) or with organ dysfunction such as kidney (acute kidney injury), lungs (acute respiratory distress syndrome), heart (myocarditis), liver (hepatitis) and central nervous system (meningitis). the mortality of scrub typhus in untreated patients range from 0% to 30% and tends to vary with age and region of infection. the involvement of lungs has been described which range from bronchitis and interstitial pneumonitis to ards. ards is one of the serious complications of scrub typhus, which has high morbidity and mortality. the occurrence of ards is high in scrub typhus patients who were diagnosed late and received antibiotics late. this study may be useful in the early diagnosis of scrub typhus (using clinical features and initial lab values) so that the early initiation of specific antibiotic (doxycycline) can prevent the occurrence or reduce the severity of ards. after informed consent either from patient or patient attender (in case where patient is not able to give consent) and ethical clearance from the institutional ethical committee, a prospective observational study was conducted on 109 patients with febrile illness and thrombocytopenia during a period of 12 months between june 2012 and may 2013 in acute medical care unit of a super specialty hospital. all 109 patients were tested with both immune - chromatography test (ict) and weil - felix test (wft). patients having either ict / wft (of 1:80 titer and above) positive have been included and considered as scrub typhus positive whereas negative for both immune - chromatography and wft were excluded from this observational study. 51 patients were excluded as they were negative for both ict / wft and remaining 58 patients were positive for either ict / wft. area of residence (andhra pradesh), detailed clinical examination, presence or absence of eschar, rash and lymphadenopathy were noted. tropical and other co - endemic diseases such typhoid, dengue, leptospirosis and malaria tests were done in all patients. additional tests such as ultrasonography of abdomen, arterial blood gas analysis, chest x - ray, electrocardiogram, two - dimensional echocardiogram, cerebrospinal fluid study, electroencephalography, computed tomography / magnetic resonance imaging brain, blood, and sputum / bronchoalveolar lavage cultures, etc., were sent as indicated. apache - ii and sequential organ failure assessment (sofa) were calculated for all these patients. onset within 1-week of a known clinical insult or new or worsening respiratory symptoms, bilateral lung opacities - not fully explained by effusions, lobar / lung collapse, or nodules, pulmonary edema not fully explained by cardiac failure or fluid overload need objective assessment (echocardiography) to exclude hydrostatic edema if no risk factor present, pao2 /fio2 300 mm hg with positive end expiratory pressure or continuous positive airway pressure 5 cm h2 o ). various complications associated with disease (aki, hypotension, ards, hepatitis, meningitis) were managed accordingly. among 58 patients proved to be having positive for scrub typhus, 24 patients had ards and 34 patients had no ards. the incidence of aki, acute hepatitis and delay in starting treatment with doxycycline were significant in ards scrub typhus group compared to non ards scrub typhus group. the clinical characteristics of scrub typhus patients with or without ards table 2 shows pao2 /fio2 ratio, intensive care unit (icu) length of stay, and severity of infection. in our observational study among 24 patients with ards (mild ards : 8 patients, moderate ards : 16 patients, and severe ards : 2 patients) the lung injury score, intubation rate, ventilator days, icu length of stay, apache and sofa score were statistically significant as compared to non ards scrub typhus group. pao2/fio2 ratio and lis with mortality the clinical features of scrub typhus patients at the time admission have been shown in table 3. the most common clinical feature in both groups were fever, headache, nausea, vomiting, myalgia, generalized weakness, pain abdomen, chest pain, conjunctival congestion, hepato - splenomegaly, eschar and macula - papular rashes. the clinical feature such as dyspnea, cough, low blood pressure (mean arterial pressure < 65 mmhg), and inferior vena cava collapsibility of > 50% by ultrasound (hypovolemia) were statistically significant in scrub typhus patients with ards. the clinical features of scrub typhus patients with or without ards table 4 shows the initial laboratory parameters of scrub typhus patients at the time admission. the laboratory parameters such as decreased hemoglobin, hematocrit, serum albumin, and increased serum creatinine, serum total bilirubin, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, lactate dehydrogenase (ldh), creatine phosphokinase (cpk), and serum lactate were statistically significant (p < 0.0001) in scrub typhus patients group with ards. the laboratory parameters (at admission) of scrub typhus patients with or without ards the correlation between wf and ict of scrub typhus patients at the time admission have been shown in table 5. in our observational study all patients were positive for scrub typhus by ict but wf titers were insignificant (< 1:80) in 7 of them. the mortality of scrub typhus in untreated patients range from 0% to 30% and tends to vary with age and region of infection. generally seen in people whose occupational or recreational activities bring them into contact with ecotypes favorable with vector chiggers. scrub typhus manifest as either nonspecific febrile illness or multi - organ dysfunction such as aki, ards, myocarditis, hepatitis, and meningo - encephalitis. in our prospective observational study conducted over 12 months, the involvement of lungs has been described which range from bronchitis and interstitial pneumonitis to ards. the pathogenesis of ards in scrub typhus is not known, thought to be immunological response of the lung to previous o. tsutsugamushi infection without direct invasion of the organism and diffuse alveolar damage without evidence of vasculitis. when the patients presented with atypical symptoms, initially they were not diagnosed (delay in diagnosis) as scrub typhus at outside hospital or when the patient came to our hospital it was average 11.333 0.570 days and 5.264 0.431 days from the onset of signs and symptoms in scrub typhus patients with and without ards, respectively. probably delay in treatment might have caused a high incidence of ards. chest x - ray showing acute respiratory distress syndrome in patient with scrub typhus table 2 shows pao2 /fio2 ratio, icu length of stay and mortality. the majority of patients in ards group had moderate ards, the number of ventilator days and icu mortality were high which was statistically significant (p < 0.05). eschar is a vesicular lesion at the site of mite feeding is the first sign of disease and also pathognomonic of scrub typhus often found in the groin, axilla, genitalia, and neck. this is rarely seen in south east asia and indian subcontinent. in our observational study, 6 out of 34 scrub patients without ards and 4 out of 24 scrub patients with ards had eschar. figure 2 shows the eschar seen near right axilla in one of our patient coming from an endemic area. eschar over anterior chest wall near axilla - on right side table 3 shows the clinical features of scrub typhus patients with or without ards. fever, headache, conjunctival congestion, myalgia, generalized weakness, vomiting, and pain abdomen were the common clinical features. in ards group, the hypovolemia was more common which was statistically significant (p < 0.05). table 4 shows the laboratory parameters (at admission) of scrub typhus patients with or without ards. patients in ards group had more severe disease in the form of deranged liver parameters, increased serum creatinine, elevated ldh, cpk, and serum lactate. serology is the mainstay of diagnosing scrub typhus, immuno - florescence antibody test or indirect immuno - peroxidase assay is the gold standard. due to high - cost factor and nonavailability of above two tests we have done both ict and wft in all 109 patients. based on either ict / wft positivity patients we considered as scrub typhus positive. remaining 58 patients were positive for interstitial cystitis test whereas 7 patients who are positive for ict had wf titer < 1:80. this shows that the wft, which is cheap and commonly used in resource limited countries, has a sensitivity of 30% and specificity of 100% at titer breakpoint of 1:80 in detection of scrub typhus. wft among patients of immuno - chromatography proven scrub typhus with or without ards have been shown in table 5. the higher titers of wf can be correlated with more severe form of the disease according to our observation. treatment of scrub typhus was initiated with doxycycline in 56 patients and combination of doxycycline and azithromycin was used in 2 patients with ards and multiple organ dysfunction syndrome (mods). most of the patients responded to doxycycline therapy within 2 - 3 days except 2 patients who died due to severe mods (four organ involvement) and they were referred fairly late to our hospital. azithromycin has shown to have comparable efficacy when compared to doxycycline in a small trial. rifampicin can be used in combination with azithromycin or doxycycline in cases of poor response to doxycycline alone. all 34 scrub typhus patients without ards recovered completely and discharged. among 24 scrub typhus patients with ards, 22 patients recovered, and 2 patients died (refractory septic sepsis with severe mods). early diagnosis using ict in a patient from the rural with a history of fever, thrombocytopenia and multiorgan dysfunction and treatment with doxycycline in these patients can help to reduce mortality.

: scrub typhus is one of the differential diagnoses for fever with thrombocytopenia. ards associated with scrub typhus has high morbidity and mortality.aims:to evaluate clinical features, lab values, and outcome in patients with scrub typhus and comparison in patients with or without ards.methods:a prospective observational study was conducted on 109 patients with febrile illness and thrombocytopenia during a period of 12 months. all 109 patients were tested with both immune - chromatography test and weil felix test. patients having either immune - chromatography test / weil felix test positive have been included and considered as scrub typhus positive whereas negative for both immune - chromatography and weil felix test were excluded. clinical features, lab parameters, and outcome were evaluated in all patients with scrub typhus. statistical analysis used in this study was t-test.:among 58 patients who were included (after exclusion of 51 patients among total of 109 patients) 34 patients had no ards and 24 patients had ards. the clinical feature like dyspnoea, cough, low blood pressure (map<65 mmhg), ivc collapsibility (by ultrasound) and laboratory parameters like decreased hemoglobin, hematocrit, serum albumin, and increased serum creatinine, serum total bilirubin, sgot, sgpt, ldh, cpk, and serum lactate were statistically significant (p < 0.0001) in scrub typhus patients group with ards. the higher titers of weil - felix can be correlated with more severe form of disease according to our observation. all 34 scrub typhus patients without ards recovered completely. among 24 scrub typhus patients with ards, 22 patients recovered, and 2 patients died.:scrub typhus is an important differential diagnosis in a patients having fever with thrombocytopenia. scrub typhus associated with ards has high morbidity and mortality. early diagnosis and treatment with doxycycline can prevent the occurrence of ards

study design and inclusion / exclusion criteria have been described previously. in brief, for this prospective cohort study all adult allograft recipients between august 2001 and july 2003 who survived with a functioning allograft beyond the first year after transplantation were eligible to participate at their next visit to our outpatient clinic. a total of 606 from an eligible 847 rtr (72% consent rate) signed written informed consent. we excluded 105 recipients with existing diabetes (defined as fasting plasma glucose 7.0 or antidiabetic medication) at baseline from analysis. baseline data were collected between august 2001 and july 2003, and rtr were followed - up for several years. the groningen renal transplant database contains information on all renal transplantations performed at our center since 1968. relevant transplant recipient characteristics such as age, sex, and date of transplantation were extracted from this database. we found current medication information in the medical record and obtained information on employment status, living situation, smoking and alcohol consumption, and cardiovascular history by self - report questionnaire. standard immunosuppressive treatment consisted of the following: prednisolone and azathioprine (100 mg / day) from 1968 to 1989; cyclosporine standard formulation (trough levels of 175 to 200 mg / l in the first 3 months, 150 mg / l between 3 and 12 months after transplantation, and 100 mg / l thereafter ; sandimmune ; novartis pharma b.v ., arnhem, the netherlands) and prednisolone (starting with 20 mg / day, rapidly tapered to 10 mg / day) from january 1989 to february 1993; cyclosporine microemulsion (trough levels idem ; neoral ; novartis pharma b.v .) and prednisolone from march 1993 to may 1997; and mycophenolate mofetil (2 g / day ; cellcept ; roche b.v . , woerden, the netherlands), which was added from may 1997 to present date. in some specific situations, cyclosporine was converted to tacrolimus in the event of acute rejection, hypertrichosis, gingival hypertrophy, or intolerance of cyclosporine. waist circumference was measured on bare skin midway between the iliac crest and the 10th rib. blood pressure was measured after a 6-min rest in the supine position as the average of three automated measurements at 1-min intervals (omron m4 ; omron europe b.v .). plasma glucose, insulin, hdl cholesterol, ldl cholesterol, high - sensitivity c - reactive protein, and serum creatinine were measured as described previously. homeostasis model assessment (homa) was calculated as: /22.5. in this study, metabolic syndrome (ms) was defined according to the definition of the national cholesterol education program expert panel (ncep - atp). mercodia - proinsulin elisa is a solid phase, two - site enzyme immunoassay based on the sandwich technique, in which two monoclonal antibodies are directed against separate antigenic determinants on the proinsulin molecule. proinsulin in the sample reacts with antiproinsulin antibodies bound to microtitration wells and peroxidase - conjugated anti - insulin antibodies in the solution. cross - reactivity for insulin is < 0.03% and cross - reactivity for c - peptide is < 0.006%. the international expert panel meeting proposed recommendations to define nodat based on the american diabetes association criteria 2003. the diagnosis of nodat was based on one of the following criteria: symptoms of diabetes (classic symptoms of diabetes include polyuria, polydipsia, and unexplained weight loss) plus casual plasma glucose concentration 200 mg / dl (11.1 mmol / l); fasting plasma glucose 126 mg / dl (7.0 mmol / l); or use of antidiabetic medication. fasting is defined as no caloric intake for at least 8 h. nodat was recorded until april 2012. data were analyzed with spss version 16.0 (spss, chicago, il), stata version 11 (statacorp lp), and graphpad prism version 4.03 (graphpad software, san diego, ca). for analyses, we combined quartiles 13 as one group and compared this group with quartile 4. differences between groups were tested for statistical significance with student t test for normally distributed variables, mann - whitney test for skewed distributed variables, and test for categorical variables. we performed multivariate cox regression analyses to investigate whether proinsulin is independently associated with nodat. in subsequent multivariate analyses, we investigated whether the association of proinsulin is independent of age, sex, use of cyclosporine, tacrolimus, dose of prednisolone, trough levels of cyclosporine and tacrolimus, homa, or components of the metabolic syndrome. patients were censored at date of last follow - up or death. because there are no validated clinical models for the prediction of nodat in rtr this clinical model includes the following: sex, smoking, waist circumference, hypertension, and family history of diabetes. to assess the added value of proinsulin, we examined improvement of diabetes prediction in terms of discrimination and integrated discrimination improvement (idi). discrimination was evaluated using the harrell c - index for censored data, a statistic similar to the area under a receiver - operating characteristic curve. in general , discrimination refers to the ability of a model to distinguish well between individuals with and without incident diabetes; a value of 1 implies a perfect discrimination and a value of 0.5 implies performance no better than chance. we used idi as a continuous measure of reclassification, calculated by subtracting the mean difference of predicted risk between the clinical model and the model including different biomarkers. study design and inclusion / exclusion criteria have been described previously. in brief, for this prospective cohort study all adult allograft recipients between august 2001 and july 2003 who survived with a functioning allograft beyond the first year after transplantation were eligible to participate at their next visit to our outpatient clinic. a total of 606 from an eligible 847 rtr (72% consent rate) signed written informed consent. we excluded 105 recipients with existing diabetes (defined as fasting plasma glucose 7.0 or antidiabetic medication) at baseline from analysis. baseline data were collected between august 2001 and july 2003, and rtr were followed - up for several years. the groningen renal transplant database contains information on all renal transplantations performed at our center since 1968. relevant transplant recipient characteristics such as age, sex, and date of transplantation were extracted from this database. we found current medication information in the medical record and obtained information on employment status, living situation, smoking and alcohol consumption, and cardiovascular history by self - report questionnaire. standard immunosuppressive treatment consisted of the following: prednisolone and azathioprine (100 mg / day) from 1968 to 1989; cyclosporine standard formulation (trough levels of 175 to 200 mg / l in the first 3 months, 150 mg / l between 3 and 12 months after transplantation, and 100 mg / l thereafter ; sandimmune ; novartis pharma b.v ., arnhem, the netherlands) and prednisolone (starting with 20 mg / day, rapidly tapered to 10 mg / day) from january 1989 to february 1993; cyclosporine microemulsion (trough levels idem ; neoral ; novartis pharma b.v .) and prednisolone from march 1993 to may 1997; and mycophenolate mofetil (2 g / day ; cellcept ; roche b.v . , woerden, the netherlands), which was added from may 1997 to present date. in some specific situations, cyclosporine was converted to tacrolimus in the event of acute rejection, hypertrichosis, gingival hypertrophy, or intolerance of cyclosporine. waist circumference was measured on bare skin midway between the iliac crest and the 10th rib. blood pressure was measured after a 6-min rest in the supine position as the average of three automated measurements at 1-min intervals (omron m4 ; omron europe b.v .). plasma glucose, insulin, hdl cholesterol, ldl cholesterol, high - sensitivity c - reactive protein, and serum creatinine were measured as described previously. homeostasis model assessment (homa) was calculated as: /22.5. in this study, metabolic syndrome (ms) was defined according to the definition of the national cholesterol education program expert panel (ncep - atp). mercodia - proinsulin elisa is a solid phase, two - site enzyme immunoassay based on the sandwich technique, in which two monoclonal antibodies are directed against separate antigenic determinants on the proinsulin molecule. proinsulin in the sample reacts with antiproinsulin antibodies bound to microtitration wells and peroxidase - conjugated anti - insulin antibodies in the solution. cross - reactivity for insulin is < 0.03% and cross - reactivity for c - peptide is < 0.006%. the international expert panel meeting proposed recommendations to define nodat based on the american diabetes association criteria 2003. the diagnosis of nodat was based on one of the following criteria: symptoms of diabetes (classic symptoms of diabetes include polyuria, polydipsia, and unexplained weight loss) plus casual plasma glucose concentration 200 mg / dl (11.1 fasting is defined as no caloric intake for at least 8 h. nodat was recorded until april 2012 . mercodia - proinsulin elisa is a solid phase, two - site enzyme immunoassay based on the sandwich technique, in which two monoclonal antibodies are directed against separate antigenic determinants on the proinsulin molecule . proinsulin in the sample reacts with antiproinsulin antibodies bound to microtitration wells and peroxidase - conjugated anti - insulin antibodies in the solution . cross - reactivity for insulin is < 0.03% and cross - reactivity for c - peptide is < 0.006% . the international expert panel meeting proposed recommendations to define nodat based on the american diabetes association criteria 2003. the diagnosis of nodat was based on one of the following criteria: symptoms of diabetes (classic symptoms of diabetes include polyuria, polydipsia, and unexplained weight loss) plus casual plasma glucose concentration 200 mg / dl (11.1 fasting is defined as no caloric intake for at least 8 h. nodat was recorded until april 2012 . data were analyzed with spss version 16.0 ( spss, chicago, il), stata version 11 (statacorp lp), and graphpad prism version 4.03 (graphpad software, san diego, ca). recipient - related characteristics were analyzed separately for quartiles of proinsulin. for analyses, we combined quartiles 13 as one group and compared this group with quartile 4. differences between groups were tested for statistical significance with student t test for normally distributed variables, mann - whitney test for skewed distributed variables, and test for categorical variables. we performed multivariate cox regression analyses to investigate whether proinsulin is independently associated with nodat. in subsequent multivariate analyses, we investigated whether the association of proinsulin is independent of age, sex, use of cyclosporine, tacrolimus, dose of prednisolone, trough levels of cyclosporine and tacrolimus, homa, or components of the metabolic syndrome. patients were censored at date of last follow - up or death. because there are no validated clinical models for the prediction of nodat in rtr this clinical model includes the following: sex, smoking, waist circumference, hypertension, and family history of diabetes. to assess the added value of proinsulin, we examined improvement of diabetes prediction in terms of discrimination and integrated discrimination improvement (idi). discrimination was evaluated using the harrell c - index for censored data, a statistic similar to the area under a receiver - operating characteristic curve. in general , discrimination refers to the ability of a model to distinguish well between individuals with and without incident diabetes; a value of 1 implies a perfect discrimination and a value of 0.5 implies performance no better than chance. we used idi as a continuous measure of reclassification, calculated by subtracting the mean difference of predicted risk between the clinical model and the model including different biomarkers. the study cohort was composed of 487 rtr (55% men) aged 50 12 years at a median time of 6.0 (interquartile range, 2.611.5) years after transplantation. baseline characteristics of the rtr according to the two groups of proinsulin are shown in table 1. high proinsulin was positively associated with use of -blocker, use of statin, history of bmi, high - sensitivity c - reactive protein, glucose, insulin, homa, and proinsulin - to - insulin ratio. we found other differences in lipid - profile with lower hdl and ldl cholesterol and higher triglycerides in subjects with high proinsulin. no differences were found in other components of immunosuppressive treatment. out of the 487 rtr, 309 (75%) fulfilled the criteria for ms. prevalence of ms was 101 (86%) in the highest quartile of proinsulin compared with 208 (57%) in the lowest three quartiles (p < 0.001). recipient characteristics according to groups of proinsulin nodat developed during median follow - up for 10.1 (interquartile range, 9.710.4) years in 76 (16%) rtr. incidence of nodat was 42 (35%) in the highest quartile compared with 34 (9%) in the lowest three quartiles of proinsulin (p < 0.001) (fig . 1). cumulative percentages of nodat at 1, 3, 5, and 10 years after baseline were 1.2, 4.6, 7.4 and 14.8%, respectively. kaplan - meier curve of de novo diabetes in quartiles of proinsulin tested with log - rank test (p < 0.001). cut - off points for quartiles of proinsulin were as follows: quartiles 13, 2.424.4 (pmol / l), and quartile 4, > 24.5 (pmol / l). subsequently, we proceeded with prospective analyses for proinsulin and development of nodat during follow - up. proinsulin (hazard ratio , 2.29 ; 95% ci, 1.852.83 ; p < adjustment for age and sex did not materially influence the associations ( model 2). we adjusted for use of cyclosporine, tacrolimus, and prednisolone dose in model 3. this adjustment also did not materially influence the association of proinsulin with nodat development. of note, use of tacrolimus was significantly associated with nodat development (hr, 2.84 ; 95% ci, 1.375.89 ; p = 0.005) in this cox regression model. this was independent of proinsulin, age, sex, use of cyclosporine, and prednisolone dose. we found no significant association of use of cyclosporine with development of nodat. in further analyses in which we additionally adjusted for trough levels of tacrolimus and cyclosporine (model 4) however, trough levels of cyclosporine were associated with increased risk for higher concentrations (hr, 1.07 ; 95% ci, 1.011.13 ; p = 0.02). in further analyses, it appeared that the association between proinsulin and nodat was independent of homa (model 5). interestingly, in this model, hr for homa also was associated with nodat independent of proinsulin (hr, 1.23 ; 95% ci, 1.091.40 ; p = 0.001). adjustments for factors of ms (model 6) slightly weakened the association, but proinsulin remained independently associated with nodat. waist circumference (hr, 1.02 ; 95% ci, 1.011.04 ; p = 0.01), triglycerides (hr, 1.21 ; 95% ci, 1.051.40 ; p = 0.01), and glucose (hr, 2.26 ; 95% ci, 1.633.11 ; p < 0.001) were significantly associated with nodat, independent of proinsulin. proinsulin independently predicts nodat in rtr evaluation of prognostic value of proinsulin is summarized in table 3. harrell c - index of discrimination improved from 0.71 (interquartile range, 0.650.77) to 0.80 (interquartile range, 0.750.85 ; p < 0.01) after adding proinsulin to a clinical prediction model including sex, smoking, waist circumference, hypertension, and family history of diabetes. idi analysis shows that the clinical risk score with proinsulin predicted nodat more accurately than the clinical risk score alone. we found similar when homa or glucose was added to the clinical model. when proinsulin was added on top of glucose, idi was positive and remained significant. these show that proinsulin is a promising biomarker for predicting nodat beyond established clinical risk predictors in rtr. proinsulin levels predicted development of nodat in rtr after adjustment for risk factors for nodat. our findings emphasize the importance of -cell dysfunction in the pathophysiology of nodat in rtr. in our study, adjustment for ms (waist circumference, triglycerides, hdl cholesterol, blood pressure, and glucose concentration) attenuated the association of proinsulin with nodat. this supports the notion that ms and particularly waist circumference, triglycerides, and glucose, partly contribute to this association. factors of the ms could be modified by regular physical activity and a healthy diet, which shows the importance of lifestyle interventions after renal transplantation. the association between proinsulin and nodat was independent of homa, which suggests that the relationship is driven by -cell dysfunction. it has become clear that both insulin resistance and -cell dysfunction are present early in the natural history of diabetes. there is a hyperbolic relationship between insulin sensitivity and insulin secretion that depends on a negative feedback loop. pancreatic -cells compensate for changes in insulin sensitivity. in healthy subjects, plasma glucose levels are maintained near to normal, even with low insulin sensitivity, as a consequence of a compensatory increase in insulin secretion. showed that obesity, waist - to - hip ratio, and prednisolone treatment are the predominant determinants of insulin resistance after transplantation. furthermore, proinsulin can be used as a marker of pancreatic -cell dysfunction. in this light, increased circulating levels of proinsulin are seen as a marker of -cell stress when insulin demands required for maintenance of glycemic control are relatively high for the prevailing -cell capacity. this is accompanied by increased spill - over of proinsulin. calcineurin inhibitors impair insulin secretion, produce -cell toxicity, cause insulin resistance, and thereby contribute to an increased risk for nodat. various studies comparing cyclosporine and tacrolimus showed that use of cyclosporine is associated with a significantly lower incidence of nodat than tacrolimus after renal transplantation. interestingly, proinsulin levels tended to be lower in rtr receiving tacrolimus, despite the fact that use of tacrolimus was significantly associated with increased risk for nodat development. use of tacrolimus increased the risk for nodat by almost three - fold (hr, 2.84 ; 95% ci, 1.375.89 ; p = 0.005). this observation could point to the known interference of tacrolimus with insulin production by pancreatic -cells at the level of synthesis rather than at the level of conversion of proinsulin to insulin. the low variation in steroid doses in the population we investigated did not allow for us to find a relationship between steroid dose and nodat. based on the from the united kingdom prospective diabetes study, it was suggested that -cell dysfunction was reduced up to 50% at time of diagnosis. therefore, proinsulin can be used to identify patients at risk for nodat development several years later. proinsulin can still bind to the insulin receptor and has a glucose - lowering effect of 1020% compared with insulin. because of this minor but evident effect, patients with -cell dysfunction do not always have diabetes diagnosed. although no cut - off points for proinsulin are described in the literature, there is one study in which cut - off values are given for defining insulin resistance. data from the iris - ii (study on insulin resistance and insulin sensitivity) show that elevated proinsulin levels (> 10 pmpl / l) are a good indirect marker for insulin resistance. in our study, 80% of the rtr had proinsulin levels above this threshold of > 10 pmpl / l. this may reflect the fact that -cell function is impaired in almost all rtr because of increased metabolic demands on the -cells and the chronic exposure to immunosuppressive drugs, which places rtr at high risk for development of nodat. sharif et al. validated important insulin resistance indexes in rtr using tacrolimus. rodrigo et al. analyzed the performance of two general population risk scores for prediction of diabetes in rtr. recently developed a pretransplant risk score for the prediction of post - transplant diabetes. was modest, with areas under receiver - operating curves varying between 0.70 and 0.72. our study is the first to include a marker of -cell dysfunction in addition to insulin resistance indexes in the prediction of nodat in rtr. in our analyses , we found that both proinsulin as a marker of -cell dysfunction and homa as a marker for insulin resistance are independently associated with increased risk for nodat. the prediction model with proinsulin had good discrimination, showing that 80% of the rtr were adequately classified as at risk for nodat. proinsulin is a promising marker for early detection of patients at risk for nodat and, possibly, future studies also may identify it as useful in the clinic to monitor -cell function early after transplantation. monitoring -cell functions could allow for early intervention and treatment strategies to preserve -cell function. recently showed in a randomized controlled study that basal insulin therapy may be a good strategy to reduce hba1c and may decrease the incidence of nodat, presumably by protection of the -cells. patients were randomized to immediate postoperative isophane insulin (treatment group) or short - acting insulin with or without oral antidiabetic agents (standard care). the treatment group had 73% lower odds for nodat and hba1c was 0.38% lower than in the control group. besides pharmacological strategies, lifestyle interventions could play an important role in prevention of nodat beyond the first year after transplantation. exercise training decreases insulin resistance and the risk of diabetes in the general population, and it can be assumed that it has similar effects in rtr. sharif et al. showed that lifestyle modification is beneficial for high - risk rtr with glucose intolerance. intensive lifestyle modification (dietitian, exercise program, and weight loss advice) ed in 15% improvement in 2-h postprandial glucose. lifestyle interventions targeting physical activity and diet after transplantation as well as individualized choice of immunosuppressive agents could help in the prevention of nodat. rtr in this study were closely monitored through regular check - ups at our clinic, which provide complete information on patient status. our study population is a cross - cut of all the rtr who visited our outpatient clinic, giving a variation of rtr with different times after transplantation and including stable rtr late after transplantation. however, our study is limited by the heterogeneousness of our study population, with variable time after transplantation and immunosuppressant medication. this modeling can not fully correct for the fact that rtr without nodat late after transplantation may be a healthier group. our highlight the role of -cell dysfunction in the pathophysiology of nodat in rtr. considering that the development of nodat is associated with a higher risk of complications and worse survival, identifying rtr at increased risk for development of nodat proinsulin as a marker of -cell dysfunction has potential value for identification of rtr at increased risk for nodat. prevention of nodat by lifestyle interventions, early identification of patients at risk, and choice of immunosuppressive medication are all important to control and manage nodat in rtr.

objectivechronic exposure to calcineurin inhibitors and corticosteroids poses renal transplant recipients (rtr) at high risk for development of new - onset diabetes after transplantation (nodat). pancreatic -cell dysfunction may be crucial to the pathophysiology of nodat and specific markers for -cell dysfunction may have additive value for predicting nodat in this population. therefore, we prospectively investigated whether proinsulin, as a marker of pancreatic -cell dysfunction, is associated with future development of nodat and improves prediction of it.research design and methodsall rtr between 2001 and 2003 with a functioning graft for 1 year were considered eligible for inclusion, except for subjects with diabetes at baseline who were excluded. we recorded incidence of nodat until april 2012.a total of 487 rtr (age 50 12 years, 55% men) participated at a median time of 6.0 (interquartile range , 2.611.5) years after transplantation. median fasting proinsulin levels were 16.6 (iqr, 11.024.2) pmol / l. during median follow - up for 10.1 (iqr, 9.110.4) years, 42 (35%) rtr had development of nodat in the highest quartile of the distribution of proinsulin versus 34 (9%) in the lowest three quartiles (p < 0.001). in cox regression analyses, proinsulin (hazard ratio, 2.29 ; 95% ci, 1.852.83 ; p < 0.001) was strongly associated with nodat development. this was independent of age, sex, calcineurine inhibitors, prednisolone use, components of the metabolic syndrome, or homeostasis model assessment.in , fasting proinsulin is strongly associated with nodat development in rtr. our highlight the role of -cell dysfunction in the pathophysiology of nodat and indicate the potential value of proinsulin for identification of rtr at increased risk for nodat.

tuberculosis (tb), caused by mycobacterium tuberculosis (m. tuberculosis ( mtb) ), is still a major health problem around the world. it is second only to aids as the leading cause of death from infectious diseases. the world health organization estimates that there were 9 million new cases of tb and 1.4 million tb - related deaths in 2011. the effectiveness of the only available tb vaccine, bacillus calmette - gurin (bcg), is limited. protection in children has only been demonstrated for disseminated tb and tuberculosis meningitis, while the efficacy for adult pulmonary tb is very variable. in fact, a controlled trial of bcg failed to demonstrate its effectiveness in protecting against the development of tb. lack of efficacy of bcg for adult tb is a serious problem in controlling the disease and may be partly related to the fact that the efficacy of bcg vaccinations declines over time. the of several epidemiological studies suggest that the effectiveness of bcg vaccinations lasts for around twenty years. this decline of efficacy would be associated with a decrease of cell - mediated immunity (cmi) to mtb. an assessment of the degree of cmi over time would require objective, repeatable, and economical assays. the tuberculin skin test (tst), an assay for type iv - delayed hypersensitivity reaction, is the primary screening method used to determine tb exposure. the presence or absence of induration following the tst injection can be used as objective proof of active cmi against mycobacterial infection. however, the tst can not distinguish mtb from bcg or nontuberculous mycobacteria (ntm). therefore, an in vitro (or ex vivo) interferon- (ifn-) release assay (igra), which utilizes antigens within the region of difference 1 (rd1) of m. tuberculosis, is used in conjunction with the tst. these antigens, such as early secreted antigenic target 6 kda (esat-6) (rv3875), culture filtrate protein 10 kda (cfp-10) (rv3874), and tb7.7 (rv2654c), are not present in bcg or most environmental ntm. these antigens are also very potent inducers of cmi, even in an in vitro setting , which enables the establishment of an elisa - based assay system to measure ifn- secreted into the culture supernatant after antigenic stimulation. quantiferon - tb gold (qft - g) is an igra that utilizes two mtb - specific antigens, esat-6 and cfp-10. its successor, the quantiferon - tb gold in - tube test (qft - git), incorporated tb7.7 in addition to esat-6 and cfp-10. a simultaneous and longitudinal comparison of the qft - g and qft - git assays among healthcare workers showed that qft - git is the more sensitive of the two, probably due to the addition of tb7.7. however, t-spot.tb uses only two antigens, esat-6 and cfp-10, to stimulate samples. from previous methods were significantly affected by the lymphocyte count in the peripheral blood of each patient. therefore, we aimed to analyze precise epitopes of mtb - specific antigens in cd4 + cells, which are the predominant cell type that secretes ifn- upon stimulation with mtb antigens. we developed an elispot assay (elispot) using multiple, overlapping peptides from three proteins, esat-6, cfp-10, and tb7.7, to stimulate memory t cells. in this study, the sensitivity of two igra assays, qft - git and elispot, was compared using blood samples from the same tb patients and the same tuberculosis antigen sets to determine the activity of cmi in patients. elispot, using the same antigen peptide sets, was then used to analyze t cell response to mtb - specific antigens to identify restricted epitopes. finally, mtb - specific cd4 + lymphocytes were segregated to evaluate their phenotype in tb patients. patients of tokyo national hospital in tokyo, japan, were consecutively enrolled in the study, after giving informed consent, from april 2010 to april 2011. a total of 177 japanese patients (age : 58.5 18.2 yr ; male : 68.5%) were recruited. the following information was obtained from all patients at the time of enrollment: history of prior tb disease, work history in any healthcare settings or recent exposure to a patient with active tb, and other tb risk factors such as taking immunosuppressive drugs. information on previous medical history, any clinical symptoms and signs, and radiological and microbiological data were also collected. the patients were then divided into three categories: active disease: patients having positive symptom(s) and positive smear and/or positive demonstration of mtb in culture; past disease: previously diagnosed with tb, treated, and currently free from symptom(s); and latent tb infection (ltbi): no symptoms with normal chest x - ray but having positive from an igra. among the 177 patients recruited, 56 (32%) had active disease, 103 (58%) were in the past disease group, and for those patients classified as having the disease in the past, the average time period since disease onset was 2605 605 (mean se) days. the research protocol was approved by the institutional review board of tokyo national hospital and by the research ethics committee of the national institute of infectious disease, tokyo, japan. the qft - git assay was performed using fresh whole blood in accordance with the manufacturer's instructions (cellestis, chadstone, australia). the were interpreted with software provided by cellestis. were scored as positive if the ifn- concentration in the tube with tb - specific antigen was > 0.35 iu / ml after subtracting the value of the nil control and at least > 25% of the negative control value. if the net ifn- response was < 0.35 iu / ml for the antigens and the response to the mitogen - positive control was > 0.5 iu / ml, the response was considered peptides 15 amino acids in length, with nine overlapping residues, were synthesized to cover the entire length of esat-6, cfp-10, and tb7.7. imajoh - ohmi in the medical proteomics laboratory, institute of medical science, university of tokyo, tokyo, japan. the purity of the peptides was > 95% after purification with reversed - phase hplc. the cfp-10 peptides were labeled as c1 to c16, esat-6 peptides as e1 to e15, and tb7.7 peptides as t1 to t13. supplemental table 1 (see supplementary material available online at http://dx.doi.org/10.1155/2014/764028) lists the sequence of each peptide. peripheral blood mononuclear cells (pbmcs) were separated from heparinized blood samples by density centrifugation using bd vacutainer cell preparation tubes (becton, dickinson and company, franklin lakes, nj, usa). genomic dna was isolated from pbmcs with qiaamp dna blood kits (qiagen, germantown, md, usa) for hla typing using the luminex multi - analyte profiling system (xmap ; luminex, austin, tx, usa). the ifn- elispot assay was performed to compare the sensitivity between igras and to find an immunological epitope to tuberculosis - specific antigens. the peptide mixture of each protein was used to obtain sensitivity measurements, while a single peptide was applied to each well for epitope determination. pbmcs were seeded into precoated ifn- elispot plates (becton, dickinson and company, franklin lakes, nj, usa) with 2.5 10 cells per well in aim - v medium (gibco) and incubated with one of a series of peptides (10 m) or a peptide mixture (10 m) of each antigen at 37c in 5% co2 for 16 h. a negative control (no mitogen or antigen) and a positive control (phytohemagglutinin, pha, 5 g / ml) were also included. after incubation , the wells were washed and developed with a conjugate against the antibody used and an enzyme substrate. spot - forming units were counted using a ks elispot imaging system (carl zeiss, hallbergmoos, germany) as spot - forming cells (sfc). elispot were interpreted according to the following criteria: the test was positive when the negative control had 05 spots and the (antigen spot count) (negative control spot count) was greater than six. the test was negative if the above criteria were not met and the positive control was valid. hla class ii tetramers conjugated with apc - labeled streptavidin were provided by the tetramer core laboratory at the national institutes of health, bethesda, md, usa. pbmcs were incubated with class ii tetramers for 2 h at room temperature. the following fluorescence - labeled monoclonal antibodies (mabs) were used in this study: anti - cd3-apccy7 (hit3a), anti - cd8-percp - cy5.5 (mab11) (biolegend, san diego, ca, usa), and anti - cd4-pacific blue (okt4) (ebioscience, san diego, ca, usa), anti - pd-1-pe (bd bioscience). anti - klrg-1-alexa488 was kindly provided by professor h. pircher (university of freiberg, germany). where necessary, the relevant isotype control mab was used. cell viability was assessed using the live / dead kit (invitrogen, carlsbad, ca, usa). following a 30 min incubation at 4c, the cells were washed and acquired using a facs canto ii flow cytometer (bd bioscience). facs data were reanalyzed using flowjo software, version 8.8.7 (treestar, san carlos, ca, usa). group medians and distributions were analyzed using the wilcoxon matched - pairs signed - rank test and the mann - whitney u test. all analyses were performed using graphpad prism software, version 5 (san diego, ca, usa). all patient samples (n = 177) were analyzed by qft - git and 115 were analyzed by ellispot. as expected, when the from both assays were compared, there was a positive correlation between qft - git values and the number of spots obtained in the elispot assay (r = 0.532 ; p < 0.001) (figure 1). the detection rate of each assay was compared across the three groups of patients: active disease, past disease, and ltbi. qft - git failed to detect many patients with active and past disease; elispot detection was more consistent (overall detection rate of 93.9% versus 65.5%) (table 1). the average duration from disease onset to the date of the assay was 2, 086 743 days in qft - git - negative past tb patients and 4, 920 3042 days in elispot - negative past tb patients. when both assays were compared in the same patients, 31 of 37 qft - git - negative cases (84%) were positive by elispot (table 2), suggesting that the elispot assay developed in our laboratory was better at detecting a broader range of tb+ patient populations. consequently, the elispot was chosen to carry out the detection of antigenic peptides in mtb - specific proteins. each mtb - specific antigen used in the igra comparison was further evaluated to identify the peptide(s) that most efficiently induced antigenicity to mtb and activation of host cmi. we found that 77% of patients responded to esat-6 peptides and 66% to cfp-10, while no single case responded to tb7.7 (data not shown). elispot data provided an indication of the prevalence and strength of the esat-6 and cfp-10 mtb antigens. the next step was to map the precise epitope(s) of the mtb - specific antigens recognized by cd4 + lymphocytes. two sets of peptides were synthesized, 15 overlapping esta-6 peptides (e1e15) and 16 overlapping cfp-10 peptides (c1c16) (supplemental table 1), to evaluate patient samples using elispot. among the 15 peptides from esta-6, e1, e4, e5, e10, and e13 elicited a response in multiple patients (figure 2(a) ). the same peptides induced a higher average number of spot - forming cells (sfc) in the elispot assay (figure 2(b) ), suggesting that these are major epitopes that stimulate cd4 + cells in peripheral blood. similarly, peptides c1, c5, c9, c10, and c13 from cfp-10 were identified as responsible epitopes that stimulated multiple patients (figure 3(a) ) to induce a stronger ifn- response (figure 3(b) ). although cmi to tb involves both cd4 + and cd8 + positive lymphocytes , the primary source of ifn- as measured by elispot is cd4 + cells. cd4 + t lymphocytes are activated by specific antigens presented by antigen presenting cells (apcs) through major histocompatibility complex (mhc) class ii molecules (or hla - dr in humans). therefore, to ascertain specificities to each esat-6 and cfp-10 peptide, human hla drb1 haplotypes were examined by employing a high - resolution luminex - based method. as shown in table 3, the proportion of drb1 identified in tb patients was most similar to that found in the japanese population (n = 916). the predominant haplotype was drb1 * 0405 (13.5%), followed by drb1 * 0901 (12.9%), drb1 * 1502 (12.6%), and drb1 * 0803 (8.0%). however, some haplotypes were significantly higher or lower than those in the japanese control population. they included drb1 * 0101 (1.8% in tb versus 4.8% in control), drb1 * 0406 (7.1% versus 3.2%), and drb1 * 1502 (12.6% versus 8.7%). the ability of each esat-6 and cfp-10 peptide to induce ifn- in elispot was examined and its relation to each hla drb1 haplotype was analyzed. haplotypes that showed characteristic are illustrated in figure 4, in which positive spots as described in section 2.5 are shown in dark grey. among the esat-6 peptides identified in the previous analysis (figures 2 and 3), e4 showed a strong association with drb1 * 0405 (28 out of 31 cases) and weak associations with drb1 * 1501 (5 out of 17 cases) and drb1 * 1502 (4 out of 21 cases) (figure 4, left panel). likewise, the c10 peptide from cfp-10 showed strong associations with drb1 * 1501 (17 cases) and drb1 * 1502 (21 cases). peptide - mhc binding is the most selective of the events that determine t cell epitopes. thus, peptide - mhc binding motif profiles can be used to predict the identity of t cell epitopes. we predicted hla drb1 * 0405-restricted t cell epitopes using in silico analysis of peptide - mhc binding profiles derived from peptides known to bind the relevant mhc molecules. a given peptide bound to a specific hla molecule was considered a potential cd4 + t cell epitope when its binding score ranked within the top 3% percentile of scores obtained for 1000 random 9-mer peptides (average amino acid composition of proteins in the swissprot database), using the same profile. peptide - mhc class ii binding profiles only predict the 9-mer core that fits in the binding groove. however, we also provided the three most proximal n - terminal and c - terminal residues, as they can also be the target of t cell recognition. as a , the minimal core region of the e4 peptide although elispot is a highly sensitive method for the identification of tb patients, it is still difficult to differentiate active patients from past patients using igras. based on the epitope information described above, an esat-6-specific tetramer (qgnvtsihslldegk) was synthesized with hla drb1 * 0405. another tetramer (pvskmrmatpllmqa) provided by the nih tetramer facility was used as the negative control. the pmbcs of one active and one past tb patient were stained with drb1 * 0405 esat-6 tetramer using a modification of a previous method. pbmcs were gated with ssc and fsc to isolate the lymphocyte fraction after deletion of doublets using fsc - a and fsc - h. lymphocytes were stained with anti - cd3, cd4, cd8, and live / dead dye to eliminate dead cd4 + t cells (figure 5(a) ). viable cd4 + lymphocytes were then gated with esat-6-specific tetramer (figures 5(b) and 5(c), left panels ). a lower proportion of esat-6-specific cd4 + lymphocytes was found in the past tb patient than in the active tb patient (0.148% versus 0.939%, figures 5(b) and 5(c), left panels ). (pd-1) and antikiller cell lectin - like receptor g1 (klrg-1) were then used to distinguish the proliferation and cytokine production phenotypes of the cd4 + cells. pd-1 expressing cd4 + lymphocytes possess proliferative capacity, while klrg-1 expressing cd4 + lymphocytes are relatively short lived but have cytokine secretion capacity. although pd-1 positive and klrg-1 positive cd4 + t cells were identified in both cases, pd-1 and klrg-1 double positive cd4 + lymphocytes were detected only in the past tb patient (figure 5(c) versus figure 5(b), right panels ). in this study, we first compared the ability of two igras to detect tb infections in order to evaluate cmi to tb. both assays were mutually acceptable for this purpose, but the elispot was more sensitive than qft - git. the elispot was then used to determine antigenic dominant regions in mtb - specific proteins. several epitopes of mtb - specific antigens were found in cd4 + lymphocytes. using these epitopes , we made an esat-6-specific mhc class ii tetramer to detect the kinetics of tuberculosis - specific cd4 + lymphocytes. there are many studies comparing qft - git and elispot, especially for individuals suspected of having tuberculosis or a latent tb infection. in most cases, elispot or the commercially available t-spot.tb was more sensitive than qft - git with a similar specificity. moreover, in this study, we were able to detect specific epitopes of the mtb - specific antigens. a time lapse between bacterial growth and appearance of the adaptive immune response has been observed in mice as well as in humans. around 30% of active patients were negative by qft - git, suggesting that early evaluation of tb might lead to misdiagnosis. next, past tb patients were recruited to investigate the continuation of the host immune response to mtb. qft - git and elispot exhibited differences in time course when the igra were negative (5 yr versus 13 yr). however, the age factor could not be adjusted because of the small population size. indeed, among 18 ltbi cases, 4 were diagnosed by qft - git only and 6 by elispot; only 8 cases were diagnosed by both. with the elispot assay established in our laboratory , we used overlapping peptides from antigenic proteins, not an empirical mixture of peptides, to detect distinct antigenic regions in esat-6 and cfp-10. identified similar regions; however, as the ethnicity of the two groups of subjects was considerably different, the regions were not expected to be identical. * 0405 is the most prevalent in the japanese population and, as expected, the most frequent population in tb patients. none of the patients responded to the tb7.7 peptides, which was consistent with the of a us study. this was surprising, given that this antigen is a recent addition to qft - git tests. this finding suggests that the increased sensitivity of qft - git tests is due to stimulation by the mixture of antigens in the same tube rather than addition of the novel tb7.7 antigen. pd-1 and klrg-1 were used as phenotypic markers for esat-6-specific cd4 + lymphocytes. in the lcmv model, pd-1 expression on cd8 + lymphocytes is a marker of cell exhaustion; however, in the tuberculosis model, cd4 + lymphocytes that express pd-1 have proliferative potential, suggesting that these are effector cd4 cells. examined several other activation markers such as cd44, cd62l, cd27, and cd127 (il-7r) and found that they do not differ between the klrg-1- and pd-1- expressing cell populations during tuberculosis infection. our suggest that, in the active phase, esat-6-specific cd4 + lymphocytes, expressing either pd-1 or klrg-1, are dominant. however, in the chronic phase, pd-1 expression might decline and klrg-1 + or pd-1+/klrg-1 + cd4 + lymphocytes are dominant, although a recent mouse study showed that a significant portion of esat-6-specific pd-1 expressing cd4 + lymphocytes also express klrg-1. further precise study with human peripheral blood is required with a larger population of participants. the elispot assay was more sensitive than qft - git in evaluating the adaptive immunity to tb. in addition, the use of overlapping peptides revealed the association between epitopes of two mtb peptides in cd4 + lymphocytes and mhc class ii haplotypes. finally, a significant difference in the expression of esat-6-specific cd4 + lymphocytes was discovered based on stage of treatment.

tuberculosis remains a major global health problem worldwide, and hence there is a need for novel vaccines that better induce cellular - mediated immunity (cmi). in search of a better vaccine target , the quantiferon - tb gold in - tube test (qft - git) and the interferon- elispot assay (elispot) were used to compare the magnitude of cmi in patients. of the elispot assay led to the discovery of specific epitopes within the early secreted antigenic target 6 kda (esat-6) and culture filtrate protein 10 kda (cfp-10) proteins. both peptides showed a strong association with several hla class ii drb1 molecules in the japanese population. using esat-6-specific hla class ii tetramers , we determined that the expression of esat-6-specific cd4 + lymphocytes was significantly decreased in treated patients compared with active patients. in addition, programmed death-1 (pd-1)/killer cell lectin - like receptor g1 (klrg-1) double positive cells were found only in treated patients and not in those with active tb. these data could provide clues for the development of novel tuberculosis vaccines.

the preservation of teeth to support an attachment - retained prosthesis whether fixed or removable is an appropriate and stable alternative to extractions and complete dentures. combinations of fixed and removable partial dentures using precision / semi - precision attachments represent one high - tech solution in the field of prosthodontics. combined fixed / removable partial denture prosthesis usually refers to the use of precision attachments, double crowns and sometimes overdentures with root attachments. tooth - supported overdentures can be retained with the help of precision attachments and can improve both retention and stability while simultaneously reducing alveolar bone resorption. they may also be more cost - effective and maintain more dental proprioception than implant supported overdentures. it incorporates one component into the removable partial denture, and the connecting component is traditionally incorporated into a cast crown or a fixed partial denture, sometimes referred to as patrix and matrix. the classic indication for precision attachments is in patients with natural anterior teeth and unilateral or bilateral distal extension cases for whom high esthetic demands must be met. attachment - retained cast partial dentures facilitate both esthetic and functional replacement of missing teeth. studies have shown a survival rate of 83.35% for 5 years, of 67.3% up to 15 years, and of 50% when extrapolated to 20 years. this case report describes the rehabilitation of a partially edentulous patient by use of preci - vertix attachment in the maxillary arch and stud attachments in the mandibular arch. a 50-year - old female patient in good general health presented with poor esthetics and compromised masticatory function to the department of prosthodontics and implantology. the clinical examination revealed several missing teeth both in the mandibular and maxillary arch with no loss of vertical dimension. maxillary lateral incisors were found to be supra - erupted, buccally inclined with grade ii mobility. the remaining maxillary canines and 1 premolars presented good periodontal support and mandibular canine / premolar presented with reasonable bone support. radiographs were made , diagnostic casts were articulated at the existing occlusal vertical dimension, and the treatment was carefully planned taking into account patient's esthetic demand and economical condition. inter - arch space was found to be 14.03 mm , adequate for the use of precision attachments in both the arches. treatment plan included extraction of hopeless teeth (i.e., maxillary lateral incisors and second premolar in the second quadrant), followed by rehabilitation of maxillary arch with combined fixed / removable prosthesis (using preci - vertix precision attachment) and overdenture with stud attachment in the mandibular arch. (a) pre- treatment patient presentation (b) pre treatment orthopantomogram of the patient showing healthy abutments after extraction of hopeless teeth digital vernier caliper showing available inter - arch space diagnostic impressions were made and mounted on semi adjustable articulator using a face bow, following which diagnostic wax - up was done on the mounted casts. a putty matrix (express std putty ; 3 m espe, st . paul, minn .) was made over the completed diagnostic wax - up for evaluation of the existing space for the extra - coronal resilient attachments. maxillary canines and 1 premolars were prepared to receive porcelain - fused - to - metal crowns. impression was made in polyvinyl siloxane impression material (affinis, coltene / whaledent, altsttten, switzerland) and the cast was poured in die stone (kalrock, kalabhai karson, mumbai). crowns were waxed to full contour and milled in wax for maximum guiding plane surface. burnout plastic male (preci - vertix standard attachment) with built in paralleling mandrel was attached to the distal surface of the waxed abutment using a dental surveyor, lingual to the center of proximal contour. this ensured that the bulk of the matrix does not interfere with esthetics of the buccal cusp of replacing a tooth. the height of the standard plastic male was 5.0 mm which was sufficient to provide lateral stabilization to the prosthesis. eight unit fixed partial denture along with a male part of preci - vertix attachment was cast in ni - cr alloy. porcelain build - up of the 8 unit fixed partial denture was completed and tried in the patient's mouth. after the cementation of the bridge, impression was made in polyvinyl siloxane impression material (affinis, coltene / whaledent, altsttten, switzerland) and poured in die stone. wax - up of the cast framework was completed on the master cast, and the entire cast partial framework was cast in co - cr alloy. patient was instructed regarding insertion and removal of the prosthesis. (a) abutments prepared to recieve 8 unit pfm bridge (b) prepared canine and premolar teeth to recieve cast pivots with short copings burn - out plastic male with paralleling mandrel attached to distal surface of waxed 8 unit segment (a) casting with opaque layer: 8 unit metal framework with male preci - vertix attachment (b) try - in of 8 unit pfm bridge with male attachment (a) acrylized cast partial framework (b) acrylized combined prosthesis showing orientation of hader polypropylene clips with eight unit fpd similarly, mandibular abutments (canine and premolar) were prepared to receive short copings. postspace was prepared and the impression made in polyvinyl siloxane impression material (aquasil lv, dentsply, caulk, germany) for indirect technique. plastic post with sphere were placed in prepared root space and checked for parallelism with the help of ney's surveyor. wax patterns of the pivots were cast in ni - cr alloy (mealloy, dentsply, uk). retentive nylon caps were placed over master cast, wax block out completed and cast partial framework was fabricated in co - cr alloy. maxilla - mandibular relationships were recorded, and occlusion was evaluated. (a) wax milling of copings with plastic posts and sphere to achieve parallelism (b) wax block out of master cast with retentive nylon caps (a) wax pattern cast partial framework over the refractory cast (b) try - in of co - cr cast framework with retentive nylon caps (a) post insertion picture with combined prosthesis seated (b) overdenture prosthesis with nylon retentive caps overdenture prosthesis for the mandibular arch and cast partial denture prosthesis for the maxillary arch was fabricated in heat - cure acrylic resin. balanced occlusion was achieved and home care instructions regarding insertion and cleaning of the prosthesis were given to the patient. attachments have always been surrounded by an aura of mystery, primarily because of a lack of familiarity and experience. glossary of prosthodontic terms 8 edition defines attachment as a mechanical device for the fixation, retention, and stabilization of a prosthesis or as a retainer consisting of a metal receptacle and a closely fitting part; the former (the female component) is usually contained within the normal or expanded contours of the crown of the abutment tooth and the latter (the male component), is attached to a pontic or the denture framework. attachments may be classified as either precision or semi - precision, depending on the method of fabrication and tolerance of fit. precision attachments have prefabricated, machined components with precisely manufactured metal - to - metal parts with close tolerances. these attachments have a long track record of more than 50 years and have been preferred in cases of reduced tooth support. attachments have a number of desirable qualities that indicate their use in place of conventional clasp retained removable partial dentures. conventional clasp assemblies and rests may be visible and unesthetic whereas preci - vertix and stud attachments get enclosed within contours of the part of the prosthesis. a major advantage of the use of attachments is that the point of force application to the tooth is more apical than for occlusal or incisal rests, thus shortening the lever arm and decreasing torqueing forces. attachments may also allow better cross - arch force transmission and stabilization than clasps, but this is determined by the type of attachment used, the number of guiding surfaces and the design and adaptation of the framework and the attachment. attachment alignment is not as critical in highly resilient extracoronal attachments due to the omniplanar motion possible. poor dental motivation and manual dexterity of the patient may in earlier failure than with the use of conventional clasping. a minimum of 4 mm of vertical space is necessary for most attachments. in this particular case preci - vertix (ceka) attachments and stud attachments (ot cap, rhein 83 inc ., usa) were used that provided frictional retention to the maxillary cast partial denture as well as for the mandibular overdenture. preci - vertix (ceka) attachments are extra - coronal devices in which exchangeable plastic layers of various sizes are used in the female elements to vary the retention force. the female plastic insert (made up of polypropylene) used in this case provided standard retention to the prosthesis. moreover, cross arch splinting of the upper canines and premolars provided better stress distribution thus reducing the rate of alveolar bone resorption. preci - vertix resilient attachments permit vertical movement during mastication reducing stress transfer to the abutments (stress breaking function) and direct the forces to the residual ridge acting as stress redirectors. these attachments are based on a broken stress philosophy, thus help to distribute forces equally between soft and hard tissues and are advocated in kennedy class i situations. due to reduced tooth support provided by the mandibular arch and to reduce the masticatory load over canine / premolar teeth, it was decided to use resilient stud attachments that will redirect the forces to the residual ridge thus preventing the torqueing of the abutment teeth, justifying the use of resilient attachments in both the arches. according to feinberg classification of precision attachments, preci - vertix is categorized as passive (free moving, stress - breaking action type of attachment). these attachments are passive, and free - moving that dissipates destructive lateral forces, preventing their infliction on the abutment teeth. thomas forde, in the principles and practice of oral dynamics, theorizes that vertically directed forces drive the hydraulic system of dentitional blood supply to the periodontal structures, whereas rocking or rotational forces disrupt the dentitional blood supply, causing force - induced mouth degeneration and loss of teeth. the tissue under a passive, free - moving attachment case is generally pink and healthy as a of the vertically - directed physiologic stimulation during function. various stud attachments available are selected based on vertical space available, crown / root ratio, type of coping, number of teeth support, amount and quality of bone support, location of abutments, type of opposing dentition, angulation of the root to the occlusal plane, chewing pattern and the musculature of the patient and patient desire. rheins stud attachments to retain mandibular overdenture were used in this case due to their simplicity in design, ease in maintenance and minimum leverage. canines are the most important proprioceptive organs, the shape and strategic position, and the larger periodontal attachment area make them ideal abutments. the metal denture fabricated to serve as overdenture is less subject to breakage and denture supporting tissues respond more favorably to metal base which may be related to greater ease in maintaining cleanliness of metal base and to effective transmission of thermal changes through the metal base. various cases with esthetic and retention challenges can be solved with correct selection of attachment. whether the need for treatment revolves around health, function or esthetics, attachment retained prosthesis have the capacity to impact patients in life - changing ways. attachment - retained dentures provide long - term prosthetic stability compared to conventional clasp retained removable partial dentures along with support to the oral and facial soft tissue, which can bolster patient's confidence and alleviate insecurity.

satisfactory restoration in a patient with a partially edentulous situation can be challenging especially when unilateral or bilateral posterior segment of teeth is missing. successful restoration can be done with various conventional and contemporary treatment options. one such treatment modality is attachment - retained cast partial dentures. a key to success for an attachment retained cast partial denture is the strategic selection of teeth for retention. this clinical report discusses rehabilitation of a patient with the help of a combined prosthesis in the upper arch and stud retained overdenture in the lower arch.

evidence from randomized trials of medical therapy versus carotid endarterectomy (cea) for symptomatic stenosis of the carotid artery has led to the recommendation that cea should be performed in patients with symptomatic carotid artery stenosis to reduce the long - term risk of recurrent stroke or tia. the combined rate of stroke or death at 30 days following cea in nascet, ecst, and the va trials was 7.1% (95% ci 6.3 to 8.1%). however, the primary endpoints of these trials did not include cranial nerve palsy (cnp) or haematoma. although less extensively studied, the surgical complications of cnp and haematoma have long been recognized following cea, and have been associated with an increased risk of stroke or death. nerves affected include the mandibular branch of the facial nerve, vagal, glossopharyngeal, hypoglossal, and accessory nerves, and therefore cnp has the potential to cause significant postoperative morbidity. carotid angioplasty and stenting (cas) was developed as an alternative to cea, in part to avoid these hazards of a surgical incision. however, the of recent large randomized trials, including the international carotid stenting study (icss), have consistently shown that cas carries a higher risk of non - disabling stroke than cea within 30 days of the procedure, with no significant difference in the rates of disabling stroke or death. icss was a randomized controlled multicentre open clinical trial that randomized patients with symptomatic carotid stenosis to cea or cas. in this study, the incidence and severity of cnp and haematoma in icss, and risk factors for their development, were studied, to identify groups of patients at higher risk and determine whether these complications merit consideration in selection of a revascularization procedure. patients over 40 years old were eligible for randomization in icss if they had more than 50% recently symptomatic carotid stenosis suitable for either cas or cea, and were clinically stable. patients were excluded if they had a major stroke with poor recovery of function, if their vascular anatomy rendered cas or cea unsuitable, if the stenosis was caused by non - atheromatous disease, if cardiac bypass was planned within 1 month of the revascularization procedure, or if there had been previous revascularization of the symptomatic artery. carotid endarterectomy in icss was performed according to the surgeon's usual practice: local, general, or combined anaesthesia was allowed for the procedure. the type of arterial reconstruction to be carried out was not specified in the protocol, nor was the choice of peri - procedural medication. technical details of the surgical procedure and the occurrence of cranial nerve palsy or haematoma were reported by trial investigators. all patients were then re - assessed at 1 month after the procedure by a neurologist or investigator under their supervision. cnps were adjudicated internally at the icss trial office and judged to be disabling if the patient's score on the modified rankin scale (mrs) increased to 3 or more at 30 days after the procedure, where that increase was attributable to the cnp. investigators were additionally asked to complete a questionnaire (appendix i) giving details of the clinical consequences of cnp and whether or not the lesion resolved during subsequent follow - up in the trial. haematoma was classified as severe if it required re - operation, transfusion, or prolonged hospital stay. the data were analysed per - protocol; only patients in whom the randomly allocated procedure was initiated were included in this analysis. a procedure was deemed to have been initiated if the patient underwent either local or general anaesthesia prior to commencement of surgery. risk factors for cnp and haematoma were examined sequentially in a univariable binomial regression analysis using maximum likelihood estimation. wald tests were used for continuous and binary predictors, with an overall likelihood ratio test for categorical predictors of more than two levels. stata statistical software: release 12. college station, tx: statacorp lp ). patients over 40 years old were eligible for randomization in icss if they had more than 50% recently symptomatic carotid stenosis suitable for either cas or cea, and were clinically stable. patients were excluded if they had a major stroke with poor recovery of function, if their vascular anatomy rendered cas or cea unsuitable, if the stenosis was caused by non - atheromatous disease, if cardiac bypass was planned within 1 month of the revascularization procedure, or if there had been previous revascularization of the symptomatic artery. carotid endarterectomy in icss was performed according to the surgeon's usual practice: local, general, or combined anaesthesia was allowed for the procedure. the type of arterial reconstruction to be carried out was not specified in the protocol, nor was the choice of peri - procedural medication. technical details of the surgical procedure and the occurrence of cranial nerve palsy or haematoma were reported by trial investigators. all patients were then re - assessed at 1 month after the procedure by a neurologist or investigator under their supervision. cnps were adjudicated internally at the icss trial office and judged to be disabling if the patient's score on the modified rankin scale (mrs) increased to 3 or more at 30 days after the procedure, where that increase was attributable to the cnp. investigators were additionally asked to complete a questionnaire (appendix i) giving details of the clinical consequences of cnp and whether or not the lesion resolved during subsequent follow - up in the trial. haematoma was classified as severe if it required re - operation, transfusion, or prolonged hospital stay. the data were analysed per - protocol; only patients in whom the randomly allocated procedure was initiated were included in this analysis. a procedure was deemed to have been initiated if the patient underwent either local or general anaesthesia prior to commencement of surgery. risk factors for cnp and haematoma were examined sequentially in a univariable binomial regression analysis using maximum likelihood estimation. wald tests were used for continuous and binary predictors, with an overall likelihood ratio test for categorical predictors of more than two levels. stata statistical software: release 12. college station, tx: statacorp lp ). patients over 40 years old were eligible for randomization in icss if they had more than 50% recently symptomatic carotid stenosis suitable for either cas or cea, and were clinically stable. patients were excluded if they had a major stroke with poor recovery of function, if their vascular anatomy rendered cas or cea unsuitable, if the stenosis was caused by non - atheromatous disease, if cardiac bypass was planned within 1 month of the revascularization procedure, or if there had been previous revascularization of the symptomatic artery. carotid endarterectomy in icss was performed according to the surgeon's usual practice: local, general, or combined anaesthesia was allowed for the procedure. the type of arterial reconstruction to be carried out was not specified in the protocol, nor was the choice of peri - procedural medication. technical details of the surgical procedure and the occurrence of cranial nerve palsy or haematoma were reported by trial investigators. all patients were then re - assessed at 1 month after the procedure by a neurologist or investigator under their supervision. cnps were adjudicated internally at the icss trial office and judged to be disabling if the patient's score on the modified rankin scale (mrs) increased to 3 or more at 30 days after the procedure, where that increase was attributable to the cnp. investigators were additionally asked to complete a questionnaire (appendix i) giving details of the clinical consequences of cnp and whether or not the lesion resolved during subsequent follow - up in the trial. haematoma was classified as severe if it required re - operation, transfusion, or prolonged hospital stay. the data were analysed per - protocol; only patients in whom the randomly allocated procedure was initiated were included in this analysis. a procedure was deemed to have been initiated if the patient underwent either local or general anaesthesia prior to commencement of surgery. risk factors for cnp and haematoma were examined sequentially in a univariable binomial regression analysis using maximum likelihood estimation. wald tests were used for continuous and binary predictors, with an overall likelihood ratio test for categorical predictors of more than two levels. of 858 patients randomized to cea, the allocated procedure was initiated in 821 (95.7%). four of 821 patients (0.5%) died between initiation of the procedure and 30 days post - procedure. forty - five of 821 patients (5.5% of initiated ceas) were reported to have cnp within 30 days of the procedure. the of adjudication of which cranial nerves were affected are presented in table 1. a total of 50 cnps were reported: facial (n = 23), vagus, hypoglossal, glossopharyngeal, accessory, and trigeminal; in two patients it was not possible to determine which cranial nerve was affected. one cnp was judged to be disabling with an mrs of 3 at 1-month follow - up. this patient had glossopharyngeal nerve palsy with impairment in swallowing requiring placement of a naso - gastric feeding tube. two cranial nerve palsies were reported in each of five trial participants. in those patients with cnp where symptomatic resolution was confirmed (n = 20), the median duration of symptoms before resolution was 30 days (minimum 2 days, maximum 520 days). the exact duration of symptoms in the remainder was undetermined. in only two of 821 patients (0.2%) were the symptoms reported to have not resolved during follow - up of 6.4 and 3.1 years, respectively. one of these patients experienced voice hoarseness caused by vocal cord paresis following vagal nerve injury. the other experienced uplifting of the mouth on one side as a of facial nerve injury. 1. statistically significant predictors of cnp in univariable analysis were female sex (risk ratio 1.90, 95% ci 1.08 to 3.36, p = .03) and a high degree of contralateral carotid artery stenosis. other demographic and technical factors, including the type of arterial reconstruction, type of anaesthesia or shunt use did not predict cnp. independent predictors of cnp in multivariable analysis, summarised in table 2, were cardiac failure (rr 2.66, 95% ci 1.11 to 6.40, p = .03), female sex (rr 1.80, 95% ci 1.02 to 3.20, p = .04), the degree of contralateral carotid stenosis, and time to operation of > 14 days after the day of randomization (rr 3.33, 95% ci 1.05 to 10.57, p = .04). of 821 patients in whom the surgical procedure was initiated, 50 (6.1%) developed neck haematoma. the of univariable regression analysis for the risk factors for haematoma development are presented in appendix ii. statistically significant predictors of increased risk of haematoma were: anticoagulant prescription preoperatively (rr 1.83, 95% ci 1.04 to 3.23, p = .04), previous cardiac bypass graft surgery (cabg) (rr 2.46, 95% ci 1.37 to 4.42, p < .01), atrial fibrillation (rr 2.29, 95% ci 1.08 to 4.85, p = .03), and the duration of arterial clamping in minutes (rr per each extra 20 minutes 1.13, 95% ci 1.04 to 1.24, p < .01). factors associated with a decreased risk of postoperative haematoma were shunt use (rr 0.54, 95% ci 0.29 to 0.99, p = .05), antiplatelet agent prescription prior to the procedure (rr 0.44, 95% ci 0.21 to 0.93, p = .03) and each 1 mmol / l increase in cholesterol at baseline (rr 0.69, 95% ci 0.55 to 0.88, p < .01). the of multivariable analysis of predictors of risk for haematoma are presented in appendix iii. independent predictors of increased risk were being female (rr 2.03, 95% ci 1.13 to 3.62, p = .02), having atrial fibrillation (rr 2.38, 95% ci 1.07 to 5.27, p = .03), and the prescription of anticoagulant pre - procedure (rr 1.86, 95% ci 1.01 to 3.42, p = .05). independent factors reducing the risk of haematoma were shunt use (rr 0.40, 95% ci 0.21 to 0.80, p < .01) and each 1 mmol / l increase in the patient's baseline cholesterol level (rr 0.68, 95% ci 0.54 to 0.86, p < .01). twelve of 45 (26.7%) patients with cnp also suffered haematoma, versus 38/776 (4/9%) of patients without cnp. there was a significant association between these complications as detailed in table 3 (p < .01, fisher 's exact test). table 4 details the impact of adding cnp to the combined incidence of stroke, myocardial infarction (mi), or death in icss in a post - hoc analysis comparing cea with cas. there was no significant difference in the combined risk of stroke, mi, death, or cnp, nor was there a significant difference in the incidence of disabling stroke, disabling cnp, or death between the two trial arms. of 858 patients randomized to cea, the allocated procedure was initiated in 821 (95.7%). four of 821 patients (0.5%) died between initiation of the procedure and 30 days post - procedure. forty - five of 821 patients (5.5% of initiated ceas) were reported to have cnp within 30 days of the procedure. the of adjudication of which cranial nerves were affected are presented in table 1. a total of 50 cnps were reported: facial (n = 23), vagus, hypoglossal, glossopharyngeal, accessory, and trigeminal; in two patients it was not possible to determine which cranial nerve was affected. one cnp was judged to be disabling with an mrs of 3 at 1-month follow - up. this patient had glossopharyngeal nerve palsy with impairment in swallowing requiring placement of a naso - gastric feeding tube. two cranial nerve palsies were reported in each of five trial participants. in those patients with cnp where symptomatic resolution was confirmed (n = 20), the median duration of symptoms before resolution was 30 days (minimum 2 days, maximum 520 days). the exact duration of symptoms in the remainder was undetermined. in only two of 821 patients (0.2%) were the symptoms reported to have not resolved during follow - up of 6.4 and 3.1 years, respectively. one of these patients experienced voice hoarseness caused by vocal cord paresis following vagal nerve injury. the other experienced uplifting of the mouth on one side as a of facial nerve injury. 1. statistically significant predictors of cnp in univariable analysis were female sex (risk ratio 1.90, 95% ci 1.08 to 3.36, p = .03) and a high degree of contralateral carotid artery stenosis. other demographic and technical factors, including the type of arterial reconstruction, type of anaesthesia or shunt use did not predict cnp. independent predictors of cnp in multivariable analysis, summarised in table 2, were cardiac failure (rr 2.66, 95% ci 1.11 to 6.40, p = .03), female sex (rr 1.80, 95% ci 1.02 to 3.20, p = .04), the degree of contralateral carotid stenosis, and time to operation of > 14 days after the day of randomization (rr 3.33, 95% ci 1.05 to 10.57, p = .04). of 821 patients in whom the surgical procedure was initiated, 50 (6.1%) developed neck haematoma. the of univariable regression analysis for the risk factors for haematoma development are presented in appendix ii. statistically significant predictors of increased risk of haematoma were: anticoagulant prescription preoperatively (rr 1.83, 95% ci 1.04 to 3.23, p = .04), previous cardiac bypass graft surgery (cabg) (rr 2.46, 95% ci 1.37 to 4.42, p < .01), atrial fibrillation (rr 2.29, 95% ci 1.08 to 4.85, p = .03), and the duration of arterial clamping in minutes (rr per each extra 20 minutes 1.13, 95% ci 1.04 to 1.24, p < .01). factors associated with a decreased risk of postoperative haematoma were shunt use (rr 0.54, 95% ci 0.29 to 0.99, p = .05), antiplatelet agent prescription prior to the procedure (rr 0.44, 95% ci 0.21 to 0.93, p = .03) and each 1 mmol / l increase in cholesterol at baseline (rr 0.69, 95% ci 0.55 to 0.88, p < .01). the of multivariable analysis of predictors of risk for haematoma are presented in appendix iii. independent predictors of increased risk were being female (rr 2.03, 95% ci 1.13 to 3.62, p = .02), having atrial fibrillation (rr 2.38, 95% ci 1.07 to 5.27, p = .03), and the prescription of anticoagulant pre - procedure (rr 1.86, 95% ci 1.01 to 3.42, p = .05). independent factors reducing the risk of haematoma were shunt use (rr 0.40, 95% ci 0.21 to 0.80, p < .01) and each 1 mmol / l increase in the patient's baseline cholesterol level (rr 0.68, 95% ci 0.54 to 0.86, p < .01). twelve of 45 (26.7%) patients with cnp also suffered haematoma, versus 38/776 (4/9%) of patients without cnp. there was a significant association between these complications as detailed in table 3 (p < .01, fisher 's exact test). table 4 details the impact of adding cnp to the combined incidence of stroke, myocardial infarction (mi), or death in icss in a post - hoc analysis comparing cea with cas. there was no significant difference in the combined risk of stroke, mi, death, or cnp, nor was there a significant difference in the incidence of disabling stroke, disabling cnp, or death between the two trial arms. in icss, cnp developed in 5.5% of icss patients undergoing cea, and haematoma in 6.1%. the largest available series of patients studied pre- and post - operatively, in the european carotid surgery trial (ecst), found a motor cnp rate of 5.1% and a long - term cnp rate of 0.5% at 4 months. likewise, nascet reported an overall risk of postoperative cnp of 8.6%, of which the majority were mild in severity, suggesting that cnp rates remain constant over time. patients should be made aware of these common complications and the likely clinical effects, including sensory and possible motor consequences. they can be reassured from the evidence presented here that postoperative cnp is rarely disabling (risk around 1 in 1000 operations), but should be warned that the symptoms of the cnp may persist for several weeks or longer. there are several other findings of interest from this study: to the authors' knowledge, the association between female sex and cnp has not previously been described, and this finding is worth confirming in another cohort of patients. one possible explanation for higher risk in female patients may be more challenging surgical anatomy and the smaller average diameter of the carotid artery. combined with other reports of a higher perioperative stroke risk in symptomatic women undergoing cea versus men, and evidence that the net benefit of cea in women is lower than in men, an increased incidence of cnp should be borne in mind when advising female patients of their risk of complications following cea. other risk factors for cnp identified in this analysis, including the degree of contralateral carotid stenosis, are statistically significant predictors but are more difficult to link clinically with the outcome and have not, to the authors' knowledge, been reported by other groups. the association of neck haematoma with pre - operative anticoagulation is not surprising, but emphasizes the importance of careful surgical technique to mitigate the risk of haematoma in anticoagulated patients. a risk of haematoma in icss of 6.1% is similar to the rate of wound complications seen in large case series and a severe haematoma risk of 3.4% is similar to the reported risk of re - exploration of the surgical wound required in patients undergoing cea while on antiplatelet medication. however, although there is concern about perioperative bleeding in patients on dual antiplatelet therapy, the incidence of haematoma was actually decreased in this study in patients taking these medications, perhaps because surgeons took more care with haemostasis in these patients. some systematic reviews have included cnp in a composite outcome event of death or neurological complications up to 30 days after treatment. icss compared cas with cea on the assumption that if cas could avoid neck incision, cnp, and haematoma with no excess risk of stroke, then it could provide a beneficial alternative to cea for the prevention of recurrent stroke in patients with symptomatic carotid stenosis. in icss, as reported here, the total number of events in the composite cluster of any stroke, mi, death, or cnp was greater after cea compared with cas, but the numbers in the cluster of disabling stroke, disabling cnp or death were greater after cas than after cea. it is concluded, therefore, that it would not be appropriate to base treatment considerations concerning the choice of cas versus cea on the basis of composite short - term endpoints including cnp. information regarding baseline risk factors was unavailable, and information about the duration of cnp symptoms was limited. multiple comparisons without statistical correction raise the possibility of obtaining a type i (false positive) error. this is not a randomized comparison of surgical techniques or perioperative processes of care, and it is possible that unmeasured confounders are associated with the risk of cnp or haematoma. cnp remains a relatively common complication of cea, but in many patients is transient. haematoma is similarly common, and there is a statistical association between haematoma and cnp. fortunately, prolonged disability or permanent symptoms as a of haematoma or cnp are rare, and thus, in the authors' opinion, do not warrant inclusion in composite endpoints for future trials of carotid revascularization, but nevertheless one in 821 cea patients in icss had permanent impairment of swallowing caused by cranial nerve palsy. information regarding baseline risk factors was unavailable, and information about the duration of cnp symptoms was limited. multiple comparisons without statistical correction raise the possibility of obtaining a type i (false positive) error. this is not a randomized comparison of surgical techniques or perioperative processes of care, and it is possible that unmeasured confounders are associated with the risk of cnp or haematoma. cnp remains a relatively common complication of cea, but in many patients is transient. haematoma is similarly common, and there is a statistical association between haematoma and cnp. fortunately, prolonged disability or permanent symptoms as a of haematoma or cnp are rare, and thus, in the authors' opinion, do not warrant inclusion in composite endpoints for future trials of carotid revascularization, but nevertheless one in 821 cea patients in icss had permanent impairment of swallowing caused by cranial nerve palsy. icss was funded by the uk medical research council (mrc) and managed by the uk national institute for health research (nihr) on behalf of the mrc - nihr partnership (grant numbers g0300411 and eme 09/800/14 respectively). the views and opinions expressed herein are those of the authors and do not necessarily reflect those of the nihr health services research programme of the department of health. additional funding was supplied by grants from the stroke association (grant numbers tsa2005/01 and tsa2007/12), sanofi - synthelabo and the european union. mmb's chair in stroke medicine is supported by the reta lila weston trust for medical research. this work was undertaken at university college london, which received a proportion of funding from the uk department of health's national institute for health research biomedical research centres funding scheme. the funders and sponsors of the study had no role in study design, data collection, data analysis, data interpretation or the writing of this paper.

objectivecranial nerve palsy (cnp) and neck haematoma are complications of carotid endarterectomy (cea). the effects of patient factors and surgical technique were analysed on the risk, and impact on disability, of cnp or haematoma in the surgical arm of the international carotid stenting study (icss), a randomized controlled clinical trial of stenting versus cea in patients with symptomatic carotid stenosis.materials and methodsa per - protocol analysis of early outcome in patients receiving cea in icss is reported. haematoma was defined by the surgeon. cnp was confirmed by an independent neurologist. factors associated with the risk of cnp and haematoma were investigated in a binomial regression analysis.of the patients undergoing cea, 45/821 (5.5%) developed cnp, one of which was disabling (modified rankin score = 3 at 1 month). twenty - eight (3.4%) developed severe haematoma. twelve patients with haematoma also had cnp, a significant association (p < .01). independent risk factors modifying the risk of cnp were cardiac failure (risk ratio 2.66, 95% ci 1.11 to 6.40), female sex (rr 1.80, 95% ci 1.02 to 3.20), the degree of contralateral carotid stenosis, and time from randomization to treatment > 14 days (rr 3.33, 95% ci 1.05 to 10.57). the risk of haematoma was increased in women, by the prescription of anticoagulant drugs pre - procedure and in patients with atrial fibrillation, and was decreased in patients in whom a shunt was used and in those with a higher baseline cholesterol level.cnp remains relatively common after cea, but is rarely disabling. women should be warned about an increased risk. attention to haemostasis might reduce the incidence of cnp. icss is a registered clinical trial: isrctn 25337470.

the transobturator approach to treat female stress urinary incontinence by means of a midurethral sling (mus) placement was first described by delorme to potentially prevent complications associated with the retropubic mus placement originally described by ulmsten (delorme, 2001 ; ulmsten et al ., 1996). a similar transobturator passage utilizing an inside - out approach, as opposed to delorme s outside - in approach, was later described by de leval, aiming to further reduce the potential injury to the urethra and bladder (de leval, 2003). a recent meta - analysis found similar cure rates between the 3 most commonly used surgical approaches for treating stress urinary incontinence, namely the retropubic, outside - in transobturator, and inside - out transobturator mus procedures (latthe et al ., 2010). multiple studies have discussed the anatomical trajectories of both obturator approaches and confirmed their anatomical safety from a neurovascular perspective (bonnet et al ., 2005 ; achtari et al ., 2006 ; hinoul et al . , 2007 ; zahn et al ., 2007 ; reisenauer et al ., 2006). the large body of clinical evidence supports these findings. to date, anatomical studies have focused on the mus s trajectory in relation to the obturator foramen, the muscles and especially the neuro - vascular structures. traditional anatomical dissections do not allow the study of the mus s configuration in relation to the urethra, as a progressive dissection of the entire tape s trajectory would be required, inherently leading to a distortion of the relevant anatomy. the aim of this cadaveric study was to compare the specific relationship between the tape and the urethra in both the inside - out and outside - in approach. to allow such a comparison, ct - scan image analysis was performed after tantalum wire marked mesh slings were implanted in a series of cadavers. all 10 cadavers were operated upon, by independent surgeons, one experienced with the inside - out (in - out) and one with cadavers were positioned in the dorsal supine lithotomy position with the legs in abduction and at a 100 flexion position (at the level of the hips) on the in - outside and at 90 flexion on the out - in side. the surgical technique for the transobturator mus procedures were performed according to the instructions for use as provided by the respective manufacturers (out - in : monarc, american medical systems, minnetonka, mn and in - out : tvt - obturator system, ethicon inc ., all cadavers were catheterized using a foley catheter with its lumen filled with a radio - opaque solution ( hypaquemeglumine 60%, amersham health, princeton, nj) to allow subsequent identification of the urethral position. both the in - out and the out - in transobturator procedures were performed on each cadaver, alternating the type of procedure and tape between the right and left sides in the consecutively operated cadavers. to be able to do this with a single tape in each cadaver, a device was conceived for this study in which the tape on one side consisted of an in - out mus device, while an out - in mus device was attached to the other side at the midpoint. to allow radiographic visualization of these tapes, a double tantalum wire was threaded through each side of both midurethral tapes as shown in figure 1. this design allowed for an intra - cadaveric comparison between 10 independent in - out transobturator mus trajectories and 10 out - in transobturator mus trajectories. care was taken to have a flat positioning of both tape ends at the end of the procedures. the vaginal incision sites were closed and the mus tapes were cut 2 cm from the level of the skin. to exclude the possible influence of the surgeon s right handedness, an equal number of both procedures was performed on the right and left sides; bmi of the cadavers ranged between 15 and 33 kg / m2 (mean : 25.3 ; median : 26). post capture image analysis was performed using the vitrea core 5.1.1618.1808 image analysis software (vital, 5850 opus parkway, suite 300, minnetonka, mn 55343 - 4414). plane that included the urethra at the point where it crossed the urethra and the two arms of the sling extending from the urethra to the point where it crossed the obturator membrane. this was done to prevent parallax effects that might alter the measured angle (hoyte and ratiu, 2001). on the mips view of the images, the relative angulations between a line through the urethral axis and the slope drawn through each mus tape s mean upward pathway (forming the hammock part of its trajectory) were measured (table i). the angle inside the upward trajectory of the tape constituted the angle under consideration for this study and is referred to as the tape s hemi inner angle, as only half a procedure was performed on each cadaver (fig . for the study s purposes the assumption was made that a symmetric, bilateral placement of a full length tape would yield the full inner angle of the mus hammock corresponding to double the hemi - inner angle measurement . 2a . ventro - dorsal view ( frontal view ; l = left, r = right). note: dotted lines schematically demonstrates the hemi - inner angle but do not correspond to the mips view used to measure the actual angles. statistical analyses were performed using the wilcoxon signed - rank test for matched paired data for non - parametric testing of 2 dependent samples. statistical analyses were performed using the wilcoxon signed - rank test for matched paired data for non - parametric testing of 2 dependent samples. three cadavers were noted to have stage 2 prolapse and 1 had a notably high vaginal sulcus. the 3-dimensional ct images revealed that the mus tapes lie as a band posterior / dorsal to the urethra rather than inferior. the out - in muss were more closely wrapped around the inferior ischio - pubic ramus than the in - out muss. qualitative assessment of the images demonstrated a safe distance from the tapes to the obturator canal for both techniques. distances of the tapes trajectories to the individual obturator nerve branches could not be determined. the mean full inner angle in a strict antero - posterior view formed by the in - out mus measured 122 (95%ci : 107-136); versus 144 (95%ci : 131-151) for the out - in mus (p = 0.02). the (paired) differences between the tapes inner angles were significantly different; with a mean difference of 19.8 (median 19.0), (wilcoxon signed rank test : p = 0.008). correlation analysis only showed a trend between the out - in and the in - out mus angle (correlation coefficient : 0.6, p = 0.08). a similar trend was observed between the patient s bmi and the in - out mus angle (correlation coefficient : 0.6, p = 0.08), but not for bmi and the out - in mus angle (correlation coefficient : 0.08, p = 0.8). there was no correlation between the infra - pubic angle measurement and the out - in mus angle nor the in - out mus angle. the use of tantalum wires threaded through the polypropylene-/ prolene - tape in combination with three - dimensional ct - scanning yielded a unique method to visualize midurethral tapes throughout the entirety of their trajectory. this technique can be applied to different types of non radio - opaque implants, allowing for a better spatial understanding of the anatomical position within the pelvis. this technique circumvents the technical problems encountered by ultrasound where visualization is partially obstructed by the bony pelvis and the fact that traditional anatomical techniques rely on a progressive dissection intrinsically only allowing visualization at a single level each time. this is the first controlled anatomical study to compare the spatial relationships between the in - out and out - in mus tapes and the female urethra. the difference could be objectified by measuring the angle formed between the two legs of the mus tape. the in - out approach angulated around the urethra in a more acute fashion than the out - in approach, which seemed to follow a more horizontal, flatter, trajectory (122 versus 142). these findings were counter - intuitive as the exit - points of both devices at the level of the skin would suggest the opposite. the three dimensional radiographic visualization of the tape s trajectory also allowed for an appreciation of the mus tapes spatial configuration in relation to the bony pelvis and the urethra. contrary to the belief that the urethra is suspended in a cranially directed u - shaped band, both obturator mus approaches were demonstrated to be u - shaped bands that head backwards or dorsally. the tape is not positioned caudally but lies as a band behind the urethra, suggestive of its passive (non obstructive) role in achieving continence. as observed in the different measurements of the tapes angles, there seemed to be a higher variability in the in - out s than the out - in trajectory (58 versus 40 variation respectively), as previously described by hinoul et al. in a traditional anatomic study, underscoring the need for strict adherence to the prescribed operative technique (hinoul et al ., 2007). the importance of this becomes clear when comparing it to two ultrasound studies investigating the angles formed by the two arms of mus tapes. lin et al described an angle at rest for the in - out tape to be 115 (sd13) while chene et al, measured the same angle at rest also using ultrasound to be 138(sd7) (lin et al . the differences in outcomes obtained in these studies demonstrate that ( inter- and intra - individual) comparisons may prove to be difficult to interpret. the cadaveric study design was also the study s limitation as no clinical findings could be correlated to the static anatomic findings. a direct meta - analysis comparing both routes of transobturator tapes by madhuvrata et al. reported no significant differences in the objective and subjective efficacy but showed that the inside - out route was associated with significantly fewer vaginal angle injuries but with a non - significanttrend towards higher risk of postoperative groin pain. in a small retrospective study, out - in mus tapes were more frequently associated with a finding of superficial placement at the level of the lateral vaginal sulcus, described as paraurethral banding (cholhan et al ., 2010). coincidentally, the only cadaver with a notably high vaginal sulcus was found to have a very palpable out - in mus tape post placement. increased rates of dyspareunia and tape exposures after the out - in compared to the in - out approach , but these finding were not confirmed in an rct by abdel - fattah et al. the more horizontal positioning of the out - in tape s trajectory, taking the mesh closer to the vaginal wall, as described in this study could explain these clinical observations. the radiographic images explain the increased risk of button - holing during an out - in procedure because the more acute in - out mus trajectory buries the tape deeper inside the internal adductor muscles. on the other hand , this study also confirmed that the out - in mus trajectory stayed closer to the ischiopubic ramus. the slightly more lateral in - out mus trajectory causes the tape to be seated in more muscular tissue. the corresponding inflammatory reaction might explain the trend to a slightly higher risk of transient thigh pain. the clinical implications from this cadaveric imaging study are that surgeons must pay more attention to the 2nd half of the helical pass, whichever way they chose to go. for the in - out passage , one must rotate hard and carefully around the ischiopubic ramus and not get lazy with the rotation; while for the out - in pass, care must be taken that dissection of the peri - urethral tunnel provides for a full thickness vaginal epithelial layer. the advent of three - dimensional ultrasonography will allow for similar measurements of the different tapes trajectories in vivo in a dynamic setting, possibly allowing correlation of these findings to nuances in clinical outcomes. ultrasound will not be able to provide the same holistic visualization within the bony pelvis, but mesh - tape beyond the obturator membrane probably does not contribute to the mus functional effect or possible adverse events at the level of the vagina. recently, a new approach, incorporating iron particles into polymer - based implants, allowed in vivo visualization of mesh by magnetic resonance imaging (mri) following inguinal hernia surgery (hansen et al ., 2013). approval of these types of products for human use in the future will bring new insights into the action mechanism of mesh in the pelvic floor as well as a better understanding of the mesh- tissue interaction.

the three - dimensional configuration of mid - urethral sling tapes is difficult to demonstrate in traditional anatomical dissections or imaging studies. the aim of this study was to test the utility of a novel technique using mesh tapes to assess spatial differences between the in - out and out - in transobturator mid - urethral slings. two independent surgeons performed their usual transobturator mid - urethral sling placement on 10 fresh thawed cadavers, alternating sides in the consecutive cadavers. tantalum wires threaded through the polypropylene - tapes rendered them radio - opaque. following placement, ct scans were obtained to generate 3-d and mips images for analysis. showed that the mean angle formed by the in - out sling measured 122 (95%ci : 107-136); versus 144 (95%ci : 131-151) for the out - in sling (p = 0.02). the paired differences between the tapes inner angles were significantly different; with a mean difference of 20 (median 19.0), (p = 0.008). there was no significant correlation between either approach and bmi or angle of the pubic arch. the images revealed that the tapes lie as a band posterior / dorsal to the urethra rather than inferior. in : marking mesh with tantalum wire, in combination with 3-d and mips ct - scan reconstruction images, provided a unique method to visualize the entire sling trajectory. the clinical implications of the more horizontal positioning after the out - in approach remain to be determined.

neurocutaneous melanosis (ncm) is a rare neurocutaneous syndrome characterized by the presence of multiple congenital melanocytic nevi (cmn) and the proliferation of melanocytes in the central nervous system, usually involving the leptomeninges. a 32-year - old man suffering from chronic partial epilepsy presented with multiple cmn on his trunk and scalp. brain mri demonstrated a focal lesion in the right amygdala that was consistent with interictal epileptiform discharges in the right temporal region on electroencephalography (eeg). an anterior temporal lobectomy was performed, and the pathology investigation revealed numerous melanophages in the amygdala. we report a patient with ncm presenting as chronic partial epilepsy who was successfully treated by anterior temporal lobectomy. neurocutaneous melanosis (ncm) is a rare congenital, noninheritable neurocutaneous syndrome, characterized by the presence of large and/or multiple congenital melanocytic nevi (cmn) associated with melanosis or melanoma that usually appear in the leptomeninges. although the clinical presentations of ncm vary with age, ncm is generally accompanied by significant neurological deficits. however, ncm as a cause of epilepsy without any neurological deficit is extremely rare. we report a case of chronic partial epilepsy caused by pathologically proven parenchymal ncm in the brain without leptomeningeal involvement, that was successfully treated surgically. a 32-year - old right - handed man visited our epilepsy clinic due to drug - resistant partial epilepsy. his seizures started at the age of 22 years, since when he had suffered from frequent episodes of disgusting odor and light - headedness despite antiepileptic drug treatment. also, he had occasionally experienced motionless staring followed by generalized tonic - clonic seizures when he had not complied with treatment. his familial and own medical histories were not remarkable, and the findings of a neurological examination were normal. electroencephalography (eeg) showed frequent right temporal sharp waves with maximal negativity in the ipsilateral nasopharyngeal electrode. brain mri showed a small focal lesion in the lateral part of the right amygdala. the lesion was hyperintense on a t2-weighted image and hypointense on a t1-weighted image without gadolinium enhancement (fig . 1), which suggested a benign tumor.1 there were multiple large black pigmented nevi on the back, scalp, and posterior neck area, which had existed from birth. the largest black nevus was on his back, covering 9% of his body surface area (1.82 m). the amygdala lesion was a grayish - to - black friable soft tissue without definite mass formation. the skin biopsy of the scalp lesion showed that it was an intradermal nevus, as expected (fig . 2). he was seizure - free (including aura) during the 15-month postsurgery follow - up period. kadonaga and frieden2 proposed the following criteria in 1991 that are currently used to diagnose ncm: 1 ) large and/or multiple cmn in association with meningeal melanosis or melanoma; 2 ) no evidence of cutaneous melanoma, except in patients with histologically benign meningeal lesions; and 3 ) no evidence of meningeal melanoma, except in patients with histologically benign cutaneous lesions. the second and third definitions exclude the possibility of metastases from the skin to the central nervous system (cns) and vice versa, respectively. cmn are classified into large, medium, and small nevi according to their size:3 larger than 20 cm in an adult, 1.5 to 19.9 cm, and smaller than 1.5 cm, respectively. kadonaga and freiden2 found that 66% of ncm patients had large nevi, and the remaining 34% had numerous pigmented lesions in the absence of a single large congenital melanocytic nevus. in their study, all ncm patients had either posterior midline nevi or head and neck lesions, which suggests that the posterior axial distribution is an important risk factor for developing ncm. embryologically, melanocytes originate from the neural crest and migrate to various sites in the body, such as the basal layer of the epidermis, pia mater, leptomeninges, medullary reticular formation, and substantia nigra of the midbrain.4 it is thought that cmn and ncm are caused by the arrest or aberrancy of this normal developmental pattern. in ncm, pigmented thickening of the leptomeninges is most frequent in areas with melanocytes.4 parenchymal melanosis is less common than leptomeningeal melanosis, and caused by primary involvement of the melanin - containing macrophages and melanocytes, or secondary spread via virchow - robin spaces from the leptomeninges. the pathology investigation of our patient showed melanin - containing macrophages, and revealed parenchymal melanosis without leptomeningeal melanosis. the neurological manifestations of ncm vary with age.2 before the age of 2 years, the most common initial clinical signs and symptoms of ncm are related to increased intracranial pressure, including headache (35%), vomiting (42%), generalized seizures (48%), increased head circumference (23%), cranial nerve palsies (26% ; in particular vi), papilledema (10%), and meningeal signs (3%).6 the subset of patients with a discrete intracranial mass becomes symptomatic when older (mean age, 12.8 years ; range, from birth to 65 years) and is more likely to develop focal seizures, localized sensorimotor deficits, difficulties with speech, or psychiatric symptoms.6 our patient became symptomatic with partial seizures when he was 22 years old. most of the 33 ncm patients in a study involving giant cmn7 demonstrated the first neurologic symptom when they were younger than 5 years. eighty - two percent of the patients died by the time of reporting, and the median age at death was 3 years. while symptomatic ncm is estimated to occur in less than 3% of patients with giant cmn,7 one study found mri evidence of cns melanosis in 10 (23%) of 42 asymptomatic giant - cmn patients,8 among whom only 1 had subsequently developed symptomatic disease during an average follow - up of 5 years. because the mri findings of these 10 patients showed parenchymal ncm without diffuse leptomeningeal involvement, the prognosis of parenchymal ncm appears to be much better than that of typical ncm with diffuse leptomeningeal involvement or melanoma. melanin pigment is inherently paramagnetic, and hence melanin deposits in ncm show a high signal intensity on t1-weighted mri and a low signal intensity on t2-weighted mri. this pattern of parenchymal melanosis appears to be most evident and frequent at the amygdala and adjacent anterior temporal cortex.8 in our patient, brain mri showed the typical location but an atypical pattern of hyperintensity on t2-weighted images and isointensity on t1-weighted images. some cases with chronic epilepsy related to ncm have been reported, but most of them had other conditions such as cns melanoma, mental retardation, psychosis, and diffuse leptomeningeal involvement with neurologic deficits.9 to our knowledge, the only surgical case with ncm similar to our patient was a 29-year - old woman with normal psychomotor development and intelligence who had right temporal - lobe epilepsy due to amygdaloid melanosis and underwent right temporal resection.10 here we have reported a surgically successful case of chronic partial epilepsy caused by pathologically proven parenchymal ncm without leptomeningeal involvement. therefore, ncm occurring in the brain parenchyma should be considered as a cause of chronic partial epilepsy.

neurocutaneous melanosis (ncm) is a rare neurocutaneous syndrome characterized by the presence of multiple congenital melanocytic nevi (cmn) and the proliferation of melanocytes in the central nervous system, usually involving the leptomeninges. chronic partial epilepsy as a sole manifestation is rare in ncm.case reporta 32-year - old man suffering from chronic partial epilepsy presented with multiple cmn on his trunk and scalp. brain mri demonstrated a focal lesion in the right amygdala that was consistent with interictal epileptiform discharges in the right temporal region on electroencephalography (eeg). an anterior temporal lobectomy was performed, and the pathology investigation revealed numerous melanophages in the amygdala. the patient was seizure - free after surgery.we report a patient with ncm presenting as chronic partial epilepsy who was successfully treated by anterior temporal lobectomy.

maternal thyroid function changes during pregnancy and inadequate adaptation to these changes in thyroid dysfunction. some of these alterations in thyroid function occur due to increased thyroid hormone - binding globulin (tbg) concentration, increased iodine clearance in the kidneys, and thyrotrophic effect of human chorionic gonadotropin (hcg). in previous studies, the prevalence of overt hypothyroidism was 1% to 1.5%, and prevalence of subclinical hypothyroidism was 5% to 8%. the main pregnancy complications of hypothyroidism were anemia, preeclampsia, prematurity, low - birth weight (lbw), fetal distress in labor, fetal death, and congenital hypothyroidism, and neurocognitive deficits in children. subclinical hypothyroidism might be associated with preterm delivery and low apgar score. overt hyperthyroidism and subclinical hyperthyroidism affects about 0.2% to 0.8% and 0.4% to 1% of pregnancies, respectively. maternal hyperthyroidism may cause preterm delivery, intrauterine growth restriction (iugr), and neonatal thyrotoxicosis. according to the last published guidelines on 2012 for the management of thyroid dysfunction during pregnancy and postpartum, " universal screening of healthy women for thyroid dysfunction before pregnancy is not recommended " and " the committee could not reach agreement with regard to screening recommendations for all newly pregnant women. " increased knowledge about the interaction between the thyroid and pregnancy has changed our view about the definition and diagnosis of thyroid dysfunction in pregnancy; for example, we have recently found that thyroid stimulating hormone (tsh) level of 2.5 miu / l in the first trimester has been accepted as the upper limit of normal range. this fact has an important implication in interpretation of the previous literature as well as a critical effect on diagnosis of clinical hypothyroidism. considering these changes, the american thyroid association has published a new guideline in 2011 for an accurate diagnosis and management of thyroid disease in pregnancy. this guideline determined the normal limits of tsh in the second trimester as 0.2 to 3 miu / l and free thyroxin (ft4) as 12 to 30 pmol / l. subsequent to these changes in definition of thyroid diseases in pregnancy, it is necessary to reevaluate the accurate prevalence of thyroid diseases in pregnancy and their mentioned effects in previous studies. recent changes in definition of thyroid diseases in pregnancy has ed in changes in interpretation of previous studies about the prevalence and effect of thyroid diseases on pregnancy outcomes after 2011. in addition, due to lack of sufficient data about the prevalence and pregnancy outcomes of thyroid disease in iran, this prospective study aimed to evaluate the prevalence of thyroid diseases and its outcomes in pregnancy in fars province, south of iran. this prospective study included 600 singleton pregnant women who were in consecutive follow - up at shiraz university of medical sciences obstetric hospitals (zeinabieh hospital and hafez hospital), shiraz, iran. shiraz is the capital city of fars province in the south of iran. in 2000, iran was considered as an iodine sufficient country by the world health organization and in previous reports, iranian pregnant women were also iodine sufficient. informed consent was obtained from each patient included in the study and the study protocol conforms to the ethical guidelines of the 1975 declaration of helsinki as reflected in a priori approval by the shiraz university of medical sciences human research committee. inclusion criteria were healthy singleton pregnancy, residents in fars province, and pregnancy with gestational age of 15 to 28 weeks. in the first visit, individuals at high risk for thyroid illness according to their medical history, physical examination, or prior biochemical information had been considered and the women were excluded if they had known chronic diseases such as thyroid diseases, usage of thyroid drugs, diabetes mellitus, and hypertension. data about maternal age, parity, obstetric history, gestational age (determined by last menstrual period), medical history of thyroid drug usage, and physical examination of the mother were collected twice: in the second trimester of pregnancy and at delivery. physical examination of the neonate was done in delivery room to determine apgar score, resuscitation at birth, weight, head circumference, and length in the hospital. preeclampsia was diagnosed when a pregnant woman develops both of the followings: 1 ) systolic blood pressure > 140 mm hg and/or diastolic blood pressure > 90 mm hg at two separate visits with at least six hour interval in a previously normotensive woman; and 2 ) proteinuria 0.3 g/24 hours. iugr was defined as birth weight 10th percentile for its gestational age. preterm delivery was defined as the delivery before the end of 37th week of gestational age. apgar score was determined by evaluating the newborn on five simple criteria on a scale from zero to two and summing up the five values. the five criteria are appearance, pulse, grimace, activity, and respiration. the first minute apgar score < 7 was defined as low apgar score. for thyroid function tests (tft), 10-ml blood sample of pregnant women was drawn at the first visit in the second trimester; then it was centrifuged and stored in aliquots at -70 until assays, which were done after delivery. tft were assessed by quantitative analysis of serum tsh and ft4 (electrochemiluminescence, cobase411, japan). the inter - assay and intra - assay coefficients of variation were respectively < 8.6% and < 8.7% for tsh; in addition, these values were < 2.9% and < 6.6% for ft4, respectively. tsh was mentioned as miu / l and ft4 as pmol / l. according the last guideline of american thyroid association (ata) for the diagnosis and management of thyroid disease during pregnancy and postpartum, the reference range for the second trimester tsh and ft4 was 0.2 to 3 miu / l and 11.84 3.86 pmol / l, respectively. considering this recommendation and according to ata guideline, we categorized pregnant women into five groups: category 1 (clinical hypothyroidism) was defined as an elevated tsh (> 3 miu / l) in conjunction with a low ft4. in women with tsh 10 miu / l, irrespective of their ft4 level, were also considered as clinical hypothyroidism. category 2 (subclinical hypothyroidism) was defined if serum tsh was between 3 to 10 category 3 (normal), was those with tsh of 0.2 to 3 miu / l and ft4 of 11.84 3.86 to. category 4 (subclinical hyperthyroidism) was defined as suppressed tsh (range, 0.1 - 0.2 miu / l) with normal ft4. category 5 (overt hyperthyroidism) was defined as any suppressed tsh (< 0.2 miu / l) when accompanied by high ft4 and anyone with tsh < 0.1 miu / l irrespective of ft4 level. in addition, we calculated the prevalence of isolated hypothyroxinemia according to ata guideline in which tsh was in normal range but had low ft4. analysis was done using spss 18 (spss inc ., normality of the quantitative variables was assessed by kolmogorov - simonov test . to compare difference across groups, anova was used for normally distributed variables and kruskal - wallis test was used in those without normal distribution . data were mentioned as mean sd or median . for serum tsh, free triiodothyronine ( ft3), and ft4, 2.5th, 25th, 50th, 75th, and 97.5th percentiles as previously mentioned, category 3 was normal thyroid function pregnant women and was used as control group. the influence of thyroid diseases was analyzed by comparing the frequencies of each outcome in the abovementioned categories. of analysis were mentioned as relative risk (rr) and the corresponding 95% confidence interval (95% ci). this prospective study included 600 singleton pregnant women who were in consecutive follow - up at shiraz university of medical sciences obstetric hospitals (zeinabieh hospital and hafez hospital), shiraz, iran. shiraz is the capital city of fars province in the south of iran. in 2000, iran was considered as an iodine sufficient country by the world health organization and in previous reports, iranian pregnant women were also iodine sufficient. informed consent was obtained from each patient included in the study and the study protocol conforms to the ethical guidelines of the 1975 declaration of helsinki as reflected in a priori approval by the shiraz university of medical sciences human research committee. inclusion criteria were healthy singleton pregnancy, residents in fars province, and pregnancy with gestational age of 15 to 28 weeks. in the first visit, individuals at high risk for thyroid illness according to their medical history, physical examination, or prior biochemical information had been considered and the women were excluded if they had known chronic diseases such as thyroid diseases, usage of thyroid drugs, diabetes mellitus, and hypertension. data about maternal age, parity, obstetric history, gestational age (determined by last menstrual period), medical history of thyroid drug usage, and physical examination of the mother were collected twice: in the second trimester of pregnancy and at delivery. physical examination of the neonate was done in delivery room to determine apgar score, resuscitation at birth, weight, head circumference, and length in the hospital. preeclampsia was diagnosed when a pregnant woman develops both of the followings: 1 ) systolic blood pressure > 140 mm hg and/or diastolic blood pressure > 90 mm hg at two separate visits with at least six hour interval in a previously normotensive woman; and 2 ) proteinuria 0.3 g/24 hours. iugr was defined as birth weight 10th percentile for its gestational age. preterm delivery was defined as the delivery before the end of 37th week of gestational age. apgar score was determined by evaluating the newborn on five simple criteria on a scale from zero to two and summing up the five values. the five criteria are appearance, pulse, grimace, activity, and respiration. the first minute apgar score < 7 was defined as low apgar score. for thyroid function tests (tft), 10-ml blood sample of pregnant women was drawn at the first visit in the second trimester; then it was centrifuged and stored in aliquots at -70 until assays, which were done after delivery. tft were assessed by quantitative analysis of serum tsh and ft4 (electrochemiluminescence, cobase411, japan). the inter - assay and intra - assay coefficients of variation were respectively < 8.6% and < 8.7% for tsh; in addition, these values were < 2.9% and < 6.6% for ft4, respectively. according the last guideline of american thyroid association (ata) for the diagnosis and management of thyroid disease during pregnancy and postpartum, the reference range for the second trimester tsh and ft4 was 0.2 to 3 miu / l and 11.84 3.86 pmol / l, respectively. considering this recommendation and according to ata guideline, we categorized pregnant women into five groups: category 1 (clinical hypothyroidism) was defined as an elevated tsh (> 3 miu / l) in conjunction with a low ft4. in women with tsh 10 miu / l, irrespective of their ft4 level, were also considered as clinical hypothyroidism. category 2 (subclinical hypothyroidism) was defined if serum tsh was between 3 to 10 category 4 (subclinical hyperthyroidism) was defined as suppressed tsh (range, 0.1 - 0.2 miu / l) with normal ft4. miu / l ) when accompanied by high ft4 and anyone with tsh < 0.1 miu / l irrespective of ft4 level. in addition, we calculated the prevalence of isolated hypothyroxinemia according to ata guideline in which tsh was in normal range but had low ft4. normality of the quantitative variables was assessed by kolmogorov - simonov test. to compare difference across groups, anova was used for normally distributed variables and kruskal - wallis test was used in those without normal distribution. data were mentioned as mean sd or median. for serum tsh, free triiodothyronine (ft3), and ft4, 2.5th, 25th, 50th, 75th, and 97.5th percentiles as previously mentioned, category 3 was normal thyroid function pregnant women and was used as control group. the influence of thyroid diseases was analyzed by comparing the frequencies of each outcome in the abovementioned categories. of analysis were mentioned as relative risk (rr) and the corresponding 95% confidence interval (95% ci). out of 600 enrolled women in our study, 14 refused to follow the study. ultimately, 586 women participated in our investigation to the end. the mean of participants age was 25.6 3.6 years, mean of their weight was 63.4 9.1 kg, and the mean of their height was 162.5 6.7 cm. fourteen women (2.4%) had clinical hypothyroidism, 66 (11.3%) had subclinical hypothyroidism, 2 (0.3%) had subclinical hyperthyroidism, and 7 (1.2%) had overt hyperthyroidism. general characteristics of the mothers (height, weight, and age) in each category are summarized in table 1. there was no significant difference in general characteristics of mothers in different thyroid function categories (p > 0.05). the mean of the newborns head circumference was 35.4 1.7 cm, mean of their weight was 2599 385 g, and mean of their length was 50.9 2.8 cm. the neonates had an apgar score of 8.4 1.1 at first minute of delivery. general characteristics of the neonates are categorized according to maternal thyroid function in table 2. categories are defined as follows: i, clinical hypothyroidism; ii, subclinical hypothyroidism; iii, euthyroid (control); iv, subclinical hyperthyroidism; v, over hypothyroidism. p value across groups according to the diagnosis of thyroid functions (category i - v). abbreviations: bmi, body mass index; sbp, systolic blood pressure; dbp, diastolic blood pressure; cs, cesarean section delivery. categories are defined as follows: i, clinical hypothyroidism; ii, subclinical hypothyroidism; iii, euthyroid (control); iv, subclinical hyperthyroidism; v, over hypothyroidism. p value across groups according to the diagnosis of thyroid functions (category i - v). there was no significant difference between gestational age, head circumference, weight, length, and apgar score of neonates in different thyroid function categories (p > 0.05). therefore, the rank numbers of 2.5th to 97.5th percentiles were used to estimate the lower and the upper limits of the reference interval, respectively. 2.5th, 25th, 50th, 75th, and 97.5th percentiles for ft3 and ft4, which had normal distribution, were calculated by frequency analysis. median (interquartile range) for subclinical hypothyroidism and overt hypothyroidism was 3.65 (3.18 - 4.93) and 12.1 (4.42 - 15.22), respectively. abbreviations: tsh, thyroid stimulating hormone, ft3, free triiodothyronine; and ft4, free thyroxine. prevalence rates of preeclampsia, iugr, preterm delivery and low apgar score in participants were 6.3%, 7.5%, 14.5%, and 8%, respectively. rout of delivery in 16.2% of mothers was cesarean section. in women with hypothyroidism, prevalence rate of preeclampsia, iugr, preterm delivery, and low apgar score was 7.5%, 13.7%, 21.2%, and 13.7%, respectively. prevalence rates of preeclampsia, iugr, preterm delivery and low apgar score in women with hyperthyroidism were 0%, 22.2%, 11.1%, and 11.1%, respectively. prevalence of hypothyroidism in pregnant woman was 13.7% (clinical, 2.4% ; and subclinical, 11.3%). hypothyroidism was associated with iugr (p = 0.017) and low apgar score at first minute (p = 0.04); it increased the risk of iugr by 2.2 times and low apgar score by 1.95 times. clinical hypothyroidism had no significant association with preeclampsia (p > 0.05), but was associated with preterm delivery (p = 0.045). subclinical hypothyroidism had a significant association with iugr (p = 0.028) and low apgar score at first minute (p = 0.022). it increased the risk of low apgar score by 2.15 times and iugr by 2.18 times. this regression analysis for iugr was adjusted for maternal body mass index (bmi), age of mother, and occurrence of preeclampsia. moreover, logistic regression analysis for neonates with low apgar score was adjusted for delivery gestational age, maternal bmi, age of mother, and occurrence of preeclampsia. after this adjustment, no significant change in associations was found, except for the preterm delivery. the association of clinical hypothyroidism and preterm delivery was not significant (p value = 0.116) after adjustment for confounding factors. abbreviations: iugr, intrauterine growth restriction; rr, relative risk; ci, confidence interval. p values are the of chi square analysis of the frequency of each complication between abnormal thyroid function mothers and euthyroid ones (as controls). no significant change in associations was seen after adjusting the analysis for the confounding factors except for the p value of the analysis of clinical hypothyroidism and preterm delivery, which was change to 0.116 after adjusting this analysis for maternal bmi, age, and preeclampsia. prevalence of hyperthyroidism in pregnant women was 1.5% (overt, 1.2% ; and subclinical, 0.3%). nevertheless, it did not show a significant association with preeclampsia, preterm delivery, and low apgar score. these for each complication were not significantly changed in logistic regression analysis after covariates were adjusted for abovementioned confounding factors. abbreviations: iugr, intrauterine growth restriction; rr, relative risk; ci, confidence interval. p values are the of chi square analysis of the frequency of each complication between abnormal thyroid function mothers and euthyroid ones (as controls). no significant change in associations was seen after adjusting the analysis for the confounding factors. prevalence rates of preeclampsia, iugr, preterm delivery and low apgar score in participants were 6.3%, 7.5%, 14.5%, and 8%, respectively. rout of delivery in 16.2% of mothers was cesarean section. in women with hypothyroidism, prevalence rate of preeclampsia, iugr, preterm delivery, and low apgar score was 7.5%, 13.7%, 21.2%, and 13.7%, respectively. prevalence rates of preeclampsia, iugr, preterm delivery and low apgar score in women with hyperthyroidism were 0%, 22.2%, 11.1%, and 11.1%, respectively. prevalence of hypothyroidism in pregnant woman was 13.7% (clinical, 2.4% ; and subclinical, 11.3%). hypothyroidism was associated with iugr (p = 0.017) and low apgar score at first minute (p = 0.04); it increased the risk of iugr by 2.2 times and low apgar score by 1.95 times. clinical hypothyroidism had no significant association with preeclampsia (p > 0.05), but was associated with preterm delivery (p = 0.045). subclinical hypothyroidism had a significant association with iugr (p = 0.028) and low apgar score at first minute (p = 0.022). it increased the risk of low apgar score by 2.15 times and iugr by 2.18 times. this regression analysis for iugr was adjusted for maternal body mass index (bmi), age of mother, and occurrence of preeclampsia. moreover, logistic regression analysis for neonates with low apgar score was adjusted for delivery gestational age, maternal bmi, age of mother, and occurrence of preeclampsia. after this adjustment, no significant change in associations was found, except for the preterm delivery. the association of clinical hypothyroidism and preterm delivery was not significant (p value = 0.116) after adjustment for confounding factors. abbreviations: iugr, intrauterine growth restriction; rr, relative risk; ci, confidence interval. p values are the of chi square analysis of the frequency of each complication between abnormal thyroid function mothers and euthyroid ones (as controls). no significant change in associations was seen after adjusting the analysis for the confounding factors except for the p value of the analysis of clinical hypothyroidism and preterm delivery, which was change to 0.116 after adjusting this analysis for maternal bmi, age, and preeclampsia. prevalence of hyperthyroidism in pregnant women was 1.5% (overt, 1.2% ; and subclinical, 0.3%). nevertheless, it did not show a significant association with preeclampsia, preterm delivery, and low apgar score. these for each complication were not significantly changed in logistic regression analysis after covariates were adjusted for abovementioned confounding factors. abbreviations: iugr, intrauterine growth restriction; rr, relative risk; ci, confidence interval. p values are the of chi square analysis of the frequency of each complication between abnormal thyroid function mothers and euthyroid ones (as controls). no significant change in associations was seen after adjusting the analysis for the confounding factors. we have investigated the normal distribution of tsh in our 586 pregnant women and showed that our range for the level of tsh in iran was higher than the ata ranges. our data were similar to those of previous reported from india and iran in which the ranges of tsh in the second trimester were about 0.43 to 5.78 and 0.5 to 4.1 miu / l, respectively. differences in laboratory methods, differences in kits, maternal iodine status, and ethnic, genetic, and environmental factors in our and other similar studies could explain these differences. on the other hand, other studies report a lower range for tsh level. after publishing the ata guideline, we have found some studies in which the thyroid function status was evaluated according to this guideline. the present study provides, for the first time, the data about the thyroid function status in iranian pregnant women according to this new guideline. we found that prevalence of hypothyroidism in pregnancy was 13.7% (clinical, 2.4% ; and subclinical, 11.3%). in addition, 1.4% of our pregnant women had isolated hypothyroxinemia. according to previous reports, prevalence of clinical hypothyroidism in pregnant women ranged from 1% to 3.5% and subclinical hypothyroidism ranged from 4% to 31%. prevalence of hyperthyroidism of pregnant women was 1.5% in our study (subclinical, 0.3% ; and over, 1.2%). in previous reports, prevalence of hyperthyroidism was reported as 0.2% to 2%. one of the causes of variation in prevalence of thyroid dysfunction might be variation in definition of normal range for tsh in different studies. the ata recommendations could orchestrate the investigations about the prevalence and effects of thyroid dysfunction and prevent the errors in our interpretations and clinical judgment about the detrimental effects of thyroid dysfunction on pregnancy outcomes. hypertension is one of the most important health problems related to both overt hypothyroidism and hyperthyroidism and to a lesser extent, to subclinical thyroid dysfunction. its prevalence was 6%, 7.5%, and 0% in euthyroid mothers, mothers with hypothyroidism, and mothers with hyperthyroidism, respectively. although the prevalence of preeclampsia was higher in pregnant women with hypothyroidism, this difference was not statistically significant. a few investigations have reported that overt hypothyroidism increases the incidence of preeclampsia; however most of the studies showed no significant association. prevalence of preterm delivery was 13%, 21%, and 11.1% in euthyroid mothers, mothers with hypothyroidism, and mothers with hyperthyroidism, respectively. spontaneous abortion and preterm delivery have been reported in 17% to 31% of all pregnancies. the majority of them have no known risk factor. one of the known risk factors is clinical or subclinical hypothyroidism. allan et al. showed that tsh level > 6 miu / l was significantly associated with a higher frequency of pregnancy loss; however, one recent study showed no significant associations between tsh level and the risk of preterm delivery. we showed that the association of clinical hypothyroidism and preterm delivery might be due to secondary confounding factors such as maternal bmi, age, and preeclampsia. consistent with our , previous reports revealed that subclinical hypothyroidism was associated with iugr. it might be due to the essential role of thyroid hormones in growth and maturation of many tissues of the fetus like the brain, bones, and muscles. we revealed that hypothyroidism is associated with low apgar score and mothers with subclinical hypothyroid have 2.15 times greater risk for having low apgar score neonates. showed a higher risk of fetal distress in mothers with subclinical or clinical hypothyroidism. it seems that hypothyroidism exerts irreversible influences on the placenta and fetus during pregnancy and decreases the fetal ability to tolerate stress and therefore, neonates present with low apgar scores at birth. therefore, future studies should evaluate thyroid autoantibodies in addition to tft. in our study , we found that the hypothyroidism during pregnancy, even in subclinical form, could cause iugr and low apgar score. although prevalence of hyperthyroidism in our pregnant women was very low, it could be associated with iugr. interventional studies are required to determine whether early diagnosis and treatment of thyroid diseases, even in the subclinical form, prevent their adverse effect on the fetus in iranian pregnant women.

: maternal thyroid function alters during pregnancy. inadequate adaptation to these changes in thyroid dysfunction and pregnancy complications.objectives:this prospective study aimed to evaluate the prevalence of thyroid diseases in pregnancy and its outcomes in south of iran.materials and methods: this prospective study was conducted on 600 healthy singleton pregnant women who aged 18 to 35 years old at 15 to 28 weeks of gestation. we investigated the prevalence of thyroid dysfunctions in women. multivariate analysis was performed to determine the effect thyroid dysfunction on obstetric and neonatal outcome.:thyroid stimulating hormone (tsh) levels of 0.51, 1.18, 1.68, 2.4, and 4.9 miu / l were at 2.5th, 25th, 50th, 75th, and 97.5th percentile in our population. the prevalence of clinical hypothyroidism, subclinical hypothyroidism, overt hyperthyroidism, and subclinical hyperthyroidism in all pregnant women was 2.4%, 11.3%, 1.2%, and 0.3%, respectively. in addition, 1.4% of patients had isolated hypothyroxinemia. clinical hypothyroidism was associated with increased risk of preterm delivery (p = 0.045). subclinical hypothyroidism had a significant association with intrauterine growth restriction (iugr) (p = 0.028) as well as low apgar score at first minute (p = 0.022). maternal hyperthyroidism was associated with iugr (p = 0.048).: we revealed that thyroid dysfunction during pregnancy was associated with iugr and low apgar score even in subclinical forms. further studies are required to determine whether early diagnosis and treatment of thyroid diseases, even in subclinical form, can prevent their adverse effect on fetus.

microrna refers to the category of single - stranded small noncoding rnas that are approximately 22 nucleotides in length. more than 1500 human microrna have been identified and registered via various approaches including high throughput screenings (http://www.mirbase.org/). as more than 30% of human of mrnas are regulated by micrornas, the functional impact of microrna in physiology and pathology has yet to be fully elucidated. generally speaking , microrna imposes its regulatory role by sequence - specific but incomplete complementary binding to its target mrna sequences, which are usually located at the 3 untranslated region. this binding may mediate the degradation of target mrna or the inhibition of protein translation efficiency of target mrna. however, due to the loose stringency of this kind of targeting, the exact mechanism of specific microrna function remains undefined and is an actively addressed research topic. the function and target mrnas of individual micrornas can not be reliably predicted via current bioinformatic approaches, thereby warranting continued experimental interrogation. the investigation of microrna expression and its functional relationship with various cancer types has instigated tremendous interest in employing such molecules as novel diagnostic or therapeutic modalities in oncology studies. recent developments that show correlation between plasma microrna levels and cancer have further increased enthusiasm for this approach due to the easy accessibility of blood serum and plasma specimens. similarly abundant in clinical settings are archived formalin - fixed paraffin - embedded (ffpe) human cancer specimens, which can provide a rich resource for investigating the relationship between microrna expression and cancer progression. studies on such material have the added advantage of maintaining the information content from cancer tissue morphology when in situ hybridization (ish) is used to detect microrna expression. unlike the frequently used quantitative reverse transcription polymerase chain reaction (q - rt - pcr) method, which requires extraction of rna, ish retains the microscopic topological information content with respect to microrna expression and makes it possible to be related to other factors on the same tissue section. however, formalin fixation causes cross - links between biological molecules, potentially limiting access to microrna molecules. moreover, degradation has been a constant concern for preservation of rna molecules in such tissue samples. the in situ hybridization technique for nucleic acid detection on tissue and cytological preparations was initiated decades ago , when radioisotope labeling and autoradiography were the only means to visualize positive hybridization signals. improvement and development of newer technologies, such as the advent of nonradioactive digoxigenin as probe labeling / reporter molecule and the tyramide signal amplification (tsa) system, have made in situ hybridization much more accessible. however, due to the short length of microrna molecules, in situ hybridization for microrna detection remains challenging. this paper demonstrates the feasibility and highlights key technical factors of developing a protocol for fluorescence in situ hybridization (fish) to detect microrna in archived ffpe human oral cancer tissues. formalin - fixed paraffin - embedded tissues are from lab - archived human oral cancer xenografts (10 blocks) and commercially available human oral cancer tissue microarray (tma) sections from us biomax, inc. (rockville, md), consisting of 50 tissue cores including 40 oral squamous cell carcinoma and 10 adjacent normal stratified squamous epithelia. xenograft specimen collection was approved by the animal care and use committees (northwestern university and university of missouri). surgically removed xenograft tissues were immediately fixed in 10% neutral buffered formalin for 1224 hours and passed through dehydration, clearing, and paraffin - embedding steps. sections were cut at 5 m thick and mounted on positively charged slides, baked at 65c for 2 hours, and then stored at room temperature for later use. as for samples which were used to make tma, according to the supplier, they were typically put into formalin within 1530 minutes after surgical resection. some of the tissue samples were snap frozen in liquid nitrogen and stored there for later fixation. either way, fixation in neutral buffered formalin was about 24 hours before they were processed in automatic tissue processor and embedded in paraffin. (st . louis, mo, usa) unless otherwise specified. normal goat serum and hrp - conjugated anti - mouse igg were from santa cruz biotechnologies, inc. mouse antidigoxigenin and hrp - conjugated anti - digoxigenin antibodies were obtained from roche applied science (indianapolis, in, usa). tsa - cyanine 5 kit (nel705a) was from perkin - elmer (waltham, ma, usa), and contains hrp - streptavidin, blocking reagent, amplification diluents, and cyanine 5-conjugated tyramide. lna - based microrna mir-146a antisense oligonucleotides were labeled with digoxigenin (dig) at the 5 end. lna and non - lna - modified 5 biotinylated mir-146a specific probe and scrambled probe with the same sequences were custom made from integrated dna technologies, inc. prolong mounting media containing 4,6-diamidino-2-phenylindole (dapi) was from invitrogen (carlsbad, ca, usa). an important consideration is to use diethylpyrocarbonate (depc)-treated water in all solution preparation and to exercise caution to maintain a rnase - free work environment during all procedures. to generate 20x ssc, dissolve the following in 800 ml of milli - q grade water: 175.3 g of nacl and 88.2 g of sodium citrate, adjust the ph to 7.0 with a few drops of 1 m hcl, and adjust the volume to 1 liter with additional distilled h2o. sterilize by autoclaving. to prepare 50x denhardt's solution, add the following to 900 ml distilled h2o: 10 g ficoll 400, 10 g polyvinylpyrrolidone, and 10 g bsa, then fill up to 1 liter. filter the solution prior to storage through a 0.2 m filter and store at 4c (but warm up to appropriate temperature prior to use). the prehybridization solution contains the following: 50% deionized formamide, 2x ssc, 1x denhardt's, 0.02% sds, yeast trna (0.5 mg / ml), and salmon sperm dna (0.5 mg / ml). the hybridization solution contains 50% deionized formamide, 2x ssc, 1x denhardt's, 10% dextran sulfate, yeast trna (0.5 mg / ml), and salmon sperm dna (0.5 mg / ml). for testing the effectiveness of peroxidase inhibition, two methods (hydrogen peroxide versus hydrochloric acid) after the sections were dewaxed and rehydrated, two kinds of fresh prepared solutions: 3% hydrogen peroxide in phosphate - buffered saline (pbs) and 0.024 m hydrochloric acid in ethanol applied, respectively, 200 l per slide, to two groups of slides and incubated for 10 minutes at room temperature. the cyanine 5-tyramide stock solution was diluted 1: 50 using the included 1x amplification diluent to make the cyanine 5-tyramide working solution. approximately 100 l of cyanine 5-tyramide working solution was added per slide, incubated for 10 minutes at room temperature, and washed (3 times, 5 minutes each) with pbs. slides were then air dried in the dark and mounted using prolong mounting media with dapi and coverslips. solidified fluorescent slides were observed with the wide field function of an olympus dsu spinning disc confocal microscope and slidebook software version 4.0. fluorescent filter sets used for dapi are d350/50x and et455/50 m, and for cy5 are 645/30x and et705/72 m. the typical work flow of our fluorescence in situ hybridization for mir-146a microrna detection is described below. the total procedure can be completed within 2 days and is adaptable to detecting proteins of interest at the same time with multicolor labeling. a negative control slide should always be included that will be otherwise treated equally but with scrambled probe or without any probe in the hybridization step. deparaffinize in xylene (2x 10 minutes), rehydrate in serial ethanol solutions (100%, 90%, 80%, 70%), and depc - treated water (2 minutes each) and pbs wash (2x 5 minutes). pretreatment of the slide: cross - linking during fixation can block reagent access to rna / dna molecules, so it is critical to unmask these sites using proteinases. it is also necessary to block certain endogenous active molecules that may interfere with signal development in later steps. to quench endogenous peroxidase: 0.024 m hcl in ethanol, incubate for 10 minutes, then pbs wash (2x 5 minutes). for proteinase treatment, incubate with proteinase k (20 g / ml, 37c for 10 minutes) followed by a pbs wash (2x 5 minutes), and complete with 4% paraformaldehyde (pfa) fixation for 10 minutes followed by a pbs wash for 5 minutes; 100 mm glycine incubation for 10 minutes; pbs wash (2x 5 minutes) and end with a 2x ssc wash (5 minutes). prehybridization: this is meant to block nonspecific binding of probes and minimize signals. pre - hybridize for 2 hours at 50c in prehybridization solution, cover with plastic coverslip, and place in a moist chamber. hybridize overnight (18 hours) at 50c with probe at the concentration of 25 nm (1 l 2.5 m dig labeled probe to 100 l hybridization solution), cover with plastic coverslip and place in a moist chamber. stringency wash: this step is critical to remove nonspecific probe binding and overloaded probes. wash with 2x ssc (37c for 15 minutes) followed by a high temperature 2x scc wash (at 50c with shaking). next wash with 1x ssc (2x at 37c for 15 minutes), with shaking at 50c in 1x scc for another 15 minutes, and wash with 0.02% sds in 1x ssc (2x at 37c for 15 minutes), with shaking at 50c in the same buffer for another 15 minutes. then followed by pbs - t (pbs containing 0.1% tween-20) serum block uses 10% normal goat serum in pbs for 1 hour at room temperature, followed by mouse anti - dig (1 : 250) in the above blocking solution for 0.5 hour at room temperature; rinse with pbs - t wash (4x 5 minutes). add hrp - conjugated goat anti - mouse igg (1 : 500) for 0.51 hour at room temperature. note that the use of 2 antibodies (mouse anti - dig and hrp - conjugated goat anti - mouse igg) could be replaced with hrp - conjugated anti - dig only. following antibody incubation, wash with pbs - t (4x 5 minutes) and add cy5-tyramide working solution (100 l per slide); incubate at room temperature for 10 minutes. pbs - t wash (4x 5 minutes); pbs wash (5 minutes), air - dry for 10 minutes in the dark, and apply prolong gold mounting medium with dapi and a coverslip. the need to further evaluate the expression and function of specific micrornas in cancer pathology prompted us to establish a method of detecting micrornas in archived ffpe materials. abundant concerns regarding the integrity of rna in ffpe samples have been confirmed by studies showing that mrna in ffpe materials has various degrees of degradation as well as chemical modification by the fixative, rendering downstream analysis of extracted rna difficult. interestingly however, recent studies using microarray analyses to compare rna species between paired ffpe and fresh frozen samples demonstrated closely related microrna profiles, while mrna profiles exhibited signs of degradation in ffpe materials. time to fixation, nuclease activity before fixation, chemical modification by formalin and variation in sample - processing procedures likely contribute to the decline in mrna quality in ffpe relative to fresh frozen tissue samples. however, as demonstrated in the aforementioned studies, the small size of microrna as well as its close association with large protein complexes enable the relatively better maintenance of their integrity after formalin fixation and paraffin embedding. this high degree of correlation between microrna signatures in ffpe and fresh frozen specimens encourages further exploration of microrna detection methods on archived pathology samples. after optimizing certain critical conditions , we have found that lna - based probes labeled with digoxigenin and combined with tsa amplification provided satisfactory for mir-146a fish detection in archived oral cancer tissues. specimen processing in our study was relatively well controlled; we have to acknowledge that on specimens from real - life clinical archives may exhibit greater variability. horseradish peroxidase (hrp) conjugated antibodies combined with chromogenic substrates are frequently employed for this purpose. however, some tissues and cells contain endogenous peroxidase, especially leukocytes and erythrocytes. in cancer tissues, because of abundant angiogenesis and frequent inflammatory infiltration, peroxidase - containing cells are common. this is a major concern when using the highly sensitive tsa system, the mechanism of which depends on peroxidase activity and which provides up to 1000-fold amplification in detection sensitivity, as endogenous peroxidases produce significant staining when not inhibited properly. because of this concern, we tested different quenching methods for endogenous peroxidase. hydrogen peroxide is mostly commonly used in immunohistochemistry, at a typical concentration from 0.3% to 3% diluted in methanol or pbs buffer and an incubation time of 10 to 60 minutes (lower concentrations require longer incubation times but may induce less damage to certain antigens of interest). a less known method was reported by weir and colleagues decades ago, that is, to incubate slides with 0.024 m hydrochloric acid (hcl) in ethanol for 10 minutes to efficiently destroy endogenous peroxidase. we compared the use of 3% h2o2 (figures 2(a) and 2(b) ) and 0.024 m hcl (figures 2(c) and 2(d) ) on consecutive ffpe sections. the difference is striking, wherein incubation with the hcl solution produced a nearly complete suppression of endogenous peroxidase when incubated only for 10 minutes, but in 3% h2o2-treated samples at the same incubation time, inflammatory cells and even some carcinoma cells are quite positive with highly sensitive cy5-tyramide detection (figure 2). complete suppression of endogenous peroxidase activity may not be necessary for routine immunohistochemistry as trace peroxidase will not detectably affect the chromogenic reaction. however, with the tyramide signal amplification system, trace amounts of active peroxidase can in tyramide precipitation, and this high sensitivity dictates the needs for thorough inhibition of such enzyme activity. as demonstrated (figure 2), dilute hcl solution is a convenient, low cost, and highly effective replacement for the traditional h2o2-blocking step. as for the timing of peroxidase blocking the ready availability of commercially labeled oligonucleotide - based probes enables end users to choose the tracer or reporter that best fits their protocols. biotin, digoxigenin, and fluorescein have been frequently used as reporter molecules on probes. early versions of the commercially available tsa system used streptavidin - biotin affinity for initial signal detection. in this reaction schema , a biotinylated probe hybridizes with a single - stranded target sequence, hrp - conjugated streptavidin binds to the biotin, hrp catalyzes the activation of tyramide conjugated with fluorescein, and active tyramide precipitates in the vicinity of hrp molecule. based on this protocol, we initially used biotinylated probes for in situ hybridization. however, exploratory tests confirmed that this is not appropriate for the type of tissues evaluated, as oral cancer cells are rich in endogenous biotin (figure 3). indeed endogenous biotin (or a similar streptavidin - binding activity) has been reported in many types of human tissues. to circumvent this nonspecific staining problem, digoxigenin (dig) has been long proven to be a good option as reporter molecule in nucleic acid probe design. as a heptan, the only natural source of dig is digitalis plants, significantly reducing the likelihood that the anti - dig antibody will bind any endogenous antigens in animal tissues. locked nucleic acid (lna) is a nucleic acid analog that contains at least one nucleotide monomer with a bicyclic furanose ring locked in a conformation mimicking rna. as the length of microrna is only about 22 nucleotides, many microrna molecules are differentiated from each other by only a few bases, such that it is difficult to achieve appropriate high - level probe sensitivity and specificity. lna - modified oligonucleotides demonstrate much higher thermal stability and higher melting temperatures when hybridized with target rna sequences compared to unmodified counterparts. it improves the mismatch discrimination, increasing the base - pairing selectivity and providing the needed high degree of affinity for effective microrna hybridization. using a commercially available lna - modified dig - labeled probe under the conditions outlined above ed in appropriate differential staining (figure 4). prickle cells (stratum spinosum) in most normal oral squamous epithelia were positive for mir-146a, while basal cells exhibited negative staining. this kind of good performance is consistent with other studies showing the use of lna - based probes for hybridization - based microrna detection. the introduction of tyramide conjugates as substrates for hrp has revolutionized the sensitivity of any hrp - based detection system. mechanistically, hrp reacts with hydrogen peroxide and the phenolic part of tyramide and produces a quinone - like structure with a radical on the c2 group, becoming activated. activated tyramide then rapidly and covalently binds to all nearby tyrosine residues with proximity to the initially immobilized hrp site such that signal resolution is not compromised. previous studies have shown that the tyramide signal amplification system, which was also known as catalyzed reporter deposition method (card), provides the capability to identify single copy dna or rna with conjugated fluorophore. to detect microrna in situ with probe - based approaches requires such highly sensitive signal amplification. we have tested the use of the more traditional chromogenic substrate diaminobenzidine (dab) with hrp - labeled anti - dig antibody in the context of lna - based dig - labeled probes but did not get well - differentiated staining in in situ hybridization. the caveat is that when using any tsa system, complete suppression of endogenous peroxidase is essential, as discussed above. another well - recognized way of amplifying positive signal from nucleic acid hybridization is branched dna signal amplification , which makes use of multiple layers of probes with the last layer being extensively enzyme labeled. however, for microrna detection, the primary probes still require the lna or lna - like nucleotides to enhance the initial specificity and sensitivity, and signal amplification with the branched dna procedure is less customizable than the tsa system allows. the use of branched dna signal amplification for in situ hybridization microrna detection has not been reported. ffpe tissues initially appeared to be a challenging platform for microrna detection but are actually better suited for microrna than mrna studies as recently revealed. thus, clinically archived cancer tissue specimens can represent buried treasure, as micrornas are well preserved in such materials. our study has demonstrated that after efficient inhibition of endogenous peroxidase, lna - based and digoxigenin - labeled probe, applied together with tyramide signal amplification, significantly improves the of fluorescence in situ hybridization for microrna detection. in summary, we have established a feasible in situ hybridization procedure for detecting the expression of microrna in ffpe oral cancer tissues. this detection is important for studies on the participation of microrna in oral cancer pathology and may have potential prognostic or diagnostic value as large cohort studies using such material will confirm.

the noncoding rna designated as microrna (mirna) is a large group of small single - stranded regulatory rna and has generated wide - spread interest in human disease studies. to facilitate delineating the role of micrornas in cancer pathology, we sought to explore the feasibility of detecting microrna expression in formalin - fixed paraffin - embedded (ffpe) tissues. using ffpe materials, we have compared fluorescent in situ hybridization (fish) procedures to detect mir-146a with (a) different synthetic probes: regular custom dna oligonucleotides versus locked nucleic acid (lna) incorporated dna oligonucleotides; (b) different reporters for the probes: biotin versus digoxigenin (dig); (c) different visualization: traditional versus tyramide signal amplification (tsa) system; (d) different blocking reagents for endogenous peroxidase. finally, we performed mir-146a fish on a commercially available oral cancer tissue microarray, which contains 40 cases of oral squamous cell carcinoma (oscc) and 10 cases of normal epithelia from the human oral cavity. a sample fish protocol for detecting mir-146a is provided. in summary, we have established reliable in situ hybridization procedures for detecting the expression of microrna in ffpe oral cancer tissues. this method is an important tool for studies on the involvement of microrna in oral cancer pathology and may have potential prognostic or diagnostic value.

conventional dendritic cells (cdcs) are found in almost all tissues and lymph nodes (lns) and act as sentinels capable of integrating multiple environmental signals and conveying them to cd4 and cd8 t lymphocytes. plasmacytoid dcs (pdcs) produce type i interferons and can also develop into antigen - presenting cells, particularly when stimulated by virus or self dna. human and mouse cdcs are derived from committed dc precursors (pre - cdcs) produced in the bone marrow (bm). these pre - cdcs migrate from the bm into the blood and then seed the various tissues where they develop into two distinct lineages of cdc. the existence of two distinct dc lineages is supported by the identification of lineage - defining transcription factors (tfs) required for development and/or function of cdc1 (irf8, batf3, id2) and cdc2 (irf4, zeb2) (breton et al ., 2015, grajales - reyes et al ., 2015, guilliams et al ., 2014, , 2006, schlitzer et al ., 2015, scott et al ., 2016). a separate e2 - 2-dependent progenitor with prominent pdc potential has been recently described (onai et al ., 2013). with these recent molecular insights, it is now clear that cdcs belonging to the same lineage are present in various tissues and species; however, these have been historically characterized by different surface markers. this from the fact that many murine macs can express the prototypical cdc markers cd11c or mhcii and, conversely, that cdc2 can express the mac marker f4/80 (bain et al ., 2012, , 2015, scott et al ., 2015, tamoutounour et al ., 2012, tamoutounour et al ., 2013). distinguishing dcs from macs in human tissues has been equally challenging (collin et al ., 2013, mcgovern et al ., 2015). finally, the lack of conserved markers to identify dcs hampered communication between mouse and human experts and was detrimental for fostering translational medicine. the advent of multicolor flow cytometry only aggravated the matter by yielding a seemingly ever - growing list of dc subsets based on different marker combinations. therefore, a rational approach simplifying the classification of dc subsets across tissues and species, yet still permitting the use of additional markers to study tissue- and disease - specific activation states, is urgently needed. it was recently proposed to classify dcs based on their ontogeny before subdividing them based on their micro - anatomical location or specific functional specialization (guilliams et al ., 2014). this would yield only three subsets of dcs: conventional type 1 dcs (cdc1s), conventional type 2 dc (cdc2s), and pdcs. however, due to a lack of consensus regarding how to define dc subsets experimentally, such classification remains of limited practical use (guilliams and van de laar, 2015). recent progress in the unsupervised analysis of high - dimensional flow cytometry datasets has rendered the identification process of cell subsets more objective and more reproducible (saeys et al ., 2016). however, a limitation of those approaches is that they give an equal weight to all the surface markers, not necessarily yielding the most biologically meaningful clusters. for instance, both langerhans cells (lcs) and cdc1s express cd207, cd24, mhcii, and cd11c, but they have completely different localization, ontogeny, lifespan, and functional specialization (malissen et al ., 2014). thus, the way forward has to be based on better markers to faithfully identify dc subsets alongside computational approaches that simplify the classification of dc subsets without compromising the multidimensional marker combinations necessary to grasp the fascinating functional heterogeneity of dcs. cd64 is highly expressed on macs and can be used in combination with f4/80 to discriminate these cells from cdc2s (bain et al ., 2012, gautier et al ., 2012, , 2013, scott et al ., 2015, tamoutounour et al ., 2013) (figure 1a). outgating macs on the basis of their cd64f4/80 phenotype is essential to prevent them from contaminating the cdc2 gate in most tissues (figure s1). as f4/80 is expressed on a part of the cdc2s, it should not be used alone to exclude macs, as has been proposed (gurka et al ., 2015). cd3 t cells, cd19b220 b cells, and nk1.1 natural killer (nk) cells were next excluded from further analysis using a lineage mix and the remaining cells gated for expression of mhcii molecules (lineagemhcii cells). pdcs can be found among the lineage (lin) cells and identified as 120g8(cd317)b220cd11cly6ccd11b cells (figure s1). to obviate the fact that cd11c, a classical cdc marker, can be downregulated on dcs (osorio et al ., 2014) and is lowly expressed by the double - negative , cd26 was added as an additional cdc marker, since it is highly expressed on all mouse cdcs across tissues (see below). this permitted us to identify a well - defined population of cd11ccd26 cdc across tissues (figure 1a, cyan gate) that can be further subdivided into xcr1cd172a (dark blue gate) and xcr1cd172a (green gate) cdcs. to validate that xcr1cd172a and xcr1cd172a cells corresponded to cdc1s and cdc2s, respectively, we relied on their differential expression on irf8 and irf4 (murphy et al ., 2015, sichien et al ., analysis of the co - expression of both transcription factors ( tfs) by intracellular staining revealed that xcr1cd172a cdc1 and xcr1cd172a cdc2 had an irf8irf4 or irf8irf4 profile, respectively (figure 1b), while macs and pdcs had an irf8irf4 or irf8irf4 profile, respectively (figure 1b and figure s1). the pulmonary cd11cmhciicd64f4/80cd172a monocyte - derived cells (often referred to as modcs) also had an irf8irf4 profile. as expected, cdcs were lacking in flt3l mice (figure 1c) and competitive bm chimeras showed that cdc1 development was batf3 dependent and irf4 independent in all tissues analyzed (figure 1d). cdc2s developed independently of batf3, whereas they were irf4 dependent particularly in the lns. this likely reflects a late requirement for irf4 in cdc2 survival (persson et al ., 2013, schlitzer et al ., 2013) and is consistent with the fact that irf4 deficiency blocks the migration of dermal cdc2s to cutaneous lns, but not their intradermal development (bajaa et al ., 2012). therefore, the differential dependence on subset - defining tfs and growth factors validates the correct identification of cdc1s and cdc2s using just eight surface markers. intracellular irf4-irf8 double staining can be further used to validate the correct assignment of cdc1s, cdc2s, pdcs, and macs across tissues. identifying cdc1s and cdc2s across tissues in parallel allowed us to analyze their relative abundance in all tissues (figure 1e). the network of myeloid cells found in the mouse skin and cutaneous lns is particularly complex to dissect due to the presence of lcs. lcs share many markers with cdcs (mhcii, cd207 , cd24, and cd11c) and macs (f4/80) (henri et al . presently, lc radioresistance constitutes the best way to distinguish them from radiosensitive dermal cdcs ( ginhoux et al . to distinguish lcs from cdcs, cd64f4/80 macs were first outgated and the broadly cd11ccd26 cells found among linmhcii cells ( figure 2a, cyan gate) were subdivided into xcr1cd172a and xcr1cd172a cells. owing to their cd64linmhciixcr1cd172a phenotype, lcs must be distinguished from bona fide cdc2s present in the same gate, and cd24 expression allowed this. on a cd26-cd24 dot plot, cd26cd24 cdc2s (green gate) the distinction of cdcs from lcs on the basis of this minimalistic surface marker combination was validated using bm chimeras (figure 2b). intracellular irf4-irf8 staining validated the correct identification of dcs and macrophages (macs), in that all cdc1s displayed an irf8irf4 profile, all cdc2s displayed an irf8irf4 profile, and all macs and lcs displayed an irf8irf4 profile (figures 1 and 2). therefore, our gating strategy allows cdc1s and cdc2s to be identified across mouse tissues when cd24 is added to the panel to separate lcs from cdcs in skin. the dermis has been shown to contain cdc1s with a cd103 and cd103 phenotype and double - negative cdc2s that have low expression of cd11c and cd11b (henri et al ., 2010, , cd11b expression permitted cd11bcd11c double - negative cdc2s ( olive green gate) to be distinguished from cd11bcd11c cdc2s (green gate). nonetheless, all cdc1s had a homogeneous irf8irf4 profile and were batf3 dependent and irf4 independent, whereas all cdc2s showed a homogeneous irf8irf4 profile and were batf3 independent and irf4 dependent (figures 2a and 2c). this illustrates that using a restricted set of lineage - imprinted markers facilitates the correct alignment of cdcs into cdc1s and cdc2s according to their ontogeny while still permitting the inclusion of additional markers to further gauge the presence of different subsets of cdc1s and cdc2s within a given tissue. clec9a - based fate - mapping identified a putative cdc2 subpopulation expressing cd64 in the kidney (schraml et al . consistent with this, our gating strategy revealed that, unlike in other organs, kidney cd64f4/80 cells were not homogeneously irf8irf4 ( figure 1) but contained a small population of irf8irf4 cdc2-like cells (figure 2d). these irf8irf4 cells expressed higher cd26 and cd11c and lower f4/80 and cd64 compared with irf8irf4 cells (figure 2d). furthermore, irf8irf4cd64f4/80 cells, but not the irf8irf4cd64f4/80 cells, were flt3l dependent, identifying the irf8irf4 and irf8irf4 cells as cdc2s and macs, respectively. therefore, regardless of the expression of cd64 on a small fraction of kidney cdc2s, our gating strategy unambiguously identified cdc2s in the kidney. the flow cytometry dataset from distinct tissues was subjected to the unsupervised identification method flowsom (van gassen et al ., 2015). flowsom uses a self - organizing map (som) to cluster cells in different nodes based on the expression of the distinct markers used in a given flow cytometry dataset and subsequently structures the nodes in a minimal spanning tree. we first concatenated live cd45 cells from all tissues and used flowsom to generate a single flowsom tree. to identify the node(s) corresponding to the cdc1s and cdc2s, we defined them ab initio as xcr1cd24cd26cd11cmhciicd11bcd172af4/80cd64linfscssc and cd11bcd172acd26cd11cmhciixcr1f4/80cd64linfscssc cells, respectively (figure 3). for each node, we calculated a final score indicating its correspondence with the defined cdc1 or cdc2 profile. first, a marker score was calculated for each node as the difference between the median value of the node for that marker and the minimum or maximum median node value present in the tree. then, for each marker, the score was normalized between zero and one, and the final node score was then computed as the mean of its scores for each individual marker. all nodes with a final node score of at least 0.95 times the highest score separate analysis of each tissue confirmed the presence of discrete cdc1 and cdc2 nodes in all tissues (figure 3d). finally, exporting the cells within the cdc1 and cdc2 nodes identified by flowsom on xcr1-cd172a, cd11c - cd26, and cd64-f4/80 dot plots confirmed that cdc1 and cdc2 were correctly identified across tissues (figure 3e). notably, although cdc2s are defined as f4/80, flowsom clustered f4/80 cdc2 with the f4/80 cdc2 (figure 3e), since a small difference in one marker is not sufficient to separate cells into two separate clusters. therefore, unsupervised analysis corroborated our manual gating strategy and allowed the robust and fully automated identification of cdc1s and cdc2s across mouse tissues. an additional advantage of unsupervised identification algorithms such as flowsom or tsne is that each marker on a cell is analyzed simultaneously, compared with manual techniques that rely on sequential gating using pairs of surface markers. we have recently reported that splenic cdc1s lacking xbp1 downregulate their surface expression of cd11c (osorio et al ., 2014). this in the loss of a fraction of cd11c cdc1s from the analysis when using a classical manual gating strategy (figure s2a). in contrast, since our proposed manual gating strategy includes identification of cdcs by their cd11ccd26 profile, it was easier to avoid missing the cd11c cdc1s, as these cells maintained their high cd26 profile (figure s2b). more importantly, both flowsom (figures s2d and s2e) and tsne (figure s2f) readily identified cdc1s regardless of their lower cd11c expression because the remaining xcr1cd172acd64f4/80mhciicd26 profile was sufficient to identify these cells as cdc1s in a fully unsupervised way. this demonstrates that unsupervised gating using flowsom or tsne outperformed classical manual gating for the analysis of the dc compartment of xbp1-deficient mice. we next aimed to align dcs across mouse, macaque, and human tissues (figures 4 and s3). we first had to overcome a few obstacles, such as the species - specific expression pattern of some markers (cd64 in human and macaque) and the lack of cross - reactive antibodies (cd26 for macaque and xcr1 for human and macaque). in human and macaque , cd64 can not be used, as cdcs also express some cd64 (figure s3e). therefore, we opted to exclude monocytes and macs on the basis of cd14 and cd16 expression as classically used. in mouse, human, and macaque and in all organs tested (spleen, liver, lung for mouse and spleen, blood, lung for human and macaque), cdcs were thus defined as cd45linmhciicd11c cells that are f4/80cd64 in mouse and cd14cd16 in human and macaque. as in the mouse, cd26 although mouse cdc1s and cdc2s were both cd11ccd26, human cdc1s were cd26cd11c, whereas cdc2s were cd26cd11c (figure s3b). we investigated whether cadm1 could be used as a cdc1 marker instead of xcr1 since cadm1 is expressed on porcine and macaque cdc1s (dutertre et al . 2015). to compare the degree of overlap between the expression of xcr1 and cadm1 , we used a chimeric protein consisting of human xcl1the ligand of xcr1and of the mcherry fluorescent protein (mcherry - xcl1 vaccibodies) to detect xcr1 on human and macaque spleen cells (figure s4). all cadm1cd172a cdc in human, macaque, and mouse spleen displayed a high xcr1 expression and also strongly expressed irf8, but not irf4 (figure s4). thus, cdc1s can be defined as cadm1cd172a cdc in mouse, human, and macaque. we next evaluated the use of this panel in multiple mouse, human, and macaque tissues in combination with classical human dc markers such as cd1c (bdca1) (figure 4). while cadm1cd172a cells comprised only irf4irf8 bona fide cdc2s in mouse, two populations of cadm1cd172a cells were detected in human and macaque lung: a population of bona fide cdc2s with a cd1cirf4irf8 phenotype and a population of cd1c cells showing the typical irf4irf8 expression observed for macs (figures 4c and s4a). therefore, in the case of human and macaque, monocytes or macs were not properly outgated using cd14 and cd16 expression, and cd1c expression was further required to define cdc2s hence, across tissues and species, cdc1s and cdc2s could be identified as cadm1cd172acd11ccd26irf8irf4 and cadm1cd172acd1ccd11cirf4irf8 cells, respectively. akin to mouse pdcs, human and macaque pdcs (defined as cd45cd11chladr cells) were irf8irf4 across tissues (figures s4b s4d). finally, in the human and macaque skin, the gating strategy required an additional marker, cd1a, to identify and outgate cd1acd11c lc before cdc1s and cdc2s could be faithfully identified (figure s4). therefore, using a limited number of flow cytometry markers, it is possible to align cdc subsets across several human, macaque, and mouse tissues, including the skin. we next used tsne to perform an unsupervised analysis of the flow cytometry dataset generated from different tissues of mouse, human, and macaque (figure 5). for each species (figures 5a, 5e, and 5i), cd45linmhcii cells of the distinct tissues were exported, concatenated, and displayed in a single tsne contour plot (tsne_dim1-tsne_dim2). heatmap representations of the expression of various markers that define macs, cdcs, and pdcs defined clusters of cells corresponding to the different cell subsets (figures 5b, 5f, and 5j). cells falling in the cdc1 (blue), cdc2 (green), and (for human and macaque) pdc (pink) tsne cell clusters were overlaid on classical contour plots, indicating that the dc subsets automatically delineated on tsne fit the criteria used to define them by manual gating (figures 5c, 5 g, and 5k). comparison of the different tissues showed that equivalent dc subsets always fell in the same tsne regions (figures 5d, 5h, and 5l). additionally, tsne analysis confirmed that, in human lung, cd14cd16hladrcd172acd1cirf4irf8 cells, identified by manual gating (figures 4 and s4a), were indeed related to monocytes or macs since they clustered with cd14cd16 monocytes (figures s4c and s4d). to elucidate the heterogeneity among the dc subsets, mass cytometry (cytof) data were acquired from different mouse and human tissues and total dc events were analyzed using one - sense (one - dimensional soli - expression by nonlinear stochastic embedding) (cheng et al ., 2015). here, manually chosen lineage - imprinted markers define the first dimension, and the other markers define the second dimension (figure 6). this ed in the generation of multiple clusters of cadm1cd26 cdc1 (blue), cd172acd11b (mouse), or cd172acd1c (human) cdc2s (green) and siglechb220 (mouse) or cd123cd303 (human) pdcs (pink) (figures 6a and 6d). binned frequency heatmaps were generated for each dimension. in human tissues, the lineage dimension validated the established dc subsets and also revealed contaminating cd172acd1c cells (region delineated by an orange rectangle), separating them from the bona fide cd172acd1c cdc2s (figure 6d). as before, these contaminating cells were confirmed to be cd11ccd26cadm1cd172acd1c cells when visualized in classical two - dimensional (2d) contour plots (figure 6e). the marker dimension gave key information concerning the degree of heterogeneity that exists among dc subsets (figures 6a and 6d). such heterogeneity can be primarily accounted for by tissue imprinting (figures 6b and 6f). heatmaps (figures 6c and 6 g), 2d contour plots, or histograms (figure s5) illustrated the mean expression of the list of differentially expressed markers for each of the three dc subsets. in mouse, this analysis confirmed that esam cdc2s (cluster 3) were mainly found in the spleen, while spleen cdc1s (cluster 5) had the highest cd8 and the lowest cd103 expression compared with the majority of cdc1s in the lung and gut (clusters 4 and 6, figure 6c). in humans, cdc phenotypic heterogeneity was also mostly explained by the tissue of residence (figure 6f). furthermore, some heterogeneity could be observed for human pdcs with a subset expressing higher cd141, cd56, cla, cd62l, cd5, cxcr3, cd2, cx3cr1, and cd39 (cluster 6) being observed in the spleen and in lower proportions in the blood and lung. therefore, our analysis reveals a previously unappreciated phenotypic heterogeneity in both mouse and human dcs that can now be mined for functional relevance. to demonstrate the usefulness of our approach in inflammatory settings, we utilized one - sense to track the activation of cdc1s, cdc2s, and monocyte - derived cells upon lps - induced inflammation in mice. linmhciicd11c cells from the lung and mediastinal lns harvested 1, 2, or 3 days following intranasal treatment with lps were profiled using cytof followed by one - sense analysis (figures 7 and s6a). linmhciicd11c cells from the different time points post - lps were exported and concatenated, yielding a single one - sense analysis for the lung (figure 7a) and for the ln (figure 7f). f4/80 and cd64 were added to the lineage markers to analyze monocyte - derived cells, yielding separated clusters of cd26cadm1cd11bcd172a cdc1 (blue clusters), cd11bcd172af4/80cd64 cdc2 (green clusters), and cd11bcd172af4/80cd64 monocyte - derived cells (orange clusters). these were then further subdivided into multiple smaller clusters along the marker dimension, revealing variations linked to the duration of lps treatment (figures 7b, 7c, 7 g, and 7h), including progressive phenotypic changes in expression of costimulatory receptors and inflammatory cytokines (figures 7d, 7e, 7i, and 7j). this analysis also revealed striking differences in the proportion of cdcs and monocyte - derived cells in both organs after lps challenge. day 1 (d1) following lps - treatment, cdc1s and cdc2s were strongly reduced in the lung, which was paralleled by a massive accumulation of monocyte - derived cells that represented 95% of all lung linmhciicd11c cells by d3 after lps. monocyte - derived cells appeared later in the ln, and their frequency gradually increased from below 1% at d0 and d1 to 36% at d3. at d0, both cdc subsets found in the lung and ln were not activated (low cd40, cd80, and cd86 expression), and cdcs reached maximal activation only after migration to the lns (see migratory cdc2 # 3, cdc1 # 6, and # 7 at d1 in the ln). lung monocyte - derived cells upregulated the costimulatory molecules cd80 and cd86, as well as the inhibitory receptor pdl1 (see monocyte - derived cells # 3 at d3). we also observed a progressive increase of fcri, bst2, and sca1 expression by monocyte - derived cells in the lung post - challenge (expression : # 1 < # 2 < # 3 ; figures 7d and 7e). ln monocyte - derived cells likely represent cells directly recruited from the bloodstream, as previously described (nakano et al ., 2009). both lung and ln monocyte - derived cells expressed inflammatory cytokines il-12p70, tumor necrosis factor (tnf), and interleukin-6 (il-6), whereas high costimulatory molecule expression was limited to those in the lung. altogether, this analysis demonstrates the power of our approach in which cdcs and monocyte - derived cell activation can be tracked automatically during an inflammatory response. this open - ended approach could, in the future, include phospho - stat signaling analysis or a wider range of cytokines, chemokines, or costimulatory receptors. recent developments in computational methods permit more robust analysis of flow cytometry and cytof data. relying on objective mathematical principles to define cellular clusters, automated analyses increase the reproducibility of flow analysis by circumventing manual gating (saeys et al ., 2016). this constitutes a major improvement, since manual gating is one of the largest variables in the analysis of flow cytometry experiments (mair et al ., 2015). furthermore, automated analyses assess the expression of all markers simultaneously and are not influenced by the order in which cells are gated, as in manual sequential pairwise comparisons. finally, these techniques simplify the visualization of the multidimensional datasets, which is particularly important when analyzing such data with more than 30 markers. however, application of these techniques for the study of dcs has remained limited. here, we have defined lineage - imprinted surface markers that permit the faithful identification of cdc1s and cdc2s across species and tissues by adding cd26 as a cdc marker complementary to cd11c, combining cd64 and f4/80 to ensure a better distinction between dcs and macs in mice, using cadm1 or xcr1 in combination with cd172a to separate cdc1s from cdc2s, and using irf8-irf4 staining to validate the identification of cdc1s and cdc2s. unsupervised computational techniques, such as flowsom and tsne, confirmed the robustness of our strategy for defining cdc1s and cdc2s across mouse, human, and macaque tissues. these techniques could also identify cdc1 and cdc2 populations automatically, paving the way toward the reliable analysis of the dc compartment of many mutant mice in a high - throughput, unsupervised, and standardized manner. importantly, automated identification can outperform classical manual gating in the analysis of mutant strains when markers change their expression profile, as was exemplified here with the correct identification of cd11c xbp1-deficient cdc1s that were missed by the classical manual gating strategy. 2009 ) and is highly expressed on human and macaque cdc1s, as demonstrated with xcl1-mcherry vaccibodies, no commercial anti - human or anti - macaque xcr1 antibody is currently available. similarly, although cd26 is expressed by macaque dcs at the rna level (data not shown), none of the commercial anti - cd26 antibodies tested showed cross - reactivity with macaque. generating cross - species reactive anti - xcr1 and anti - cd26 antibodies will be invaluable, as it will allow further simplification of the minimalistic and universal cdc phenotyping panel proposed here. ideally, we would need one or two surface markers that allow the identification of all macs (including lcs) across tissues and species, an essential issue to avoid contamination of the cdc2 population since macs are also xcr1cd172a. as confirmed by the addition of additional markers to our standard minimalistic panel for instance, mouse skin xcr1cd172airf8irf4 cdc1s comprise both cd103 and cd103 cells (henri et al ., 2010). in mouse, the foundations of dc subset specialization are imprinted in the bm, as pre - cdcs already contain cells committed toward the cdc1 or cdc2 lineage before colonizing peripheral tissues (grajales - reyes et al ., 2015, these lineage - imprinted programs include the mutually exclusive expression of xcr1 versus cd172a and of irf8 versus irf4 . to acquire the phenotype of terminally differentiated tissue cdc1s and cdc2s , precursors likely further integrate tissue - associated programs on the top of those lineage - imprinted foundations . in some organs , cdcs occupy distinct micro - anatomical compartments that can provide distinct environmental cues to developing cdc1s and cdc2s . for example, only those cdc2s that are localized in the splenic bridging channels specifically express cd4 . this subset of splenic cdc2s requires notch2 for their terminal differentiation ( caton et al . , 2007, lewis et al ., 2011) and depends on the expression of chemotactic receptor ebi2 for their localization in the bridging channels (gatto et al ., 2013). analysis by flow cytometry of the splenic dc compartment of ebi2-deficient or notch2-deficient mice using only xcr1 and cd172a yields a less dramatic phenotype than with the inclusion of cd4, because a significant fraction of cd172acd4 cdc2s are found in these mice. use of additional, tissue - imprinted markers such as cd4 is therefore required to fully appreciate the functional heterogeneity of the splenic cdc compartment. as such the one - sense approach we have described fulfills such a requirement, as it first aligns the dc across tissues based on lineage - imprinted markers yielding a simplified lineage dimension and subsequently analyzes the heterogeneity ing from the tissue - imprinted programs through an unsupervised marker dimension. it simplifies the classification of dcs into cdc1s and cdc2s across tissues and species based on conserved lineage - imprinted markers without losing the power that multi - dimensional analyses offer. on that basis, we have gauged the differences that exist in cdc1s and cdc2s according to their tissue of residence and following an inflammatory insult. this type of analysis can be readily expanded to track any parameters of interest, such as the production of inflammatory mediators, or to dissect particular signaling pathways using phospho - flow approaches. in , the herein described methodology should provide a useful framework to analyze the complexity of the dc compartment, paving the way toward the identification of the best dc subset(s) to target for specific therapeutic applications such as the development of next - generation vaccines. c57bl/6 mice used in france and belgium were obtained from harlan or janvier laboratories. flt3l and c57bl/6 mice for the experiment in singapore were from the biological resource center (brc), agency for science, technology and research (astar). all animals were housed under specific pathogen - free conditions in individually ventilated cages in a controlled day - night cycle and were given food and water ad libitum. all animal experiments performed were approved by the local animal ethics committee (vib - ugent ; institutional animal care and use committee of the biological resource center, astar ; ciml) and were performed according to the guidelines of belgian, french, and european animal protection law and of the agri - food and veterinary authority and the national advisory committee for laboratory animal research of singapore. see supplemental experimental procedures for additional information about the generation of bm chimeras, the intranasal lps treatment, and the digestion of mouse tissues. c57bl/6 mice were treated or not intranasally with 5g lps (invivogen) and were euthanized 1, 2, or 3 days later. 8 hr before euthanasia, mice were injected intraperiotoneally with 0.25 mg of brefeldina (sigma) in 200 l pbs. human samples were obtained with approval from singapore singhealth and national health care group research ethics committees. samples were prepared as described previously for skin (mcgovern et al ., 2014), lung (schlitzer et al ., 2013), and colon (watchmaker et al ., 2014). the antibodies and reagents used for facs analyses of mouse tissues are listed in tables s1 (extracellular panel) and s2 (irf4 and irf8). the antibodies used for facs analyses of human tissues were all mouse anti - human monoclonal antibodies, except the chicken anti - human cadm1 igy primary mab. the list of antibodies and reagents used for human and macaque flow cytometry experiments are listed in table s3, and details of the antibody combinations (panels) can be found in table s4. , purified antibodies were obtained from invitrogen, becton dickinson, biolegend, ebioscience, bioxcell fluidigm, r&d biosystems abd, and abd serotec using clones as listed in table s5 for mouse experiments and in table s6 for human experiments. the data were exported as a traditional flow cytometry file (.fcs) format, and cells for each barcode were deconvoluted using boolean gating. one - sense analysis was performed as recently described (cheng et al ., 2015). the automated analysis was performed by the flowsom algorithm (van gassen et al ., 2015) or the tsne algorithm ( becher et al., 2014, wong et al.,; clinicians for helping to access samples, multiple advice, and discussion, j.k.y.c., c.n.m.; formal analysis, m.g., c.- a.d., c.l.s., n.mcg.,

summarydendritic cells (dcs) are professional antigen - presenting cells that hold great therapeutic potential. multiple dc subsets have been described, and it remains challenging to align them across tissues and species to analyze their function in the absence of macrophage contamination. here, we provide and validate a universal toolbox for the automated identification of dcs through unsupervised analysis of conventional flow cytometry and mass cytometry data obtained from multiple mouse, macaque, and human tissues. the use of a minimal set of lineage - imprinted markers was sufficient to subdivide dcs into conventional type 1 (cdc1s), conventional type 2 (cdc2s), and plasmacytoid dcs (pdcs) across tissues and species. this way, a large number of additional markers can still be used to further characterize the heterogeneity of dcs across tissues and during inflammation. this framework represents the way forward to a universal, high - throughput, and standardized analysis of dc populations from mutant mice and human patients.

in june 2004, a 58-year - old asian female presented with anxiety, palpitations and breathlessness. differential leukocyte count revealed blasts, promyelocytes, myelocytes, and metamyelocytes with decreased leukocytes. liver function test (lft) and kidney function test (kft) were normal. the patient was started on imatinib 400 mg and folic acid 5 mg once daily in august 2004. peripheral smear remission was seen in 2 months, i.e., tlc returned to normal value and hemoglobin and platelet counts normalized. one year later, the patient presented with bilateral redness of eyes and facial swelling which was treated with antihistamines. following this, 1 month later, the patient presented with bilateral parotid swelling which was not evaluated. parotid swelling resolved of its own within a month without any treatment. in august 2005, bone marrow biopsy revealed normocellular marrow spaces with adequate representation of all three marrow elements, i.e., complete hematological remission. triiodothyronine (t3), thyroxine (t4), and thyroid - stimulating hormone were normal, but thyroid microsomal autoantibodies (tma) antibody was positive. fine needle aspiration cytology was planned, but the patient did not consent for the investigation. most common adverse events reported with imatinib are edema, nausea, muscle cramps, musculoskeletal pain, diarrhea, rash, fatigue, and headache. this case illustrates a possible occurrence of imatinib - induced autoimmune thyroiditis. in a case of thyroiditis, the differential diagnosis of thyroid pain includes acute, subacute thyroiditis, chronic thyroiditis, hemorrhage into a cyst, malignancy including lymphoma, and rarely, amiodarone - induced thyroiditis or amyloidosis. the absence of small tender asymmetric goiter, fever, dysphagia, and erythema over thyroid rules out bacterial or fungal causes of thyroiditis. the patient had bilateral parotid swelling 6 months before the presentation which resolved of its own. the absence of fever and presence of tma ruled out mumps as a cause of thyroiditis. finally, the positive response to treatment with corticosteroids also ruled out infection and malignancy as a cause of pain in thyroid. thus, only possible cause can be autoimmune thyroiditis which responded well to the treatment with corticosteroids. imatinib was started in august 2004, and the patient presented with the thyroiditis in november 2005 establishing the temporal association of thyroiditis with imatinib. use of naranjo's probability scale and who causalty scale assessment indicated imatinib as a possible cause of thyroiditis because the idiopathic cause of autoimmune thyroiditis can not be ruled out. sunitinib, a tk inhibitor, a drug of same class has been implicated as a cause of lymphocytic and destructive thyroiditis. a review the literature of studies of thyroid dysfunction induced by tk inhibitors showed that thyroid dysfunction is not a rare entity with the use of tk inhibitors although not so common with the use of imatinib. as there was possibility of progression of cml when drug is withheld, drug withdrawal was not done to check the possible causation of thyroiditis by imatinib. second, there was no increase in the thyroid hormone levels and the severity of thyroiditis did not mandate drug withdrawal. hence, a possibility of activation of autoimmune diseases with the use of imatinib can not be ruled out. hence, clinicians should be aware of the possibility of thyroiditis with imatinib and should have an eye not to miss the event.

here, we present a case of chronic myeloid leukemia for which imatinib therapy was initated. triiodothyronine (t3), thyroxine (t4), and thyroid - stimulating hormone was normal, and thyroid microsomal autoantibodies (tma) were positive and patient was diagnosed as thyroiditis treated with corticosteroids for 1 months which lead to resolution.

allergic conjunctival diseases (acd) are conjunctival inflammatory disorders, characterized by an immediate hypersensitivity reaction associated with antigen - specific ige antibodies. patients with acd are usually categorized into the following types based on clinical criteria: seasonal allergic conjunctivitis (sac), perennial allergic conjunctivitis (pac), vernal keratoconjunctivitis (vkc), atopic keratoconjunctivitis (akc), and giant papillary conjunctivitis (gpc). sac and pac are characterized by conjunctival hyperemia, conjunctival edema, and papillary hyperplasia of tarsal conjunctiva and are classified as mild acd. in contrast, vkc is characterized by the development of proliferative lesions of the conjunctiva, including giant papillary proliferation of the tarsal conjunctiva and gelatinous cell infiltration of the limbal conjunctiva. vkc also complicates corneal disorders, such as shield ulcer and punctate corneal keratitis, and is classified as a severe acd, because the visual prognosis may be poor. akc usually develops in older patients with atopic dermatitis and also complicates severe ocular surface diseases, including giant papillary conjunctivitis, shield ulcer, and dry eye. in shield ulcers of patients with vkc and akc, depositions of major basic protein and eosinophil cationic protein (ecp), which comprise specific granules of eosinophils, have reportedly been observed histologically in the corneal ulcerative lesions. furthermore, ecp levels in the tears of vkc and akc patients are reportedly increased, in comparison with those in controls. therefore, the pathophysiological characteristics of these severe acd, including vkc and akc, include eosinophilic inflammation in the conjunctiva. eotaxin is a member of the cc chemokine family and is divided into three subfamilies, namely, ccl11/eotaxin-1, ccl24/eotaxin-2, and ccl26/eotaxin-3. eotaxin-1, eotaxin-2, and eotaxin-3 interact with the cc chemokine receptor 3 (ccr3). representative inflammatory cells expressing ccr3 on their cell surfaces are eosinophils, type-2 helper t cells (th2), and basophils. il-13, which is a th2-derived cytokine, induces eotaxin in vitro through activation of the il-4r receptor / stat-6 pathway. therefore, eotaxin-1, eotaxin-2, and eotaxin-3 are thought to be allergic inflammation - related chemokines. in the eotaxin subfamily, it has been reported that increased eotaxin-2 levels in tears and expression of ccl24 (eotaxin-2) mrna on the ocular surface are more common in vkc patients than those of eotaxin-1 and eotaxin-3. however, the relationship between the expression levels of ccl24 (eotaxin-2) mrna on the ocular surface and the severity of severe acd, including vkc and akc, has not been fully investigated. in this study , we evaluated the clinical efficacy of using ccl24 (eotaxin-2) mrna expression levels on the ocular surface in patients with severe acd, including vkc and akc, as a biomarker for severe acd. this study was approved by the institutional review board of the nihon university school of medicine and adhered to the tenets of the declaration of helsinki. this study included 18 consecutive patients with vkc or akc treated at the department of ophthalmology, nihon university itabashi hospital, tokyo, japan, from august 2012 to january 2015 (akc / vkc group), and 12 healthy volunteers who did not have any personal or family history of atopic disease, were not affected by ocular surface diseases, or have a history of wearing contact lenses as controls (control group). objective diagnoses were made in vkc and akc patients by means of slit - lamp clinical examination and serum examination for antigen - specific ige antibodies, according to the japanese guidelines for acd. patients with ocular surface disease other than acd, including lagophthalmos, blepharospasm, conjunctival chalasis, dry eye, infectious conjunctivitis, infectious keratitis, stevens - johnson syndrome, and ocular pemphigoid, were excluded. nontreated patients or patients treated with antiallergic ophthalmic solutions alone, such as mast cell stabilizers, histamine h1 receptor antagonists, corticosteroids, and immunosuppressive agents, were included in the study. patients who used oral medicines or injections for treating allergic diseases and those who received immunotherapy were excluded from the study. clinical scores of objective findings in the akc / vkc group were determined using the 5 - 5 - 5 exacerbation grading scale for acd. the akc / vkc group was divided into two subgroups depending on the clinical score: the active stage subgroup with clinical scores of 100 points or more (n = 6) and the stable stage subgroup with 100 points or less (n = 12). modified impression cytology was performed after instillation of topical oxybuprocaine 0.4% (benoxil, santen, osaka, japan). schirmer's test strips (schirmer tear production measuring strips, showa yakuhin kako, tokyo, japan) were applied to the upper tarsal conjunctiva, pressed gently using a glass rod, and then removed. the membrane was preserved in rnalater rna stabilization reagent (qiagen, hilden, germany) until analysis. total rna was harvested from each schirmer tested paper using an rneasy mini kit (qiagen, hilden, germany) following the manufacturer's instructions. cdna was then synthesized using a high - capacity cdna reverse transcription kit (life technologies japan, tokyo, japan), according to the manufacturer's instructions. to detect expression of ccl24 (eotaxin-2) mrna, real - time reverse transcription polymerase chain reaction (real - time rt - pcr) was performed using a commercial pcr master mix (taqman universal pcr master mix ; life technologies, tokyo, japan) and predesigned primers (life technologies) for ccl24 (eotaxin-2 ; hs00171082_m1). samples were analyzed using the step one plus real - time pcr system (life technologies) and comparative threshold (ct) values were obtained. target ct values were normalized to those of gapdh (hs99999905_m1) in the same sample. differences between akc / vkc and control groups were identified using welch's t - test or the chi - square test. the for ccl24 (eotaxin-2) mrna expression on the ocular surface were evaluated using the nonparametric steel - dwass test. spearman's rank correlation coefficient was used to evaluate whether ccl24 (eotaxin-2) mrna expression correlated with the clinical score for ccl24 (eotaxin-2) mrna expression on the ocular surface, 6 of 6 patients in the active stage subgroup of akc / vkc group were ccl24- (eotaxin-2-) positive, with median (range) levels of 133 (27.6232). eleven of 12 patients in the stable stage subgroup of akc / vkc group were ccl24- (eotaxin-2-) positive, with median (range) levels of 5.99 (0.14033.2); the remaining patient had levels below the lower limit of detection. eight of 12 patients in the control group were ccl24- (eotaxin-2-) positive, with median (range) levels of 0.98 (0.0820.2), while 4 patients had levels below the lower limit of detection. the expression levels of ccl24 (eotaxin-2) mrna on the ocular surface were significantly higher in the active stage than in the stable stage akc / vkc subgroup and the control group (both p < 0.01 ; steel - dwass test ; figure 1). the median value (range) of the clinical scores in the active and stable stage subgroups of akc / vkc group were 13 and 128, respectively. in patients with akc / vkc , clinical scores were significantly correlated with the levels of (ccl24) eotaxin-2 mrna expression on the ocular surface (= 0.795, p < 0.01, spearman 's rank correlation coefficient ; figure 2). a 10-year - old boy was diagnosed as having vkc and had been under treatment for vkc by a local doctor for 6 years. his clinical observation of vkc repeated exacerbation and remission, and his severity of vkc was different in right and left active giant papillae and exfoliative epithelial keratopathy was present in his right eye; his clinical score was 212, and his ccl24 (eotaxin-2) mrna expression levels on the ocular surface were 203.2. in his left eye, papillary lesions and hyperemia at the upper palpebral conjunctiva were observed, and the clinical score was 2, while the ccl24 (eotaxin-2) mrna expression level on the ocular surface was 17.2 (figure 3). a 10-year - old girl was diagnosed as having vkc and had been under treatment for vkc by a doctor for 1 year. she experienced pain in her right eye and also had recurrence of corneal plaque and was referred to our hospital. at her first examination, she demonstrated shield ulcer in her right eye, giant papillae, and palpebral conjunctiva with a velvety appearance. topical 0.1% tacrolimus ophthalmic suspension (talymus ophthalmic suspension 0.1%, senju pharmaceutical, co., ltd ., osaka, japan) and 2% sodium cromoglicate ophthalmic solution (intal ophthalmic solution 2%, sanofi, tokyo, japan) were administered. at commencement of treatment with tacrolimus ophthalmic suspension, her clinical score was 213 and the level of ccl24 (eotaxin-2) mrna expression on the ocular surface was 349.6. during the first 10 weeks of treatment, the clinical score was 3 points and the ccl24 (eotaxin-2) mrna expression level on the ocular surface was 9.6 (figure 4). in this study, we assessed the usefulness of ccl24 (eotaxin-2) mrna expression levels on the ocular surface as a biomarker of the severity of acd. we found that these levels correlated well with the clinical score reflecting objective findings in patients with akc and vkc. as a method for sampling the ocular surface to test the expression levels of ccl24 (eotaxin-2) mrna this method entailed a membrane biopsy technique, as used for impression cytology, but used filter paper instead of nitrocellulose membrane for the biopsy. in the conventional impression cytology method, the specimen obtained using a nitrocellulose membrane is examined histologically. however, the major concern of this method is the ocular sensation of a foreign body and the ocular pain experienced after the examination; repeated examinations for follow - up of the biomarker may cause discomfort for the patients. changing to the filter paper for impression cytology sampling and using a quantitative method (real - time rt - pcr) allowed assessment of the biomarker on the ocular surface. the specimens obtained by modified impression cytology most likely included conjunctival epithelial and invading inflammatory cells, in addition to tears and mucin. it was therefore considered useful for investigating inflammation - associated factors expressed by conjunctival epithelial cells and inflammatory cells as potential biomarkers of allergic inflammation at the ocular surface. in this study , we elucidated a significant correlation between clinical observations and ccl24 expression on the ocular surface of akc / vkc patients with or without treatment with ophthalmic solutions. these suggest that ccl24 expression on the ocular surface is suitable as a biomarker of severe allergic conjunctival diseases. in previous reports , it has been demonstrated that eotaxin-1 plays a critical role in eosinophilic infiltration in the conjunctiva and cornea of patients with acd. however, the concentration of eotaxin-2 in tears has been reported to be higher than those of eotaxin-1 in acd patients. therefore, in this study, we investigated the usefulness of ccl24 (eotaxin-2) mrna expressed on the ocular surface as a biomarker for patients with akc / vkc. we found that expression levels of ccl24 (eotaxin-2) mrna on the ocular surface were significantly increased and correlated with the clinical score in the active stage of vkc. eotaxin-1 is reportedly produced by corneal keratinocytes , conjunctival fibroblasts, cd68-positive cells in the conjunctiva, and eosinophils in the conjunctiva. previously, we have reported that the tear levels of eotaxin-2 correlated significantly with those of eosinophil cationic protein and that epithelial cells in conjunctival smears of patients with vkc expressed eotaxin-2, based on immunohistochemistry. leonardi and colleagues reported that tear levels of eotaxin-1 and eotaxin-2 significantly correlated with the percentage of eosinophils in tears. therefore, conjunctival epithelial cells and eosinophils are thought to be candidate eotaxin-2-producing cells on the ocular surface. furthermore, expression levels of ccl24 (eotaxin-2) mrna in modified impression cytology may be a good biomarker for evaluating allergic inflammation in the conjunctivas of patients with akc / vkc. in these case reports , we showed differences between the right and left eye in the severity of vkc based on the quantitative analysis of ccl24 (eotaxin-2) mrna levels. furthermore, in the 10-year - old girl with vkc, we were able to show the therapeutic effect of treatment by tacrolimus instillation by the reduction of both clinical scores and expression levels of ccl24 (eotaxin-2) mrna. therefore, in akc / vkc patients undergoing treatment, monitoring of the expression levels of ccl24 (eotaxin-2) mrna may provide a useful index of exacerbation and therapeutic response. the limitation of this study included the small sample size, and a lack of patients with mild acd, such as sac and pac. further investigation is necessary for verifying the usefulness of ccl24 (eotaxin-2) mrna levels as a biomarker of acd in a large sample that includes patients with sac and pac. another limitation of this study was that akc / vkc patients not receiving treatment and those only receiving treatment with ophthalmic solutions were enrolled. the ccl24 mrna expression on the ocular surface may be affected by treatment with ophthalmic solutions. however, the efficacy of the therapeutic agent is one of the items measured by a biomarker. therefore, further investigation on the therapeutic effect of antiallergic treatment in a large cohort, including untreated patients with allergic conjunctival diseases, using eotaxin-2 expression as a biomarker, will be necessary in the future. expression levels of ccl24 (eotaxin-2) mrna on the ocular surface are a useful biomarker of the clinical severity of akc / vkc.

purpose. this study aimed to evaluate the clinical efficacy of using expression levels of ccl24 (eotaxin-2) mrna on the ocular surface as a biomarker in patients with vernal keratoconjunctivitis (vkc) and atopic keratoconjunctivitis (akc). methods. eighteen patients with vkc or akc (vkc / akc group) and 12 control subjects (control group) were enrolled in this study. the vkc / akc clinical score was determined by objective findings in patients by using the 5 - 5 - 5 exacerbation grading scale. all subjects underwent modified impression cytology and specimens were obtained from the upper tarsal conjunctiva. expression levels of ccl24 (eotaxin-2) mrna on the ocular surface were determined using real - time reverse transcription polymerase chain reaction. . the vkc group was divided into two subgroups, depending on the clinical score: the active stage subgroup with 100 points or more of clinical scores and the stable stage subgroup with 100 points or less. ccl24 (eotaxin-2) mrna expression levels in the active vkc / akc stage subgroup were significantly higher than those in the stable vkc / akc subgroup and the control group. clinical scores correlated significantly with ccl24 (eotaxin-2) mrna expression levels in the vkc group. . ccl24 (eotaxin-2) mrna expression levels on the ocular surface are a useful biomarker for clinical severity of vkc / akc.

the pedant genome database was first announced in a short note entitled pedantic genome analysis which appeared in trends in genetics in 1997 and reported computational analysis of seven completely sequenced and two partial genomes available at that time. from the very beginning the main mission of pedant was defined as filling the gap between manually curated high quality protein sequence databases, such as uniprot / swiss - prot, and the enormous amounts of other protein sequences produced by genome sequencing projects at an ever increasing pace. over the past decade the pedant genome database was produced by systematically applying an automatic annotation pipeline to genome data released in the public domain. these efforts ed in one of the most comprehensive currently available genome databases which includes 468 organisms from all three domains of life. we report on three new features of the pedant web server: (i) an all - new graphical user interface (gui), (ii) availability of web services and (iii) rule - based detection of annotation errors. the current version of the pedant software familiar to most users was introduced several years ago and is known as version 2. in 2006 we were deploying and testing the all - new version 3 which represents a complete re - implementation of the pedant software in the java programing language. server application for enterprise - scale molecular sequence analysis with advanced features such as highly dynamic workflow - based process management, direct interface to computing grids, support for essentially all existing sql database management systems, open architecture for easy integration of additional algorithms, and powerful scripting interfaces (d. frishman et al ., manuscript in preparation). migration of pedant genomes to the new version 3 is currently in progress and will be accomplished by mid-2007. so far we have completed a pilot study to annotate 40 genomes to demonstrate the advantages of pedant3. the new version of the pedant gui can be viewed by selecting any genome with a the pedant gui has been made user - configurable and supports four main types of windows: (i) sequence analysis: information about the complete analysis with links to orf lists of the individual algorithms, (ii) gene report: information about a selected gene, (iii) contig report: information about a selected contig and (iv) genetic element report (where available): information about a selected nonprotein coding genetic element. on selection of a pedant3 genome, the analysis window is opened displaying a list of genes and their best - blast hits. the left - hand panel contains a context - dependent navigation tree that makes available different choices dependent on the particular view the user is working with. the report page (figure 1) is split into different sections (overview, protein function, protein structure, protein location, general properties and export) and lists the analysis for a particular gene product, which are also available for download in different formats. the protein viewer displays several panels for various types of evidence that can be configured by selecting the dna viewer allows for easy navigation along the chromosomes and is capable of displaying an unlimited number of features at different zoom levels. navigation tree and report page for the hypothetical secreted protein of helicobacter pylori (gi_15645712). this figure also illustrates the application of association rule mining for finding potential annotation errors (see text). the interpro domain ipr001440 and pfam domain pf00515 were erroneously assigned to this gene product based on a weak similarity hit to a single tpr_1 repeat whereas the domain definition requires at least three repeat copies. note that all other features marked as suspicious are in fact annotated correctly; the method does not detect annotation errors as such, but rather incompatible feature combinations which might include annotation errors. any pedant3 dataset can be searched using sequence ids, sequences, prosite - like patterns or free text as the query option. web services technology is becoming increasingly popular within the bioinformatics community as a means to exploit the large amounts of data, software programs and computing power available at various institutions. according to the world wide web consortium (w3c) a web service is a software system designed to support interoperable machine - to - machine interactions over a network . this technology is based on the extensible markup language (xml) and open standards, and is platform and programing language independent. this enables clients for a particular service to be written in many languages, such as java or perl, irrespective of the language the service was written in. a web service has an interface that is described in a machine processable format using the xml based web services description language (wsdl). wsdl provides a format for the description of a web service interface, including parameters and data types in sufficient detail for a programer to write a client application for that service. tools are available for various programing languages to generate the required client classes, such as apache axis's wsdl2java . the client programs interact with the web service using messages based on the simple object access protocol (soap). as with wsdl, soap messages are xml based, permitting the interoperability of web services. for the transport layer itself , web services typically use the hypertext transfer protocol (http), preventing problems sending the soap messages through firewalls. bioinformatics users can avoid keeping local copies of databases and software and use a client program instead to access remote databases and software via web services. the pedant web service allows the user to query the database in an automated way from client programs and workflows. we provide a number of data retrieval methods in our data retrieval service (table 1). for example, to fetch the functional and structural annotations of a particular protein, the client program can call the getreportbydbcodeandcontig method. methods available in the data retrieval service currently this web service only retrieves data from pedant2 analyses; the pedant3 web service is in preparation. the raw (unparsed) output from various bioinformatics methods a protein can be uniquely identified with just the database name and protein code. for unfinished genomes, where the proteins were predicted using orpheus, it is necessary to provide the database name, protein code and contig name to uniquely identify a protein. a list of the bioinformatics methods available for each genome can be obtained by calling the getmethodsbydb method. we have generated a java client program using the apache axis software (wsdl2java) which has an example class demonstrating how to make calls to the dataretrievalservice. we have also included pedant web service client functionality in our prompt workbench as a use case to demonstrate the various advantages of the web services. prompt is a standalone application which enables a user to compare protein sequence sets, revealing statistically significant differences in their annotation features; . genome annotation produced by automatic pipelines such as pedant is notoriously prone to various kinds of errors. while every effort is being made to select the most reliable, carefully benchmarked bioinformatics tools and to avoid spurious similarity hits by setting conservative similarity thresholds, automatically produced function predictions are still not on a par with the of manual curation of sequence data in high - quality databases such as uniprot. we estimate that only 5% of all known proteins have been manually annotated and, given the progress in superfast sequencing technologies, it is becoming increasingly clear that the overwhelming majority of sequence data will not be processed by human experts. we have recently developed a technology to improve the quality of automatically generated annotation using data mining techniques. the entire body of annotation available in the pedant database can be considered as a collection of records of variable length, one for each gene product, containing functional and structural attributes, such as predicted functional categories, domain assignments and the like. using a large collection of pedant records as input, we apply an unsupervised learning algorithm called association rule mining to derive protein features that occur frequently together. specifically, implications in the form a&b c are derived which mean that the majority of proteins possessing features a and b also possess c. some rules have the strength 1.0 which means that they are always fulfilled, while other rules may be true only in a certain percentage of cases. an exception from a reasonably strong rule will have features a and b, but not c, which may be caused either by over - annotation (features a or b ascribed erroneously), or by under - annotation (feature c missed). we have shown that 70% of exceptions from strong association rules found in pedant data point to incompatible or missing annotation and thus may be instrumental in identifying annotation errors. however, since strong association rules detect not annotation errors as such but only incompatible feature combinations, it is not possible to automatically correct errors. instead, we highlight suspicious features and add the features which were putatively missed in a separate section at the bottom of the report page (figure 1). in a recent pilot project we applied this approach to analyze the pedant annotation of 10 model genomes arabidopsis thaliana, aeropyrum pernix, bacillus subtilis, escherichia coli, helicobacter pylori, mycobacterium tuberculosis, parachlamydia, saccharomyces cerevisiae, synechocystis and thermoplasma acidophilum containing a total of 55 123 protein entries. in the course of 2007 we intend on providing rule - based correction for all pedant genomes. the current version of the pedant software familiar to most users was introduced several years ago and is known as version 2. in 2006 we were deploying and testing the all - new version 3 which represents a complete re - implementation of the pedant software in the java programing language. server application for enterprise - scale molecular sequence analysis with advanced features such as highly dynamic workflow - based process management, direct interface to computing grids, support for essentially all existing sql database management systems, open architecture for easy integration of additional algorithms, and powerful scripting interfaces (d. frishman et al ., manuscript in preparation). migration of pedant genomes to the new version 3 is currently in progress and will be accomplished by mid-2007. so far we have completed a pilot study to annotate 40 genomes to demonstrate the advantages of pedant3. the new version of the pedant gui can be viewed by selecting any genome with a the pedant gui has been made user - configurable and supports four main types of windows: (i) sequence analysis: information about the complete analysis with links to orf lists of the individual algorithms, (ii) gene report: information about a selected gene, (iii) contig report: information about a selected contig and (iv) genetic element report (where available): information about a selected nonprotein coding genetic element. on selection of a pedant3 genome, the analysis window is opened displaying a list of genes and their best - blast hits. the left - hand panel contains a context - dependent navigation tree that makes available different choices dependent on the particular view the user is working with. the report page (figure 1) is split into different sections (overview, protein function, protein structure, protein location, general properties and export) and lists the analysis for a particular gene product, which are also available for download in different formats. the protein viewer displays several panels for various types of evidence that can be configured by selecting for example certain panels can be hidden, resized and the color scheme can be changed. the dna viewer allows for easy navigation along the chromosomes and is capable of displaying an unlimited number of features at different zoom levels. navigation tree and report page for the hypothetical secreted protein of helicobacter pylori (gi_15645712). this figure also illustrates the application of association rule mining for finding potential annotation errors (see text). the interpro domain ipr001440 and pfam domain pf00515 were erroneously assigned to this gene product based on a weak similarity hit to a single tpr_1 repeat whereas the domain definition requires at least three repeat copies. note that all other features marked as suspicious are in fact annotated correctly; the method does not detect annotation errors as such, but rather incompatible feature combinations which might include annotation errors. any pedant3 dataset can be searched using sequence ids, sequences, prosite - like patterns or free text as the query option. web services technology is becoming increasingly popular within the bioinformatics community as a means to exploit the large amounts of data, software programs and computing power available at various institutions. according to the world wide web consortium (w3c) a web service is a software system designed to support interoperable machine - to - machine interactions over a network . this technology is based on the extensible markup language (xml) and open standards, and is platform and programing language independent. this enables clients for a particular service to be written in many languages, such as java or perl, irrespective of the language the service was written in. a web service has an interface that is described in a machine processable format using the xml based web services description language (wsdl). wsdl provides a format for the description of a web service interface, including parameters and data types in sufficient detail for a programer to write a client application for that service. tools are available for various programing languages to generate the required client classes, such as apache axis's wsdl2java . the client programs interact with the web service using messages based on the simple object access protocol (soap). as with wsdl, soap messages are xml based, permitting the interoperability of web services. for the transport layer itself , web services typically use the hypertext transfer protocol (http), preventing problems sending the soap messages through firewalls. bioinformatics users can avoid keeping local copies of databases and software and use a client program instead to access remote databases and software via web services. the pedant web service allows the user to query the database in an automated way from client programs and workflows. we provide a number of data retrieval methods in our data retrieval service (table 1). for example, to fetch the functional and structural annotations of a particular protein, the client program can call the getreportbydbcodeandcontig method. methods available in the data retrieval service currently this web service only retrieves data from pedant2 analyses; the pedant3 web service is in preparation. the raw (unparsed) output from various bioinformatics methods can be obtained using the methods getrawhitsbydbcodeandmethod and getrawhitsbydbcodecontigandmethod. for completely sequenced genomes, a protein can be uniquely identified with just the database name and protein code. for unfinished genomes, where the proteins were predicted using orpheus, it is necessary to provide the database name, protein code and contig name to uniquely identify a protein. a list of the bioinformatics methods available for each genome can be obtained by calling the getmethodsbydb method. we have generated a java client program using the apache axis software (wsdl2java) which has an example class demonstrating how to make calls to the dataretrievalservice. we have also included pedant web service client functionality in our prompt workbench as a use case to demonstrate the various advantages of the web services. prompt is a standalone application which enables a user to compare protein sequence sets, revealing statistically significant differences in their annotation features; . genome annotation produced by automatic pipelines such as pedant is notoriously prone to various kinds of errors. while every effort is being made to select the most reliable, carefully benchmarked bioinformatics tools and to avoid spurious similarity hits by setting conservative similarity thresholds, automatically produced function predictions are still not on a par with the of manual curation of sequence data in high - quality databases such as uniprot. we estimate that only 5% of all known proteins have been manually annotated and, given the progress in superfast sequencing technologies, it is becoming increasingly clear that the overwhelming majority of sequence data will not be processed by human experts. we have recently developed a technology to improve the quality of automatically generated annotation using data mining techniques. the entire body of annotation available in the pedant database can be considered as a collection of records of variable length, one for each gene product, containing functional and structural attributes, such as predicted functional categories, domain assignments and the like. using a large collection of pedant records as input, we apply an unsupervised learning algorithm called association rule mining to derive protein features that occur frequently together. specifically, implications in the form a&b c are derived which mean that the majority of proteins possessing features a and b also possess c. some rules have the strength 1.0 which means that they are always fulfilled, while other rules may be true only in a certain percentage of cases. an exception from a reasonably strong rule will have features a and b, but not c, which may be caused either by over - annotation (features a or b ascribed erroneously), or by under - annotation (feature c missed). we have shown that 70% of exceptions from strong association rules found in pedant data point to incompatible or missing annotation and thus may be instrumental in identifying annotation errors. however, since strong association rules detect not annotation errors as such but only incompatible feature combinations, it is not possible to automatically correct errors. instead, we highlight suspicious features and add the features which were putatively missed in a separate section at the bottom of the report page (figure 1). in a recent pilot project we applied this approach to analyze the pedant annotation of 10 model genomes arabidopsis thaliana, aeropyrum pernix, bacillus subtilis, escherichia coli, helicobacter pylori, mycobacterium tuberculosis, parachlamydia, saccharomyces cerevisiae, synechocystis and thermoplasma acidophilum containing a total of 55 123 protein entries. in the course of 2007 we intend on providing rule - based correction for all pedant genomes.

the pedant genome database provides exhaustive annotation of 468 genomes by a broad set of bioinformatics algorithms. we describe recent developments of the pedant web server. the all - new graphical user interface (gui) implemented in java allows for more efficient navigation of the genome data, extended search capabilities, user customization and export facilities. the dna and protein viewers have been made highly dynamic and customizable. we also provide web services to access the entire body of pedant data programmatically. finally, we report on the application of association rule mining for automatic detection of potential annotation errors. pedant is freely accessible to academic users at.

the increased use of radiotherapy techniques such as intensity - modulated radiotherapy and stereotactic radiosurgery has generalized the use of small fields. also, some treatment units as the gammaknife or the cyberknife are based on the use of small fields. it is therefore important to have methods for the precise measurement of the dose for these kinds of fields. the measurement of the dose distributions for smalls fields have beam geometries that are quite different from the reference conditions stated in the dosimetry protocols. the radiation field does not produce constant dose volumes to encompass the volume of conventional detectors. this finite detector volume has been identified as the major reason for the measurement of low dose values in the non - constant dose regions. moreover, the spectrum exhibits a dependence on the field size and the dose rate becomes smaller as the field size decreases, due to the loss of lateral electronic equilibrium. it is therefore necessary to use small volume dosimeters, with negligible energy and dose rate dependencies. ionization chambers (ics) have a small dependency on the energy of the radiation for the range of energies found in radiotherapy. however, in order to obtain a good signal to noise ratio, small sensitive volumes are not recommended. in this way the volume effect is not negligible for small field sizes, and the measurement in these non - constant dose regions is limited in accuracy. given the high spatial resolution of radiochromic (rc) films, the volume effect is not a concern in the dosimetry of radiotherapy beams. however, the response of rc films has a poor spatial homogeneity, and this may be their main drawback for dosimetric applications. lynch et al., shows that important lateral inhomogeneities are introduced by a flatbed scanner, and these inhomogeneities are function of the optical density. this work uses a method for small fields two - dimensional (2d) measurements with rc films based on the reduction of the spatial inhomogeneity of the film and the film reading. by means of averaging several measurements of a field, , the calibration curve is obtained after averaging several measurements of each dose calibration value. the film pieces used for each dose value are selected from different locations of one sheet of film. in this way the positive characteristics of rc film are then exploited to measure output factors (ofs), dose profiles, and 2d dose distributions. the obtained with the rc on ofs are compared with the measurements carried out with a pinpoint ic and a semiflex ic, whereas the measured profiles are compared with profiles calculated by monte carlo methods. the ics used in this work were a 0.015 cm pinpoint thimble - type 31006 (ptw, freiburg, germany) and a 0.125 cm semiflex thimble - type 31010. the rc film used was the ebt-2 film (international specialty products, wayne, usa). after irradiation the films were digitized in a flatbed scanmaker 9800xl (microtek, hsinchu, taiwan). the red channel of a 48 bits rgb digitization was then extracted and used as the digitizer reading. ics and films were irradiated with 6 and 15 mv photon beams produced from a varian 2100-dhx linac (varian medical systems). films and ics were placed in a pmma phantom of size 30 30 20 cm. the field sizes investigated were 0.5 0.5 cm, 0.7 0.7 cm, 1 1 cm, 2 2 cm, 3 3 cm, 6 6 cm, and 10 10 cm. ofs for fields ranging from 0.5 0.5 cm to 10 10 cm, and beam profiles and 2d dose distributions for fields of sizes 0.5 0.5 cm, 0.7 0.7 cm, and 1 1 cm were measured with the secondary jaws delimiting the field size, at a depth of dmax (1.5 cm for the 6 mv beams and 2.5 cm for the 15 mv beams) and 100 cm source - to - surface distance (ssd). all measurement were performed with a gantry angle of 0 and a dose rate of 300 mu / min that correspond to 3 gy / min for a 10 10 cm field, at dmax and 100 cm ssd. the pinpoint and the semiflex ics were placed with their stems perpendicular to the beam axis. the maximum lengths of the sensitive volumes of both chambers are 5 and 6.5 mm, respectively; these lengths being equal or slightly larger than the smallest field lateral dimension, and larger than the constant dose region for some of the fields measured. a number of segments of one sheet of film are used to carry out a calibration of the film response. calibration segments of film are irradiated separately to known doses of 50, 170, 250, 320, 400, and 520 cgy. then the segments are digitized and the readings obtained are used to fit a power function relating the digitizer reading to the dose. for each dose value 10 segments of film are read, and the readings are averaged before the curve fitting. the reading for each film calibration segment is obtained as the median of the pixel values in a roi of 1.5 1.5 cm in the center of the imaged segment. a sheet of film is cut into segments of 2 2 cm, conforming a 2d array. for each dose value 10 segments of film are selected, so that each pair of segments is in different columns and rows of the array. the film segments are irradiated separately and 6 h after irradiation the films are arranged in the same way before being cut and read. in this way, after averaging all the pieces belonging to a dose value the effect of the spatial inhomogeneity is minimized. six calibration fields (50, 170, 250, 320, 400, and 520 cgy) together with four fields of sizes 0.5 0.5 cm, 0.7 0.7 cm, 1 1 cm, and 2 2 cm were used (these fields can be seen, for instance, in first row and columns 7, 8, 9 and 10 respectively). each field was used to irradiate 10 film segments, and the readings of these 10 segments are averaged to minimize film inhomogeneities for each small field (0.5 0.5 cm, 0.7 0.7 cm, and 1 1 cm), the images obtained after digitizing the pieces of films are registered. after averaging the registered images, the pixel values are converted to dose by applying the calibration curve. then contour plots and transversal profiles are obtained. monte carlo calculations of the 1 1 cm fields were carried out with the penelope code and the spectra data for the 6 and 15 mv photon beams were taken from daryoush and rogers. a simplified simulation was carried out, aimed to simulate the effect of the collimator jaws in the penumbra of the small fields. the photon beam was modeled by a photon point source with the energy spectrum given by daryoush and rogers. figure 2 shows the calibration curve relating the readings of the films and the irradiation doses. it is a power function y = ax + c, where y stands for the dose and x stands for the normalized pixel value. pv = pixel value figure 3 shows the reduction of uncertainty when applying the method of averaging of readings. this figure shows 100 curves (solid lines), randomly chosen from 100,000 possibilities. each curve is obtained by fitting the readings of five pieces of film to the doses 520, 400, 320, 170, and 50 cgy. the curve obtained by fitting the averaged readings is also shown (bold and dashed line). using this curve, the dose estimation for the films irradiated to 250 cgy (averaged readings) is 249.6 cgy. on the other hand, the standard deviation for the estimation based on one single piece of film for each dose value is 19 cgy. calibration curves using one piece of film for dose values (solid lines) and averaging 10film pieces for each dose value (dashed line) figure 4 shows contour plots of film measurements for the fields 0.5 0.5 cm, 0.7 0.7 cm, and 1 1 cm. the isodoses shown are 80, 50, and 20% of the maximum dose for each of the 6 and 15 mv fields. the isodose curves represent the 80, 50, and 10% of the maximum dose in each field figure 5 shows the transversal profiles measured with films for the fields of sizes 0.5 0.5 cm, 0.7 0.7 cm, and 1 1 cm, and the energies 6 and 15 mv. the dashed lines correspond to the in - plane direction (gun - target direction) and the continuous lines correspond to the cross - plane direction (transversal to the in - plane). this figure shows the differences in the shape of the dose profiles for the in - plane and the cross - plane directions. in the clinac 2100 the jaws delimiting the field in the in - plane are closer to the target, and transversal profiles measured with films for the 0.5 0.5 cm, 0.7 0.7 cm, and 1 1 cm fields. the dotted lines correspond to the in - plane direction, while the solid lines correspond to the cross - plane direction figures 6 and 7 show the transversal profiles for the field of size 1 1 cm and the energies 6 and 15 mv, respectively. the dashed lines correspond to the monte carlo calculations and the continuous lines correspond to the film measurements. film measurements (solid lines) and monte carlo calculations (dashed lines) transversal profiles for the 1 1 cm 15 mv field. film measurements (solid lines) and monte carlo calculations (dashed lines) the measurements of the field sizes (determined as the full width half maximum) and penumbras 2080% for the 6 and 15 mv beams are shown in tables 1 and 2, respectively. the monte carlo calculations for the field of size 1 1 cm are also presented. field sizes (full width half maximum) and penumbras 20 - 80% in the in - plane (ip) and cross - plane (cp) directions for the fields shown in figure 5a (6 mv beams) field sizes (full width half maximum) and penumbras 20 - 80% in the in - plane (ip) and cross - plane (cp) directions for the fields shown in figure 5b (15 mv beams) figure 8 shows the measured ofs. the right side shows the measurements for the 6 mv energy beams, while the left side shows the measurements for the 15 mv beams, the error bars in the rc film measurements correspond to 1 standard deviation. in both cases a good agreement is found between both types of chambers (semiflex and pinpoint) and the rc films for field sizes of 2 2 cm or larger. rc = radiochromic, pp = pinpoint, sf = semiflex the measurement of the of for the 6 mv field of size 1 1 cm with the pinpoint and semiflex chambers are 10 and 14% lower, respectively, than the of measured with the rc films. in the case of 15 mv, the corresponding differences are 10 and 18%, respectively. for the smallest fields the differences increase. for the 0.7 0.7 cm, the differences with the rc measurements are 19 and 35% for the 6 mv beams and 17 and 33% for the 15 mv, respectively. in the case of the 0.5 0.5 cm field, the differences with the rc measurements are 38 and 52% for the 6 mv beams and 32 and 50% for the 15 mv beams, respectively. in this work a method to measure small radiotherapy fields by means of rc films has been presented. ofs, beam profiles, and 2d dose distributions are accurately measured, taking advantage of the dosimetric properties of the rc media. the small energy and dose rate dependencies shown by the rc films and their high spatial resolution are exploited for measuring small fields. on the other hand, the method presented in this work shows how to deal with the main drawback of rc films: the spatial inhomogeneity of their response. this problem is even bigger when reading the film with a flatbed scanner. in this work, the inhomogeneity is mostly canceled by means of averaging the measurements over a number of segments taken from different parts of one sheet of film. one advantage of using rc films is the possibility of accurately measuring the dose gradient regions. in - plane and cross - plane profiles are shown and the differences in penumbras are obtained, together with the full width at half maximum. good agreement is found between the measured profiles and the monte carlo calculated profiles for the field size of 1 1 cm. for the fields of size 1 1 cm or smaller, the measurements of the ofs by means of the pinpoint and semiflex chambers is affected by the volume effect. the sensitive volume has a length of 5 mm for the pinpoint chamber and of 6.5 mm for the semiflex chamber. those lengths are larger than the constant dose regions of the in - plane profiles. the measurement of the ics averages the dose on these volumes, and therefore underestimates the dose in the central axis central axis (cax). the obtained for the ofs measurements show a good agreement between rc films and the pinpoint and semiflex chambers when the field size is 2 2 cm. this agreement and the goodness of fit shown by the calibration curve gives confidence on the accuracy of the method. the ics are accurate when measuring fields of size 2 2 cm or larger. for these fields, the ofs measured with rc films match the ofs measured with the ics (being the differences smaller than 1.0%). it can be argued that, for large fields, the rc films measurements are also accurate. moreover, as reducing the field size is not expected to change the accuracy of the rc films measurements, as demonstrated through the extensive use of the rc films in the measurements of the ofs, the accuracy of the rc film measurements can be extrapolated to the small fields. the same can be said about the measurement depth; the material media, water instead of pmma; the collimation system, multileaf collimator (mlc) instead of jaws; or even the irradiation unit. we expect, therefore, that the presented method can be used to perform accurate measurements of small fields in a wide range of situations.

the small fields in radiotherapy are widely used due to the development of techniques such as intensity - modulated radiotherapy and stereotactic radio surgery. the measurement of the dose distributions for small fields is a challenge. a perfect dosimeter should be independent of the radiation energy and the dose rate and should have a negligible volume effect. the radiochromic (rc) film characteristics fit well to these requirements. however, the response of rc films and their digitizing processes present a significant spatial inhomogeneity problem. the present work uses a method for two - dimensional (2d) measurement with rc films based on the reduction of the spatial inhomogeneity of both the film and the film digitizing process. by means of registering and averaging several measurements of the same field, the inhomogeneities are mostly canceled. measurements of output factors (ofs), dose profiles (in - plane and cross - plane), and 2d dose distributions are presented. the field sizes investigated are 0.5 0.5 cm2, 0.7 0.7 cm2, 1 1 cm2, 2 2 cm2, 3 3 cm2, 6 6 cm2, and 10 10 cm2 for 6 and 15 mv photon beams. the ofs measured with the rc film are compared with the measurements carried out with a pinpoint ionization chamber (ic) and a semiflex ic, while the measured transversal dose profiles were compared with monte carlo simulations. the obtained for the ofs measurements show a good agreement with the values obtained from rc films and the pinpoint and semiflex chambers when the field size is greater or equal than 2 2 cm2. these agreements give confidence on the accuracy of the method as well as on the obtained for smaller fields. also, good agreement was found between the measured profiles and the monte carlo calculated profiles for the field size of 1 1 cm2. we expect, therefore, that the presented method can be used to perform accurate measurements of small fields.

transient receptor potential vanilloid subtype 1 (trpv1) is a well - known pain - mediating ion channel expressed in sensory neurons including dorsal root ganglionic (drg) neurons, trigeminal ganglionic (tg) neurons, and vagal neurons. in response to various harmful stimuli, trpv1 pore opens and cationic flux through the pore into the nerve terminal causes electrical depolarization which may lead to action potential generation. when propagated and transmitted into the brain, the signals finally in the perception of pain. because of its extreme polymodality compared to other known peripheral sensor molecules that allows trpv1 to cover a large spectrum of pain qualities from chemical through thermal ones and of its famous activator capsaicin which has been traditionally utilized for pain research even before trpv1 discovery, trpv1 has garnered a great deal of attention as a peripheral pain - modulating target. topical application of a trpv1 modulator is currently the mainstream for trpv1-targeting analgesic strategies while systemic approaches have been dropped due to a potential for hyperthermic adverse effect because body temperature regulation is perturbed by antagonism of vagal trpv1. although the initial report about trpv1 finding suggested that it is specifically expressed in sensory neurons, its wider distribution in various regions including certain regions of the central nervous system (cns) and nonnervous tissues has been surmised (for review,). using rodent and human brains, mezey et al. verified the existence of trpv1 protein and mrna in the spinal cord, amygdala, medial and lateral habenula, hippocampus, striatum, hypothalamus, centromedian and paraventricular thalamic nuclei, substantia nigra, reticular formation, locus coeruleus, cerebellum, inferior olive, and certain cortical areas. soon after, valtschanoff et al., focusing on the spinal cord, showed that both presynaptic (from the central terminal of sensory neurons) and postsynaptic regions (from the dendrites of spinal cord dorsal horn neurons) exhibit trpv1 positivity, especially in the superficial laminae i and ii, which are the first relaying stations in the pain sensory pathway. using dorsal rhizotomy, they were able to histologically show that postsynaptic trpv1 expression levels were highly dependent on the presence of peripheral inputs, indicating that spinal cord trpv1 expression and function may be dynamically controlled by sensory states. since that time, cns and spinal cord expression of trpv1 these suggested that trpv1 may play a role in the central areas and in this review we more focus on pain transmission in the spinal cord. altered expression of a protein depending on disease states often implies its importance in disease progression. upregulation of trpv1 in drg or tg neurons under a proinflammatory state has been reported. differential regulation of trpv1 expression occurs in neuropathic pain states. at the scale of whole drg neuronal collection, including both damaged and undamaged neurons, the amount of trpv1 was reduced in many different neuropathy models including those of sciatic nerve axotomy, partial nerve ligation, chronic constriction injury (cci), spinal nerve ligation, and diabetic neuropathy. the loss of total trpv1 expression appears to be at least partially due to the degeneration of damaged trpv1-positive drg neurons. interestingly, in the spinal cord dorsal horn, trpv1 is upregulated in a cci neuropathic pain model. when looking at uninjured drg neurons, higher trpv1 expression was detected even in the neurons at different spinal levels from that for damaged ones. different from the peripheral neuropathy models mentioned above, in the spinal cord injury models, an increase in trpv1 proteins or mrnas was consistently detected in the drg. the molecular and cellular mechanisms for such increased trpv1 expressions in undamaged neurons in diverse injury models remain undetermined and we dealt with those in unsolved issues below. briefly, the uninjured drg neurons may be affected by inflammatory processes, for example, via increased secretion of inflammatory mediators such as nerve growth factor (ngf) from recruited immune components around adjacent damaged drg or spinal cord regions. indirect synaptic mechanisms via collaterals or descending circuits also likely participate in trpv1 upregulation in drgs at different spinal levels. the of elevated trpv1 levels indicate that peripheral trpv1 expression can be controlled upon injury conditions and that increased amplification of pain signals may involve upregulation of trpv1, which might serve as a potential leverage for therapeutic modulation. besides expression , outcomes from pharmacological manipulation of spinal cord trpv1 activity have also consistently emphasized its crucial role in pain transmission and therapeutic advantages. in particular, industrial field hypothesized that selective antagonism to spinal trpv1 could be one option for avoiding adverse malignant hyperthermia since cns trpv1 seems to be free from the hyperthermic mechanism. not only demonstrated increased trpv1 levels in the spinal cord of cci rats but also produced a promising analgesic from intrathecal administrations of the trpv1 antagonist bctc. in the hundreds of nanomolar range, mechanical allodynia and calcitonin gene - related peptide - like immunoreactivity and substance p - like immunoreactivity were attenuated in the spinal cord from the cci - injured rats researchers at abbott labs used three different inflammatory pain models with complete freund's adjuvant, capsaicin, and sodium monoiodoacetate injections. their cns - penetrable version, a-784168, more effectively blocked pain than a less penetrable a-795614, despite being similar in terms of in vitro profiles for trpv1 antagonism. watabiki et al. at astellas pharma demonstrated that mechanical allodynia in their mouse spinal nerve ligation (snl) model was alleviated by intrathecal injection of either of bctc or their own trpv1 antagonist as1928370. wu et al. demonstrated that trpv1 antagonism using intrathecal amg9810 reversed mechanical and thermal hypersensitivities in a contusive spinal cord injury model. two research groups independently demonstrated that intrathecal agonist (capsaicin or 9-hydroxyoctadecadienoic acid) injections induced mechanical allodynia. since trpv1 is not a mechanosensitive ion channel and thus trpv1 in the periphery has no role in mediating mechanical phenotypes, the mechanical hypersensitivity is purely due to central trpv1 activity on transmission. intrathecal injections of resiniferatoxin (rtx), a potent trpv1 agonist, deactivated voltage - dependent components or ablated trpv1-positive neuronal terminals. interestingly, in a carrageenan - induced inflammation model, the thermal threshold but not the mechanical threshold was normalized with this strategy. although the model involved an agonistic challenge, body temperatures of the treated animals were largely unaffected. collectively, the from expression dynamics and pain pharmacology commonly raise the importance of the existence of spinal cord trpv1. in turn, several groups have begun to explore the next question: how, differently from peripheral trpv1, spinal trpv1 intervenes pain transmission in pathologic states. spinal synaptic plasticity is a central concept that accounts for pathologic transition from a normal acute pain to a chronically morbid one. both long - term potentiation and depression (ltp and ltd) paradigms and technical progress for brain slice electrophysiology from learning and memory research mostly using the hippocampal area were imported to the pain field. those concept and technology have contributed to the understanding of the pathological pain transmission in the spinal cord and to finding painkilling targets: ionotropic and metabotropic glutamate receptors, n - type and t - type voltage - gated ca channels, upstream and downstream signaling molecules of the nitric oxide synthesis pathway, calcium / calmodulin - dependent kinases, neuropeptides and their receptors, and so forth. for validating the spinal trpv1 mechanism, , three important aspects of the roles of spinal trpv1 have been reported in recent years. the discussions about the detailed follow. gone through simple traditional observations where spontaneous excitatory postsynaptic currents (sepscs) were facilitated by the presynaptic actions of capsaicin to understand the circuitry under normal conditions , researchers became interested in trpv1's role in pathologic states. xu et al. found that trpv1 in the central terminal of drg neurons plays an important role in exaggerating pain in an inflammatory state during their analyses of the effects of endogenous proresolving lipids. when they recorded the lamina ii neurons of transverse slices of the murine lumbar spinal cord with a patch clamp technique, sepscs were increased upon tumor necrosis factor- (tnf-) exposure. because the frequency but not the amplitude of epscs was increased, tnf- seemed to elevate glutamate release by acting at presynaptic terminals. this perfusion with tnf- ex vivo may represent an inflammatory or neuropathic pain state in vivo. simultaneous treatment of capsazepine reversed this tnf- effect: the changes were limited to numbers of sepsc frequencies. consequently, the of this study provide multiple implications: the primary action site of the acute tnf- effect is presynaptic sensory neurons; trpv1 had been presumed to be a major effector of tnf- action, at least in the periphery , and the confirmed it and extended to the central terminals; regarding capsazepine - affected parameters, trpv1-mediated mechanism may be more important in presynapses. despite being out of the scope of this review, resolvin e1, a potent endogenous proresolving lipid, appears to disturb the presynaptic signaling between tnf- and trpv1 via its g - protein - coupled receptor (gpcr) activation, as a part of its painkilling mechanisms. more recently, the wei and dong labs revisited the presynaptic role of trpv1 from the viewpoint of descending excitatory modulation when they tried to explain secondary hyperalgesia that occurs from neighboring but uninjured receptive field under neuropathic conditions. they developed a knock - in mouse line in which the expression of gcamp3 encoding a ca indicator protein is driven by the promoter of pirt (phosphoinositide - interacting regulator of transient receptor potential channels), a pan - drg / tg marker. with this mouse model , changes in intracellular ca levels in cell bodies and peripheral and central terminals can be discerned in drg and tg neurons, even under ex vivo conditions surrounded by other tissue types or buried in complex synaptic circuits. they also created a cheek mechanical hyperalgesia model using cci of the infraorbital nerve, which is the major branch of the maxillary (v2) tg nerve. accordingly, the trigeminal subnucleus caudalis (vc), which is analogous to the spinal dorsal horn in terms of the sensory synaptic circuit, was observed for presynaptic trpv1 functions. although only v2 tg nerve had undergone the cci procedure, heightened pain sensitivities occurred in the cheek, jaw, and ear, the latter two of which are mandibular (v3) tg nerve territories. when intracellular ca fluorescence level due to gcamp3 was analyzed as a surrogate measure for excitability, both v2 and v3 central terminals in vc exhibited larger ca increases than under normal conditions. furthermore, these elevated sensitivities were commonly observed in terminals from superficial through deep laminae, suggesting that not only injured but also adjacent undamaged nerve fibers became hyperactive and that this situation consequently led to secondary hyperalgesia and allodynia. based on their previous observations, wei and dong's group hypothesized that the rostral ventromedial medulla (rvm) in the brainstem relays 5-hydroxytryptamine (5-ht, serotonin) dependent excitatory input to uninjured nerves. indeed, 5-ht immunoreactivity was elevated near the gcamp3-positive central sensory terminals of vc. moreover, antagonistic manipulations including treatment using the 5-ht3 receptor antagonist in vc or 5-ht depletion in rvm using rna interference against its biosynthesis alleviated both hyperactivity of trpv1-mediated ca signals and hypersensitive behaviors. since 5-ht receptor - mediated trpv1 facilitation was confirmed in the central presynaptic terminals, the descending excitatory axons from rvm seem to constitute axoaxonal contacts. collectively, trpv1 in the presynapse of sensory neurons that covers the undamaged regions participates in secondary pain amplification through a descending facilitation mechanism (figure 1). previous positive of the spinal cord expressions of trpv1 strongly implicated a functional role of trpv1 in the spinal postsynaptic neurons. the oh lab focused on the spinal cord inhibitory synapse regarding the role of trpv1. in fact, loss of gabaergic or glycinergic inhibitory control in the spinal synaptic network has long been proposed as a cause of central pain sensitization. different from sepscs, evoked epscs have been reported to decrease after capsaicin perfusion. trpv1 activation acutely induced increases in frequency in spontaneous inhibitory postsynaptic currents (sipscs) in the dorsal horn neurons via gabaergic or glycinergic connections. notably, in a mouse model with ablation of trpv1-positive drg neurons, a significant proportion of mechanical hypersensitivity remained following intrathecal capsaicin administration, indicating that postsynaptic trpv1 also contributes to the pain state. indeed, trpv1 expression and agonist - dependent activation in postsynaptic dorsal horn neurons were verified, and over 75% of gabaergic interneurons were trpv1-positive whereas ~75% of non - gabaergic postsynaptic neurons were trpv1-negative. surprisingly, in their epsc profiling of the gabaergic neurons in response to electrical stimulation of the dorsal root entry zone, ltd occurred after postsynapse - specific trpv1 activation. trpv1 activation - induced ltd was dependent on an increase in the intracellular ca concentration and the reduction of alpha - amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (ampa) receptor activities, similar to other typical ltd processes. accordingly, ltd caused less excitability and reduced gaba release of the gabaergic interneurons, and therefore the secondary projection neurons (spinothalamic tract neurons in this study) received less inhibitory input, ing in enhanced relay of pain signals to higher brain areas (figure 1). the analgesic effects of spinal trpv1 antagonism in mice cci model were successfully repeated without malignant hyperthermia, but the authors proposed a novel mechanism engaging ltd of trpv1-positive inhibitory neurons. because the studies on the contribution of trpv1 to the transmission of spinal pain circuit are relatively in their early stage, much of biological information is still unavailable. here, we focus on several important issues as follows. detailing how dynamically the spinal trpv1 expression alters depending on the pathologic process may provide more sophisticated explanation of spinal pain mechanisms and help fine - tuning analgesic strategies. as mentioned above, a promising from trpv1 expression in the spinal cord has been demonstrated by kanai et al.. in their cci rat model, trpv1 expression of the ipsilateral superficial dorsal horns was gradually elevated for two weeks. this expression location indicates an increase in trpv1 in the presynaptic terminals by neuropathic insult, supporting the enhanced capsaicin sensitivity of the central terminals in the novel trigeminal cci neuropathy model. detected elevated trpv1 levels in the dorsal horn presynaptic regions in a carrageenan - inflammation model. in a relatively chronic inflammation model using complete freund's adjuvant, despite a statistically ambiguous elevation on days from 1 to 2 after injection, over 50% increased trpv1 levels were maintained for 1 to 3 weeks. the increase may depend on the mediator effect including ngf, which is similar to well - known mechanisms for increases in trpv1 expression of the peripheral terminals. however, information on trpv1 dynamics of interneurons or projection neurons in the presence or absence of injury is still lacking. although trpv1 is known as a major heat sensor in the body, its thermosensory functions and related thermoregulatory feedback mechanisms are unlikely to be exerted in the spinal cord because this region only experiences a limited range of temperatures, close to the core body temperature. evidence mentioned above suggests that pharmacological antagonism for trpv1 in the spinal cord does not cause thermal effects. however, that situation leads to another question: what conditions else activate trpv1 there? inflammatory peptides including tnf-, which was tested by xu et al., and neurotransmitters such as 5-ht, which proved to be a descending modulator, may utilize trpv1 for a downstream effector. for the tnf--trpv1 signaling axis, hypotheses of prostaglandin production for increased trpv1 activity and extracellular signal - regulated kinase (erk) activation for elevated trpv1 expression were raised by studies on drg neurons. extracellular prostaglandins are known to enhance trpv1 sensitivity via phosphorylation by protein kinase a or c through their g - protein - coupled receptor - mediated signaling, but it remains elusive whether this paracrine signaling cascade also works for tnf- axis. even if this prostaglandin - mediated mechanism is true, the mechanism can only sensitize but not activate trpv1. by this sensitization, heat threshold for in addition, while only erk itself has been shown to participate, there has not been sufficient clarification of the further downstream signal in this pathway. moreover, it also needs to be explored in the presynapse whether the releases of neurotransmitter vesicles are facilitated simply by increased intracellular ca ions through trpv1 opening - induced depolarization, or other unknown molecular downstream signals are involved. the 5-ht3 receptor is a major receptor that receives descending excitatory input from rvm. interestingly, 5-ht3 receptor is a cation channel that may be functionally redundant regarding the trpv1 outcome to depolarize the presynaptic area. whether these two cation channels are additive or otherwise functionally or physically coupled for a synergistic action, as shown in recently published observations at the peripheral terminals about trpv1-trpa1 and trpv1-anoctamin cooperation , requires further examination. transient receptor potential ankyrin subtype 1 (trpa1) is comparable to trpv1 in terms of the importance of covering pain modalities and transductory roles to initiate nociceptor depolarization in the periphery. if both trp channels share central locations of their expressions, their redundancy, cooperativity, or compensation in the roles in synaptic transmission could not be ignorable. indeed, the presynaptic facilitative role of trpa1 in the spinal cord has been addressed. the effects of antagonistic challenges against spinal trpa1 function were examined and the treatment displayed analgesic outcomes in diverse pain models including snl, rapid eye movement sleep deprivation, capsaicin - paw injection, formalin - paw injection, and diabetic neuropathy. it was further demonstrated that the pain - facilitating effect of descending excitatory inputs from rvm stimulation or spinal cord 5-ht3 receptor activation was all blunted by trpa1 antagonism. unlike trpv1 , the analgesic mechanism does not seem to employ the postsynaptic gabaergic disinhibition mechanism, which seems to be inconsistent when comparing the earlier and the most recent observations that spinal trpa1 activation facilitates not only the frequency and amplitude of sepscs but also those of sipscs. as mentioned, kim et al. although this can form analgesic proof of concept from a therapeutic viewpoint, it is still poorly understood why the gabaergic neurons need to express trpv1 regarding their ordinary transmission and what naturally stimulates trpv1 in neuropathic conditions. regarding the polymodality of trpv1 kim et al. suggested that 12(s)-hydroperoxyeicosatetraenoic acid (12(s)-hpete ) may be a natural trpv1 activator candidate. gibson et al. demonstrated that anterograde metabotropic glutamate receptor activation in postsynaptic 12(s)-hpete production and that this lipoxygenase metabolite retrogradely diffuses and activates presynaptic trpv1, causing ltd in the hippocampal ca1 synapses. application of this paradigm to the spinal cord may narrow down the candidate mechanisms to a gpcr - lipoxygenase cascade but measurement of which substances in the spinal cord are a major metabolite is still required, for example, hepoxilins and hydroxyoctadecadienoic acids. trpa1 also has a wide spectrum for sensing endogenous reactive substances the levels of which are frequently elevated under injury conditions in tissues or within active synapses. those are reactive oxygen and nitrogen species and lipid peroxidation products that are known to covalently bind and activate trpa1. normalizing imbalanced local production of atypical excitatory substances for trp channels by developing specific enzyme inhibitors might be another analgesic strategy to utilize the central trp channel - mediated synaptic mechanism. stemming from the recent accomplishments in spinal trpv1 research, unexplored mechanisms connecting newly uncovered trpv1's roles and related hypotheses are being raised. like pharmacological approaches, a series of studies using local rna interference techniques have given new lines of firm evidence for the roles of trpv1 in pathologic pain progress and also for its action in the spinal cord. as tools to improve the efficiency of interfering gene delivery and to lessen safety concerns are being developed, expectancy about future utility of gene editing therapies for chronic pain modulation is currently forming. in the last year, hirai et al. showed that intrathecal administration of adenoassociated virus serotype 9 (aav9) vector carrying short - hairpin rna (shrna) against trpv1 ed in long - term suppression of thermal hyperalgesia in a mouse spared nerve injury model. viral delivery may confer long - term stable generation of shrna and limited exposure to immune protection mechanisms outside of the cns region may help time scale of the effect further since shrnas in themselves have tough permeability to blood brain barrier (bbb). the confinedness by intrathecal injection may also reduce potential adverse effects from nontarget tissues. interestingly, despite spinal targeting, only thermal hyperalgesia, not mechanical or cold allodynia, was blunted, which typically occurs in drg - specific trpv1 impairment. conversely, the parameters of shrna abundance and trpv1 mrna reduction were significantly better in the spinal cord than in the drgs. differential translational compensation or the presence of trpv1 isotypes free from the target sequence may be conceivable regarding the broad analgesic spectrum of the above circuit research. since trpv1 expression is absent in glial components, it is not likely that the effects of pharmacological modulations of spinal trpv1 have a direct link to glial contributions. however, in a follow - up study of kim et al., the wei lab demonstrated indirect participation of trpv1. although they did not measure trpv1 activity, guo et al. repeated presynaptic stimulation by mimicking activation of the descending 5-ht pathway, which subsequently activated microglia and astrocytes. by showing pharmacological and histological evidence, they suggested that fractalkine released from the presynapses stimulates microglia and then interleukin-18 from activated microglia boosts astrocyte function, from which released interleukin-1 finally enhances excitability of the dorsal horn neurons by inducing phosphorylation of an n - methyl - d - aspartate receptor subunit. nitric oxide (no) is of general importance as a retrograde signal for modifying brain synaptic strength. in pain synapses, postsynaptic no plays a central role for presynaptic activation of the guanylyl cyclase - cyclic guanosine monophosphate- (cgmp-) protein kinase g pathway. interestingly, trpv1 activity seems to be tolerant of pkg action or even downregulated by pkg. not only direct pkg action, but also effects of known substrates of pkg, for example, inositol trisphosphate receptor and myosin light - chain kinase, appear to be independent of the amplification of trpv1 activity. calcium / calmodulin - dependent protein kinase ii (camkii) plays a crucial role in ampa receptor facilitation in the postsynapse. it has been reported that phosphorylation of trpv1 of drg neurons by camkii is important for maintaining its sensitivity to ligands, which can be explored regarding the postsynaptic paradigm. -opioid receptor - induced ltp in the spinal cord appears to occur in trpv1-positive presynapses and it might be a future issue whether changes in trpv1 activity in the central terminal are practically involved in opioid signaling. two of many reasons why peripheral trpv1 has received much attention from industry in the decade since its gene discovery seem to be its polymodality integrating painful inputs with diverse qualities and its peripheral location. for the latter, a central advantage of targeting peripheral trpv1 is that its ligand avoids the adverse effects of the cns when it is designed to be bbb - impenetrable. however, assuming that the aim is cns administration and considering the novel nociceptive roles of trpv1 in the spinal circuit, one may need to conceive similar adverse situation as already observed or predicted for other cns analgesic candidates because the target is possibly expressed in other central regions and may have differential actions. as mentioned above, the bulbospinal circuit receives descending input from periaqueductal gray (pag) and confers descending excitatory inputs as well as inhibitory ones. earlier, mcgaraughty et al. demonstrated that trpv1 activation of dorsal pag gave a hyperalgesic phase via the rvm circuit. however, in ventrolateral pag (vl - pag), the neighboring region, it has been suggested that when -opioid receptor is simultaneously activated trpv1 activation contributes to facilitation of glutamatergic interneuronal activity, which offers gabaergic inhibition of descending excitatory on cells in the rvm circuit, leading to a reduction of nociceptive transmission in the spinal cord. decreased evoked ipscs, increased miniature ipscs and epscs, and contribution of the cannabinoid receptor have also been proposed by a different intra - pag recording study. because this circuit was examined only under acute pain conditions, the analgesic aspect of trpv1 action in vl - pag needs to be further addressed for pathologic pain conditions. exploring the mechanism of analgesic effects of acetaminophen, researchers have found out that trpv1 activation in the central nervous system is involved. when acetaminophen is systemically administered, its metabolites are formed in the brain by the action of fatty acid amide hydrolases. those appear to directly activate brain trpv1, leading to analgesia in formalin - induced pain and acute thermal or mechanical pain. antagonism by intracerebroventricular injection of an antagonist and virtual localization of the drug using methylene blue injection argue that supraspinal trpv1, but not spinal cord trpv1, may participate in the central analgesic action. interestingly, some of electrophilic and toxic metabolites produced during the acetaminophen metabolism, different from the metabolites that activate trpv1, were reported to activate spinal presynaptic trpa1, ing in acute antinociception via subsequent inactivation of adjacent presynaptic voltage - gated na and ca channels. other adverse situations due to the potential presence of trpv1 in cns regions might be possible. for example, whether inadvertent diffusion of an antagonist in the ventricular regions may enable access to untargeted areas and affect other brain functions including memory or mood needs to be carefully evaluated. whether trpv1 activation can gain specific delivery of local anesthetics to trpv1-positive nociceptor is being considered as a novel analgesic strategy. some membrane - impermeable hydrophilic derivatives of lidocaine species are able to permeate into neurons through dilated trpv1 pore when trpv1 is activated and once inside, they can block voltage - gated na channels, with or without their permeant blockade of trpv1 itself. trpv1-negative neurons should be inert to the blocking effects of these drugs since the drugs can be admitted only through trpv1, which may allow avoidance of common adverse effects of the local anesthetics including numbness and motor defects via interfering nonpain pathways. such approaches of specific application may be taken into consideration in the future for modulation of spinal cord trpv1. among trpv1-targeting pain therapies, the rationale is based on the functional incapacitation of the peripheral sensory terminals by agonist - induced trpv1 desensitization and mitochondrial permeability transition which leads to terminal ablation. the same mechanism might be possible in the spinal region: analgesia via defunctionalization of trpv1-specific pre- and postsynapses by agonist - induced effects. however, as mentioned, intrathecal administration of trpv1 agonists ed in some pain phenotypes in animal studies. this appears to be predictable because it is an unavoidable side effect that topical capsaicin application in its early treatment stage evokes pain via initial trpv1 activation in humans. one possible option to overcome this hurdle is currently thought to be a substitution by a nonpungent capsaicin analogue free from the initial pain induction. although such a class of trpv1 agonists were recently developed, they have not been thoroughly tested regarding their analgesic effects. in the early stage, more attention was given to the sensory involvement of trpv1 in the peripheral terminal of the nociceptor neurons to harmful environments and to its contribution to neurogenic inflammation. however, revisiting traditional capsaicin pharmacology, assessments regarding trpv1 expression patterns and their dynamics in diverse neural regions have provided clues of other nociceptive roles for trpv1. particularly, recent accumulation of knowledge on trpv1 functions in the spinal presynaptic and postsynaptic locations connecting to exacerbation mechanisms for neuropathic pain has begun to address its roles in the central nervous system. this heightened understanding and new hypotheses also appear to raise the possibility of developing new proof of concept targeting spinal trpv1. such attempts for new approaches could be extended to other cns locations and other polymodal trp channels such as trpa1. further analyses regarding the role of trpv1 in plastic changes for pain synapses at the individual level including supraspinal circuits will shed light on the collective contribution to exacerbation of pathologic pain and its analgesic utility.

trpv1 is well known as a sensor ion channel that transduces a potentially harmful environment into electrical depolarization of the peripheral terminal of the nociceptive primary afferents. although trpv1 is also expressed in central regions of the nervous system, its roles in the area remain unclear. a series of recent reports on the spinal cord synapses have provided evidence that trpv1 plays an important role in synaptic transmission in the pain pathway. particularly, in pathologic pain states, trpv1 in the central terminal of sensory neurons and interneurons is suggested to commonly contribute to pain exacerbation. these observations may lead to insights regarding novel synaptic mechanisms revealing veiled roles of spinal cord trpv1 and may offer another opportunity to modulate pathological pain by controlling trpv1. in this review, we introduce historical perspectives of this view and details of the recent promising . we also focus on extended issues and unsolved problems to fully understand the role of trpv1 in pathological pain. together with recent findings, further efforts for fine analysis of trpv1's plastic roles in pain synapses at different levels in the central nervous system will promote a better understanding of pathologic pain mechanisms and assist in developing novel analgesic strategies.

in bacteria, the phosphoenolpyruvate - carbohydrate phosphor - transferase system (pts) is a chief carbohydrate transport system (deutscher et al ., a large number of carbohydrates are phosphorylated by the pts generally consisting of cytoplasmic proteins, enzyme ei ( ei) and heat stable phosphoryl carrier protein (hpr), and a membrane - associated carbohydrate - specific permease complex enzyme ii (eii). in general, eii complexes are composed of hydrophilic enzymes, eiia and eiib, and hydrophobic integral membrane proteins, eiic (and eiid in a mannose- and fructose - specific pts). the catalytic function of ei, hpr and eiia depends on a functional histidine residue transiently phosphorylated during enzyme reaction. the active sites of eiib subunits include either a cysteine or a histidine as a catalytic residue. the phosphoryl group which will be transferred to various carbohydrates is obtained from phosphoenolpyruvate by ei. hpr becomes phosphor - hpr by accepting the phosphoryl group from ei. at the next step, the phosphoryl group from phospho - hpr is transferred to a sugar - specific eii complex. in general, ei and hpr are not selective and are shared by different pts systems. however, there are many different types of eiis present for uptake of different carbon sources, and they are not interchangeable. a phylogenetic analysis of the eii proteins show that the pts permease families are classified into seven families as follows (marchler - bauer et al ., 2015); the (i) glucose (including glucoside) (glc), (ii) fructose (including mannitol) (fru), (iii) lactose (including n, n - diacetylchitobiose) (lac), (iv) galactitol (gat), (v) glucitol (gut), (vi) mannose (man), and (vii) l - ascorbate (asc) families. in addition, pts components directly interact with their target proteins which carry out various cellular functions such as transport proteins or transcription regulators. an open reading frame of e. coli str. mg1655 codes for a frwd gene (ncbi refseq : np_418388.1) which is annotated as putative pts eiib protein (eceiib) of 12.6 kda. it belongs to the ncbi conserved domain cd05569 (pts_iib_fructose) which includes subunit iib of eii of the fructose specific pts (barabote and saier, 2005). a psi - blast search (http://www.ncbi.nlm.nih.gov) of this sequence revealed around six hundred proteins with sequence identity above 35% (fig . most of the homologous sequences are annotated as fructose - like specific pts system eiib or fused forms, eiibc or eiiabc . the biochemical study revealed that at least 15 different eii complexes are found in escherichia coli ( saier and reizer, 1994). several putative fructose - like iib components are also detected in the e. coli pts system. since the molecular structure and the functional implication of this eceiib family have not been reported, we have determined the x - ray crystal structure of eceiib. based on the crystal structure analysis, molecular functions of eceiib have been inferred and discussed in this paper. details of the preliminary x - ray study (shin, 2008) and the structure determination (joo et al ., 2015) of eceiib had been previously published. in brief, eceiib crystals were grown using 20% (w / v) peg mme2000 in the presence of 10 mm nickel (ii) chloride hexahydrate. the x - ray dataset of eceiib was collected to 2.28 resolution and the crystal belonged to the primitive trigonal space group p32 with unit - cell parameters a = b = 33.11, c = 154.38. molecular replacement (mr) was performed using phenix (adams et al ., 2010) with highly accurate protein 3d models generated by the recently proposed method called got (global - optimization - based template - based modeling of proteins) (joo et al ., 2015). a relatively good electron density map was obtained after automated model - building and refinement with the phenix autobuild wizard (afonine et al ., 2012). the electron density map clearly showed the presence of two protomers of eceiib in the asymmetric unit. interestingly, they form a dimer bridged by a nickel ion contained in the crystallization solution. the final model exhibited good stereochemical geometry and was refined to r- and r - free values of 22.5% and 29.9%, respectively (joo et al ., 2015). the atomic coordinates and structure factors of the eceiib structure have been deposited in the rcsb protein data bank under the accession code 4tn5. details of the preliminary x - ray study (shin, 2008) and the structure determination (joo et al ., 2015) of eceiib had been previously published. in brief, eceiib crystals were grown using 20% (w / v) peg mme2000 in the presence of 10 mm nickel (ii) chloride hexahydrate. the x - ray dataset of eceiib was collected to 2.28 resolution and the crystal belonged to the primitive trigonal space group p32 with unit - cell parameters a = b = 33.11, c = 154.38. molecular replacement (mr) was performed using phenix (adams et al ., 2010) with highly accurate protein 3d models generated by the recently proposed method called got (global - optimization - based template - based modeling of proteins) (joo et al ., 2015). a relatively good electron density map was obtained after automated model - building and refinement with the phenix autobuild wizard (afonine et al ., 2012). the electron density map clearly showed the presence of two protomers of eceiib in the asymmetric unit. interestingly, they form a dimer bridged by a nickel ion contained in the crystallization solution. the final model exhibited good stereochemical geometry and was refined to r- and r - free values of 22.5% and 29.9%, respectively (joo et al ., 2015). the atomic coordinates and structure factors of the eceiib structure have been deposited in the rcsb protein data bank under the accession code 4tn5. the crystal structure of eceiib has been determined at the resolution of 2.4 (supplementary fig . the structure of the eceiib protein is composed of a central six - stranded parallel open twisted -sheet, which is flanked by three -helices on the concave side and two (2, g1) on the convex side (fig . , the crystal structure shows an artificial dimer seemingly being triggered by one nickel ion coordinated to two neighboring his109 residues located on the c - terminus of each protomer ( fig . this coordination may induce a loop - to - strand conversion of the c - terminal loop ing in the formation of the last -strand 6 . as a , a twelve stranded -sheet composed of two contiguous -sheets is formed in the dimer . however, eceiib is a monomer as indicated in the monodisperse peak with molecular weight of 13 kda from dynamic light scattering ( dynapro 99, proterion corporation, usa) (shin, 2008) and this dimeric form is not found in the other crystal structures of its closest homologues as mentioned below. however nickel ions improved the diffraction limits of crystals (shin, 2008). in the crystal structure, two histidine residues and two water molecules coordinated the nickel ion. during the refolding step, various metals were tried but a dimeric size was not detected in size exclusion chromatography and dynamic light scattering. furthermore, the cd spectrum showed the same secondary structure contents regardless of the presence of metal ions including zinc and nickel ions (oganesyan et al ., 2005). however, the metal induced dimerization of eceiib can not be thoroughly excluded and its biorelevance should be further studied inside bacterial cells. eceiib belongs to a sequence family with more than 5000 sequence homologues having 2599% amino - acid sequence identity obtained by a psi - blast search (fig . the 3d structure alignment search with dali ( holm and rosenstrom, 2010) reveals that the crystal structure of eceiib determined in this study, along with the closest homologues (z - scores above 14), 2r48 (mannose - specific eiib from bacillus subtilis), 2r4q (fructose - specific eiib from b. subtilis), 2m1z (hypothetical protein lmo0427 from listeria monocytogenes) and 2kyr (fructose - like enzyme iib from e. coli), forms a unique fold family not found in other protein structures. it is composed of 1, 1, 2, 3, 2, 4, g1, 5, 3 and 4. the root - mean - square (rms) deviations of matching c atom positions of eceiib compared with its closest homologues are 0.727 (70 c atoms of 2r48) 0.965 (71 of 2r4q), 1.090 (86 of 2m1z) and 0.989 (80 of 2kyr), respectively. it is noteworthy that the c - terminus of eceiib is longer than its closest homologues and forms an additional -strand 6 whereas the shorter c - termini of the other members are unstructured (fig . the central -sheet of eceiib has the topology of 6 5 4 1 2 3 instead of 5 4 1 2 3 found in the other members . the dali search revealed that the topology of the six - stranded -sheet, 6 5 4 although this additional -stand is stabilized in our crystal form by an inter - molecular antiparallel -bonding and chelation of a nickel ion by the neighboring two his108 ( fig . 2a), it is not yet clear whether the last -strand is an intrinsic structure or an artificial . in fact, the predicted secondary structure of the c - terminus of eceiib calculated with psipred (http://bioinf.cs.ucl.ac.uk/psipred/) (buchan et al ., 2013) is a loop. therefore, the state of the c - terminus in solution and the possibility of a transition from disordered to ordered or vice versa related to its function are still under investigation. the next homologue group of eceiib is dna - binding response regulators with z - scores above 8. however, this family has a different -sheet topology from eceiib and contains unique active site residues. the structure of the eiib subunit of fructose permease (eiib), another homologue of eceiib, also has a different -sheet topology (3 2 4 1 5 6 7) with the active site residue, his15 (schauder et al ., 1998). interestingly, the dali search revealed that the core -sheet topology, 21 4 5, of eceiib is closer to that of enzyme iib from e. coli (eciib, pdb : 1iib) (van montfort et al ., 1997) with the z - score of 7.2 (fig . it contains a -sheet with the topology of 2 13 4 lacking two -strands at each end of the sheet . eciib homologues comprises a protein family with the enzyme gatb of the galactitol - specific phosphoenolpyruvate - dependent phosphotransferase system from e. coli ( 1tvm), the cytoplasmic b domain of the mannitol transporter iiabc from e. coli (1vkr), and ptxb from streptococcus (3czc). they have the -sheet topology of 2134 with a conserved cysteine (cys10) and a threonine / serine (thr17) located at the active site constructed by a p - like loop (fig . the cysteine residue functions via a cysteine - phosphate intermediate and the threonine residue assists the binding of phosphate ( ab et al ., since these residues are critical in transferring a phosphoryl group in eciib homologues, cys10 and thr17 of eceiib may function in a similar way . as for eciib homologues, the phosphorylation site of eiib is located on this conserved cysteine residue at the n - terminal end of the p - like loop . this cysteine receives the phosphoryl group from eiia and transfers it to the specific carbohydrate when bound at the catalytic site of eiic . the catalytic activity of the cysteine is aided by the p - like loop structurally similar to that of the phosphate binding p - loop of the phosphotyrosine protein phosphatase ( ptpase) superfamily (consensus sequence cxxxxxr(s / t) ) (su et al ., 1994). the p - loop forms an anion - binding motif together with the helix dipole of helix 1 and thus provides a favorable environment to generate deprotonated cysteine as a nucleophile and to accommodate a negatively charged phosphoryl group. however, the p - like loop of eciib homologues lack the conserved arginine residue at the corresponding position of the p - loop of the ptpase superfamily and thus the insufficient positive charge is thought to be compensated by their partner molecules (lei et al ., 2009). as mentioned above, the architecture of the core structure of eceiib is quite similar to the overall structure of eciib together with the conserved residues, cys10 and thr17. when the core structural elements of eceiib are superimposed with those of eciib homologues (fig . 3b), the rms deviations of aligned c atoms are 0.822 (25 c atoms of 1iib) 0.929 (28 of 1tvm), 1.030 (32 of 1vkr) and 1.253 (10 of 3czc), respectively. however, unlike eciib, eceiib homologues contain a conserved histidine his16 which may assist accommodating a negatively charged phosphoryl group. the spatial position occupied by his16 in the p - like loop is almost equivalent to that of the functional arginine of the p - loop of the ptpase superfamily (fig . consequently, the eceiib family has a unique consensus sequence of cxxgxaht at the p - like loop . interestingly, the crystal structures of the nucleotide binding subunit b of a1a0 atp synthase ( sankhala et al ., 2014) and human pir1 (tadwal et al ., 2012) revealed that they also contain a catalytic histidine residue in their p - loops which interacts with a phosphate moiety. in summary though the overall fold is quite similar to different iib enzymes and other fold families, eceiib homologues still consist of an independent subfamily due to their unique topological connection. based on the sequence alignment and structural comparison of its homologues, a unique motif cxxgxaht comprising a p - loop like architecture therefore, we proposed that the conserved cysteine on this loop may be deprotonated to act as a nucleophile to transfer phosphoryl moiety to a specific carbohydrate. the bigger size of eceiib homologues together with the presence of extra catalytic histidine over eciib suggests that though their core molecular function is similar, their biological partners and substrates may be different. a further study is going on to find a biological partner and substrate of eceiib.

we have solved the crystal structure of a predicted fructose - specific enzyme iibfruc from escherichia coli (eceiibfruc) involved in the phosphoenolpyruvate - carbohydrate phosphotransferase system transferring carbohydrates across the cytoplasmic membrane. eceiibfruc belongs to a sequence family with more than 5,000 sequence homologues with 2599% amino - acid sequence identity. it reveals a conventional rossmann - like -- sandwich fold with a unique -sheet topology. its c - terminus is longer than its closest relatives and forms an additional -strand whereas the shorter c - terminus is random coil in the relatives. interestingly, its core structure is similar to that of enzyme iibcellobiose from e. coli (eciibcel) transferring a phosphate moiety. in the active site of the closest eceiibfruc homologues, a unique motif cxxgxaht comprising a p - loop like architecture including a histidine residue is found. the conserved cysteine on this loop may be deprotonated to act as a nucleophile similar to that of eciibcel. the conserved histidine residue is presumed to bind the negatively charged phosphate. therefore, we propose that the catalytic mechanism of eceiibfruc is similar to that of eciibcel transferring phosphoryl moiety to a specific carbohydrate.

chronic kidney disease (ckd) is a worldwide public health problem that affects millions of people from all over the world. diabetic nephropathy is a common complication in patients with diabetes and the leading cause of end - stage renal disease (esrd). over the last decade, the prevalence of diabetes has increased worldwide, as a of the continuous rise in type 2 diabetes incidence. the pathogenesis of this drastic complication is not clearly understood, but available data suggests that multiple factors such as hemodynamic alterations, metabolic abnormalities, various growth factors, and genetic factors contribute to the pathogenesis of diabetic nephropathy. in experimental and human diabetic nephropathy, these hemodynamic changes may be explained in part by alterations in the renin - angiotensin system. consequently, genes involved in the renin - angiotensin system have been suggested as potential genetic predispositions for the development of diabetic nephropathy. many previous publications suggest that genetic predisposition plays a role in the development of diabetic nephropathy which clusters within families, both in type 1 (iddm) and type 2 (niddm) diabetes mellitus. dna polymorphism, so far, is known to exist for the great majority of human genes. in diabetes mellitus , several models can be figured out to represent different concepts of the pathogenesis of diabetic nephropathy and to incorporate genetic factors. angiotensin - i converting enzyme (ace) is one of the key enzymes in the renin - angiotensin - aldosterone system (raas) and the insertion (i)/deletion (d) polymorphism of this gene has been studied extensively with renal and cardiovascular complications of diabetic nephropathy. the ace, which was originally discovered in equine plasma, is a membrane - bound dipeptidyl carboxypeptidase ectoenzyme located in the endothelial lining of blood vessels throughout the body where it plays an important role in proliferation of vascular smooth muscle cells through the conversion of angiotensin - i to angiotensin ii and bradykinin inactivation. the ace is found as a membrane - bound enzyme in endothelial cells and different types of epithelial and neuroepithelial cells as well as in circulating form in biological fluids, such as plasma, cerebrospinal fluid, amniotic fluid, and seminal fluids. the mechanisms that lead to the biosynthesis of the circulating form of ace are unclear, but available data indicates that its structure is very similar to that of the cellular form. the circulating form is virtually identical to the cellular form except for the lack of the transmembrane and intracellular sequence but whether it arises from specific proteolytic cleavage from the cell surface or nonspecifically from senescent cells has yet to be determined. ace genes have been cloned in the human, mouse, and rabbit and the enzyme was the product of one gene in man. most somatic forms of ace appear to be transcribed from all exons except exon 13 which encodes the unique n - terminal region of the testicular form. cloning of the ace gene had made it possible to identify a 287 bp insertion / deletion polymorphism in intron 16 (ace i / d polymorphism) that appears to affect the level of serum ace activity. the genotype with deletion of the 287 base pair ed in higher plasma ace levels. the plasma ace was predominantly derived from tissue vascular endothelial cells suggesting that patients with the dd genotype might have higher tissue angiotensin ii. angiotensin ii might modulate the growth of smooth muscle cell and induce myointimal hyperplasia after endothelial injury, and administration of ace inhibitors prevented myointimal proliferation after vascular injury. plasma ace concentrations are stable when measured repeatedly in a normal subject but large interindividual variations make it difficult to interpret plasma ace levels in a given patient. the study conducted in a large sample of healthy families showed an intrafamilial resemblance between ace levels and also suggested that they were subject to the effect of major gene. the ace i / d polymorphism accounts for over 40% of interindividual variability of serum or tissue ace activity. patients homozygous for the deletion had the greatest serum ace activity, whereas those homozygous for the insertion had the lowest level. an insertion / deletion (i / d) polymorphism of intron 16 of the angiotensin - i converting enzyme (ace) gene is largely responsible for variations in plasma ace levels and for explaining approximately 50% of the variability in serum ace activity observed. using the linkage - segregation analysis of the plasma ace , cambien has shown that the ace i / d polymorphism was a marker for an unknown functional polymorphism (ace s / s) which appeared to be a new independent risk factor for myocardial infarction. the ace i / d polymorphism is thought to be a marker for functional polymorphism which regulates circulating and tissue ace activity. since 1990, its association with diabetic nephropathy has been extensively investigated and more than 300 studies have explored genetic associations of this polymorphism in more than 100 conditions including diabetic nephropathy. up to now, the i / d polymorphism of ace gene is the most extensively studied marker for association with diabetic nephropathy. while the initial study by marre et al. found a protective effect of the ii genotype on the development of nephropathy in iddm patients, subsequent studies have yielded inconsistent . over the past decades , many studies have been conducted to examine the association between ace i / d polymorphism and nephropathy in type 1 and type 2 diabetes, but the still remain inconsistent. however, recently conducted meta - analysis showed consistent association between ace d allele or dd genotype and esrd risk in diabetic nephropathy patients. in 2005, ng et al. reported a meta - analysis of 14727 diabetic patients including 47 studies (8,663 cases, 6,064 controls) from 1994 to 2004. they confirm a statistically significant protective role of the ii genotype in the development of diabetic nephropathy; the effect was most pronounced in asians with type 2 diabetes, followed by caucasians with types 1 and 2 diabetes. they suggested that these findings may have implications for the management of diabetic nephropathy using ace inhibitors especially among type 2 diabetic asians. in 2012, they conducted a comprehensive meta - analysis on 63 published studies from 1994 to 2010 with 14,108 cases and 12,472 controls relating variants of the ace i / d polymorphism to the risk of developing diabetic nephropathy. they included all of the studies that determined the genotype distribution of ace i / d polymorphism in cases with type 1 or type 2 diabetes and nephropathy and in diseased controls or in healthy controls. the overall analysis showed a significant association between the ace i / d polymorphism and the risk of diabetic nephropathy for all genetic models (i d versus ii : or = 1.12, 95% ci 1.021.24 ; dd versus ii : or = 1.27, 95% ci 1.131.44 ; allele contrast : or = 1.15, 95% ci 1.081.23 ; dominant model : or = 1.18, 95% ci 1.071.31 ; and recessive model : or = 1.18, 95% ci 1.081.30, resp .). in stratified analysis by ethnicity and diabetes mellitus type, they further found that the asian group with type 2 dm showed a significant association for all genetic models (i d versus ii : or = 1.25, 95% ci 1.071.47 ; dd versus ii : or = 1.57, 95% ci 1.241.98 ; allele contrast : or = 1.30, 95% ci 1.151.46 ; dominant model : or = 1.37, 95% ci 1.101.69 ; and recessive model : or = 1.34, 95% ci 1.151.56, resp .). however, they failed to find any significant effects for different genetic models in other subgroups. the authors suggested that the ace i / d polymorphism may contribute to nephropathy development, especially in the asian group with type 2 diabetes mellitus. in 1996, yoshida et al. including 168 japanese patients with niddm followed over 10 years have shown that ace dd genotype has a high prognostic value for progressive deterioration of renal function. they also showed that patients with stable renal function had no significant difference in the distribution of ace genotype between patients with albuminuria and patients without albuminuria. analysis of the clinical course of the three ace genotypes revealed that the majority (95%) of patients with the dd genotype who had albuminuria progressed to end - stage renal disease within 10 years of diagnosis of diabetes. moreover, the ace dd genotype had the increased mortality in patients on dialysis and the prevalence of the dd genotype in patients on chronic dialysis decreases year by year. next year, schmidt et al. reported 658 caucasian patients with type ii diabetes, 347 without diabetic nephropathy and 311 with various stages of diabetic nephropathy, and determined the i / d polymorphism of the ace gene. they compared patients at the extremes of renal risk, that is, normotensive patients without antihypertensive treatment and without nephropathy (n = 144) versus patients on dialysis (n = 61), differed with respect to genotype (dd 36.8% versus 57.4% ; p = 0.007) and allele frequencies (d 0.59 versus 0.76 ; p < 0.001). in this study, patients with the highest renal risk more frequently had the dd genotype. this would be compatible with a greater risk of (or rate of) progression to end - stage renal failure. also compared the ace genotype distributions in 79 caucasian iddm patients with esrd and 82 control patients without microalbuminuria after fifteen years of iddm. the ace genotype distribution in patients was not in accordance with the hardy - weinberg equilibrium due to a significant overrepresentation of the dd genotype (= 8.9, p = 0.01). the presence of the dd genotype increased the risk of end - stage renal failure two - fold compared to the other genotypes (odds ratio 2.1, 95% ci 1.14.0). they concluded that the risk of end - stage renal failure in patients with iddm is two - fold increased in patients with the dd genotype as compared to patients with other genotypes. we also studied the impact of insertion / deletion (i / d) genotypes on the progression of diabetic nephropathy in 239 korean patients with type 2 diabetes (group 1, 99 patients with stable renal function ; group 2, 140 patients with declining renal function). the frequency of the dd genotype was significantly greater in group 2 compared with group 1 (30.7% versus 9.1% ; p < 0.05). patients with the dd genotype reached the end point (serum creatinine > 2.0 mg / dl) faster than those with the other genotypes (dd, 11.38 4.08 years ; i d, 13.85 4.04 years ; ii, 14.04 4.06 years, resp ., p < 0.05) and took significantly less time to reach dialysis therapy (dd, 13.10 4.45 years ; i d, 16.21 4.74 years ; ii, 15.13 4.09 years, resp . in multiple logistic regression analysis, systolic blood pressure and dd genotype showed significant correlations with the progression of diabetic nephropathy . in patients with the dd genotype, the odds ratio was 3.881 ( 95% confidence interval, 1.564 approximately 9.628 ; p = 0.003) compared with that with the ii genotype. we suggested that the ace dd genotype might be a significant risk factor for the progression of diabetic nephropathy. recently, yu et al. published meta - analysis of 12 previous studies containing 4,015 diabetic nephropathy patients (981 cases, 3,034 controls). in this study , they showed that in overall populations, there was a notable association between d allele or dd genotype and esrd susceptibility (d : or 1.32, 95% ci 1.111.56, p = 0.002 ; dd : or 1.67, 95% ci : 1.252.21, p = 0.004). in the subgroup analysis according to ethnicity, d allele or dd genotype was associated with esrd risk in asians. in caucasians, the association of dd genotype with esrd risk was observed, but the d allele was not. furthermore, ace i / d gene polymorphism was associated with esrd risk in patients with diabetic nephropathy due to type 2 dm, but the association was not found for patients with diabetic nephropathy due to type 1 dm. they concluded that d allele or dd genotype is associated with the esrd susceptibility in diabetic nephropathy patients. earlier studies repeatedly showed that patients with dd genotype or d allele have elevated circulating and tissue ace activity compared to patients with i allele. this may contribute to the interindividual variability in the antiproteinuric responses to inhibition of the raas using either ace inhibitors (aceis) or angiotensin ii type 1 receptor blockers (arbs). raas blockers are the first - line drugs for the treatment of hypertension associated with diabetes in the fact that these drugs not only lower systemic blood pressure but also reduce intraglomerular pressure. imanishi et al. showed that the mechanism by which an ace inhibitor caused a short - term decrease in albuminuria in early diabetic nephropathy involved a glomerular hemodynamic change, namely, a decrease in intraglomerular capillary pressure. these drugs have greatly improved the renal prognosis and survival of diabetic patients with nephropathy over the last decade. however, despite therapy targeting elevated blood pressure, albuminuria, hyperglycemia, and lipid abnormalities, patients with diabetic nephropathy still on average have a rate of decline in renal function three to six times that seen in individuals without renal disease. consequently, diabetic nephropathy is still one of the principal causes of end - stage renal disease, leading to dialysis and death in developed countries. although current therapeutic strategies have alleviated the huge burden of diabetic nephropathy, many patients still progress to esrd. one reason for the inadequacy of current antiproteinuric (renoprotective) therapy and the persistent poor renal prognosis is the large interindividual variation in response to first - line therapy including antihypertensive drugs blocking the renin - angiotensin - aldosterone system. furthermore, overt proteinuria seen in diabetic nephropathy is itself a risk factor that may adversely affect renal function, and it is associated with a faster rate of renal disease progression. therefore, the reduction of proteinuria is an important tool for retarding the progression of renal disease in diabetic nephropathy patients. traditionally, ace inhibitors (aceis) and angiotensin ii type 1 receptor blockers (arbs) have been widely used in everyday clinical practice of nephrology for the purpose of reducing proteinuria in patients with various renal diseases including diabetes mellitus. however, the antiproteinuric effect of ace inhibitors on proteinuria is variable and the percentage of reducing proteinuria is in the range of 2080% in a variety of renal diseases. the antiproteinuric effect of ace inhibitors and/or angiotensin ii type 1 receptor blockers may be related to a number of factors: the type or dose of the raas blockers, the duration of therapy, the level of sodium intake, and the type of patient's ace genotype. the antiproteinuric mechanisms of raas blockers are thought to decrease intraglomerular capillary pressure by reducing both afferent and efferent renal arteriolar resistance, predominantly dilating efferent arteriole and systemic blood pressure. as demonstrated in previous studies, raas blockade has been superior to other antihypertensive agents in reducing albuminuria and slowing rate of decline in gfr despite similar blood pressure controls. besides the nongenetic factors, the basic concept of pharmacogenomics is a drug interacting with its target, for instance, an enzyme or a receptor. when genetic polymorphism leads to modified target availability or function, the drug response is modified as well. it has been reported that ace i / d genotypes appeared to be a major determinant of plasma and tissue ace activities. individuals with the dd genotype have the greatest and those with ii genotype have the least ace concentrations. so, it is expected that the differences in plasma and tissue ace activities associated with ace i / d genotype might affect the antiproteinuric (renoprotective) responses to raas inhibition. i / d polymorphism in diabetic nephropathy were reported so far. however, the antiproteinuric (renoprotective) effect of raas blockers and ace i / d genotype in diabetic nephropathy is inconclusive. these may be due to incomplete blockade of raas by suboptimal doses and/or compensatory mechanisms occurring during long - term treatment with raas blockers. after initiation of acei treatment, angiotensin ii level in plasma is lowered initially. however, during long - term treatment of acei, angiotensin ii level tends to increase as a of ace escape and angiotensin ii generation through non - ace dependent pathways such as chymase or other serine proteases. incomplete raas blockade during chronic acei therapy may be overcome by inhibiting the action of angiotensin ii at the site of the angiotensin ii type 1 receptor by an arb. on the contrary, in treatment with arb , there also tends to be a compensatory increase in renin and angiotensin ii levels, thereby increasing the competition at the angiotensin ii type 1 receptor site. so, in this situation, we can reduce compensatory increased angiotensin ii by adding acei. several previous studies suggested that ace genotype may predict the response of patients to antiproteinuric and renoprotective effect with ace inhibitors (aceis). , in their observational followup study of type 1 diabetic patients with diabetic nephropathy receiving ace inhibitor captopril (n = 35 and n = 169), have reported that the dd genotype reduces the long - term beneficial effect of ace inhibition on the progression of diabetic nephropathy in patients with iddm. jacobsen et al. tested the potential role of ace i / d polymorphism on the early antiproteinuric responsiveness in an observational followup study with sixty young hypertensive type 1 dm nephropathy patients. they showed more albuminuria reduction with captopril therapy in ii genotype than in i d or in dd genotypes. data from a eurodiab randomized controlled trial lisinopril in iddm showed that the i / d polymorphism of the ace gene modulates the therapeutic effect of ace inhibition on the progression of urinary albumin excretion in iddm patients. patients with the ii genotype showed the fastest rate of aer progression on placebo but had an enhanced response to lisinopril. albumin excretion rate (aer) at 2 years (adjusted for baseline aer) was 51.3% lower on lisinopril than placebo in the ii genotype patients (95% ci, 15.7 to 71.8 ; p = 0.01), 14.8% lower in the i d group (7.8 to 32.7 ; p = 0.2), and 7.7% lower in the dd group (36.6 to 37.6 ; p = 0.7). only in the ii group the difference was statistically significant even after adjustment for gender, baseline and followup bp, and metabolic control. the authors concluded that knowledge of ace genotype may be of value in determining the likely impact of ace inhibitor treatment on the albuminuria reduction. , which included 169 iddm patients with overt nephropathy, showed that the d allele of the ace i / d polymorphism in addition to nongenetic risk factors independently accelerated progression of diabetic nephropathy during ace inhibition. also, in this study of patients with type 1 diabetes, the i allele was associated with a slower progression to doubling of serum creatinine or esrd. taken together, in type 1 diabetes with nephropathy, the i allele increases the responsiveness to the antiproteinuric (renoprotective) effect of ace inhibitor therapy. data in type 2 dm nephropathy in relation to ace i / d polymorphism and antiproteinuric (renoprotective) effect of ace inhibitors are scarce. our group investigated the antiproteinuric effect of ace inhibition (benazepril 10 mg / day or perindopril 4 mg / day) in relation to ace i / d polymorphism in a short - term observational followup study in 83 niddm patients with overt nephropathy (urinary protein excretion over 500 mg / day) classified into three groups in accordance with ace genotypes (17 dd ; 33 i d ; 33 ii) and prospectively followed up for 3 months. before entry, previously used ace inhibitors were withdrawn for at least 2 weeks and baseline proteinuria was measured. our study showed that the percentage reduction in proteinuria for dd genotype was significantly higher than in i d and in ii genotypes (50.9 19.2% versus 19.2 16.0%, 20.2 20.4%, p < 0.05). there were no statistically significant correlations between the levels of baseline proteinuria and the magnitudes of the reduction of proteinuria under ace inhibition (p > 0.05). these indicated that ace dd genotype is more susceptible than ace ii and i d genotypes to the antiproteinuric effect of ace inhibitors. they found that, in 2089 chinese patients with normoalbuminuria, microalbuminuria, or macroalbuminuria over a median period of 44.6 months, ace inhibitor therapy decreased mortality and renal end point which was defined as death due to renal failure, dialysis, egfr of < 15 ml / min/1.73 m or more than 50% loss of egfr, more effectively in i allele carriers than in dd. this study was the only study in type 2 diabetes with adequate power and followup. however, we have to be cautious in interpreting this finding because the favorable effects of the i allele were restricted to those with normoalbuminuria or microalbuminuria. another study regarding raas gene polymorphisms and renal responsiveness to raas inhibition in type 2 diabetic asian indians they enrolled 810 north indian type 2 diabetics treated with ace inhibitor or arb and were followed up for 3 years. they observed that the ace ii genotype and cumulative genetic risk score of < 1 was associated with better renoprotective response to ace inhibitor in type 2 dm with normoalbuminuria. however, there was no significant difference in renoprotective effect in type 2 diabetics with nephropathy based on ace i / d genotypes with 3-year ace inhibitor therapy (table 1). data on angiotensin ii type 1 receptor blocker (arb) in relation to ace i / d polymorphism and antiproteinuric (renoprotective) effects in type 1 diabetes with nephropathy are also scarce. they showed that the antiproteinuric effect of 36- and 4-month arb (losartan) had similar beneficial renoprotective and antiproteinuric effect in 54 hypertensive white type 1 dm nephropathy patients with ace ii and dd genotypes. same was reported by haneda et al. in 127 japanese type 2 dm nephropathy patients. they found that arb (candesartan) is useful in reducing proteinuria in type 2 dm nephropathy patients compared with placebo and seems to be effective in subjects with both the ii and dd genotypes of the ace gene. more data on the interactions between ace i / d polymorphism and arb therapy have been provided by analyses of the reduction of endpoints in niddm with the aii antagonist losartan (renaal) study, a double blind, multicenter, prospective, randomized, and placebo controlled clinical trial designed to evaluate the renal effects of losartan in 1513 type 2 diabetic patients with overt nephropathy. ace i / d data were available in 1435 of the 1513 renaal study patients. available ace i / d data showed that compared with placebo losartan reduced the risk of reaching the composite end point of doubling of serum creatinine, esrd, or death by 5.8%, 17.6%, and 27.9% among those with the ii, i d, and dd genotypes, respectively. this study demonstrated that the deletion allele of the ace gene had a harmful impact on the composite endpoint. the beneficial effects of losartan were greatest in the ace dd genotype group and intermediate in the ace i d genotype group for nearly all endpoints. the novel clinical importance of this study is that those patients who have the greatest need for renoprotective treatment have the best effect of losartan (dd and i d genotype), whereas those patients with a better renal prognosis (ii genotype) also derived renal benefit. first, this is the largest trial evaluating the association of the ace / id polymorphism on renal outcome and death during angiotensin ii - receptor blockade in diabetic nephropathy; the study may well be underpowered in relation to the individual components of composite endpoint. second, the i d alleles were not in hardy - weinberg equilibrium in the black patients. lastly, despite randomization, higher baseline proteinuria was present in the ii genotype in the losartan group. in spite of the several limitations of this study , the deletion allele of the ace i / d polymorphism showed unfavorable renal prognosis in patients with proteinuric type 2 diabetes, which can be improved by losartan treatment. another recent study by cheema et al. also reported that, in type 2 diabetics with nephropathy, ace dd genotype and a genetic risk score of > 6 were associated with better renoprotective response to arb. they suggested that ace i / d genotypes individually and in interaction with other ras single nucleotide polymorphisms (angiotensinogen and angiotensin ii type 1 receptor gene) modulate renoprotective efficacy of ace inhibitor and arb in type 2 diabetics depending on the status of proteinuria (table 2). the association between the raas activation and the development and progression of diabetic nephropathy has been known for a long period of time. ace is the key enzyme of the raas - cascade that plays a central role of blood pressure regulation and volume homeostasis in the body. despite the large amount of studies looking for candidate genes, the ace i / d polymorphism remains the unique and well - characterized locus clearly associated with development and progression of diabetic nephropathy. , numerous studies have addressed the role of ace i / d polymorphism in the development and progression of diabetic nephropathy. data reported so far showed that the ace i / d polymorphism directly influences circulating levels of ace, the ii genotype protects against the development of diabetic nephropathy, the dd genotype predicts poor renal response to raas inhibitors (the current strategy of raas inhibition in patients with the dd genotype may be insufficient to block an activated raas), and angiotensin ii type 1 receptor blockers (arbs) can ameliorate the adverse effect of the d allele (no difference between genotypes is observed when patients are treated with an arb). evaluating the ace i / d polymorphism in diabetic nephropathy is a reliable tool to identify diabetic patients at risk and identify patients who may benefit from antiproteinuric (renoprotective) therapy with ace inhibitors and/or arbs. this may guide pharmacologic therapy in individual patients and help to identify the patients with more aggressive use of raas blockers such as supramaximal dose of individual raas blocker and double or triple blockade of the raas. in case of an insufficient response to raas blockers, we also consider other treatment strategies such as glycemic control, low salt intake, more aggressive proteinuria reduction strategy, and hypercholesterolemia control that halt progression of diabetic nephropathy.

approximately 2040% of diabetic patients develop nephropathy which is the leading cause of esrd in developed countries. the ace i / d polymorphism is thought to be a marker for functional polymorphism which regulates circulating and tissue ace activity. while the initial study found a protective effect of the ii genotype on the development of nephropathy in iddm patients, subsequent studies have addressed the role of ace i / d polymorphism in the development and progression of diabetic nephropathy. raas blockers are the first line drugs for the treatment hypertension associated with diabetes and have been widely used in everyday clinical practice for the purpose of reducing proteinuria in patients with various renal diseases. however, the antiproteinuric effect of raas blockers is variable and the percentage of reducing proteinuria is in the range of 2080%. the antiproteinuric effect of raas blockers may be related to a number of factors: the type or the dose of raas blockers, the duration of therapy, the level of sodium intake, and the type of patient's ace i / d genotype. besides the nongenetic factors , drug responses, can be influenced by ace gene polymorphism. in this review, we discuss the relationship between ace i / d polymorphism and diabetic nephropathy and therapeutic response of raas blockers.

as per the united nations environment programme (unep), global incidence of non - melanoma skin cancer exceeds 2 million each year 2. with a 10% decrease in stratospheric ozone, seventy - five percent of the non - melanoma skin cancers are basal cell carcinomas (bcc), which are usually effectively treated with excision. treatment options for scc vary depending on the severity of the disease, but surgical excision alone is generally adequate treatment. however, for high risk scc patients, which include those with inadequately excised and recurrent lesions, lymph node metastasis, and immunosuppression, the current treatment recommendation is combined surgery and radiation 3. patients with high risk cutaneous scc includes those with inadequately excised and recurrent lesions, lymph node metastasis, and immunosuppression 3. at the molecular level, over expression of egfr is considered a predictive factor for identification of high - risk scc 3. the combination of radiation treatment and surgery has reduced the rate of locoregional relapse by 20 - 25% 4. as per porceddu et al , patients with high - risk pathologic features such as multiple nodes, extra nodal spread, positive margins, and perineural and vascular invasion will benefit from concurrent platinum - based chemotherapy and radiation treatment 5. but, this is frequently a patient population with advanced age and multiple co - morbidities, in which platinum - based regimens are least tolerated. cetuximab is a chimeric human / murine monoclonal antibody that binds competitively to egfr and prevents activation of the receptor 6. considering the low side effect profile, interest in cetuximab as a treatment for non - melanoma skin cancer is growing area. most adverse skin reactions expected with this treatment are cutaneous and include acneiform eruption, xerosis, paronychia, hair changes, telangectasia and hyperpigmentation. the most common adverse effect is acneiform rash which is seen in 50 - 100% of patients 7. this is assumed to be due to interference with the physiological role of egfr in the epidermis. there are reported cases of severe acneiform rash, mucositis and dermatitis in people treated with combined radiation and cetuximab 8;9. there is also one reported case of toxic epidermal necrolysis caused by cetuximab plus minocycline in a patient receiving chemoradiation for recurrent squamous cell carcinoma of head and neck which evolved in the 5th week of treatment with cetuximab 10. here , we evaluate eight cases of advanced squamous cell carcinoma or basal cell carcinoma patients treated with cetuximab. among the eight patients, four were basal cell carcinoma and four were squamous cell carcinoma. a retrospective analysis was performed using the records of the siu - s between the years 2007 - 2011. local institutional board approval (exempt approval on 01/21/2010) eight patients were identified who had received cetuximab for treatment of skin carcinoma. collected from the case records were demographic data including age, co - morbidities, and family history. tumor characteristics included histology, stage and treatment factors included previous interventions, dose and frequency of cetuximab, and adverse effects of cetuximab. assessment of disease status was based on physical examination documented in the clinic notes and imaging studies. complete and partial remission (cr & pr) has been determined based on recist criteria except the fact that patients may or may not been continuing the treatment during that time 11. among the eight patients analyzed, four had basal cell carcinoma and four had squamous cell carcinoma. three of the four patients with bcc had gorlin syndrome with a significant family history of basal cell carcinoma. the age group of these patients varied from 45 - 87, including three above the age of 80. all of the octogenarians had multiple medical problems and required multiple surgeries and excisions of basal cell carcinomas that had ed in deformities in the head and neck area. except for one scc patient who had received carboplatin and docetaxel chemotherapy, the fourth bcc patient, without a history of gorlin syndrome, had an aggressive basal cell carcinoma that warranted numerous resections over several years as well as radiation therapy. he ultimately developed regional lymph node metastasis and was referred for further management of his progressive disease that was considered refractory to any further radiation or surgery. four patients, aged 38 to 89, had recurrent squamous cell carcinoma with a history of multiple recurrences in the past. one was on mycophenolate and presented with a right temple lesion that recurred four times. it was treated initially with surgery and combined cetuximab and radiation for the second recurrence. the third and fourth recurrences were also treated with surgery. the other patient presented with a metastatic squamous cell carcinoma of unknown primary with mediastinal adenopathy, neck mass and pulmonary nodules. he failed carboplatin/ docetaxel chemotherapy and developed progressive disease with a pericardial effusion, necessitating a pericardial window. his transplanted kidney was surgically removed, and all of his immunosuppression was stopped. for five out of the 8 patients, one of the patients with bcc received cetuximab 250 mg / m once a week, and the latter dose was subsequently escalated to 300 mg / m. the starting dose of cetuximab for 2 of the bcc patients was 125 mg /m once a month. the dose was gradually increased up to 250 mg / mfor one of the patients. one of the scc patients started with cetuximab 250 mg / m2 once a week for eight weeks as induction treatment. the same patient continued maintenance treatment for two and half years as cycles of once a week treatment for four weeks with two weeks off. five out of 8 patients (63%) experienced grade ii acneiform skin rash as a side effect, which was usually noted after 2 - 3 treatments. one patient was treated with steroids and minocycline because the rash recurred after each treatment. another patient required premature termination of cetuximab due to persistent skin rash despite treatment with steroids and minocycline. this latter patient was subsequently able to tolerate cetuximab with a 50% dose reduction when it was restarted for disease recurrence. for the remaining three patients, the grade ii skin rash resolved with 2 - 3 weeks of minocycline treatment. this patient later developed grade ii skin induration and grade iii skin pain after two and half years of treatment and stopped therapy. they were treated with magnesium intravenously (iv) with each treatment. among the four patients with bcc, fifty percent attained complete remission (cr) and three patients relapsed after stopping the treatment (2 cr and 1 pr) and currently have been placed back on cetuximab. one patient remains on maintenance treatment for the last 12 months after attaining an initial partial response. another patient had a local recurrence of the disease after a four - month hiatus from treatment and now remains on maintenance treatment. the third bcc patient had two recurrences after the first treatment with cetuximab, which had to be discontinued after four cycles secondary to skin rash. the first recurrence occurred two months after discontinuing cetuximab but responded to reinstitution of treatment for seven months. the second recurrence occurred after five months off therapy, but remission was obtained again with the reinitiation of cetuximab, and the patient has been on maintenance treatment for the last seven months. the fourth patient, who had a partial response with cetuximab, had a multifocal recurrence soon after being off treatment. he was then started on a multidrug chemotherapy regimen with 5-flurouracil, carboplatin, and cetuximab. he achieved a near complete response to three cycles of chemotherapy but therapy was stopped due to his advanced age and performance status. his disease recurred several months later, and he chose to receive supportive care only at that point. median disease - free survival was 1 month. with a median follow up of 12 months, overall survival was 100%. one relapsed at 6 months following active treatment, and the other two have been in remission for the past 3 years. the latter two patients had a renal transplant and had previously been on mycophenolate or other immunosuppression. the patient who had a partial response died secondary to other co - morbidities before any further follow - up. median follow - up and disease- free survival were 32.5 months and 20.5 months, respectively. this study shows that recurrent non - melanoma skin cancers can be successfully treated with extended cetuximab therapy. 62.5 percent of the study patients obtained a complete remission and a major response (cr and pr) was achieved in all of the patients. however, among those whose treatment was held, 63% of the patients subsequently relapsed. for the three patients who had a sustained remission, two have continued the maintenance treatment, and the other one has stopped. 63% and 25% of patients had grade ii skin rash and grade 1 hypomagnesemia, respectively. the skin induration and skin pain experienced by one patient were considered to be unrelated adverse events. no other adverse events were noted, and no one developed grade iii or iv toxicities with treatment. epidermal growth factor receptor is a member of tyrosine kinase growth factor receptor family. activation of this receptor in intracellular tyrosine kinase autophosphorylation, which initiates a cascade of intracellular events ultimately leading to cell cycle progression, angiogenesis, metastasis and reduced apoptosis 12. egfr is normally expressed in human cells, but higher levels of expression have been shown in many malignancies. as per krahn, 80% of scc and 57% of bcc express egfr 13. as per previous studies, aberration in egfr pathways is associated with poor prognosis in many malignancies. even though bcc also expresses egfr, the key pathology in bcc is an aberration in the hedgehog pathway 14;15. novel inhibitors of this pathway such as gdc-0449 are on trial but are not yet clinically approved 16. it has been shown that egfr signaling acts synergistically with the hedgehog pathway in the malignant transformation of cells 17;18. we were able to obtain either a cr or a pr for all of our patients. however, we had a significant relapse rate for patients when treatment was stopped, which emphasizes the importance of maintenance treatment for long - term disease control. even though median disease free survival was only one month, overall survival over the 30 month follow up period was 75%. all of these patients had refractory disease, and we were able to prolong their life with a relatively low toxicity treatment regimen. compared to currently approved chemotherapies for skin carcinomas, cetuximab is well tolerated. in our study, the most commonly experienced side effect was acneiform skin rash occurring in 63% of patents, which is consistent with a prior study 7. all of skin rashes were grade ii in severity, which were treated successfully with steroids and minocycline. concurrent radiation treatment increases the incidence of serious adverse events as per the case reports that we discussed earlier. only one of our patients had received radiation treatment along with cetuximab treatment, and he subsequently responded to single agent cetuximab therapy along with immunosuppressant modulation. there have been isolated case reports of using cetuximab in recurrent non - melanoma skin cancers with favorable responses 19 - 22. however, there have been no studies comparing efficacy of treatment with cetuximab versus currently approved chemotherapeutic agents for cutaneous malignancies. the of the above reported cases are encouraging for the use of cetuximab in refractory non - melanoma skin cancers and suggest that a randomized phase iii study between chemotherapy and cetuximab in this condition may be useful.

objectives: non - melanoma skin cancer is the most common malignancy in us, with an annual incidence of in excess of 1.5 million cases. in the majority of cases, locoregional treatment is curative and systemic therapy is not indicated. platinum - based chemotherapy regimens have been used most commonly in refractory cases. the use of cetuximab, a monoclonal antibody targeting epidermal growth factor receptor , has been reported for skin cancer treatment. this current study evaluated eight cases of locally advanced and refractory basal cell or squamous cell cancers which were treated with cetuximab.methods: this is a retrospective study on eight patients who had received cetuximab for treatment of cutaneous carcinoma since 2007 at southern illinois university school of medicine (siu - som) medical oncology clinic.: three of the four patients with basal cell carcinoma and two of the four patients with squamous cell carcinoma maintained remission on treatment.. the main side effect was acneiform rash which required termination of treatment for one patient and dose reduction in another.: the study indicates that cetuximab may have a beneficial role for patients with non - melanoma cutaneous carcinomas that are refractory to standard therapy.

development of apraxia and other cognitive deficits requires help of others with activities of daily living. unfortunately, the person with dementia sometimes does not cooperate with family or professional caregivers but actually resists care or exhibits behaviors during care that may be labeled abusive. these behaviors may be difficult to handle by family caregivers and may lead to institutionalization. in an institution, rejection of care and behaviors directed towards others may lead to injuries of the resident or staff and to staff burnout. a survey of nursing home physicians showed that the most common problem that they treated in residents with dementia was rejection of care. some of them investigated the effectiveness of pain control, others used personalized psychosocial interventions based on the unmet needs theory for treatment of agitation and found positive . however, the problem with these studies is that they consider all behavioral symptoms to be agitation and do not differentiate between agitation and rejection of care. this is due to using the cohen - mansfield agitation inventory or neuropsychiatric inventory in which the item agitation / aggression is actually measuring rejection of care. similar problems are also present in drug trials investigating effects of antipsychotics and other medications. therefore, we decided to study specifically rejection of care that is a highly relevant problem in clinical practice. we have previously described strong correlations between rejection of care and lack of understanding, depression and psychotic symptoms. behaviors directed towards others were strongly correlated with rejection of care, depression and delusions. however, these findings were based only on cross - sectional data. in this study, we performed longitudinal analyses to further explore the relationships of these modifiable factors with rejection of care and behaviors directed towards others of nursing home residents. our hypothesis was that longitudinal data would support causal relationships between these modifiable factors and rejection of care. we used minimum data set - resident assessment instrument (mds - rai) data collected by 8 dutch nursing homes and 10 residential homes. we included the data of residents within a 12-month time window for each facility separately, ing in a range from april 4, 2007, to december 1, 2008. of the records of 2,705 residents available from the 18 facilities, we selected the last records of 1,101 residents aged over 65 with alzheimer or other dementia (i1q or u), who were dependent in decision making (b4 not equal 0) and who were not comatose (b1 equal 0). four assessments from the mds within a period of 15 months were used for the analyses. the terminology was changed by the introduction of mds 3.0, which uses rejection of care instead of resist care. other new, but equivalent, items in mds 3.0 are physical behavioral symptoms directed towards others instead of physical abuse and verbal behavioral symptoms directed towards others instead of verbal abuse. presence of rejection of care and behavioral symptoms directed towards others was obtained from mds 2.0 section e with choices from not exhibited to daily. the variables physical behavioral symptoms directed towards others (e4ca) and verbal behavioral symptoms directed towards others (e4ba) were combined into one variable called behaviors directed towards others. ability to understand (c6) was rated as sometimes and rarely / never. psychotic symptoms were combined into one variable, psychosis, whose values are number of symptoms. clinical diagnosis of depression was obtained from mds item i1ee, and depression symptoms were assessed by the validated depression rating scale using other mds items: negative statements (e1a), anger (e1d), unrealistic fears (e1f), repetitive health complaints (e1i), sad expression (e1i) and crying (e1 m ; score 0 - 14). this scale correlated well with the cornell and hamilton depression scales using at least mild depression as a cutoff point, and with psychiatric diagnosis. it was also more sensitive and specific than the 15-item geriatric depression scale in detecting depression in the nursing home population. data about presence of pain were obtained from the mds item j2a with options of no, less than daily and daily. demographic data were also obtained from the mds. we investigated the longitudinal relationship between changes in rejection of care and behaviors directed towards others, and changes in lack of understanding, pain, depression and psychotic symptoms with the structural equation modeling approach. within this approach, we used latent growth models, bivariate latent growth models and longitudinal latent growth mediation models. since the studied variables were ordinal, we used latent growth models for categorical variables to study change within each variable. this model assumes the existence of latent or nonobserved trajectories, which are observed only indirectly through the repeated measures. the intercept represents the initial level at the beginning of the study, and the slope represents the rate of change. the factor loadings for the slope represent the measurement points, and they were set to the values 0, 3, 6 and 9 that represent the months at which the measurements were taken. second, we used bivariate latent growth curve models to study the relationships between rejection of care and understanding, and rejection of care and depression. these models combine two latent growth curve models that describe two separate longitudinal processes, and study the relationship between the initial level of the first process (y, e.g. rejection of care) with the initial level and rate of change in the second process (x, e.g. lack of understanding), and the relationship between the rate of change in the first process with the initial level and rate of change in the second process (fig . 1, process y and x). bivariate latent growth curve models were also used to describe the relationship between behavioral symptoms directed toward others with rejection of care and behavioral symptoms directed towards others with understanding, and depression. frequency distributions of the variables psychotic symptoms and presence of pain remained stable through the whole measurement period, and the latent growth models showed no trend. therefore, we did not use bivariate latent growth models but latent growth models that included these variables as time - dependent covariates. in the model for psychosis symptoms , we included only the measurements of the first and the fourth psychosis symptom because of the high correlation between the first and the second measurement, and the third and the fourth measurement. finally, longitudinal mediation models were used to study whether rejection of care mediated the relationship between lack of understanding and behaviors directed towards others, and whether it mediated the relationship between depression and behaviors directed towards others (fig . this means that first changes in lack of understanding ( depression) occurred, and they promoted changes in rejection of care. next, changes in behaviors directed towards others occurred as a consequence of changes in rejection of care. three sets of latent growth factors were specified, one set for the independent variable, one set for the mediator, and, finally, another set for the dependent variable. the mediation model examined whether the growth in the independent variable affected the growth trajectory of the mediating variable which, in turn, affected the growth trajectory of the dependent variable. the value, the root mean square error of approximation (rmsea), the comparative fit index (cfi), and the tucker - lewis (tli) index were used to evaluate the model. a model achieves a good fit if the rmsea is lower than 0.05, and fit is acceptable for values between 0.05 and 0.09. we used minimum data set - resident assessment instrument (mds - rai) data collected by 8 dutch nursing homes and 10 residential homes. we included the data of residents within a 12-month time window for each facility separately, ing in a range from april 4, 2007, to december 1, 2008. of the records of 2,705 residents available from the 18 facilities, we selected the last records of 1,101 residents aged over 65 with alzheimer or other dementia (i1q or u), who were dependent in decision making (b4 not equal 0) and who were not comatose (b1 equal 0). four assessments from the mds within a period of 15 months were used for the analyses. for rejection of care, we used the mds 2.0 item resist care (e4ea). the terminology was changed by the introduction of mds 3.0, which uses rejection of care instead of resist care. other new, but equivalent, items in mds 3.0 are physical behavioral symptoms directed towards others instead of physical abuse and verbal behavioral symptoms directed towards others instead of verbal abuse. presence of rejection of care and behavioral symptoms directed towards others was obtained from mds 2.0 section e with choices from not exhibited to daily. the variables physical behavioral symptoms directed towards others (e4ca) and verbal behavioral symptoms directed towards others (e4ba) were combined into one variable called behaviors directed towards others. ability to understand (c6) was rated as sometimes and rarely / never. psychotic symptoms were combined into one variable,. clinical diagnosis of depression was obtained from mds item i1ee, and depression symptoms were assessed by the validated depression rating scale using other mds items: negative statements (e1a), anger (e1d), unrealistic fears (e1f), repetitive health complaints (e1i), sad expression (e1i) and crying (e1 m ; score 0 - 14). this scale correlated well with the cornell and hamilton depression scales using at least mild depression as a cutoff point, and with psychiatric diagnosis. it was also more sensitive and specific than the 15-item geriatric depression scale in detecting depression in the nursing home population. data about presence of pain were obtained from the mds item j2a with options of no, less than daily and daily. demographic data were also obtained from the mds. we investigated the longitudinal relationship between changes in rejection of care and behaviors directed towards others, and changes in lack of understanding, pain, depression and psychotic symptoms with the structural equation modeling approach. within this approach, we used latent growth models, bivariate latent growth models and longitudinal latent growth mediation models. since the studied variables were ordinal, we used latent growth models for categorical variables to study change within each variable. this model assumes the existence of latent or nonobserved trajectories, which are observed only indirectly through the repeated measures. the intercept represents the initial level at the beginning of the study, and the slope represents the rate of change. the factor loadings for the slope represent the measurement points, and they were set to the values 0, 3, 6 and 9 that represent the months at which the measurements were taken. second, we used bivariate latent growth curve models to study the relationships between rejection of care and understanding, and rejection of care and depression. these models combine two latent growth curve models that describe two separate longitudinal processes, and study the relationship between the initial level of the first process (y, e.g. rejection of care) with the initial level and rate of change in the second process (x, e.g. lack of understanding), and the relationship between the rate of change in the first process with the initial level and rate of change in the second process (fig . 1, process y and x). bivariate latent growth curve models were also used to describe the relationship between behavioral symptoms directed toward others with rejection of care and behavioral symptoms directed towards others with understanding, and depression. frequency distributions of the variables psychotic symptoms and presence of pain remained stable through the whole measurement period, and the latent growth models showed no trend. therefore, we did not use bivariate latent growth models but latent growth models that included these variables as time - dependent covariates. in the model for psychosis symptoms , we included only the measurements of the first and the fourth psychosis symptom because of the high correlation between the first and the second measurement, and the third and the fourth measurement. finally, longitudinal mediation models were used to study whether rejection of care mediated the relationship between lack of understanding and behaviors directed towards others, and whether it mediated the relationship between depression and behaviors directed towards others (fig . this means that first changes in lack of understanding ( depression) occurred, and they promoted changes in rejection of care. next, changes in behaviors directed towards others occurred as a consequence of changes in rejection of care. three sets of latent growth factors were specified, one set for the independent variable, one set for the mediator, and, finally, another set for the dependent variable. the mediation model examined whether the growth in the independent variable affected the growth trajectory of the mediating variable which, in turn, affected the growth trajectory of the dependent variable. the value, the root mean square error of approximation (rmsea), the comparative fit index (cfi), and the tucker - lewis (tli) index were used to evaluate the model. a model achieves a good fit if the rmsea is lower than 0.05, and fit is acceptable for values between 0.05 and 0.09. the residents were mostly female, and some had diagnoses of both alzheimer's disease and other dementias (table 1). over one third of them exhibited rejection of care for at least some days, and almost one third exhibited verbal behavioral symptoms directed towards others, while about 15.0% exhibited physical behavioral symptoms directed towards others. about half of these residents (51.0%) exhibited significant symptoms indicating depression, but only 12.4% had a clinical diagnosis of depression. psychotic symptoms were present in 15.1% of residents, while almost one half of them experienced some pain (table 1). most common medications used for treatment of residents with rejection of care and behaviors directed towards others were antipsychotics that were administered at the beginning of the study to 32.2% of subjects (table 1). antidepressants were used in 18.7% of subjects, and antianxiety medications were used in 13.7% of subjects. further, all three classes of psychotropic medications were used more often in residents with depressive symptoms than in residents without depression (antipsychotics 27.5 vs. 44.0%, antidepressants 13.0 vs. 27.4%, antianxiety 10.2 vs. 19.6%). the prevalence of psychotropic drug administration did not change during the study for antipsychotics (t = 0.22, p = 0.826) and antidepressants (t = 0.81, p = 0.416), while the use of antianxiety medications increased (13.7 vs. 17.3%, t = 2.51, p = 0.012). table 2 shows the longitudinal trajectories of all the variables analyzed with latent growth models to investigate the development of the symptoms over time. based on the rmsea criteria, the model fit was good for the lack of understanding, rejection of care and behaviors directed towards others, and acceptable for depression and pain. the cfi and tli coefficients were equal to one for all the models. however, the increase was not significant for pain. the latent growth model for psychosis symptoms could not be estimated because there were almost no cases with change in psychosis scores within the study period. next, bivariate linear latent growth models were used to study the relationships between evolution in rejection of care and evolution in symptoms related to rejection of care. for psychosis and pain, we fitted a model with these variables as time - dependent covariates because previously estimated latent growth models did not show evidence of change for these variables. the model fit columns in table 3 show low rmsea values indicating a good model fit. changes in rejection of care were associated with or can be predicted by changes in lack of understanding and also by changes in depression. finally, lower initial levels of pain were associated with a stronger increase in rejection of care (c = 0.009, se = 0.004, p = 0.029). the relationships between the intercepts or initial level of rejection of care and intercepts of the other variables are not reported in table 3 because intercept parameters depend on the measurement scale of the analyzed variables. the relationships between psychosis symptoms and pain, and initial levels of rejection of care are scale free and can be interpreted. we found a strong relationship between psychosis and initial levels of rejection of care, with higher initial levels of psychosis associated with higher initial levels of rejection of care (c = 0.207, se = 0.053, p < 0.001). we also found that high initial levels of pain were associated with high initial levels of rejection of care (c = 0.116, se = 0.045, p = 0.009). then, bivariate linear latent growth models were used to describe the relationships between evolution in behaviors directed towards others and evolution in symptoms related to those behaviors. the columns reporting relationships between slopes show significant positive relationships between changes in rejection of care and changes in behaviors directed towards others. changes in understanding and changes in depression were also associated with changes in behaviors directed towards others. the relationships between intercepts for behaviors directed towards others are not reported in table 3 because of the same reasons explained above for the rejection of care analyses. regarding the relationship between initial behaviors directed towards others and initial levels in pain or psychotic symptoms, we found a significant relationship between initial behaviors directed towards others and initial psychosis (c = 0.205, se = 0.051, p < 0.001), indicating that initial psychosis was related to more frequent behaviors directed toward others. 1 ), one to investigate whether rejection of care mediated the relationship between lack of understanding and behaviors directed towards others, and the other to investigate whether rejection of care mediated the relationship between depression and behaviors directed towards others. consistent with the literature on statistical mediation, the parameters illustrating the relationships in figure 1 are called a, b and c. parameter a describes the relationship between the slope for the independent variable (x) and the slope for the mediator (m). parameter b describes the relationship between the slope of the mediator (m) and the slope in the dependent variable (y). parameter c represents the direct effect, which is the part of the effect of x on y that is independent of the mediator. the fit of the first model was good (test = 88.6, d.f . = 61, p = 0.012, rmsea = 0.021, cfi = 1.000, tli = 1.000). the relationship between lack of understanding and rejection of care was significant (a = 0.876, se = 0.124, p < 0.000). there was a significant relationship between rejection of care and behaviors directed towards others (b = 1.249, se = 0.337, p < 0.001), while the relationship between lack of understanding and behaviors directed towards others after adjusting for rejection of care was not significant (c = 0.645, se = 0.465, p = 0.166). therefore, this model suggests that the relationship between lack of understanding and behaviors directed towards others is mediated by rejection of care. to quantify the indirect effect, the product of the parameters a and b was calculated and tested with the sobel test. we found a significant mediation effect (ab = 0.979, se = 0.440, p = 0.026, with ab being the change in the outcome per one unit lack of understanding that goes through the mediator rejection of care ; fig . the fit of the second model was also good ( test = 105.1, d.f . = 58, p = 0.0002, rmsea = 0.027, cfi = 1.000, tli = 1.000). the relationship between the slope for depression and the slope for rejection of care was significant (a = 0.828, se = 0.138, p < 0.001), but there was not significant evidence that the slope in the mediator rejection of care was associated with the slope in behaviors directed towards others (b = 0.561, se = 0.306, p = 0.067). the adjusted direct effect (c = 0.427, se = 0.343, p = 0.214) and the indirect effect were also not significant (ab = 0.465, se = 0.277, p = 0.094). therefore, we can not conclude that the relationship between depression and behaviors directed towards others is mediated by rejection of care. since we did not observe changes in psychosis and pain, we did not consider mediation models for these processes. table 2 shows the longitudinal trajectories of all the variables analyzed with latent growth models to investigate the development of the symptoms over time. based on the rmsea criteria, the model fit was good for the lack of understanding, rejection of care and behaviors directed towards others, and acceptable for depression and pain. the cfi and tli coefficients were equal to one for all the models. in table 2, positive slopes show that all these variables increased with time. however, the increase was not significant for pain. the latent growth model for psychosis symptoms could not be estimated because there were almost no cases with change in psychosis scores within the study period. next, bivariate linear latent growth models were used to study the relationships between evolution in rejection of care and evolution in symptoms related to rejection of care. for psychosis and pain, we fitted a model with these variables as time - dependent covariates because previously estimated latent growth models did not show evidence of change for these variables. the model fit columns in table 3 show low rmsea values indicating a good model fit. the cfi and tli coefficients were equal to one. changes in rejection of care were associated with or can be predicted by changes in lack of understanding and also by changes in depression. finally, lower initial levels of pain were associated with a stronger increase in rejection of care (c = 0.009, se = 0.004, p = 0.029). the relationships between the intercepts or initial level of rejection of care and intercepts of the other variables are not reported in table 3 because intercept parameters depend on the measurement scale of the analyzed variables. the relationships between psychosis symptoms and pain, and initial levels of rejection of care are scale free and can be interpreted. we found a strong relationship between psychosis and initial levels of rejection of care, with higher initial levels of psychosis associated with higher initial levels of rejection of care (c = 0.207, se = 0.053, p < 0.001). we also found that high initial levels of pain were associated with high initial levels of rejection of care (c = 0.116, se = 0.045, p = 0.009). then, bivariate linear latent growth models were used to describe the relationships between evolution in behaviors directed towards others and evolution in symptoms related to those behaviors. the columns reporting relationships between slopes show significant positive relationships between changes in rejection of care and changes in behaviors directed towards others. changes in understanding and changes in depression were also associated with changes in behaviors directed towards others. the relationships between intercepts for behaviors directed towards others are not reported in table 3 because of the same reasons explained above for the rejection of care analyses. regarding the relationship between initial behaviors directed towards others and initial levels in pain or psychotic symptoms, we found a significant relationship between initial behaviors directed towards others and initial psychosis (c = 0.205, se = 0.051, p < 0.001), indicating that initial psychosis was related to more frequent behaviors directed toward others. 1 ), one to investigate whether rejection of care mediated the relationship between lack of understanding and behaviors directed towards others, and the other to investigate whether rejection of care mediated the relationship between depression and behaviors directed towards others. consistent with the literature on statistical mediation, the parameters illustrating the relationships in figure 1 are called a, b and c. parameter a describes the relationship between the slope for the independent variable (x) and the slope for the mediator (m). parameter b describes the relationship between the slope of the mediator (m) and the slope in the dependent variable (y). parameter c represents the direct effect, which is the part of the effect of x on y that is independent of the mediator. the fit of the first model was good (test = 88.6, d.f . = 61, p = 0.012, rmsea = 0.021, cfi = 1.000, tli = 1.000). the relationship between lack of understanding and rejection of care was significant (a = 0.876, se = 0.124, p there was a significant relationship between rejection of care and behaviors directed towards others ( b = 1.249, se = 0.337, p < 0.001), while the relationship between lack of understanding and behaviors directed towards others after adjusting for rejection of care was not significant (c = 0.645, se = 0.465, p = 0.166). therefore, this model suggests that the relationship between lack of understanding and behaviors directed towards others is mediated by rejection of care. to quantify the indirect effect, the product of the parameters a and b was calculated and tested with the sobel test. we found a significant mediation effect (ab = 0.979, se = 0.440, p = 0.026, with ab being the change in the outcome per one unit lack of understanding that goes through the mediator rejection of care ; fig . the fit of the second model was also good ( test = 105.1, d.f . = 58, p = 0.0002, rmsea = 0.027, cfi = 1.000, tli = 1.000). the relationship between the slope for depression and the slope for rejection of care was significant (a = 0.828, se = 0.138, p < 0.001), but there was not significant evidence that the slope in the mediator rejection of care was associated with the slope in behaviors directed towards others (b = 0.561, se = 0.306, p = 0.067). the adjusted direct effect (c = 0.427, se = 0.343, p = 0.214) and the indirect effect were also not significant (ab = 0.465, se = 0.277, p = 0.094). therefore, we can not conclude that the relationship between depression and behaviors directed towards others is mediated by rejection of care. since we did not observe changes in psychosis and pain, we did not consider mediation models for these processes. the of this longitudinal study showed that changes in lack of understanding or depression commonly precede changes in rejection of care. changes in behaviors directed towards others are related to changes in lack of understanding, depression and rejection of care. furthermore, a mediation model suggested that the relationship between lack of understanding and behaviors directed towards others was mediated by rejection of care. therefore, our support of our previous cross - sectional study, which found that lack of understanding and depression are the two main risk factors for development of rejection of care and behaviors directed towards others. however, our are more informative because longitudinal models provide more information regarding temporal relationships between the variables, and the mediation model provides information about the order in which the symptoms develop. initial psychotic symptoms and pain were also related to increased rejection of care, but we did not observe changes in the number of psychotic symptoms and pain within our study period. regarding to the absence of observed changes in pain, hendriks et al. found a high proportion of patients with pain persistence. that precluded inclusion of psychotic symptoms and pain in the mediation model. a relationship of depression to resistive behavior (rejection of care) and aggression (abusive symptoms) was already reported by lyketsos et al. and by leonard et al.. they found, in agreement with our , that psychotic symptoms play a minor role in the development of these behaviors. the lack of understanding is caused by the progression of dementia, and it increases the prevalence of rejection of care; this increase in rejection of care contributes to the increasing behaviors directed towards others. it may be improved by communication skills training of professionals and family caregivers that includes verbal skills, nonverbal and emotional skills, behavioral management skills, usage of tools and theoretical knowledge. such a program would be useful also in a hospital where many dementia patients are transferred. the most important intervention is to teach the staff that most people with dementia are not aggressive; they just defend themselves against unwanted attention from the caregivers and may consider the caregivers to be the aggressors. there is a clear - cut distinction between rejection of care and agitation, and the term agitation should be reserved for behaviors that occur when the resident is solitary, e.g. restlessness, repetitive movements, crying out. we identified depressive symptoms using the mds depression scale, which uses 7 mds items. this scale correlated well with the cornell and hamilton depression scales using at least mild depression as a cutoff point, and with psychiatric diagnosis. this is comparable to 47.4% detected in a large study of a long - term care facility. high prevalence of depression in individuals with alzheimer's disease can be expected because alzheimer's disease causes serotoninergic deficit, and some data indicate that this deficit may be related to aggressive behaviors. the relationship between serotoninergic function and aggressive behavior may not be unique to alzheimer's disease because decreased serotoninergic activity is present also in frontotemporal dementia. our suggest that depression contributes to both rejection of care and behaviors directed towards others and that the relation between depression and behaviors towards others is not mediated by rejection of care. depression in dementia could be treated with psychological interventions as shown in a recent review. antidepressants could be the first line of medication for individuals exhibiting these behaviors directed towards others if psychotic symptoms are not present. although some studies found improvement of behavioral symptoms after treatment with antidepressants, other studies were negative. this was documented by the depression in alzheimer disease study, which found that treatment with sertraline decreased behavioral disturbance and caregiver distress only in patients whose depression responded to antidepressant treatment. antidepressant treatment was found effective in decreasing behavioral symptoms of dementia in a cochrane data analysis and in a recent citalopram study, but antidepressants may sometimes require augmentation with atypical antipsychotics. the of this study show that the psychotropic medications administered most frequently to the participants in this study were antipsychotics. it is possible that these drugs somewhat reduced rejection of care by their sedative effects. similar prevalences of antipsychotic use were reported from a survey of nursing home physicians in the us and a dutch survey. in that study, 32% of subjects received antipsychotics, which is comparable with reports from sweden (38%) and from germany (32.5%). antipsychotics are used frequently for treatment of behavioral symptoms of dementia despite multiple reports that found serious side effects including increased mortality in residents treated with these medications. efforts are made in several countries to decrease the use of antipsychotics in dementia. in the us, the prevalence of antipsychotic use in nursing homes is about 25%, and the use of antipsychotics was recently reduced by 9.1%. in the uk, antipsychotic use in people with dementia decreased from 19.9% in 1995 to 7.4% in 2011, concomitant with an increase of antidepressants from 10.7 to 25.3%, indicating that antidepressants may be substituted for antipsychotics. our data were derived from mds records that were completed mostly by nurses. however, these were experienced clinicians, and mds data correlated well with other scales. we have studied the relationship of only four factors in abusive behavior of nursing home residents with dementia. however, there are probably also other factors that may be involved, e.g. physical causes such as thirst, hunger or inappropriate environmental temperature. using a discomfort scale in future studies would provide information about the importance of these other factors. our data could not evaluate these causes. a model with all predictors controlling for the influence of each variable would provide more evidence for causality than our models. another limitation is that we did not have data about specific drugs and drug dosages that may have been modified in residents with rejection of care and behaviors directed towards others even when the prevalence of their use did not change. the of this study together with the of previous investigations indicate that lack of understanding and depression are important factors in development of rejection of care and behaviors directed towards others, and that rejection of care may escalate into behaviors directed towards others. furthermore, the relationship between lack of understanding and behaviors directed towards others may be mediated by rejection of care. therefore, improved communication between caregivers and persons with dementia and perhaps also effective treatment of depression may prevent or ameliorate rejection of care and behaviors directed towards others.

aimsthe aim of this study was to analyze factors related to rejection of care and behaviors directed towards others in nursing home residents with dementia.methodsthe relationship of lack of understanding, depression, psychosis and pain with rejection of care and behaviors directed towards others was explored using four assessments from the minimum data set (mds) within a period of 15 months on 1,101 residents with dementia in dutch nursing homes. presence of depressive symptoms was ascertained using a validated mds scale, and presence of lack of understanding, rejection of care, psychosis and pain through the individual mds items. a structural equation modeling approach and latent growth models were used to investigate the longitudinal relationship between changes in rejection of care and physical or verbal behaviors directed towards others, and changes in lack of understanding, pain, depression and psychotic symptoms.changes in lack of understanding predicted changes in rejection of care, and there was also a relationship between changes in depression and rejection of care. changes of behaviors directed towards others were related to changes in lack of understanding and depression. pain and behaviors directed towards others were unrelated, and psychosis was rather stable throughout. a mediation model suggested that the relationship of lack of understanding with behaviors directed towards others was mediated by rejection of care.these indicate that lack of understanding and depression are important factors in development of rejection of care and behaviors directed towards others. the relationship between lack of understanding and behaviors directed towards others is mediated by rejection of care. improvement in communication between residents and caregivers, and perhaps also effective treatment of depression may prevent or ameliorate these behaviors directed towards others.

miriplatin, a cisplatin derivative with a high affinity for iodized ethyl esters of fatty acids from poppy seed oil, is a novel chemotherapeutic agent designed for use in the transarterial treatment of hepatocellular carcinoma (hcc). miriplatin inhibits cell proliferation in a similar manner as cisplatin and has superior solubility in ethyl esters of iodized fatty acids from poppy seed oil; releases its platinum constituent continuously by remaining at local tumor sites together with ethyl esters of iodized fatty acids from poppy seed oil (sustained release); and alleviates adverse effects as it maintains an antitumor effect owing to its sustained release as well as its minimal presence in the general circulation. despite such characteristics, however, many aspects of its antitumor effects are not clear. we experienced a case of advanced hcc that showed marked decrease in tumor markers following treatment with transcatheter arterial infusion (tai) with miriplatin, despite being refractory to tai with epirubicin. the patient, a 66-year - old man, had been diagnosed with chronic hepatitis c and had been followed - up at another hospital for the past 5 years. he had never smoked cigarettes and occasionally drank alcohol. during follow - up for chronic hepatitis c at the other hospital, the levels of tumor markers an advanced hcc with portal vein tumor thrombus (pvtt) in the right lobe was observed on the dynamic computed tomography (ct) scan, and he was admitted to our hospital for medical treatment in july 2010. the patient's height was 157 cm, body weight was 46 kg, blood pressure was 134/74 mm hg, body temperature was 36.6c, heart rate was 84 bpm, and no significant findings were observed upon physical examination. laboratory data are shown in table 1. a marked increase in the levels of tumor markers dynamic ct showed an irregularly shaped tumor, 5.6 3.8 cm in size, which was enhanced in the early phase (not enhanced in some areas) and indicated a region of deficiency in the delayed phase, in liver segment 8/7. the tumor had invaded the right anterior branch of the portal vein, and was cut off at the portion indicated by the arrow in fig. 1 (i.e. vp2). in segment 5, adjacent to the gallbladder, a classical hcc 2 cm in size was observed, which was enhanced in the early phase and indicated a region of deficiency in the delayed phase. as an initial clinical course beginning in late july 2010, abdominal angiography (fig . 2) was performed. digital subtraction angiography (dsa) of the superior mesenteric artery showed disruption of the right anterior branch of the portal vein. the lumen of the posterior branch of the postal vein was narrowed. in the dsa of the common hepatic artery, a large irregular tumor stain in the right lobe of the liver and a nodular tumor stain, 2 cm in size, were found in segment 5. an emulsion of epirubicin (50 mg) and lipiodol (8 ml) was infused primarily from the right hepatic artery. after tai, a transient fever over 38c was observed; however, the fever was easily managed using only antipyretics, and there were no other adverse events. conducted in late september revealed a marked increase in tumor marker levels (alpha - fetoprotein from 2,116 ng / ml to 4,622 ng / ml, and des - gamma - carboxy protein from 373 mau / ml to 8,394 mau / ml). the second clinical course was begun in early october 2010, during which abdominal angiography was performed. next, an emulsion of miriplatin (80 mg) and lipiodol (8 ml) was infused primarily from the right hepatic artery because the tumors were refractory to tai with epirubicin. one month after tai with miriplatin, the afp level was 1,507 ng / ml and the dcp level was 9,457 mau / ml, indicating that the therapy was not effective. however, 2 months after tai, the afp level was 63.9 ng / ml and the dcp level was 64 mau / ml. a marked decrease in the levels of the tumor markers was observed. because of the good response to tai with miriplatin, an emulsion of miriplatin (100 mg) and lipiodol (5 ml) was again infused primarily from the right hepatic artery in early january 2011. one month after the 2nd tai with miriplatin, the levels of the tumor markers improved to a nearly normal range (afp, 16.3 ng / ml and dcp, 34 mau / ml). dynamic ct performed 3 months after the 2nd tai with miriplatin showed that the size of the main tumor in segment 8/7 had markedly decreased, ing in less obstruction of the right branch of the portal vein (fig . 4). because the tumor in segment 5 exhibited poor response to the treatment, percutaneous therapy to the nodule several intra - arterial chemotherapy regimens using epirubicin, mitomycin, adriamycin, fluorouracil, fluorodeoxyuridine, mitoxantrone, and cisplatin as treatment for hcc have been reported. however, the optimal regimen for intra - arterial chemotherapy for hcc is still unknown. in the case described, the patient obtained a marked decrease in tumor marker levels following treatment with tai and miriplatin, despite being refractory to tai with epirubicin. considering the clinical courses used, it was thought that the main tumor caused an elevation in the tumor marker levels. the tumor enhancement pattern seen on the dynamic ct indicated that the main tumor contained an undifferentiated region (irregular enhancement with deficit regions in certain areas) in the early phase. considering the effects of the applied treatment (i.e. remarkably effective for the former tumor and ineffective for the latter tumor), tai with miriplatin may be effective for treating undifferentiated tumors. it is noteworthy that in the ineffective treatment of the tumor in segment 5 as well as in the markedly effective treatment of the main tumor, most of the administered lipiodol was washed out after treatment with tai and miriplatin. tai treatment typically in tumor necrosis, which is normally evaluated by lipiodol accumulation and ct because lipiodol accumulation in the area of the tumor corresponds to the necrotic area of the tumor. the liver cancer study group of japan recommends that the area of lipiodol accumulation should be calculated when evaluating the effects of tai. however, 1 case has been reported in which sufficient treatment effects were obtained despite washing out of most of the infused lipiodol as was observed for the main tumor in this case. the relationship between the degree of lipiodol accumulation and tumor necrosis should be verified. in , tai with miriplatin is a safe and effective treatment option for advanced hccs refractory to tai with epirubicin.

miriplatin, a cisplatin derivative with a high affinity for iodized ethyl esters of fatty acids from poppy seed oil, is a novel chemotherapeutic agent designed for use in the transarterial treatment of hepatocellular carcinoma (hcc). here, we describe transcatheter arterial infusion (tai) using miriplatin to treat a case of advanced hcc with portal vein tumor thrombus (pvtt) refractory to tai with epirubicin. a 66-year - old man with advanced hepatitis c virus - related hcc with pvtt in the right lobe of the liver was treated with tai with epirubicin suspended in iodized oil; however, tumor marker levels (alpha - fetoprotein and des - gamma - carboxy protein) did not decrease. next, he was treated twice with tai with miriplatin suspended in iodized oil. the tumor marker levels markedly decreased to a nearly normal range and the size of the main tumor was markedly reduced according to dynamic computed tomography. no serious adverse events occurred during the course of treatment with tai and miriplatin. therefore, we suggest that tai with miriplatin is a safe and effective treatment option for advanced hccs refractory to tai with epirubicin.

polyps in the second part of the duodenum are uncommon, however, up to 22% are adenomas. a proportion of these have high - grade dysplasia and need some form of intervention to prevent the progression to carcinoma. there are a range of options to manage the adenomas, from less invasive endoscopic resections to surgical resection and pancreaticoduodenectomy. this paper describes the management and outcome of two patients who presented with high - grade villous adenomas of the ampulla. the aim is to highlight the potential pitfalls of a minimally invasive approach to resecting adenomas with high - grade dysplasia involving the ampulla of vater. a 66-year - old woman with familial adenomatous polyposis (fap) had a colectomy and ileorectal anastomosis in 1961, followed by a completion proctectomy in 2004. as part of the follow - up, a gastroscopy was performed and demonstrated multiple adenomas carpeting the duodenum with high - grade dysplasia on biopsy. a computed tomography scan confirmed that the disease was localised but was associated with mild bile duct dilatation. because of the number and the extent of the lesions, it was elected to perform a pancreaticoduodenectomy. the patient made an uneventful post - operative recovery and was discharged 14 days after surgery. on histology multiple discrete and merging polypoid lesions were seen to stud the duodenal mucosa, ranging in size from 3 mm to 22 15 5 mm, including a 10 mm polyp in the periampullary region. histologically these demonstrated multiple tubular and villous adenomas with focally high - grade glandular dysplasia (fig . 1). both high- and low - grade dysplasia extended down the common bile duct at the ampulla of vater for a distance of 12 mm and down the separate pancreatic duct for a distance of 8 mm (fig . low - grade dysplasia involved the jejunal margin but not the pancreatic or bile duct margins . gastroscopy revealed a villous adenoma of the second part of the duodenum and histology confirmed a villous adenoma with low - grade dysplasia . an ercp in october 2004 showed progression of the adenoma, then being 10 cm in length, almost circumferential ( fig . the patient had significant co - morbidities, including ischaemic heart disease and a coronary artery bypass graft, chronic renal failure and a renal transplant, hypercholesterolaemia, hypertension, gastro - oesophageal reflux disease and recurrent urinary tract infections. despite this the post - operative course was prolonged and complicated by a pancreatic anastomotic leak, ing in a 4-week hospital stay. on histology, a periampullary villous adenoma was present (macroscopically 90 60 17 mm) featuring high- and low - grade glandular dysplasia. both high- and low - grade dysplasia extend down the common bile duct at the ampulla for a distance of 7 mm, and there was colonisation of periampullary glands by dysplastic epithelium without invasion ( fig. the diagnosis of duodenal polyps is likely to increase in frequency as their association with hereditary neoplastic syndromes, such as hereditary non - polyposis colorectal cancer and fap, becomes more recognised. research into the natural history of duodenal polyps is inconclusive, with some authors indicating rapid progression of polyps to carcinoma, and a high incidence of carcinoma at diagnosis, whilst others describe a benign course. pancreaticoduodenectomy is the procedure of choice for malignant lesions of the duodenum, ampulla of vater or head of the pancreas. because of the considerable morbidity and mortality , there has been increased interest in less extensive operations for the management of benign disease. alternate strategies include endoscopic resection, piecemeal, snaring , or fulguration, argon plasma coagulation, trans - duodenal resection, and pancreas - preserving duodenectomy. these procedures limit the complications of surgery or post - operative morbidity, whilst attempting to maintain adequate disease control. in all of these situations, except pancreas - preserving duodenectomy, where a biopsy of the bile duct is obtained at the time of surgery the role of endoscopic ultrasound is not well established but may add information regarding invasion and/or ductal involvement. the two cases described here demonstrate one of the pitfalls of treating patients with dysplastic villous adenomas of the second part of the duodenum, involving the ampulla of vater, with a limited resection as the dysplasia extended up the bile duct in both these cases. this would potentially have been missed in a trans - duodenal, endoscopic and pancreas - preserving resection of the duodenum. the first patient presented with multiple duodenal polyps secondary to fap. the migration of dysplastic adenomatous tissue into the ampulla of vater may explain some of the high recurrence rate of these adenomas after local excision. the second patient had just one large villous adenoma, which was not related to a known hereditary polyposis syndrome. in this situation, dysplastic adenomatous tissue had again migrated up the ampulla of vater. in we present two cases with villous adenomas of the duodenum, both of which displayed migration of the dysplastic adenoma up the bile duct. this would indicate that duodenal adenomas involving the ampulla of vater, with features of dysplasia, should be treated with definitive surgery.

duodenal adenomas are uncommon, however, when present a proportion have dysplasia associated with the adenoma and therefore require treatment. the options range from less invasive endoscopic treatments to a pancreaticoduodenectomy. this case report describes two patients with adenomas involving the ampulla of vater. one patient had familial adenomatous polyposis, the other was a renal transplant patient with a large adenoma. both patients adenomas contained high - grade dysplasia. both patients underwent a pancreaticoduodenectomy. histology of both specimens demonstrated that the adenoma had migrated up the bile duct for at least 7 mm, and the pancreatic duct for 8 mm in one patient. limited resection of ampullary adenomas may leave residual adenomatous tissue in the bile duct with the risk of recurrent adenomatous disease and malignant transformation.

supplementary material is available for this article at 10.1007/s13659 - 013 - 0032 - 9 and is accessible for authorized users.

three new homo - adamantanyl type natural products were derived from polyprenylated polycyclic acylphloroglucinol. hypercohones a - c, along with five other known hypercohones, were isolated from the aerial parts of hypericum cohaerens. the structures of 13 were elucidated on the basis of comprehensive spectroscopic analysis. the inhibitory activities of these isolates against five human cancer cell lines in vitro were tested. electronic supplementary materialsupplementary material is available for this article at 10.1007/s13659 - 013 - 0032 - 9 and is accessible for authorized users.

conception is unusual in women treated with conventional dialysis, with estimates ranging from 0.3% to 1.5% per year. the diagnosis of pregnancy has often been made late in gestation because it is unexpected and because amenorrhea and nausea are common in dialysis patients. a review from 1987 found the average gestational age at diagnosis of pregnancy to be 16 weeks. it has long been recognized that beta hcg levels are elevated in both pregnant and non - pregnant dialysis patients. slightly elevated levels occasionally cause problems such as postponement of surgery while waiting to demonstrate that levels are not rising. high levels of beta hcg in pregnant dialysis patients rarely caused a problem in pregnancies diagnosed late because ultrasound was used to determine gestation age, but at least one pregnancy was terminated because of a suspected hydatidiform mole. the elevated levels become more important when the index of suspicion is higher and pregnancy is diagnosed earlier. we present a case in which misinterpretation of beta hcg levels led to consideration of terminating a viable pregnancy. in august 2005, a 25-year - old woman on hemodialysis was referred to our renal clinic with a positive pregnancy test based on serum beta hcg. she had end - stage renal disease secondary to type 1 diabetes and had started dialysis 9 months prior to her visit. she had had two previous pregnancies, the first ing in spontaneous abortion in the first trimester and the second ing in the delivery of a baby at 30 weeks gestation. her past medical history was significant for diabetes since age 9 with retinopathy and nephropathy. she was not aware of any kidney disease until she reached the point of needing dialysis in september 2004 and had been on thrice weekly dialysis since then. on exam, her bp was 112/67 mm hg and she did not have any edema. her beta hcg levels were 13 270 miu / ml at the time of her pregnancy test and increased to 30 232 miu / ml within 3 days. the plan was to increase her dialysis regimen to more than 20 h per week and she was referred to the care of high - risk obstetrics. she had a transvaginal ultrasound scan, which showed an intra - uterine gestational sac of 5.3 weeks, but no fetal heart tone could be detected. given that she had hcg levels consistent with 7 or more weeks gestation without visible cardiac activity, she was thought to have a non - viable pregnancy and surgical termination was contemplated once her blood sugars were well controlled. beta hcg levels were followed with the expectation that they would drop in the setting of a non - viable pregnancy. her beta hcg levels are charted below: weeks gestationpatient s beta hcgnormal beta hcg5 weeks13 2707526005 weeks 3 days30 32385020 8005 weeks 5 days44 68585020 8007 weeks133 7084000100 000 a transvaginal ultrasound was repeated in 10 days and showed an intra - uterine pregnancy consistent with a gestational age of 7 weeks and with good fetal heart tones at a rate of 128 bpm. although, in the second trimester, she had an abnormal triple antigen screening test for fetal abnormalities, she delivered a healthy baby weighing 3.5 kg at 32 weeks without any further complications. pregnancy in dialysis patients is a major challenge to physicians involved in care, given the rarity of occurrence, complications involved and the distressing observation that < 50% of pregnancies in patients receiving conventional dialysis in surviving infants. outcomes in patients receiving nocturnal hemodialysis have been more encouraging, making accurate interpretation of beta hcg levels more important. serum beta hcg levels should be interpreted with caution in dialysis patients as levels tend to be slightly elevated even in non - pregnant patients and can be erroneously interpreted as intact pregnancy in a non - pregnant patient or a non - viable pregnancy in a pregnant patient as in our case. hcg is secreted in small amounts by all cells, and since the hormone is largely excreted by the kidneys, its level can be elevated in dialysis patients even if not pregnant. in a study conducted by schwarz et al. , beta hcg levels were increased by 10-fold in two of the 19 non - pregnant women who were on dialysis with post - dialysis levels being significantly higher than pre - dialysis levels. this observation has an important implication in clinical practice when monitoring serial beta hcg levels (pre- or post - dialysis blood sample should be specified when are reported). further studies need to be conducted in pregnant dialysis patients as data are limited on pre- and post - dialysis values of hcg. since serum beta hcg levels are not reliable in dialysis patients, pregnancy is normally confirmed by an ultrasound. fetal cardiac activity can be first detected at 5.56 weeks and can be missed if imaging is done earlier. if a non - viable pregnancy is suspected in a dialysis patient early in the first trimester, the diagnosis should be confirmed by measuring serial beta hcg as it decreases in a non - viable pregnancy, albeit more slowly than in a woman with normal renal function. the triple antigen test which is used for screening fetal abnormalities might not be reliable in dialysis patients. the patient reported in our case would have had a therapeutic abortion in her first trimester if not for the poorly controlled diabetes. pregnancy in a dialysis patient might represent the last opportunity for child bearing and every effort should be made to ensure a successful outcome.

conception is rare in patients on chronic dialysis and diagnosis is often delayed as it is least expected. serum beta hcg levels are elevated in both pregnant and non - pregnant dialysis patients, and pregnant dialysis patients have slightly higher beta hcg levels when compared with normal pregnancy. this can be erroneously interpreted as non - viable pregnancy and so should be viewed with caution. serial beta hcg levels must be followed when non - viable pregnancy is suspected in a dialysis patient before contemplating termination of pregnancy as described in our case.

periodontitis is primarily a bacterial - induced disease that can be modified by tooth related local factors. enamel, which is normally restricted to the anatomical crown of human permanent teeth, may be found ectopically on the root either as enamel pearl or enamel projections. among the anatomical factors cervical enamel projections (ceps) are a common tooth anomaly that can act as a contributing factor in the development and progression of periodontitis. ceps are flat, ectopic deposits of enamel apical to the normal cemento - enamel junction (cej) level in molar furcation areas. these enamel deposits usually have a triangular shape and a tapering form, extending apically into furcation areas. they are most commonly found at the buccal surfaces of mandibular molars. a possible relationship between cep and periodontal breakdown he suggested that the anatomy and location of cep might act as probable causes for rapid pocket formation. , the gingival tissue adjoining the cep is attached to the tooth by epithelial attachment, which is less resistant to the insult of bacterial plaque. this, together with a reduced access for oral hygiene measures and the proximity to the furcation dome, might enhance periodontal breakdown in the furcation. the clinician must be aware of the presence of such aberrations and their importance in the etiology of local periodontal breakdown. although reports vary, about 15 - 24% of mandibular molars and 9 - 25% of maxillary molars have ceps. considering both arches, they are most likely to be found on buccal surfaces of second molars. swan and hurt, in their study on indian skulls, noticed a direct association between the contour of the marginal bone and the position of cep. they also found widening of the periodontal ligament adjacent to the alveolar crest, which allowed the apical portion of the cep to merge toward the furcation. they concluded that it was the bone's excellent remodeling properties that enabled it to adjust to the varying tooth surface anatomy. masters and hoskins reported the incidence of ceps in extracted human teeth and suggested their possible implication in isolated furcation involvement (fi). they feel that even a slight gingival inflammation could produce a deep constricted periodontal pocket almost overnight. this could explain why destruction of the periodontium occurs at one given location, while bypassing adjacent areas however, an attempt by leib et al. to statistically associate these enamel extensions with the purpose of this study is to re - evaluate the incidence and distribution of ceps and to correlate their possible relationship with fis present in dry human indian skulls. a total of 944 upper and lower first, second and third permanent molars were examined from 89 dry human indian skulls for the presence of cep dipping into the root furcation area. ceps were investigated from the buccal aspect of the tooth and classified according to a system given by masters and hoskins. classification of cervical enamel projection grade i: the enamel projection extends from the cej of the tooth toward the furcation entrance. fi was measured horizontally from the buccal aspect into the furcation with a graduated probe to the nearest millimeter. male and female skulls were identified on the basis of the following anatomical landmarks: male skulls typically have more prominent supraorbital ridges, a more prominent glabella and more prominent temporal lines. female skulls generally have rounder orbits and narrower jaws.male skulls on average have larger, broader palates, squarer orbits, larger mastoid processes, larger sinuses and larger occipital condyles than those of females.male mandibles typically have squarer chins and thicker, rougher muscle attachments than female mandibles. male skulls on average have larger, broader palates, squarer orbits, larger mastoid processes, larger sinuses and larger occipital condyles than those of females. male mandibles typically have squarer chins and thicker, rougher muscle attachments than female mandibles. the data collected were subjected to statistical analysis using a chi - square test for determining the incidence of each variable and tabulated in tables 15. distribution of cervical enamel projections in relation to maxillary and mandibular molars distribution of cep with furcation involvement according to their grade number of cervical enamel projections and furcation involvements in study sample distribution of molars involving cep & furcation according to the sex distribution of cep with furcation according to the side of the arch correlation of cervical enamel projection and furcation involvement presence of furcation involvement without presence of cervical enamel projection presence of cervical enamel projection without involving the furcation the incidence of ceps was found to be 11.9% (112 out of 944 molars examined). cep and furcation were present on 98 (10.3%) of the 944 molars examined. a total of 308 (32.6%) of the 944 molars examined, were with fi. cep without furcation were present on 14 (1.4%) of the 944 molars examined. ceps were found almost twice as frequently in the mandibular molars as in the maxillary molar, which was found to be highly significant (p = 0.000). tooth with grade iii cep shows the highest number of fi as compared with grade ii and grade i cep, which was found to be highly significant (p = 0.000). number of molars with cep and fi were 98 out of 944 (10.3%) which was statistically significant (p = 0.000) cep with fi was significantly more in male skulls as compared with female skulls (p = 0.001). there was no significant difference between the right and left maxillary and mandibular teeth with cep and fi (p = 0.751). periodontitis is primarily a dental plaque induced inflammatory disease but the local factors that facilitate the accumulation of bacteria may contribute to the progression of the disease. factors such as tooth anatomy and restorative / endodontic considerations have been linked to gingival inflammation and attachment and tooth loss. of all anatomic factors, the cep is probably the most common and associated with attachment loss in the molar furcation area. this study investigated the incidence and distribution of cep and attempted to correlate their possible relationship to fis present in dry human indian skulls. cep were present on 112 (11.9%) of the 944 molars examined (224 maxilla, 720 mandible). in previous reports, the incidence of cep ranged from 15% to 30%. a possible explanation for the low incidence in the present investigation may be the difference in racial origin of the specimens. as shown in table 1, ceps were found almost twice as frequently in the mandibular molars as in the maxillary molars. the predominance in the mandibular molars is in general agreement with previous reports. the incidence of cep varied between the first, second, third molars. mandibular second molars showed the highest incidence (14.7%), followed by maxillary second molar (14.6%). this pattern of frequency is in accordance as that reported by swan and hurt, and bissada et al. we found that 87.5% of molars with enamel projections (98 of 112) had an associated fi. such a high percentage of association between the enamel projection and fi supports the view of a possible relationship, which may lead to the progression of periodontal disease. whether or not, there is a cause and effect relationship, requires further investigation. a total of 14 molars out of 112 molars (2.5%) with cep had no associated fi. this was to be expected since there are a number of factors such as the presence of occlusal facets (trauma from occlusion), thickness of alveolar housing, variations in the root - trunk length and severity and extension of gingival inflammation which can contribute to periodontal breakdown in these areas. master and hoskins, suggested a classification system based on the extent of cep into the furcation area. they used the adjacent cej and the furcation area as reference points to classify cep as grade i, ii and iii. hou and tsai and swan and hurt, reported cep grade iii to be the most commonly found cep. in the present specimens, grade iii was the most prevalent (83.7%) and was associated with fi which shows the maximum potential of grade iii cep for the fi. this can be explained by the mechanism that connective tissue can not form an attachment to enamel. instead the junctional epithelium is present in these areas and consists of hemidesmosomes and basal lamina. as a , when enamel forms on roots (more in grade iii), it may predispose the area to increased probing depth in the presence of gingival inflammation. together with its plaque one more new found in the present study was the significantly more prevalence of cep in male skulls (77.4%) than in female (20.4%). no significant difference was found between the right and left maxillary and mandibular teeth with cep and fi. exact reason behind this finding is unclear, but the possible role of genetics should be ruled out in future studies as bilateral occurrence of cep was reported by one case report. these are not comparable as none of the previous studies have evaluated this aspects of cep. since bissada et al. had experienced great difficulty in detecting selected teeth with cep radiographically, in the present study the authors have not included such an identification in this manner; in fact, through clinical examination is more reliable for their detection. a total of 944 maxillary and mandibular molar teeth present in indian human dry skulls were examined for the incidence and location of cep. under the limitations of the present study , we have drawn the following : the prevalence of ceps in molars was 11.9%ceps were found more frequently in the mandibular than in the maxillary molars (2:1)the highest prevalence of cep was found in the mandibular second molar (14.7%) followed by the maxillary second molar. the mandibular third molar showed the lowest incidence (5.5%)the association between cep and fi (87.5%) was statistically significant. this favors the view of the possible role played by such anomalies in the progression of periodontal diseasescep in male skulls (77.4%) was significantly more prevalent than in female skulls (20.4%)no significant difference was found between the right and left maxillary and mandibular teeth with cep and fi. the prevalence of ceps in molars was 11.9% ceps were found more frequently in the mandibular than in the maxillary molars (2:1) the highest prevalence of cep was found in the mandibular second molar (14.7%) followed by the maxillary second molar. the mandibular third molar showed the lowest incidence (5.5%) the association between cep and fi (87.5%) was statistically significant. this favors the view of the possible role played by such anomalies in the progression of periodontal diseases cep in male skulls (77.4%) was significantly more prevalent than in female skulls (20.4%) no significant difference was found between the right and left maxillary and mandibular teeth with cep and fi.

: the objectives of this study were to investigate the incidence of cervical enamel projection (cep) in molars of indian dry human skulls and to evaluate its relationship with furcation involvement (fi).materials and methods: the material consisted of 944 upper and lower first, second and third permanent molars from 89 indian dry human skulls. ceps were investigated from the buccal aspect of the tooth and classified according to a system describeddescribed by masters and hoskins. fi was measured horizontally from the buccal aspect into the furcation with a graduated probe to the nearest millimeter. any measurement 2 mm was considered to have positive fi.:the showed that ceps was found more frequently in the mandibular than in the maxillary molars (2:1). the highest incidence of cep was found in the mandibular second molar (14.7%) followed by the maxillary second molar (14.6%). the mandibular third molar showed the lowest incidence (5.5%). the association between cep and fi (87.5%) was statistically significant. this favors the view of the possible role played by such anomalies in the progression of periodontal diseases. cep in male skulls (77.4%) was significantly more prevalent than in female skulls (20.4%). no significant difference was found between the right and left side of maxillary and mandibular teeth with cep and fi.:the findings suggested the role of ceps as a local contributing factor in localized chronic periodontitis and fi in molars. detailed examination as well as early diagnosis of periodontal disease at the region of furcation is clinically very important.

we had 7 040 dairy herds with 121 945 cows in latvia at the 2010. production level of milk products has been increased from 93.9 million lvl at 2002 to 218.2 million lvl at 2008. enhanced nutrition qualities, taste, and health benefits have all been advocated as reasons for increase interest in raw milk consumption. unfortunately, milk is a good source of nutrients and edible energy not only for humans but also for numerous microorganisms, which thus can grow in milk. these microorganisms are primarily bacteria, but some moulds and yeasts can also grow in milk. the presence of several species of microorganisms in raw milk is undesirable, either because the organisms can be pathogenic, or because their growth in undesirable transformations in the milk. it is known that bacterial toxins may act as very danger food poisoning substances. the often listed pathogens in raw milk are staphylococcus aureus, escherichia coli, salmonella spp. , yersinia enterocolitica, aeromonas hydrophila, brucella abortus, campylobacter jejuni, bacillus cereus, and listeria monocytogenes (l. monocytogenes), among them. monocytogenes, l. ivanovii, l. innocua, l. seeligeri, l. welshimeri, and l. grayi. the first contains l. monocytogenes, l. innocua, and l. welshimeri, the second group l. ivanovii and l. seeligeri, and the third l. grayi. only two, l. monocytogenes and l. ivanovii, are pathogenic for humans and animals. the cellular behaviour of l. ivanovii is quite similar to that of l. monocytogenes, and the virulence gene cluster of l. monocytogenes is present in the other pathogenic species l. ivanovii. reveal that the ability to grow in the host cytoplasm cause vasodilator - stimulated phosphoprotein, which is characteristic both for l. monocytogenes and l. ivanovii. l. monocytogenes is a high adaptable environmental bacterium capable of existing both as animal pathogen and plant saprophyte with powerful array of regulated virulence factors. at the same time it is potentially lethal foodborne pathogen commonly found in dairy cows' environment in cow feces, silage, soil, water, and so forth. it is proved that l. monocytogenes grows into biofilms attached to the surfaces in food - processing plants and milking systems in dairy farms. the common treatment of surfaces is not effective to eliminate this dangerous foodborne pathogen, and it easily can pass into raw milk. l. monocytogenes can cause a rare but serious disease called listeriosis, especially among pregnant women, the elderly, or individuals with a weakened immune system. l. monocytogenes is more likely to cause death than other bacteria that cause food poisoning. 20 to 30% of foodborne listeriosis infections in high - risk individuals may be fatal. the presence of pathogens depends on ingestion of contaminated feed followed by amplification in bovine hosts and fecal dissemination in the farm environment. the final outcome of this cycle is a constantly maintained reservoir of foodborne pathogens that can reach humans by direct contact, ingestion of raw contaminated milk or cheese, or contamination during the processing of milk products. therefore it is essential to gather information about microbial risk factors and hazards associated with raw milk production. risk assessment and microbial monitoring will continue to play important role in ensuring food safety. critical control point management programmes created for individual milk production farms based upon risk analysis, total quality management and hazard analysis, and critical control point principles are essential for obtaining safe and healthy raw milk for consumers and for processing. the objective of this study was to clarify incidence of bacteria from the genus listeria int. foodborne pathogen l. monocytogenes in the feed and raw milk from one organic and three conventional dairy farms in latvia. the research was carried out from june 2008 to may 2010. in total, 130 feed samples and 244 bulk tank milk samples from organic farm grantskalni and three conventional farms palsa, lacplesa piens, and robeznieki were analyzed. l. monocytogenes from feed and milk samples were isolated in accordance with international standard lvs en iso 11290 - 1+a1 microbiology of food and animal feeding stuffs horizontal method for detection and enumeration of listeria monocytogenes presumptive l. monocytogenes isolates were purified and confirmed by the fourier transform infrared spectroscopy (ft - ir) technique. identification of listeria species using ft - irsample preparation and measurement of ft - ir spectra were performed according to manufacturer's instructions using an infrared spectrometer tensor 27 and software opus version 6.5 (bruker optic gmbh, germany). the bacterial strains were subcultured on tryptone soya agar (tsa, oxoid) by incubating the plates at 37c for 24 h. bacteria inoculum was transferred from the tsa preculture onto the surface of the tsa plate with a loop and spread with spatula until homogeneity bacterial lawn (one half of the agar plate is enough for each strain). prepared plates were incubated for 24 h at 30c. after incubation suspensions with 2 full loops of the bacteria scraped from the confluent lawn in 100 l distilled water in the eppendorf tubes were prepared. afterwards we homogenized this suspension with a vortex for 10 sec., transferred 25 l of the suspension on the microtiter plate, and dried about 45 min. at 42c. for the ft - ir absorption measurements we used 32 scans, 6 cm resolution, 24 phase resolution, and repeated position measurements between 4,000 and 500 identification of bacteria was based on bruker's spectral library of listeria species which includes reference strains of l. monocytogenes, l. innocua, l. ivanovii, l. welshimeri, and l. seeligeri. sample preparation and measurement of ft - ir spectra were performed according to manufacturer's instructions using an infrared spectrometer tensor 27 and software opus version 6.5 (bruker optic gmbh, germany). the bacterial strains were subcultured on tryptone soya agar (tsa, oxoid) by incubating the plates at 37c for 24 h. bacteria inoculum was transferred from the tsa preculture onto the surface of the tsa plate with a loop and spread with spatula until homogeneity bacterial lawn (one half of the agar plate is enough for each strain). prepared plates were incubated for 24 h at 30c. after incubation suspensions with 2 full loops of the bacteria scraped from the confluent lawn in 100 l distilled water in the eppendorf tubes were prepared. , transferred 25 l of the suspension on the microtiter plate, and dried about 45 min. at 42c. for the ft - ir absorption measurements we used 32 scans, 6 cm resolution, 24 phase resolution, and repeated position measurements between 4,000 and 500 cm. identification of bacteria was based on bruker's spectral library of listeria species which includes reference strains of l. monocytogenes, l. innocua, l. ivanovii, l. welshimeri, and l. seeligeri. l. monocytogenes prevalence were analyzed with statistical package for social sciences 17.0 for windows (spss inc ., the bacteria of listeria species were detected in feed samples ( n = 130) in 38 cases or 29.2%. in 12 (9.2%) these were l. innocua, l. ivanovii, and l. seeligeri, but in 26 cases (20.0%) l. monocytogenes. bacteria of listeria genus can be widely found in nature in soil, on plants, in waters, on animal hair and birds' bodies, and so forth.. l. ivanovii shares certain characteristics with l. monocytogenes (e.g., hemolysis) and is occasionally associated with abortion in ruminants. therefore incidence of these two species in animal feed is a risk factor for presence of listeria in the farm environment, cow infections, their presence in milk and thus also in human body causing infections. animal and human pathogen l. monocytogenes was isolated from 26 feed samples (20.0%). milk plays important role in l. monocytogenes epidemiology; therefore it must be kept in mind that these dangerous bacteria are brought in the farm environment by contaminated feed and thus also on cow hair, udder, and teat skin and then also in milk. l. monocytogenes is known as cow mastitis, conjunctivitis, and other disease - causing pathogen microorganisms. it has been proved that listeria strains isolated from infections have been found also in farm environment in feces and silage which means that l. monocytogenes strains found in nature are virulent. as it can be seen, the data summarized in table 1 show that none of 14 different grass samples contains bacteria of listeria species. thus, grass has not been in contact with dung, wild animal feces, which can cause the presence of listeria in the soil and on plants. silage is considered as the main source of l. monocytogenes and other listeria genus bacteria in farm environment. however, neither silage (n = 48) nor haylage (n = 16) were highly contaminated in our study; it ranges from 1.5 to 4.7% (table 1). dry food products were contaminated with l. monocytogenes: feed mixture prepared in the farm1.5%, different fodder products (different corns, rape cakes, etc.)9.3%, straw1.5%, and hay1.5% of all tested samples (the tested straw and hay were stored at the field during winter therefore exposed to long - term impact of environment . hay used as fodder might have been contaminated on the field by bird and animal feces . listerias are gram - positive bacteria with different structure and chemical content of its cell wall and thus more resistant than gram - negative ones . listeria cell wall protects the inner content of the cell against impact of external mechanical and osmotic force, insufficient humidity and ph level, and other damaging growing and reproduction factors . the cell wall of gram - positive bacteria consists of peptidoglycan which is associated with teichoic acids and lipoteichoic acids in complex multilayer structure, while the cell wall of gram - negative bacteria consists of one peptidoglycan layer which is covered with a membrane . thus, the cell wall of gram - positive bacteria, including listeria is tenfold thicker than the cell wall of gram - negative bacteria, and they are much viable in external environment, as well as considerably resistant to disinfectants use for cow teat treatment before milking . it is able to grow at temperatures from + 1c, they can be considered as psychrophilic microorganisms capable of surviving, growing, and multiplying in feed also in autumn and winter, when straw, hay roll, and hay are on the field or under open sheds . table 2 shows summarized obtained data on incidence of listeria spp . and l. monocytogenes in organic and conventional farms and feed used in different seasons . the data summarized in table 2 show that feed was free of listeria during summer in both organic and conventional farms . is most likely isolated from feedstuff in winter and autumn than it is in summer . l. monocytogenes are found more often in feed prepared in organic farm ( correspondingly 14.8% and 29.6%) than in feed used in conventional farms (correspondingly 5.2% and 13.1%). are logical as, according to the legislative acts of the european union, organic farms should use as less chemical substances as possible; for example, ferments, yeast, and bacteria should be used as silage additives instead of chemical preservatives thus limiting multiplication of pathogenic bacteria. although different feed samples contained l. monocytogenes, no such bacteria were found in samples of bulk milk from organic farm (n = 33), but in samples of bulk milk from conventional farm l. monocytogenes was found three times or in 1.4% of all cases (fernandez et al . has isolated l. monocytogenes from 3.0% ( n = 140), jayarao and henning from 3.8%, but vilar et al. from 6.1% of bulk milk samples. the obtained show that accurate observing of hygiene standards concerning treatment of cows' udder and teats, as well as proper washing and cleaning process of milking system pipe lines and cooling tanks, protects the milk against bacteria. different type of feed is a risk factor for poisoning the farm environment thus also poisoning fresh milk with pathogenic microorganisms of listeria genus species in both organic and conventional farms. listeria ivanovii, listeria innocua, and listeria seeligeri were isolated from 9.2%, but listeria monocytogenes from 20.0% of feed samples. most often different feed concentrates (9.3%) and silage (4.7%) were contaminated with listeria monocytogenes. listeria genus species were isolated more often from feed prepared and used in organic dairy farm than from the feed used in conventional dairy farms, correspondingly 44.4% and 18.3%. no listeria monocytogenes were found in bulk milk samples from organic dairy farm (n = 33), but they were found three times in samples of conventional dairy farms (n = 211).

feed is a risk factor for poisoning the farm environment thus also fresh milk with pathogenic microorganisms of listeria genus species. listeria ivanovii, listeria innocua, and listeria seeligeri were isolated from 9.2%, but listeria monocytogenes from 20.0% of feed samples. most often different fodders (9.3%) and silage (4.7%) were contaminated with listeria monocytogenes. listeria genus species were isolated more often from feed prepared and used in organic dairy farm than from that used in conventional dairy farm, correspondingly 44.4% and 18.3%. no listeria monocytogenes was found in bulk milk samples of organic dairy farm.

mucous membrane pemphigoid (mmp) is a putative autoimmune, chronic inflammatory, subepithelial blistering disease predominantly affecting mucous membranes. mucous membranes that may be involved include the oral cavity, conjunctiva, nasopharynx, larynx, esophagus, genitourinary tract and anal canal. a 37-year - old male patient presented with complaints of redness and pain in his left eye since two years, painful erosions in the oral cavity since six months, and ulcers over the scrotum and right knee since two months. on ocular examination, there was congestion of the left sclera mainly on the lateral side along with a fibrotic band connecting the upper palpebral conjunctiva with the bulbar conjunctiva near the lateral canthus. on examination of the oral cavity there were widespread erosions involving the inner side of lips, gingival, and labial mucosa. detailed examination revealed a single depigmented macule over the glans penis , which according to the patient appeared insidiously around 8 years ago and remained static. a tzanck smear from the oral erosions, done to rule out pemphigus vulgaris, failed to show any acantholytic cells. in addition there was involvement of nasal mucosa, larynx, scrotal skin, perianal skin, and right knee. a skin biopsy from the right ear showed subepidermal bullae and fibrosis beneath it. the direct immunofluroscence of perilesional uninvolved skin showed strong linear deposits of igg along the basement membrane zone. detailed hematological, biochemical, and imaging studies did not reveal any evidence of malignancy. the long - standing depigmented macule over the glans penis did not show any evidence of atrophy, ruling out lichen sclerosus et atrophicus. mmp lesions affecting the left eye mmp lesions affecting the oral mucosa, scrotum and knee (clockwise) classical vitiligo affecting the glans penis subepidermal bulla with fibrosis beneath it, black arrow demonstrates the site of blistering (h and e, 10) subepidermal bulla (h and e, 40) mmp is described as a heterogeneous group of chronic, inflammatory, mucous membrane - dominated, subepithelial blistering diseases that manifest a varying constellation of oral, ocular, skin, genital, nasopharyngeal, esophageal, and laryngeal lesions. life - threatening airway obstruction and sight - threatening ocular scarring can occur in this condition, also known as cicatricial pemphigoid, benign mmp and incorrectly as ocular pemphigoid. these include bullous pemphigoid antigen 1 (bpag1, 230 kda), bullous pemphigoid antigen 2 (bpag2, 180kda), laminin 5, laminin 6, 6-integrin subunit, 4-integrin subunit, collagen vii, and other proteins of unknown identity and/or function. scarring is common at non - oral sites of involvement, contributing to disease - related morbidity. the choice of agents for treatment of mmp is based upon the sites of involvement, clinical severity, and disease progression. found there is an association with autoimmune diseases, both organ and non - organ - specific in a group of 34 patients with cicatricial pemphigoid suggesting a possible genetic basis for the association. autoimmune mechanisms with an underlying genetic predisposition are the most likely causes of vitiligo, although neurohumoral and autocytotoxic hypotheses are alternative theories or contributing mechanisms. previously association of vitiligo with bullous pemphigoid either alone or along with other autoimmune diseases have been reported. gaspar et al. reported a case of a 68-year - old woman with vitiligo, primary hypothyroidism and cicatricial pemphigoid with severe laryngeal involvement necessitating tracheostomy. a thorough search failed to reveal any other report of co - existence of these two conditions in the published literature. we believe that this is the first case report of vitiligo coexistent with mucous membrane pemphigoid from india. the co - existence of these two conditions is probably is related to their common autoimmune etiology.

mucous membrane pemphigoid describes a rare heterogeneous group of chronic, inflammatory, mucous membrane - dominated, subepithelial blistering diseases that manifest a varying constellation of oral, ocular, skin, genital, nasopharyngeal, esophageal, and laryngeal lesions. life - threatening airway obstruction and sight - threatening ocular scarring can occur in this condition, which is rarely reported in indian literature. vitiligo is another acquired autoimmune disorder characterized by loss of melanocytes. vitiligo is associated with a number of disorders also considered to be autoimmune. here we report a very rare coexistence of mmp and vitiligo, the first such report from india.