Although adjuvant gemcitabine (GEM) chemotherapy for pancreatic cancer is standard, the quality of life (QOL) in those patients is still impaired by the standard regimen of GEM. Therefore, we studied whether mild dose-intensity adjuvant chemotherapy with bi-weekly GEM administration could provide a survival benefit with acceptable QOL to the patients with pancreatic cancer.2 of GEM chemotherapy was performed in 58 patients with pancreatic cancer for at least 12 courses (Group A). In contrast, 36 patients who declined the adjuvant bi-weekly GEM chemotherapy underwent traditional adjuvant 5FU-based chemotherapy (Group B). Careful periodical follow-ups for side effects of GEM and disease recurrence, and assessment of patients’ QOL using the EORTC QOL questionnaire (QLQ-C30) and pancreatic cancer-specific supplemental module (QLQ-PAN26) were performed. Retrospectively, the degree of side effects, patients’ QOL, compliance rate, disease-free survival (DFS), and overall survival (OS) in Group A were compared with those in Group B.After a phase I trial, an adjuvant bi-weekly 1,000 mg/mP < 0.05). Compliance rates in Groups A and B were 93% and 47%. There was a significant difference in the median DFS between both groups . The median OS of Group A was prolonged markedly compared with Group B . For OS between both groups, univariate analysis revealed no statistical difference in 69-year-old or under females, and T1–2 factors, moreover, multivariate analysis indicated three factors, such as bi-weekly adjuvant GEM chemotherapy, T2 or less, and R0.No severe side effects (higher than Grade 2 according to the common toxicity criteria of ECOG) were observed, except for patients in Group B, who were switched to the standard GEM chemotherapy. Patients’ QOL was better in Group A than B (fatigue: 48.9 ± 32.1 versus 68.1 ± 36.3, nausea and vomiting: 26.8 ± 20.4 versus 53.7 ± 32.6, diarrhea: 21.0 ± 22.6 versus 53.9 ± 38.5, difficulty gaining weight: 49.5 ± 34.4 versus 67.7 ± 40.5, Adjuvant chemotherapy with bi-weekly GEM offered not only the advantage of survival benefits but the excellent compliance with acceptable QOL for postoperative pancreatic cancer patients. Pancreatic cancer is increasing rapidly worldwide and the prognosis is still quite poor even if the patient undergoes curative resection . CompareThereafter, in our institution, adjuvant GEM chemotherapy with the standard regimen for patients with resectable pancreatic cancer had been used from 2001 to 2003. However, the compliance rate was very low, 38%, due to severe side effects, such as gastrointestinal discomforts and emaciation, even though the adjuvant chemotherapy was adjusted according to the reduction and extension criteria. The majority of pancreatic cancer patients, over 40 cases, failed to continue the adjuvant GEM chemotherapy with the standard regimen, and died without obtaining the beneficial effects of GEM. From the bitter experience of the low compliance rate in our institution, we hypothesized that the dose intensity of adjuvant GEM chemotherapy with the standard regimen was too heavy and harmful for people of Asian descent, and a milder dose-intensity of adjuvant GEM chemotherapy with bi-weekly administration might be more suitable and lead to prolongation of DFS and/or OS without impairing patients’ QOL.Thereby, after a phase I study, we studied whether mild dose-intensity adjuvant chemotherapy with bi-weekly GEM administration could provide prolonged disease-free survival (DFS) and/or overall survival (OS) without impairing patients’ quality of life (QOL).2), a medium dose , and a low dose (800 mg/m2). Each dose group consisted of at least six patients; the compliance rate in the high dose group was low, 47%, while the rates in the middle and low dose groups were 96% and 97%. Moreover, there was no significant difference in the side effects between the middle and low dose groups according to the common toxicity criteria of the ECOG. Consequently, the appropriate dose-intensity of adjuvant bi-weekly GEM chemotherapy was set at 1,000 mg/m2 in our institute. During a five-year period between 2004 and 2009, 128 patients with ductal pancreatic carcinomas were treated with surgery in our institution. A clinical study of mild dose-intensity, adjuvant bi-weekly GEM chemotherapy was performed with a total of 58 pancreatic cancer patients (Group A), who gave informed consent (IC). On the other hand, 36 patients (Group B), who did not give IC for adjuvant bi-weekly GEM chemotherapy, chose 5FU-based chemotherapy as the traditional adjuvant treatment. The remaining 34 patients were excluded from this study due to: macroscopic non-curative surgical treatment (22 cases), previous treatment of the current disease with more than one chemotherapeutic regimen and/or radiotherapy (3 cases), major complications after surgery such as aspiration pneumonia and/or leakage of pancreatojejunostomy (2 cases), hospital death after aggressive progression of the disease (1 case), not less than 85 years old (1 case), not providing IC for adjuvant chemotherapy (1 case), and physical conditions: active infection (1 case), interstitial pneumonia or pulmonary fibrosis (1 case), myocardial infarction within 3 months (1 case), and concomitant advanced cancer (1 case).After approval from the IRB  in our university, a phase I study of adjuvant bi-weekly GEM chemotherapy was conducted in our institute using three different doses: a high dose  if the patient’s condition was favorable. One cycle of this regimen was defined as twice bi-weekly administrations of GEM. After discharge from our hospital, administration of 1,000 mg/m2 of GEM was given consecutively to the outpatients at bi-weekly intervals, for at least six full cycles or until the patient’s condition was considered tolerable. If severe side effects occurred, the 1,000 mg/m2 dosage of GEM was reduced to 800 mg/m2. These severe side effects were: leukopenia <1,000 / mm-3, decreasing platelets <20,000 / mm-3, neutrophils <1,000 / mm-3 with fever (>38°C) or infection, and non-hematologic toxicity higher than Grade 3 according to the common toxicity criteria of the ECOG except for gastrointestinal toxicity, such as nausea, vomiting, diarrhea, and stomatitis. Additionally, when there were multiple side effects, the administration of GEM was extended until the patient’s recovery. If recovery needed more than two weeks, the study was discontinued. The multiple side effects were: leukopenia <2,000 / mm-3, decreasing platelets <70,000 / mm-3, and non-hematologic toxicity higher than Grade 2 according to the common toxicity criteria of the ECOG except for gastrointestinal toxicity, such as nausea, vomiting, diarrhea, and stomatitis.In Group A, the first administration of 1,000 mg/m2 bolus of leucovorin followed by an intravenous 400 mg/m2 bolus of fluorouracil given on each 5 consecutive days every 28 days for 6 cycles. The adjuvant 5FU-based chemotherapy was also started during the third week after surgery, the same as Group A. When recurrence was identified in Group B, the adjuvant chemotherapy was converted from 5FU-based to standard GEM administration  as for the patients who had agreed with the therapy alteration.In Group B, the postoperative patients were treated with adjuvant 5FU-based chemotherapy consisting of an intravenous 20 mg/mThe patients in both groups were followed up carefully, especially for side effects from the agents, disease recurrence, and the patients’ QOL, as measured according to the European Organisation for Research and Treatment of Cancer’s (EORTC) quality of life questionnaire  and the pancreatic cancer-specific supplemental module  -13. The P < 0.05.Thus, the 58 cases in Group A were compared to the 36 cases in Group B retrospectively. Statistical analysis was performed using student’s t-test, the chi-square test, the Mann–Whitney test, the log-rank test, the Kaplan–Meier method, and the Cox hazard proportional model. Results were considered to be statistically significant when 2 of GEM, although the other four patients needed an occasional extension of administration interval from bi- to tri-weekly, not because of hematologic toxicity but because of their suboptimal physical status.The patients’ ages in Group A ranged from 34 to 82 (median 68.0) years, the patients’ other characteristics such as sex, operative procedure, primary tumor size, nodal status, resection status, and the UICC’s TNM classification  are dispThe patients’ age in Group B varied from 50 to 84 (median 69.5) years and the patients’ other characteristics are also indicated in Table Only 11 patients in Group B were able to receive continuously the second line of standard GEM chemotherapy until they collapsed; accordingly, the compliance rate of the standard GEM chemotherapy in this study was low, 36.7% (11/30). Consequently, the total compliance rate in Group B was 47.2% (17/36). Compared with the low compliance rate in Group B, most of the patients in Group A continuously received the adjuvant bi-weekly GEM chemotherapy with a 93% (54/58) compliance rate until their acceptable physical status.P < 0.05), especially for fatigue, nausea and vomiting, diarrhea, and difficulty gaining weight in the symptom scales . When OS for patients at Stages 0–IIa and IIb–IV is compared between Groups A and B, a statistical difference was observed in each of the classified stages  versus 28.7 months in Group B (n = 2). The longest survival time in Group A was over 6.7 years (80.4 months) while for Group B it was over 3.0 years (36.1 months). However, for the other patients, 51 in Group A and 34 in Group B died of recurrences of pancreatic cancer due to peritonitis carcinomatosa or multiple distant metastases in the liver, bone, and/or lung.The median DFS of Group A was significantly improved in contrast with Group B  statistically prolonged DFS and OS for pancreatic cancer patients when compared with the adjuvant 5FU-based chemotherapy (Group B) as a whole and in Stage 0–IIa and Stage IIb–IV subgroups. Thus, the adjuvant bi-weekly GEM chemotherapy was more effective than the 5FU-based treatment in resected pancreatic cancer patients even though most patients in Group B converted to the standard GEM chemotherapy due to the recurrence of pancreatic cancer. Previously, it had been reported in a Norwegian randomized study [Results from this clinical study suggest that the adjuvant bi-weekly chemotherapy with 1,000 mg/med study -9,15,16 ed study ,34. The ed study ,28. Resued study  in whichCurrently, combination chemotherapy of GEM with new agents, such as capecitabine, erlotinib, or bevacizumab, has been used to try to improve the poor survival rate of patients with advanced pancreatic cancer -41. MoreIn conclusion, the medium dose-intensity adjuvant chemotherapy with bi-weekly GEM led to improved OS with acceptable QOL in pancreatic cancer patients after surgery.5FU: 5-fluorouracil; CI: Confidence interval; CONKO: Charité Onkologie; DFS: Disease-free survival; ECOG: Eastern Cooperative Oncology Group; EORTC: European Organisation for Research and Treatment of Cancer; ESPAC: European Study Group for Pancreatic Cancer; GEM: Gemcitabine; HR: Hazard ratio; IC: Informed consent; IRB: Institutional Review Board; mo.: Months; NS: No significant difference; OS: Overall survival; JASPAC: Japan Adjuvant Study Group of Pancreatic Cancer; POD: Postoperative days; QLQ-C30: Quality of life questionnaire – core 30; QLQ-PAN26: Quality of life questionnaire – pancreatic cancer module 26; QOL: Quality of life; RTOG: Radiation Therapy Oncology Group; SD: Standard deviation; UICC: Union Internationale Contre le Cancer.The authors declare that they have no competing interests.YT designed and directed the study, and drafted the manuscript. YT, SY, RS, TU, and TN performed the majority of operations. HK, NO, RM, RI, and KS collected and analyzed the data. KY, who was involved in editing the manuscript and critically revising it, gave final approval of the version to be published. All authors read and approved the final manuscript.The subspecialty of all authors is hepato-biliary-pancreatic surgery and surgical oncology. In particular, YT, SY, RS, and TU use adjuvant chemotherapy for patients with pancreatic cancer. YT and KY are highly skilled hepato-biliary-pancreatic surgeons, approved by the Japanese Society of Hepato-Biliary-Pancreatic Surgery. KY is Professor of Surgery and Chief of Gastrointestinal Surgery at the Jikei University School of Medicine, and President of the Japan Chapter of the American College of Surgeons.


Background and Aim: T cell expression of PD1 and inhibition of T effector cells by Foxp3+-T regulatory cells are among the most powerful mechanisms for achieving a balanced immune response. Our aim was to investigate, how liver FOXP3 and PD1/PDL1 expression is regulated in chronic HBV hepatitis (CHB) on maintained long-term remission in comparison with active disease, and whether they are correlated to the expression of pro- and anti-inflammatory cytokines and apoptosis mediators, along with the degree of histological inflammation and markers of T cell effector restoration.Methods: Fifty-three HBeAg-negative CHB patients with both active (30) and completely remitted disease on long-term antiviral treatment (23) and four controls (submitted to liver biopsy due to a mild increase of aminotransferases but without liver necroinflammatory and architecture changes) were enrolled in the study. Liver mRNA levels of immunoregulatory genes , major apoptosis mediators , cytokines of effector T cell restoration , and those of IL1B, CD4, and CD8, were evaluated by quantitative real-time reverse-transcriptase PCR and were correlated with each other, along with the intensity of liver inflammation and fibrosis staging. The expression and localization of FOXP3, PD1, PDL1, CD4, and CD8 were also assessed by immunohistochemistry.Results: The expression of FOXP3, IL10, TGFB1, PD1, PDL1, FASL, and CD8 was significantly down-regulated in the remission state. In contrast, liver expression of IL2 and IFNG, along with CD4, IL1B, TNFA, and FAS did not change significantly. Moreover, FOXP3, PD1, PDL1, and CD8 transcripts were positively correlated to the intensity of liver inflammation.Conclusion: Our data indicate that in the CHB disease model, the immunosuppressive liver environment is down-regulated in the maintained on-treatment long-term remission state and correlates with the intensity of liver inflammation, but not liver T cell restoration. Howepathways , 2.+-T regulatory cells (Tregs)  and interleukin (IL)-2 production by intrahepatic lymphocytes, inducing variable levels of functional T cell restoration both in the liver and in peripheral blood, with a better functional improvement among intrahepatic T cells  molecule by T cells  and the  (Tregs) . Models  (Tregs) , 5. In Ceraction , 5. Part T cells , 7. Thei T cells  whilst,  T cells .in vitro (FOXP3 gene) induces fatal autoimmune hepatitis (AIH) in neonatal thymectomized (NTx)-PD1−/− mice, due to migration of dysregulated follicular T helper (Tfh) cells from the spleen , suggesting that might represent a bystander effect and not a causative event of chronic inflammation  in peripheral blood – 12 or lin vitro – 14. Inte spleen . In thisammation . Consideammation  claimingammation .FOXP3, characterizing mainly nTregs (IL10 (encodes IL-10) and TGFB1 (encodes TGF-β1), characterizing type I (Tr1) and T helper type 3 (Th3) inducible Tregs (iTregs) (TNFRSF6/FAS (encodes FAS), TNFSF6/FASL (encodes FASL), TNFA (encodes TNF-α), and TNFSF10/TRAIL . Furthermore, the expression of the inflammatory cytokine IL-1β (encoded by IL1B gene) and cytokines of the immune effector T cell restoration , together with the expression of CD4 and CD8 were explored. Our data provide clear evidence that in CHB HBeAg-negative disease model, the immunosuppressive liver environment is down-regulated in the maintained on-treatment long-term remission state and correlates with the intensity of liver inflammation, but not with liver T cell restoration.The aim of this study was to explore whether the long-term antiviral treatment in patients with HBeAg-negative CHB affects the abovementioned model, investigating also another important apoptosis pathway implicated in Teffs dysfunction in liver, namely the PD1/PDL1. Thus, the expressions of y nTregs , as well(iTregs) , respectLiver biopsy specimens obtained from 53 patients with CHB were examined; 30 were newly diagnosed and were evaluated before any treatment and 23 were on maintained continuous antiviral treatment response and remission for at least 240 weeks (5 years) with entecavir. Nine out of 30 newly diagnosed CHB patients were derived from a previous study of our group , since tEach liver biopsy specimen was separated into two parts. One of them was immediately fixed in 10% formalin solution for diagnostic histological examination, and the other was snap frozen and stored at −80°C until further use. Formalin-embedded sections were stained by hematoxylin-eosin and Masson’s trichrome. Two independent pathologists assessed and scored each biopsy and any discrepancy was further evaluated by an expert pathologist. The samples were blinded to the timing of biopsy and treatment assignment. Core length and number of portal tracts were taken into account to determine adequacy of biopsy specimens. Biopsy slides were graded and staged with the Ishak scoring system , 20. FurInformed consent was obtained by all participants and the study was approved by the Institutional Review Board. One of the challenges experienced in this study was the obtaining of informed consent from patients undergoing liver biopsy without a clear clinical need (patients on maintained remission), considering that they had complete virologic suppression at year 5 on continuous antiviral treatment.Total RNA was isolated from stored liver samples after homogenization, using TRI , according to the manufacturer’s instructions. Complementary DNA (cDNA) was reversed transcribed from 1 μg of the total RNA, using a random 6-mer oligonucleotide primer (50 pmol/μL)  and M-MLV reverse transcriptase (Invitrogen), according to the manufacturer’s instructions.FOXP3, IL10, TGFB1, TNFRSF6/FAS, TNFSF6/FASL, TNFSF10/TRAIL, PD1/PDCD1 (encodes PD1), PDL1/PDCD1LG1 (encodes PDL1), PDL2/PDCD1LG2 (encodes PDL2), IL2, TNFA, IFNG, IL1B, CD4 (encodes CD4), and CD8a (encodes CD8) were determined in a Quantitative real-time reverse-transcriptase PCR (qRT-PCR) using SYBR-Green PCR Supermix , in the automated thermocycler RotorGene 6000 . The B2M gene was used as an internal control for sample normalization (reference gene). An 1/20 aliquot of the cDNA reaction product was used in duplicate qRT-PCR reactions and all measurements were averaged. Primers for the amplification of the genes FOXP3, IL10, TGFB1, TNFRSF6/FAS, TNFSF6/FASL, TNFSF10/TRAIL, IL2, TNFA, IL1B, PD1/PDCD1, and IFNG were commercially obtained by Qiagen . The primers for the amplification of PDL1/PDCD1LG1, PDL2/PDCD1LG2, CD4, and CD8a were designed with the aid of the Oligo 6.0 software  and are presented in Table CT method, as described . Differences were considered statistically significant when the p-value (two sided) was <0.05.For basic statistical calculations, all gene expression levels were treated as continuous variables. Differences of gene expression between different disease statuses were analyzed by the non-parametric Mann–Whitney FOXP3, IL10, TGFB1, TNFSF6/FASL, PD1/PDCD1, PDL1/PDCD1LG1, and CD8a, as well as significantly increased levels of TNFSF10/TRAIL, as compared to patients at diagnosis with active disease. The expression levels of IL2 and IFNG were also decreased, but these alterations did not reach statistical significance  and aspartate aminotransferase (ALT) levels], and the viral load are presented in detail in Figure +-lymphocytes were mostly located in portal tracts, while CD8+-lymphocytes were found in portal tracts, limiting plates, and lobules, in an extent commensurate with their nature as effectors of necroinflammatory activity . On the other hand, TNFSF10/TRAIL displayed an opposite pattern of expression, decreasing from minimal to severe inflammation .In relation to the intensity of inflammation, n Figure . This pan Figure . The expPDL1/PDCDLG1. A similar pattern was observed for FOXP3 and PD1/PDCD1, and an opposite one for TNFSF10/TRAIL, although not reaching statistical significance . The expression of the other analyzed genes was not affected by the severity of fibrosis .The severity of fibrosis was significantly associated only with the expression of p < 0.001, r = 0.665), while the viral load was also positively correlated with both HAI score and fibrosis stage .Finally, as expected, HAI score was positively correlated with the fibrosis stage , and PDL1 (mainly attributed to infected hepatocytes and infiltrating lymphocytes) are concomitantly down-regulated in the maintained long-term remission state. However, the down-regulation of CD8, with no up-regulation of IL-2 (encoded by IL2) and IFN-γ (encoded by IFNG), is not in favor of restoration of T cell immune-responsiveness, but rather indicates reduction of CTLs and hepatocyte cytolysis when liver inflammation subsides on long-term antiviral treatment. These findings are also supported by our immunohistochemical findings  is associated with T cell dysfunction in chronic HBV and HCV infections . In thisreaction . Kassel reaction . Accordire found . On the FAS and FASL in the maintained long-term remission state in CHB patients. Considering that previous studies, including ours, have demonstrated that the contribution of Fas/FasL pathway in CHB is of utmost importance, closely related to the degree of liver inflammation (TRAIL expression with the intensity of liver inflammation and the disease stage (active vs. remission), since patients on maintained remission displayed an up-regulation of its mRNA levels in liver. TRAIL is a newly characterized TNF family member, triggering apoptosis in various tumor and virus-infected cells, by binding to certain death receptors, namely DR4 and DR5 , and it is correlated with elevated expression of apoptosis mediators FAS, FASL, and TRAIL (However, we recently observed that accumulation of Foxp3nd TRAIL . As a rend TRAIL . Should nd TRAIL . Moreovend TRAIL . Thus, sFOXP3, PD1, and PDL1 that was also correlated with a minimal intensity of liver inflammation. However, these patients did not exhibit immune restoration phenomena, as they are evident in the ex vivo human HBV infection (As mentioned above, our CHB HBeAg-negative patients on maintained on-treatment long-term remission displayed a down-regulation of the hepatic expression of nfection , 37 and nfection  and chronfection . TherefoIn conclusion, our data indicate that in the CHB HBeAg-negative disease model, the immunosuppressive liver environment is down-regulated in the maintained on-treatment long-term remission state, as compared with cases histologically, biochemically, and virologically active at diagnosis, before any treatment. In addition, the contraction of the inhibitory pathways, as measured by the down-regulation of their liver mRNA expression in long-term remission, is possibly a mere consequence of the diminution of liver inflammation, after being hyper-expressed, in order to counterbalance excessive allo- and/or auto-reactive Teffs clones.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.


Filoviruses cause severe hemorrhagic fever in humans with high case-fatality rates. The cellular factors exploited by filoviruses for their spread constitute potential targets for intervention, but are incompletely defined. The viral glycoprotein (GP) mediates filovirus entry into host cells. Recent studies revealed important insights into the host cell molecules engaged by GP for cellular entry. The binding of GP to cellular lectins was found to concentrate virions onto susceptible cells and might contribute to the early and sustained infection of macrophages and dendritic cells, important viral targets. Tyrosine kinase receptors were shown to promote macropinocytic uptake of filoviruses into a subset of susceptible cells without binding to GP, while interactions between GP and human T cell Ig mucin 1 (TIM-1) might contribute to filovirus infection of mucosal epithelial cells. Moreover, GP engagement of the cholesterol transporter Niemann-Pick C1 was demonstrated to be essential for GP-mediated fusion of the viral envelope with a host cell membrane. Finally, mutagenic and structural analyses defined GP domains which interact with these host cell factors. Here, we will review the recent progress in elucidating the molecular interactions underlying filovirus entry and discuss their implications for our understanding of the viral cell tropism. The genus Marburgvirus includes a single species, Marburg marburgvirus, which has two members, Marburg virus (MARV) and Ravn virus (RAVV). The genus Ebolavirus includes five species, each of which has a single member: Zaire ebolavirus , Sudan ebolavirus , Taï Forest ebolavirus , Bundibugyo ebolavirus  and Reston ebolavirus  [Filovirus infection causes severe hemorrhagic fever in humans and non-human primates. Outbreaks of filovirus hemorrhagic fever occur in equatorial Africa and are associated with high case-fatality rates . At pres, RESTV) ,3. Filov, RESTV) ,5 while , RESTV) ,7,8. Nev, RESTV) . Evidenc, RESTV) ,11,12,13, RESTV)  and poss, RESTV)  bats are, RESTV) ,17 and s, RESTV) ,14,18. TDeciphering filovirus pathogenesis requires the elucidation of filovirus interactions with host cells. The entry of filoviruses into target cells is the first essential step in the viral life cycle, and the viral and cellular factors involved in this process are potential targets for antiviral strategies. Infectious filovirus entry is mediated by the viral glycoprotein (GP), which is the only viral envelope protein and thus constitutes the sole target for the neutralizing antibody response. Consequently, defining which domains in GP are essential for cellular entry and can be targeted by the humoral immune response is pivotal to the design of effective vaccines. In addition, insights into the multiple, sequential interactions of GP with host cell factors required for cellular entry can define novel targets for therapeutic inhibition. Key components of the filovirus entry cascade have been identified in the recent years, and some of these discoveries have already been translated into novel antiviral approaches in cell culture and small animal models ,22,23,24The filovirus particle is predominantly of filamentous nature and consists of seven structural proteins and a non-segmented, single-stranded RNA genome of negative polarity. In the viral envelope, a single type I transmembrane glycoprotein is incorporated, which is a virulence factor  and the 0, in the secretory pathway of infected cells. Upon transport of GP0 into the Golgi apparatus, subtilisin-like proprotein convertases, in particular furin, process the precursor protein into two subunits, the surface unit GP1 and the transmembrane unit GP2 [1-GP2 heterodimers  are inserted into the cellular and the viral membrane [1,2 is heavily glycosylated and harbors numerous consensus sites for N-linked glycosylation and GP1 glycosylation has been determined on the molecular level [1, which is highly variable among filovirus species [1,2 mutants with a deleted MLR show comparable or even enhanced capacity to transduce certain cell lines (for instance Vero cells) compared to particles carrying wild type GP1,2 [The filoviral GP is synthesized as a precursor protein, GPunit GP2 . Both sumembrane . The filar level ,35. In a species ,36,37,38pe GP1,2 ,40,41. H1,2 at the atomic level identified features also present in other viral glycoproteins, termed class I membrane fusion proteins , which facilitates GP binding to host cells [1 and is relatively conserved between EBOV- and MARV-GP (47% amino acid identity) which are believed to share a cellular receptor [1 and GP2 are separated by a protease sensitive site, which is cleaved by subtilisin-like proprotein convertases, in particular furin. However, in contrast to other class I membrane fusion proteins, the furin consensus site is dispensable for cellular entry [2 contains an N-terminal fusion loop and two HRs, which are conserved between the GP2 subunits of ebola- and marburgviruses. In addition, a synthetic peptide corresponding to the C-terminal HR sequence of EBOV-GP2 inhibited both EBOV and MARV infection [As predicted for class I membrane fusion proteins, the surface subunit, GPst cells ,28,29,30receptor ,45. GP1 ar entry ,47 and pnfection , indicatnfection ,51,52,53nfection ,43, as e1,2, the N-terminal HR is hidden in the GP1-GP2 trimeric structure by the RBR [1 [1,2 for membrane fusion occurs in host cell endosomes, where pH-dependent proteases remove the glycan cap [1,2 for membrane fusion and will be discussed in detail below.In the native, pre-fusion form of GP the RBR , which ie RBR [1 ,45,55. Pycan cap ,57, whicycan cap ,58. Therycan cap ,43,59, w1,2 interactions with these factors, we will therefore provide a brief overview of the filovirus cell tropism. Filoviruses exhibit a broad cell and organ tropism in infected humans and non-human primates. However, at the early stages of the infection, cells of the mononuclear phagocyte system are mainly targeted, in particular macrophages and dendritic cells in the spleen, lymph nodes and liver [The cellular molecules involved in filovirus entry govern the spectrum of cells susceptible to filovirus infection. Before we discuss GPnd liver ,68,69,70nd liver  and ensund liver , but alsnd liver ,66,70, a1,2-induced cell rounding might result in loss of vascular integrity and hemorrhage [1,2-driven host cell entry, potentially because of lack of host cell factors required for appropriate uptake and intracellular trafficking [Secondary targets of filovirus infection are mainly fibroblasts and endothelial cells located in many different organs, including liver, kidney and testis, and these cells are permissive to robust, lytic viral replication ,73. It hmorrhage . Howevermorrhage , suggestmorrhage ,76,77,78morrhage ,67, and fficking ,82,83,841,2 at the cellular membrane and promote viral attachment to cells, signaling factors, which induce filovirus uptake through activating signaling cascades  surface molecule was initially found to be a cellular receptor for hepatitis A virus . By a bioviruses . TIM-1 ioviruses . The IgVoviruses ,108. TIMoviruses ,109,110 oviruses   family of receptor tyrosine kinases ,131. The1,2-harboring pseudotypes, which could be blocked by Axl-specific antibodies, confirming that TAM-proteins could promote filovirus entry [1,2-mediated entry [1,2-faciliated entry was dependent on Axl expression. In contrast, down-regulation of Axl‑expression in other cell lines did not compromise GP1,2-driven entry [1 and Axl. However, Axl expression was shown to augment internalization of EBOV-GP1,2-bearing pseudovirions and virus-cell fusion [1,2-mediated entry [By introduction of a cDNA library derived from permissive VeroE6 cells into non-susceptible Jurkat lymphocytes, Axl was identified as an EBOV entry factor expressed at the cell surface . The ectus entry , althouged entry ,117. A red entry . The useen entry ,115,117.l fusion , and thil fusion ,137. Indl fusion ,125. Finl fusion . WhetherIntegrins are cell surface expressed heterodimeric type I transmembrane glycoproteins, which are composed of two non-covalently linked subunits (α and β) . The int1,2 was initially shown to interfere with surface expression of various cellular membrane proteins, including α3 and ß1 integrins [1,2 expression does not reduce cell surface levels of integrins but rather sterically occludes epitopes in these proteins otherwise recognized by antibodies [1,2-bearing pseudotypes demonstrated that soluble recombinant ß1 integrin or ß1-reactive antibodies diminish GP1,2 driven entry, suggesting that GP1,2 might need to engage ß1 integrins for infectious entry [1,2 and integrins remains to be demonstrated. Work by Schornberg and colleagues provided evidence that α5β1-integrin is required for expression of the double chain forms of cathepsin B and L and for full cathepsin L activity [1,2 for membrane fusion, as discussed below. In contrast, α5β1-integrin was dispensable for GP1,2-mediated binding and uptake into target cells [1,2-driven entry by ensuring activity of GP1,2-priming cysteine proteases or by stimulating the protease maturation pathway, which might be required for viral entry. Whether physical interactions of GP1,2 with α5β1-integrin also contribute to filovirus entry remains to be determined.Expression of EBOV-GPntegrins ,124. Howtibodies . Experimactivity , an endoet cells . These oThe membrane fusion reaction driven by the GPs of enveloped viruses can be triggered by several stimuli. For some viruses, engagement of a receptor at the cell surface activates fusion with the plasma membrane at neutral pH . Alterna0 of all filoviruses excluding RESTV, which harbors an incomplete furin recognition site [Many viral GPs are synthesized as inactive precursor proteins which transit into a membrane fusion-competent state only upon proteolytic cleavage by host cell proteases, a process termed priming. A prominent example is the influenza virus, which depends on cleavage of its hemagglutinin by host cell proteases for acquisition of infectivity, and the nature of the cleavage sequences in HA determines the virulence of avian influenza viruses . The subion site . Despiteion site ,47, and 1,2 [It has long been noticed that filoviruses depend on low pH for infectious cellular entry . However1,2 . This co1,2 .Cathepsins comprise serine, aspartic and cysteine proteases and carry out diverse biological functions, including antigen processing for MHCII presentation ,169,170.1,2 and that cathepsin activity is essential for GP1,2-driven host cell entry [1,2 for membrane fusion and proposed that cathepsin cleavage of virion-associated GP1,2 occurs in a sequential fashion: First, cathepsin L and/or B cleave GP1,2 into an 18 kDa form, which is fully infectious but still requires cathepsin B activity for infectious entry. Subsequently, the 18 kDa form is processed by cathepsin B and cleavage might be sufficient to trigger membrane fusion [1,2 activation has been proposed by subsequent studies. Thus, Schornberg and colleagues demonstrated that processing of virion-inserted GP1,2 by recombinant cathepsin B and L or the bacterial protease thermolysin yielded a 19 kDa form of GP1 and was associated with a notable increase in infectivity [1 into the 19 kDa from proceeded via 50 kDa and 20 kDa intermediates and its was speculated that the 20 kDa form might differ from the 18 kDa form observed by Chandran and colleagues only in the presence of a N-linked glycan [1,2 must first be processed by cathepsins B and L before the activity of a third lysosomal factor, potentially a thiol reductase, triggers GP1,2-dependent membrane fusion [1,2 removes a glycan cap and the MLR, while the N-terminal RBR and GP2 remain in the molecule [1,2 for membrane fusion and exposes the RBR. Subsequently, an incompletely understood stimulus triggers membrane fusion and these final steps of the lysosomal escape of filoviruses critically depend on GP1,2 binding to NPC1  protein. Cote and colleagues discovered that filoviruses depend on NPC1 for cellular entry by screening a library of chemical compounds for entry inhibitors [1,2-driven entry [1,2-dependent entry harbored a defect in the NPC1 gene [NPC1 gene result in fatal, progressive neurodegenerative disorder, Niemann-Pick C1 disease, due to a defect in the export of cholesterol from lysosomes [1 [NPC1 gene into NPC1-defective, patient-derived cells or NPC1-negative CHO cells fully restored infection [1,2-mediated infection [NPC1−/+ mice were protected against filovirus infection in sharp contrast to wild type mice [An endosomal factor required for filovirus entry after GPhibitors , while Cen entry . FinallyPC1 gene . The NPCPC1 gene ,185  infection ,190, and1,2 for host cell entry [Taken together, the NPC1 protein is a filovirus receptor with unexpected characteristics: In comparison to cell surface receptors used by other viruses, NPC1 is unique in recognizing the filoviral glycoprotein only after uptake into the cell and after proteolytic processing. The identification of small molecule inhibitors directly or indirectly inhibiting NPC1-usage by GPll entry ,129 make1,2 at the cellular surface and can thereby promote infectious entry. However, lectin expression usually does not render cells susceptible to GP1,2-driven entry and a role of lectins in the cell tropism of filoviruses in the infected host remains to be demonstrated. TAM tyrosine kinase receptors and TIM-1 can augment entry of filoviruses into a subset of susceptible cell lines and into some primary cells but are not universally required for filovirus infection. In fact, neither TIM-1 nor the TAM family member Axl are expressed to appreciable amounts in human macrophages, key viral targets, and the factors regulating viral uptake into these cells remain to be elucidated. The respective studies might reveal that filoviruses can use diverse cell surface factors for uptake into TIM-1-, Axl‑negative cells, which might account for the broad cell tropism of filoviruses. In contrast to Axl and TIM-1, the broadly expressed endosomal/lysosomal protein NPC1 is required for filovirus entry into all cellular systems tested so far and seems to play a key role in filovirus entry. Further experiments will clarify whether NPC1 alone is sufficient for triggering membrane fusion or whether another host cell factor is involved. Host cell entry is the first essential step in filovirus infection. Cellular lectins can concentrate vectors bearing GP


Actinobacteria, Corynebacterium pseudotuberculosis strain P54B96, a nonmotile, non-sporulating and a mesophile bacterium, was isolated from liver, lung and mediastinal lymph node lesions in an antelope from South Africa. This strain is interesting in the sense that it has been found together with non-tuberculous mycobacteria (NTMs) which could nevertheless play a role in the lesion formation. In this work, we describe a set of features of C. pseudotuberculosis P54B96, together with the details of the complete genome sequence and annotation. The genome comprises of 2.34 Mbp long, single circular genome with 2,084 protein-coding genes, 12 rRNA, 49 tRNA and 62 pseudogenes and a G+C content of 52.19%. The analysis of the genome sequence provides means to better understanding the molecular and genetic basis of virulence of this bacterium, enabling a detailed investigation of its pathogenesis.The  Corynebacterium pseudotuberculosis  for re-centrifugation under the same conditions. The pellets were re-suspended in 1 mL TE/lysozyme . The sample was then incubated at 37oC for 30 min and then 30 µL of 30% (w/v) sodium N- lauroyl-sarcosine (Sarcosyl) was added to it, incubated for 20 min at 65oC, followed by incubation for 5 min at 4oC. Purification of DNA with phenol/chloroform/isoamylalcohol (25:24:1) was followed by precipitation with ethanol. DNA concentration was determined by spectrophotometer, and the DNA was visualized in ethidium bromide-stained 0.7% agarose gel.C. pseudotuberculosis P54B96 was obtained using the Ion Torrent PGM (Life Technologies) Sequencing Platform. A total, of 562,812 reads were generated, each with a mean size of 112 nts usable sequence (35-fold coverage). Furthermore, a hybrid de novo assembly approach was applied using 376,642 Ion filtered reads (19-fold coverage). This was carried out after quality filtering process during which reads representing an average Phred quality of less than 20, were removed. This strategy allowed closing gaps without bench work time cost [The complete genome sequence of ime cost .For homopolymer correction, an inherent problem of the Ion Torrent , CLCBio For automatic annotation, different programs were used. These include; Glimmer: gene predictor , RNAmmerThe metabolic Pathway/Genome Database (PGDB) was computationally generated using Pathway Tools software version 15.0  and MetaC. pseudotuberculosis genome are listed in The genome is 2,337,657 bp long and comprises one main circular chromosome with a 52.19% GC content. A total of 2,207 genes were predicted, among which 2,146 were protein coding genes, and 61 RNAs; 62 pseudogenes were also identified. Of the whole genome, 69.01% comprise genes that were assigned with putative functions, while the remaining genes were annotated as hypothetical proteins. The properties and statistics of the


Availability of a sensitive and rapid method to quantify multiple classes of hormones simultaneously will greatly facilitate the investigation of signaling networks in controlling specific developmental pathways and physiological responses. Due to the presence of hormones at very low concentrations in plant tissues  to analyze quantitatively the major hormones found in plant tissues within six minutes, including auxins, cytokinins, gibberellins, abscisic acid, 1-amino-cyclopropane-1-carboxyic acid (the ethylene precursor), jasmonic acid and salicylic acid. Sample preparation, extraction procedures and UPLC-MS/MS conditions were optimized for the determination of all plant hormones and are summarized in a schematic extraction diagram for the analysis of small amounts of plant material without time-consuming additional steps such as purification, sample drying or re-suspension.Rosmarinus officinalis.This new method is applicable to the analysis of dynamic changes in endogenous concentrations of hormones to study plant developmental processes or plant responses to biotic and abiotic stresses in complex tissues. An example is shown in which a hormone profiling is obtained from leaves of plants exposed to salt stress in the aromatic plant,  Hormones play a pivotal role in most physiological processes in plants. These structurally diverse compounds that act usually at nanomolar levels include five groups of the so-called "classic" hormones, comprising auxins, cytokinins, gibberellins (GA), abscisic acid (ABA) and ethylene, and several other plant growth regulators, including jasmonates, salicylates, brassinosteroids, polyamines or the very recently discovered strigolactones, which fit several of the criteria to be considered hormones -3. FurthRecent studies support the contention that hormone actions build a signaling network and mutually regulate several signaling and metabolic systems, such as auxins and GAs in growth regulation , CKs, auArabidopsis thaliana [In order to understand better the network regulation of hormone action influencing plant growth and development as well as the distribution of several hormones at the organ, cellular and sub-cellular levels, an ideal analytical method should provide a measure of multiple hormone concentrations  from a single experimental sample. Therefore several methods for the simultaneous quantification of multiple plant hormones using mass spectrometry with multiple reaction monitoring (MRM) have been developed recently. It has been reported a multiplex gas chromatography-tandem mass spectrometry (GC-MS/MS) technique for the simultaneous analysis of SA, JA, IAA, ABA and OPDA in thaliana . Howeverthaliana .Arabidopsis thaliana has been developed [An alternative to GC-MS is liquid chromatography coupled to mass spectrometry (LC-MS). A high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC/ESI-MS/MS) method for the simultaneous analysis of 15 plant hormones and metabolites from four different hormone classes  has been reported to analyze hormone regulation of thermodormancy of lettuce seeds [eveloped . To imp2 and re-suspension of the residues have been reported for plant hormone extraction [Plant hormones are structurally diverse compounds with diverse physiochemical properties. The question as to whether all plant hormones can be extracted equally well has not yet been answered. The choice of extraction methods depends not only on the target analysts but also on the matrix of the analyzed tissues. The requirements on the extraction method increase with the complexity of the sample matrix. In the literature diverse extraction solvents such as methanol, methanol-water mixtures, isopropanol, or isopropanol-water mixtures have been used with one or two extraction steps ,15,17-19traction ,20 whichRosmarinus officinalis), an aromatic Mediterranean perennial shrub rich in secondary metabolites and epicuticular waxes, was exposed to salt stress. Soil salinity is one of the most serious environmental threats for plant survival and affects many undesirable changes in plants such as hyperionic and hyperosmotic effects, increase in reactive oxygen species and metabolic toxicity. These changes lead to growth reduction, changes in biomass allocation and phenology, leaf senescence, and finally to plant death [Here we developed a new method which allows to analyze dynamic changes in endogenous concentrations of major plant hormones and to study plant development processes or plant responses to biotic and abiotic stresses in complex sample matrices. An example is shown in which rosemary .Of the 17 endogenous plant hormones investigated, Z, DHZ, 2-IP, IAA, ABA, JA, SA, ACC, GAThe extraction of plant hormones will critically determine the quality of the results obtained. Therefore the choice of the extraction solvent is very important, however, it is also challenging by the structurally diversity of plant hormones. Previously reported methods for plant hormone extraction used predominately methanol, methanol mixtures or isopropanol. Four classes of plant hormones including auxins, cytokinins, ABA and gibberellins were extracted using isopropanol:glacial acetic acid  . Methano4, GA9, IAA; 77% for d2-GA24; between 62 and 50% for JA, 2iP, ACC; and 35% for Z.Due to the structurally diversity of the 17 plant hormones a solvent alone was not able to extract all plant hormones equally well  adversely affects plant hormone contents. A 25% decrease was observed for SA and JA yields from freeze dried compared to fresh leaf material of bidopsis . A decrebidopsis . Here pl9 in four extractions; and Z, IAA, ACC, ABA, SA, GA4 and GA24 in five extractions.Undoubtedly, the requirements on the extraction methods increase with the complexity of the sample matrix. Rosemary leaves represent a complex matrix including essential oils, tannins, flavonoids, diterpenes, saponins, epicuticular waxes and resin. Five 100 mg samples (fresh weight) were extracted five times after including internal standards. Each supernatant was immediately dried under nitrogen stream, re-suspended and injected to LC-MS. Additional File 2 and re-suspension of the residues is widespread in the literature [4, GA9, GA24 and for 2iP and SA a significant loss about 70% and 50%, respectively, could be observed for the dried extract compared to the immediately injected extract  in the concentration range selected between 0.996 and 0.999 for the different analysts.In the optimum LC-MS/MS conditions calibration curves were created using solutions containing varying amounts of each unlabeled analyte compound and a known fixed amount of deuterium labeled internal standard. The obtained calibration curves showed linearity of correlation coefficients (RTo test the reproducibility of the developed method sample extracts were repeatedly injected (five times). The results show good reproducibility of elution times  and peak areas  Table  for each-1 for DHZ to 12.1 ng g-1 for GA20, and LOQ ranged from 0.24 ng g-1 for DHZ to 40.33 ng g-1 for GA20 in fresh rosemary leaves. To achieve a fully quantitative determination of plant hormones in plant tissues the necessary plant material has also to be taken into account. The minimum detectable amounts in samples analyzed were calculated regarding the limit of detection for each detectable plant hormone. The data shows that less than 0.1 mg (FW) of leaf tissue is sufficient to determine ACC, SA, JA, GA24, while the analysis of Z, ABA, IAA, GA4 and GA9 require amounts between 3 and 9 mg (FW) and DHZ and 2iP amounts between 10 and 25 mg (FW) .Sensitivity parameters are listed in Table It is quite important to develop a method that is not only simple, sensitive and rapid, but also a high throughput screening is desirable. To test the stability, analyzed sample extracts remained in the autosampler 4°C) for 48 h and were then re-injected. Table °C for 48Fv/Fm ratio  that all bring completely different matrices and therefore their specific complexity for analyses. As shown in the present study, each species will require a specific handling during extraction and analysis, and the schematic diagram presented in Figure The development of a rapid, sensitive, high throughput, cost effective method for quantification of 17 endogenous plant hormones from seven plant classes in a complex matrix is described. The use of UPLC/ESI-MS/MS with multiple reaction monitoring (MRM) allowed each sample could be analyzed within 6 minutes. The method requires minimal plant tissue, is highly reproducible and was applicable to analyze dynamic changes in endogenous concentrations of hormones in plants exposed to salt stress. Due to the structural diversity of plant hormones no one extraction solvent was capable of extract all plant hormones equally well. If we aim at reducing extraction steps to a minimum, depending on the plant hormones of interest a different solvent is recommended: methanol:isopropanol:glacial acetic acid, 20:79:1 (v/v/v) for ABA, SA, JA, IAA and GAs, methanol:isopropanol:glacial acetic acid, 60:39:1 (v/v/v) for cytokinins, and methanol:glacial acetic acid, 99:1 (v/v) for ACC. Furthermore, this method is equally applicable to fresh or freeze dried tissue. Moreover, high loss of plant hormones during drying of sample extracts under N4-ACC, d6-IPA, d5-Z, d5-ZR, d5-IAA, d6-ABA, d4-SA, d5-JA and d6-2iP were purchased from OlChemim Ltd. . Unlabeled and deuterium labeled gibberellins GA1, GA4, GA9, GA19, GA20, GA24, d2-GA1, d2-GA4, d2-GA9, d2-GA19, d2-GA20, d2-GA24 were purchased from Dr. Lewis Mander at the Australian National University .Unlabeled ACC, IPA, Z, ZR, IAA, ABA, JA, SA were purchased from Sigma-Aldrich . Unlabeled 2iP, DHZ, DHZR, and deuterium labeled dAll work was carried out with rosemary leaves. Fifteen plants were purchased from a nursery  and maintained in a greenhouse at the experimental fields of the University of Barcelona  with controlled temperature (24/18°C) and adequate water conditions by irrigating the plants with half concentration of Hoagland solution every 2 days. For the optimization procedure of sample preparation and extraction, leaves from these plants were collected, frozen in liquid nitrogen and stored at -80°C until analysis. For the salinity experiment, five plants were exposed to salt stress by irrigating with the same nutrient solution containing an extra addition of 200 mM NaCl, and compared to five control plants . Young and old leaves from the uppermost and lowest part of the plant, respectively, were collected after 8 days of treatment, frozen in liquid nitrogen and stored at -80°C until analyses.v/Fm ratio) were made by using a pulse-modulated fluorimeter Imaging-PAM  after 2 h of dark adaptation. The v/Fm Fratio was calculated as (Fm-F0)/Fm, where Fm and F0 are the maximum and basal fluorescence yields, respectively, of dark-adapted leaves.To determine the relative water content (RWC) of leaf material from the salinity experiment, young and old leaves were collected, immediately weighed (FW), re-hydrated for 24 h at 4°C in darkness (TW) and subsequently oven-dried for 48 h at 60°C (DW). The RWC was determined as 100 × (FW-DW)/(TW-DW). Measurements of the maximum efficiency of photosystem II photochemistry , the supernatant was collected and the pellet was re-extracted with 0.5 ml of extraction solvent and the extraction repeated three times again. Then, supernatants were combined and dried completely under a nitrogen stream and re-dissolved in 300 μl of methanol, centrifuged  and filtered through a 0.22 μm PTFE filter . Samples (5 μl) were then analyzed by UPLC/ESI-MS/MS. Hormones were determined in ten independent samples for each treatment. Quantification was done by the creation of calibration curves including each of the 17 unlabeled analyte compounds . Ten standard solutions were prepared ranging from 0.05 to 1000 ng ml-1 and for each solution a constant amount of internal standard (as described above) was added. Calibration curves for each analyte were generated using Analyst™ software . The limit of detection  and the limit of quantification  were also calculated with the aid of this software.Frozen leaf material (about 100 mg FW.) was ground in liquid nitrogen with the mixer mill MM400  in a 2 ml Eppendorf tube, and then extracted with 1 ml of extraction solvent  with 1% of glacial acetic acid) using ultra sonication (4-7°C). The labeled forms of the compounds d-1. Cytokinins and ACC were analyzed using method 1 (M1) and ABA, JA, SA, IAA, and gibberellins were analyzed using method 2 (M2). The gradient profile for M1 (cytokinins and ACC) was applied as follow (t (min), % A): , , , , . The gradient profile for M2  was applied as follow: (t (min), % A): , , , , . MS and MS/MS experiments were performed on an API 3000 triple quadrupole mass spectrometer . Analyses for M1 were performed using Turbo Ionspray source in positive ion mode and for M2 in negative ion mode. For both methods temperature was 400°C, nebulizer gas (N2) 10 (arbitrary units), curtain gas (N2) 12 (arbitrary units), collision gas (N2) 4 (arbitrary units) and the capillary voltage was 3.5 kV for M1 and -3.5 kV for M2, respectively. The optimized MS/MS conditions for the analysis of plant hormones are summarized in Additional File -1 into the mass spectrometer using a Model 11 syringe pump . The mass spectrometer was operated in multiple reaction mode (MRM) due to their high selectivity using precursor-to-product ion transitions because many compounds could present the same nominal molecular mass or peaks can overlap. Since more than 100 GAs with partly same molecular masses and similar retention times are found in plants special mention is needed for GAs identification in plant extracts. Additional files 1, GA4, GA9, GA19, GA20, GA24 standards. In rosemary leaf extracts GA4, GA9 and GA24 were detected. Using multiple reaction monitoring (MRM) conditions a specific precursor to one product ion transition is monitored. However, to verify the identification of GAs in rosemary leaf extracts the specific precursor ions of GA4, GA9 and GA24 to two different product ions in MRM mode were monitored. All GAs mass chromatograms from rosemary leaf extracts showed identical retention times as GA standards.The UPLC system consisted of an Aquity UPLC™ System  quaternary pump equipped with an autosampler. For the analysis of the extracts, a HALO™ C18  column  was used. Gradient elution was done with water and 0.05% glacial acetic acid (solvent A) and acetonitrile with 0.05% glacial acetic acid (solvent B) at a constant flow rate of 0.6 ml min2, 0), 2.0, 0, , using the DMS's post hoc test, and were considered significant at a probability level of P < 0.05.ABA: abscisic acid; ACC: 1-amino-cyclopropane-1-carboxyic acid; CE: collision energy: CXP: collision cell exit potential; DHZ: dihydrozeatin; DHZR: dihydrozeatin riboside; DP: declustering potencial; EP: entrance potential; ESI-MS/MS: electrospray ionization tandem mass spectrometry; FP: focusing potential; GA: gibberellin; IAA: índole-3-acetic acid; 2iP: isopentenyladenine: IPA: isopentenyladenosine; JA: jasmonic acid; LOD: limit of detection; LOQ: limit of quantification; MRM: multiple reaction monitoring; OPDA: 12-oxo-phytodienoic acid; RDS: relative deviation standard; SA: salicylic acid; UPLC: ultrahigh performance liquid chromatography; Z: zeatin; ZR: zeatin riboside.The authors declare that they have no competing interests.MM participated in the design of the study, carried out the lab work and helped to draft the manuscript. SMB conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors have read and approved the final manuscript.Effects of freeze-drying. Concentration of endogenous plant hormones (A) Z, DHZ, 2iP, IAA, ACC, and (B) ABA, JA, SA, GA4, GA9, GA24 detected in fresh weight (FW) and freeze-dried (DW) rosemary leaves.Click here for fileExtraction efficiency. Concentration of (A) Z, DHZ, 2iP, IAA, ACC, and (B) ABA, JA, SA, GA4, GA9, GA24 found in rosemary leaves after 1, 2, 3, 4 and 5 extraction procedures.Click here for fileEffects of the amount of plant material used for extraction. Capacity of extraction method to analyze different leaf amounts (20 - 200 mg FW) of rosemary leaves.Click here for fileOptimized UPLC/ESI-MS/MS parameters. Parameters are listed in multiple reaction monitoring (MRM) conditions for quantification of plant hormones.Click here for file1, GA19, and GA20 standardsFragmentation patterns of labeled and unlabeled GA. (A) GA1 (precursor m/z 347 and product m/z 273 ions) and d2-GA1 standards (precursor m/z 349 and product m/z 275 ions). (B) GA19 (precursor m/z 361 and product m/z 273 ions) and d2-GA19 standards (precursor m/z 363 and product m/z 275 ions), (C) GA20 (precursor m/z 331 and product m/z 287 ions) and d2-GA20 standards (precursor m/z 333 and product m/z 289 ions)Click here for file4, GA9, GA24 standards and identification of GA4, GA9 and GA24 in Rosmarinus officinalis extractsFragmentation patterns of labeled and unlabeled GA. (A1) Fragmentation patterns of GA4 (precursor m/z 331 and product m/z 213 ions) and d2-GA4 standards (precursor m/z 333 and product m/z 215 ions). (A2) UPLC/ESI-MS/MS chromatograms from rosemary leaf extract using multiple reaction monitoring (MRM) conditions. GA4 was identified by monitoring the precursor ion (m/z 331) to two different product ion transitions (m/z 213 and m/z 225) in MRM mode with identical retention times as the GA4 standard. (B1) Fragmentation patterns of GA9 (precursor m/z 315 and product m/z 271 ions) and d2-GA9 standards (precursor m/z 317 and product m/z 273 ions). (B2) UPLC/ESI-MS/MS chromatograms from rosemary leaf extract using multiple reaction monitoring (MRM) conditions. GA9 was identified by monitoring the precursor ion (m/z 315) to two different product ion transitions (m/z 271 and m/z 253) in MRM mode with identical retention times as the GA9 standard. (C1) Fragmentation patterns of GA24 (precursor m/z 345 and product m/z 257 ions) and d2-GA24 standards (precursor m/z 347 and product m/z 259 ions). (C2) UPLC/ESI-MS/MS chromatograms from rosemary leaf extract using multiple reaction monitoring (MRM) conditions. GA24 was identified by monitoring the precursor ion (m/z 345) to two different product ion transitions (m/z 257 and m/z 301) in MRM mode with identical retention times as the GA24 standard.Click here for file


Vitamin D is known to increase Ca absorption in adults. However, the threshold vitamin D status to benefit Ca absorption is lower than the target vitamin D status for higher bone mineral density and lower fracture risk, pointing to another pathway for vitamin D to benefit bone. One possibility is by affecting osteoblast and osteoclasts directly. Vitamin D-related bone metabolism may also be affected by soy isoflavones, which selectively bind to the estrogen receptor β and may reduce bone loss in postmenopausal women. We discuss a possible synergistic effect of soy isoflavones and vitamin D on bone by affecting osteoblast and osteoclast formation and activity in postmenopausal women. Osteoporosis, “the thinning of the bone tissue and loss of bone density over time” , is a maOlder adults, especially postmenopausal women, are more susceptible to osteoporosis due to estrogen deficiency which promotes bone resorption and inadequate intake of calcium (Ca) and vitamin D, two important nutrients to protect bone health . The tra2D), the active form of vitamin D, signal the regulation of Ca homeostasis. Ca metabolism is traditionally known to be regulated mainly through absorption, reabsorption, and resorption in the intestine, kidney, and bone, respectively, primarily to control serum Ca concentration. When Ca intake is low, the drop in serum Ca concentration is detected by the calcium sensing receptors on the parathyroid gland. This triggers parathyroid hormone secretion, which induces hydroxylation of 25(OH)D to 1,25(OH)2D, bone resorption, and renal reabsorption of Ca. Elevated serum 1,25(OH)2D levels stimulate the transcription of genes regulating Ca absorption in the intestine, such as TRPV6, calbindinD9k, and PMCA1b [i.e., TRPV5 and TRPV6, calbindinD28k and PMCA. The effect of 1,25(OH)2D on intestinal gene transcription is more effective than renal gene transcription, and consequently Ca absorption is more affected by circulating 1,25(OH)2D than reabsorption. As vitamin D and 25(OH)D are precursors of 1,25(OH)2D, it is thought that higher vitamin D intake and higher serum 25(OH)D status, indicating adequate substrate for 1,25(OH)2D production, will lead to increased Ca absorption. As a result, when dosed orally or subcutaneously with 1,25(OH)2D, Ca absorption increased in rats and humans [2D and serum Ca concentration provide a negative feedback on PTH release so that serum Ca concentration is maintained within a tight window. Once serum Ca is maintained, excess Ca from absorption is deposited in the bones or excreted through urine as PTH is suppressed. Through this process, vitamin D can stimulate Ca absorption, the primary step to increase BMD.The effect of vitamin D on Ca absorption has been well understood and thought to be the major pathway to affect bone health. Vitamin D can be obtained through oral intake or cutaneous synthesis when UV reacts with 7-dehydrocholesterol. Vitamin D is converted to 25-hydroxyvitamin D (25(OH)D) in the liver. 25(OH)D is known to be the status marker of vitamin D, due to its longer (3–4 weeks) half-life. Parathyroid hormone (PTH) and 1,25-dihydroxyvitamin D 2D, is positively correlated with Ca absorption [2D concentration [Clinical trials have pointed to a threshold 25(OH)D level of ~20 nmol/L to maximize vitamin D-mediated Ca absorption in adults. Previous research using crude methods resulted in a relatively large increase (65% higher than baseline) in fractional Ca absorption with increased vitamin D status (36.5 nmol/L increase) . Howeversorption ,15, but ntration . Though Despite the 20 nmol/L threshold for maximal vitamin D-activated Ca absorption, much data has been presented that higher vitamin D status is required to maximize BMD in adults. Vitamin D status and BMD were positively correlated in monkeys that received oophorectomy  and post2D bound to the VDR induces osteoblast, the bone-forming cell, function by activating collagen type 1α, osteocalcin and alkaline phosphatase gene transcription [2D also inhibits Runx, which stimulates differentiation of mesenchymal progenitor cells to pre-osteoblasts, and pre-osteoblasts to osteoblasts. However, mature osteoblasts are activated by 1,25(OH)2D, and the receptor for activation of NFkB ligand (RANKL) binds to RANK on pre-osteoclasts. As a result, osteoclastogenesis occurs and bone resorption is increased. In mice, overexpression of VDR in mature osteoblasts increased bone formation and decreased bone resorption in tibial cortical bone and vertebral trabecular bone, respectively [2D levels [Increasing evidence has emerged on the local action of vitamin D. The VDR and 1α-hydroxylase (1αOHase) activity has been discovered in bone cells . It has cription . 1,25D levels beyond 104 nmol/L, Fleet et al.  reportedet al. . In younet al. . Howeveret al. . Some stet al. ,31,32 buet al. ,33. Thouet al. .Some plant-derived compounds that have structural similarity to estrogen, phytoestrogens, are able to bind to the estrogen receptor (ER). The results of phytoestrogen interventions on bone health outcomes vary due to the type of phytoestrogen. Therefore, in this paper we will focus on soy-derived isoflavones genistein and daidzein. The decrease of estrogen ,36,37 or9k, an intestinal Ca transport protein [in vitro, phytoestrogens induce production of osteoprotegerin (OPG). OPG competes with RANKL and prevents maturation of pre-osteoclasts and thus, resorption [2D-induced TRAP-positive multi-nucleated cell formation, decreased resorption activity, and increased ER expression and apoptosis through caspase-8 and caspase-3 fragmentation in mono-nucleated cells [The effects of isoflavones on bone are thought to be through affecting the bone cells directly, rather than affecting Ca absorption. This is based, in part, on the lack of dose responsive effect of dietary genistein or daidzein in trans-epithelial transport of Ca in ERβ-expressing Caco-2 cell cultures . Caco-2  protein . Further protein ,45. On t protein . Low-dos protein . Geniste protein . By bindsorption . In piglsorption . In porced cells . In humaed cells . These eet al. [41Ca from pre-labeled bone in a similar population [The effect of soy isoflavones on bone in rodent and human trials has mixed results. Genistein positively affected rat mandibular subchondral bone BMD, bone volume, and trabecular bone in a dose-dependent manner . In OVX et al.  report tet al. . Others et al. . In humaet al. . In a shet al. . Commercet al.  while uppulation . Others pulation . Neitherpulation , nor US pulation  benefittpulation . Though pulation . Thus, r2D, respectively, in colon cancer cells [2D production under conditions of the low levels of substrate 25(OH)D. Though in vitro, rodent, and clinical studies suggest a relationship, more evidence must be provided to test this hypothesis and elucidate the mechanism. Another proposed mechanism is that vitamin D analogs induce ER transcription. This has been reported in the JK 1624F2-2 (JFK) analog in pre- and post-menopausal women’s bone cells [2D and 17β-estradiol increased osteoblastic MC3T3-E1 cell proliferation and viability [The possibility of soy isoflavones and vitamin D impacting Ca and bone metabolism has been investigated through cell cultures and animal models. In intestinal cancer cells, genistein up-regulates VDR transcription and VDR expression possibly through the ER and MAPK signaling pathway  but no rer cells . Intake er cells . In earler cells . The soyne cells . The syniability . Post-meiability . A similiability . VitaminThis review summarizes the evidence to support the possible benefit of the combination of vitamin D and soy phytoestrogens on post-menopausal bone loss. Clinical evidence for maximal vitamin D-mediated Ca absorption does not completely explain the optimal vitamin D status for maximal BMD or BMC and lacks correlation of vitamin D-related Ca absorption and BMD or BMC in post-menopausal women. The benefit of vitamin D on bone seems to be stronger through the direct effect on osteoblasts and osteoclasts. Soy phytoestrogens in combination with vitamin D may synergistically induce osteoblast activation and prevent pre-osteoclast and osteoclast differentiation, through the increase of vitamin D metabolites, VDR, and ER actions. More research is required to identify the causal factors and the mechanism of the benefits of vitamin D and phytoestrogens on bone in post-menopausal women.


Usher syndrome (USH) combines sensorineural deafness with blindness. It is inherited in an autosomal recessive mode. Early diagnosis is critical for adapted educational and patient management choices, and for genetic counseling. To date, nine causative genes have been identified for the three clinical subtypes . Current diagnostic strategies make use of a genotyping microarray that is based on the previously reported mutations. The purpose of this study was to design a more accurate molecular diagnosis tool.We sequenced the 366 coding exons and flanking regions of the nine known USH genes, in 54 USH patients .Biallelic mutations were detected in 39 patients (72%) and monoallelic mutations in an additional 10 patients (18.5%). In addition to biallelic mutations in one of the USH genes, presumably pathogenic mutations in another USH gene were detected in seven patients (13%), and another patient carried monoallelic mutations in three different USH genes. Notably, none of the USH3 patients carried detectable mutations in the only known USH3 gene, whereas they all carried mutations in USH2 genes. Most importantly, the currently used microarray would have detected only 30 of the 81 different mutations that we found, of which 39 (48%) were novel.Based on these results, complete exon sequencing of the currently known USH genes stands as a definite improvement for molecular diagnosis of this disease, which is of utmost importance in the perspective of gene therapy. MYO7A ; USH1C, [Swiss-Prot:Q7RTU8]; CDH23, [Swiss-Prot:Q9H251]; PCDH15-CD1, [Swiss-Prot:Q96QU1]; PCDH15-CD2, [NCBI-RefSeq:NP_001136241.1]; PCDH15-CD3, [Swiss-Prot:C9J4F3]; USH1G, [Swiss-Prot:Q495M9]; USH2A, [Swiss-Prot:075445]; VLGR1, [Swiss-Prot:Q8WXG9]; WHRN, [Swiss-Prot:Q9P202]; USH3A, [Swiss-Prot:P58418] and [Swiss-Prot:P58418-1] for "a" and "c" variants, respectively.We analyzed the nine USH genes in a cohort of 54 French patients, of whom 27 were affected by USH1, 21 by USH2, and six by USH3. From the patient and parent questionnaires, consanguinity was established for nine families  of these mutations, i.e. 27% to 100% of the mutations found in each USH gene, had not been previously reported Tables . NumerouMYO7A were found in 19 patients, specifically, eight nonsense mutations, one nucleotide duplication, five nucleotide deletions, four splice site mutations, and eight missense mutations. Seven of these mutations had not been previously reported, including two nonsense mutations , a nucleotide duplication , a nucleotide deletion c.3904delT (p.Y1302fsX97), a nucleotide substitution (c.1690+1G>A) predicted to alter the splice donor site of intron 14, and two missense mutations (p.K268R and p.P1220L) that change amino acid residues located in the motor head and the first MyTH4 domain of the myosin VIIa tail, respectively  already reported in several patients [Three distinct pathogenic or presumably pathogenic mutations in patients ,27,53, apatients .CDH23 were found in six patients, specifically, a previously reported mutation that affects splicing (c.6050-9G>A) [th, 13th and 25th cadherin repeat and the extracellular region adjacent to the transmembrane domain (3065-3085), respectively  predicted to bind Ca2+ [Eight pathogenic or presumably pathogenic mutations in 50-9G>A) , a novel50-9G>A) ,56, fourind Ca2+ , has preind Ca2+ ).PCDH15, specifically, a nonsense mutation (p.R991X) [th cadherin repeat   and a nod et al.  was not USH1G were found in three patients, specifically, an already reported nonsense mutation (p.W38X) [Three pathogenic or presumably pathogenic mutations in p.W38X) , a novel8X , a noUSH2A were found in 17 patients including three USH3 patients, specifically, five nonsense mutations, one nucleotide duplication, six nucleotide deletions [th fibronectin type III domain and the trideca-di-cysteine domain (residue 3192 to 3371) between the 18th and the 19th fibronectin type III domains , a nucleotide duplication , four nucleotide deletions , and c.17668_17669delAT/p.M5890fsX10), and five missense mutations  that all affect amino acid residues located in the large extracellular region of the protein, between the 13th and 14th β-Calx domains, in the 4th Epilepsy Associated Repeat domain, in the 32nd β-Calx domain, between the 32nd and 33rd β-Calx domains, and in the 35th β-Calx domain, respectively , and two novel missense mutations (p.S11R and p.R379W) that affect amino acid residues located in the N-terminal Ala/Gly/Ser-rich stretch (aa 9-31) and immediately after the PDZ2 domain, respectively , and on one allele in the remaining three USH1 patients. Moreover, one of these patients (U3) harboured monoallelic, presumably pathogenic mutations in two different USH1 genes (see below). Mutations in USH2A, VLGR1 and WHRN were found in 57%, 19% and 9.5% of the USH2 cases, respectively. Notably, one USH2 patient (P0486) carried biallelic mutations in MYO7A. Mutations were detected on both alleles in 15 USH2 patients (including a consanguineous family), and on one allele in the remaining four USH2 patients. Finally, as regards the USH3 patients, biallelic mutations in USH2A and monoallelic mutations in VLGR1 or WHRN were found in three patients, two patients, and one patient, respectively.We found mutations in 49 out of 54 91%) USH patients, specifically, in 24 out of 27 (89%) USH1 patients, 19 out of 21 (90%) USH2 patients, and all six (100%) USH3 patients (see Table % USH patUSH2A (p.S5030X) and a missense mutation in MYO7A (p.K268R), but his brother, who is also clinically affected, does not carry the MYO7A mutation. Patient P0432 has a c.4030_4037delATGGCTGG (p.M1344fsX42) mutation in USH2A and a missense mutation in CDH23 (p.R1189W), but his father, who has neither deafness nor retinitis pigmentosa, also carries these two mutations, and his clinically affected sister does not carry the mutation in CDH23. In the USH1 patient, we found three presumably pathogenic mutations in MYO7A (c.6657T>C), USH1G  and USH2A (c.9921T>G). Her father carries the mutations in MYO7A and USH2A without displaying symptoms of the disease, whilst her unaffected mother carries the mutation in USH1G. The mutations in MYO7A, USH1G and USH2A were not found in 666 control alleles. Of the four siblings, the affected girl is the only one who carries the mutations in MYO7A and USH1G, and, all the more, the mutations in the three genes  carried two presumably pathogenic mutations in an USH gene, plus one or two additional mutations in another USH gene. Taking into account only the 39 patients for whom biallelic mutations have been identified, 18% (7 out of 39) carry additional mutations. Specifically, five USH1 patients carried biallelic mutations in an USH1 gene plus one or two additional mutations in another USH1 (three patients) or USH2 (two patients) gene, and two USH2 patients carried biallelic mutations in USH2 genes plus one additional, presumably pathogenic mutation in an USH1 or an USH2 gene  comparing the strategy for mutation detection currently in use with the here developed USH exome sequencing (including splice sites), ii) determination of whether the phenotype can restrict the mutation screening to the USH genes corresponding to the clinical subtype in a given patient, and iii) defining the possible existence of digenic/oligogenic inheritance of the disease in some patients.MYO7A, c.238_239dupC in USH1C, and c.2299delG and c.10712C>T in USH2A. Therefore, in the process of designing any strategy for USH molecular diagnosis, taking into account the high prevalence of novel mutations appears to be of major importance.We found mutations in eight of the currently known nine USH genes, in 49 out of 54 91%) patients , and of USH2A in USH2 (62% of the cases).Previous mutation research studies performed in patients referred to medical genetic clinics showed high proportions of mutations for patients , specifis [MYO7A -64, 19%-A [CDH23 , 11%-15% [PCDH15 , and forpatients ,60,66, was minor . The preUSH3A. Yet, mutations in USH2 genes were present in all of them, and with a gene distribution similar to that observed in USH2 patients. This finding, which concerns six out of 24 patients carrying mutations in USH2 genes, calls for a revision of the USH2 clinical features. Along the same line, one patient diagnosed as USH2, because he did not have a vestibular dysfunction, carried biallelic missense mutations in an USH1 gene, MYO7A. The two mutations (p.A457V and p. K269del) affect amino acid residues located in the motor head of myosin VIIa, and have previously been reported in USH1 patients [Surprisingly, mutations were found in genes that did not fit the clinically diagnosed USH type. None of the six patients diagnosed as USH3 on the basis of the postlingual onset and progressive nature of the deafness, and the absence of vestibular dysfunction (see Subjects and methods) carried a mutation in patients . They mapatients , but notMYO7A and USH1G, and possibly USH2A. Three cases of digenic inheritance of USH1 have been reported so far [CDH23 and PCDH15, in agreement with the contribution of cadherin-23 and protocadherin-15 to the hair bundle transient lateral links and tip-links [CDH23 [PCDH15, leading to an in frame-deletion of a threonine residue (p.T1868del) [Notably, our study has revealed one case of likely oligogenic inheritance for USH1, involving d so far , all cauip-links ,36,67-69s [CDH23 ,55 is, h1868del)  within t1868del) ,70 have 1868del) , whereasush2a, myo7a, whrn) of Ciona savignyi [Therefore, even though non-monogenic inheritance of USH appears to be rare, it has to be taken into consideration in the molecular diagnosis strategy. In addition, ten patients had presumably pathogenic mutations in two different USH genes. Seven of them had biallelic mutations in one gene, and carried an additional mutation in a second and, for one of them, a third USH gene. None of these additional mutations were nonsense or frame-shifting mutations, but the conservation of the corresponding amino acid residues in the orthologous genes , born from consanguineous parents, involvement of any of the nine currently known USH genes could be excluded by segregation analysis of polymorphic markers at the corresponding loci (data not shown). In the four remaining patients, the undetected mutations might still be located in the unexplored promoter regions or intragenic regulatory sequences of these genes, but may also be located in other, still unknown USH genes, as in patient S1823. Indeed, a new locus, USH1H, at chromosome 15q22-23 , and thrPDZD7 [USH2A or VLGR1 [Direct exon sequencing of a set of specific disease genes is a reliable, easy set-up method, which remains less expensive than full exome sequencing in the patients. Based on the high prevalence of private mutations both in USH1 and USH2 patients, the substantial number of cases displaying genotype/phenotype discrepancy, and the presence of additional, presumably pathogenic mutations in a number of patients, we conclude that exon sequencing (including flanking splice sites) of all currently known USH genes is required for proper molecular diagnosis in every USH patient, both in the context of genetic counseling and in the perspective of retinal and cochlear gene therapy. The activity of the USH gene carrying biallelic mutations may indeed turn out to be only partly restored by gene therapy, and the presence of a third mutation in another USH gene may then critically impact on the benefits of the gene therapy. Moreover, as PDZD7  has receor VLGR1 , future APEX: Arrayed Primer EXtension; CDH23: Cadherin 23; DNA: DeoxyriboNucleic Acid; ESE: Exonic Splicing Enhancers; MYO7A: Myosin VIIa; PCR: Polymerase Chain Reaction; PCDH15: Protocadherin 15; SIFT: Sorting Intolerant From Tolerant; USH: Usher syndrome; USH1: USH type I; USH2: USH type II; USH3: USH type III; VLGR1: Very Large G protein-coupled Receptor; WHRN: Whirlin.The authors declare that they have no competing interests.CB and MG contributed equally to this work. FD and CP conceived of the study and participated in its design and coordination. CB and MG carried out the molecular genetic study and analysed the data. SM, BD, CV, HD, MME, AD, CC, JV, BM, DB, JD, CT, AD, CF, TM, DL, FD, VDG, MFTO, SS, AMF, PC, GC contributed to clinical and genetic evaluation of the patients. DZ, MD, DF, MP, MNG, DW, ML participated in the study of the control population. JW provided DNA sequencing facilities. CB, JPH, FD, CP wrote the manuscript. MG, SM, DW, AEA, LJ, JL, JAS participated in manuscript writing. All authors have read and approved the final manuscript.Figure S1: Sequence alignment of amino acid residues mutated in patients carrying missense mutations in USH1 genes. Representative stretches of amino acid sequences from each of the USH1 proteins in various species were aligned. Identical residues are highlighted with shading. Residues involved in missense mutations are underlined. Figure S2: Sequence alignment of amino acid residues mutated in patients carrying missense mutations in USH2 genes. Representative stretches of amino acid sequences from each of the USH2 proteins in various species were aligned. Identical residues are highlighted with shading. Residues involved in missense mutations are underlined. Orthologs of VLGR1 are not present in the genomes of invertebrates such as C. elegans and drosophila. Figure S3: Missense mutations possibly creating or disrupting a splice site. Representative stretches of amino acid sequences from each of the USH proteins in various species were aligned. Identical residues are highlighted with shading. Residues involved in missense mutations are underlined. Triangles indicate splice sites. Scores for splice sites are obtained by NetGene2 software program. Possible new splice sites are in bold. USH2A mutations in family U51Figure S4: Segregation analysis of the .Click here for fileTable S1. Presumably neutral, isocoding and intronic variants in USH genes.Click here for file


AbstractHypostomus , Hypostomus strigaticeps , Hypostomus regani , and Hypostomus paulinus ) from the seven tributaries of the Paranapanema River Basin (Brazil) by means of different staining techniques  and fluorescence in situ hybridization (FISH) to detect 18S rDNA sites. All species showed different diploid numbers: 2n=68 (10m+26sm+32st-a) in Hypostomus ancistroides, 2n=72 (10m+16sm+46st-a) in Hypostomus strigaticeps, 2n=72 (10m+18sm+44st-a) in Hypostomus regani and 2n=76 (6m+16sm+54st-a) in Hypostomus paulinus. Ag-staining and FISH revealed various numbers and locations of NORs in the group. NORs were usually located terminally on the subtelocentric/acrocentric chromosomes: on the long arm in Hypostomus strigaticeps (2 to 4) and Hypostomus paulinus (2); and on the short arm in Hypostomus ancistroides (2 to 8) and Hypostomus regani (2 to 4). Conspicuous differences in heterochromatin distribution and composition were found among the species, terminally located in some st-a chromosomes in Hypostomus ancistroides, Hypostomus strigaticeps, and Hypostomus paulinus, and interstitially dispersed in most st-a chromosomes, in Hypostomus regani. The fluorochrome staining indicated that different classes of GC and/or AT-rich repetitive DNA evolved in this group. Our results indicate that chromosomal rearrangements and heterochromatin base-pair composition were significant events during the course of differentiation of this group. These features emerge as an excellent cytotaxonomic marker, providing a better understanding of the evolutionary mechanisms underlying the chromosomal diversity in Hypostomus species.Cytogenetic analyses were performed on fishes of the genus  Hypostomus Lacépède, 1803  represent one of the most specious genus of the family Loricariidae, with 127 nominal species , and chromosome C-banding   . The speMetaphase chromosomes were obtained through the air-drying technique  and stai3 (CMA3) and 4’6-diamidin-2-phenylindole (DAPI), respectively, according to 3 for 1 h, washed in distilled water and sequentially stained with 2 µg/mL DAPI for 15 min. Slides were mounted with a medium composed of glycerol/McIlvaine buffer (pH 7.0) 1:1 supplemented with 2.5 mM MgCl2.C-banding was performed according to Prochilodus argenteus Spix and Agassiz, 1829  1,4-diazabicyclo  octane, pH 8.6), 1 μL of propidium iodate (1 μg/mL) and 1 µL of MgCl2 50 mM.The fluorescence in situ hybridization procedure was performed according to iz, 1829  was labeImages were acquired with Leica DM 4500 B microscope equipped with a DFC 300FX camera and Leica IM50 4.0 software.Hypostomus ancistroides showed a diploid number 2n=68 and a fundamental number (FN) of 104, with a karyotype formula of 10m+26sm+32st-a. One chromosome of pair 26 showed size heteromorphism  with large GC-rich blocks, and one st-a pair, probably corresponding to NOR-bearing chromosomes, and in the pericentromeric region of the first (m) pair  (m+54st-a . NORs wem+54st-a  left box(m) pair ; after D(m) pair . Heterocpair 12) .Hypostomus ancistroides  (Hypostomus ancistroides (2n=68) but with different fundamental numbers (FN) among different populations, i.e. 106, 102 and 96  and a FN of 102 and 116  in stroides , 2n=72  . The ava2 and 96  and the  and 116 , also di and 116 . On the Loricariidae. In a phylogenetic study of Loricariidae using morphological data, the genus Hypostomus was considered the most derived  an increase in the diploid number (2n) in several species, probably due to centric fissions and ii) the same 2n but with a difference in the karyotype formula, probably accounted by pericentric inversions.According to  derived , represeHypostomus species, observed after silver staining and FISH. All species showed Ag-NORs and 18S rDNA sites located in the terminal regions of st-a chromosomes, but with a significant variation in number and location among them. Hypostomus ancistroides showed up to 8 NOR sites, all located on the short arms , Hypostomus strigaticeps (pair 3), and Hypostomus paulinus (pair 1) (Hypostomus nigromaculatus by Hypostomus sp. B from the Mogi Guaçu River (Hypostomus affinis (Hypostomus cochliodon (Hypostomus nigromaculatus (Hypostomus paulinus, pair 12 proved to be well differentiated, with the long arm almost entirely heterochromatic, a feature observed only in this species. On the other hand, Hypostomus regani showed a more distinct heterochromatin distribution in relation to the other species, with a preferential location in the interstitial regions of st-a chromosomes (The chromosome banding performed in all species analyzed showed a variation in the heterochromatin distribution pattern. However, the presence of heterochromatin in some chromosomes was constant, as observed in the pericentromeric region of a metacentric pair in çu River , Hyposto affinis , Hypostochliodon , and Hypaculatus . Interesomosomes .Hypostomus species, corresponding to the NOR-bearing chromosome pair, which shows a heterochromatin block adjacent to this site (e.g. The presence of a marker chromosome that seems conserved for most ite e.g. , was alsHypostomus species. Since these characteristics do not vary among the different populations of the same species, they are significant cytogenetic markers at the species level.Karyotypes, banding patterns, number and location of ribosomal DNA sites, and repetitive DNA are important tools for the cytotaxonomy of Hypostomus species from different rivers, as well as detailed studies of satellite DNA sequences may clarify important issues of genome organization, be used as genetic markers, and provide interesting insights for the comprehension of the evolution of this genus.Further data on other


Plants are constantly exposed to a variety of environmental stresses. Freezing or extremely low temperature constitutes a key factor influencing plant growth, development and crop productivity. Plants have evolved a mechanism to enhance tolerance to freezing during exposure to periods of low, but non-freezing temperatures. This phenomenon is called cold acclimation. During cold acclimation, plants develop several mechanisms to minimize potential damages caused by low temperature. Cold response is highly complex process that involves an array of physiological and biochemical modifications. Furthermore, alterations of the expression patterns of many genes, proteins and metabolites in response to cold stress have been reported. Recent studies demonstrate that post-transcriptional and post-translational regulations play a role in the regulation of cold signaling. In this review article, recent advances in cold stress signaling and tolerance are highlighted. Arabidopsis, exhibit a variable degree of chilling tolerance and can increase their freezing tolerance during exposure to chilling and non-freezing temperatures. This process is known as cold acclimation , which is monitored by a chimera protein, formed by the fusion of aequorin to nucleaoplasmin, is also transiently increased after cold shock, and the peak of nuclear [Ca2+] is delayed at 5 to 10 s, compared to the peak of cytosolic [Ca2+] [2+] is caused by nuclear envelope, which is continuous with the endoplasmic reticulum, one of major Ca2+ stores [2+] can be propagated by cytosolic Ca2+ transients via the nuclear pore complexes  . In anim+ stores . The incomplexes . Becausen plants , as welln plants , identif2+ signature can be decoded by different pathways. Plants possess groups of Ca2+ sensors, including CaM  and CMLs (CaM-like), CDPKs (Ca2+-dependent protein kinases), CCaMK (Ca2+-and Ca2+/CaM-dependent protein kinase), CAMTA (CaM-binding transcription activator), CBLs  and CIPKs (CBL-interacting protein kinases). Genetic analysis demonstrated that CDPKs work as positive regulators   . The col alfalfa . TogetheCBF/DREB1-dependent cold signaling pathway is the best characterized and the key regulatory pathway . Ap. ApArabiOR genes . The iceabidopsis. In the ensation , suggestcription . ICE1 iscription  and sumocription   and SlP5CS (Δ1-pyrroline-5-carboxylase synthase) in tomatoes (Solanum lycopersicum) .–50.CBF/Deratures  . Th. ThArabireatment . HOS1 isreatment . The subreatment . A recen daytime . HOS1 isof HOS1-S. On the of HOS1-S. Anotherof HOS1-S.Triticum durum, the RING-finger E3 ligase TdRF1 (RING-finger protein1) interacts with another RING-finger E3 ligase, WVIP2 (wheat viviparus1 interacting protein2), and both genes are upregulated by cold treatment [TdRF1 increases the tolerance of barley cells to dehydration, suggesting that TdRF1 may function in the protection of plants from drought and freezing conditions.The overexpression of another E3 ligase, AtCHIP (carboxyl terminus of HSC70-interacting protein), causes increased sensitivity to low temperatures compared with the wild-type, suggesting its role as a negative regulator of cold response . In wheareatment . The trareatment . The ovesiz1 mutant, which is impaired in the SUMO E3 ligase [CBF3/DREB1A transcription  conjugation, or sumoylation, is one of the post-translational modifications ,135. Sum3 ligase , exhibit3 ligase , sumoylacription . Recent 5 mutant . STA1 isolerance .Low temperature affects several aspects of plant adaptation, e.g., freezing tolerance, plant growth, abiotic resistance and senescence . Among pCBF genes are also induced by exogenous ABA [VIN3, but induces the expression of another cold-regulated gene, ADH1[The ABA level increases in response to low temperatures ,165. Thenous ABA . Among cene, ADH1. These oene, ADH1,169 and Arabidopsis, cold treatment upregulates the GA 2-oxidase gene, encoding the GA-catabolizing enzyme, and represses the GA 20-oxidase gene, encoding a GA-biosynthesizing enzyme. A wheat variety, reduced height 3 (Rht3), exhibits a dwarf phenotype under 20 °C, but normal growth under 10 °C. Rht3 is defective in GA signaling, indicating the importance of GA signaling for warm temperature-dependent cell growth [CBF1/DREB1B-overexpressing plants is caused, in part, by accumulation of DELLA proteins [CBF1/DREB1B-overexpressing plants is suppressed by the application of GA and the introduction of the double DELLA mutation gai-t6 rga-24[Temperature has strong effects on the architecture of some plants. Among the above hormones, auxin and GA are directly related to cell-elongation under warm conditions . On the l growth . Hence, proteins . DELLA iproteins . Thus, gt6 rga-24. This daArabidopsis shoots, wheat and grape berry [etc. [Arabidopsis mutants, such as cpr1 (constitutive expresser of pathogenesis-related gene1) and acd6 (accelerated cell death6), in which SA is over-accumulated, exhibit a dwarf phenotype and freezing sensitivity [CBF3/DREB1A and, therefore, is one of the central regulators of cold signaling, as described above [ice1 mutant, SA-inducible genes are upregulated [CBF2/DREB1C to positively regulate cold tolerance [EDS1 to repress disease resistance [Endogenous free SA and glucosyl SA are accumulated during chilling in pe berry –176. Andry [etc. –180. Howsitivity ,181. ICEed above . In addiegulated . These rolerance  and the sistance . These rCBF2/DREB1C overexpression in Arabidopsis shows a delay of senescence in both natural and artificially induced senescence conditions. In the latter cases, an extended lifespan was observed under the darkness, ethylene or ABA treatments, although the detailed molecular mechanism still remains to be solved [Leaf senescence is a complex and tightly regulated process related to cold stress . CBF2/DRe solved .2 assimilation rate, due to changing RubisCO content and nitrogen efficiency, depending on the ambient temperature, whereas cold-sensitive species, such as cucumber, tobacco and rice, are not [Arabidopsis can modify the state transition, PSII and PSI balance, through the phosphorylation and migration of LHCII as cold responses [As described above, low temperature affects plant architecture. How cold temperatures are sensed is the great enigma for plant scientists. One hypothesis is that the energy balance in chloroplasts may function in sensing ambient temperature ,186. Alt are not . Light s are not –195. Ano are not ,197. Araesponses . Despite2+ signaling and chloroplast status, as well as clock genes. Identification of CAMTA is direct evidence to connect between Ca2+ signaling and regulation of CBF/DREB1. Because the Ca2+ concentration is transiently increased by cold shock in the cytosol and nucleus, decoding of this Ca2+ signature is required for transducing cold signaling downstream. CAMTA may be one of the candidates for a decoder. The next big challenge should be identification of cold sensors. As described above, multiple primary sensors are thought to be involved in sensing low temperatures, and each sensor may perceive a specific aspect of the stress. Perception of membrane rigidification and an imbalance between capacity of light-energy harvest and of energy dissipation may be included. The elucidation of the sensory mechanism and signaling mechanism from sensor(s) to cold signaling is an important aim in achieving a complete understanding of the cold signaling mechanism.The complex and interactive relationships among different pathways are involved in the regulation of cold acclimation. Among them, the ICE1-CBF/DREB1-dependent pathway is likely to play a central role in regulation of cold signaling, as described above . ICE1 an


Orbital mucormycosis caused by Zygomycetes is a rare and fatal infection that generally affects the patients who are immunocompromised. Despite antifungal therapy and aggressive surgical intervention, mucormycosis can cause serious and rapidly fatal infections if delayed diagnosis or therapeutic management occurs. Here, we report orbital mucormycosis in a healthy boy, with a favorable outcome after aggressive treatment. He has had no recurrence since the end of his treatment.A 2-year old healthy boy, some days after entry of dust particle to his left eye presented with swelling and redness of the eye. With diagnosis of “periorbital cellulitis” intravenous antibiotics vancomycin (40 mg/kg/day) and ceftriaxone (75 mg/kg/day) were started but no improvement was observed. The results of biopsy and tissue culture led us to a diagnosis of mucormycosis. Orbital exenteration, combined with intravenous amphotericin B (1 mg/kg/day), resulted in the patient's survival.Due to the high mortality rate of mucormycosis, early diagnosis based on clinical findings and biopsy could be effective for management of the patients suffering from this infection. Mucorales order belonging to the Phycomycetes classof which, the main pathogens belong to the Rhizomucor, Rhizopus, Absidia and Mucor species  and ceftriaxone (75 mg/kg/day) for 10 days, but no improvement was observed and referred to Nemazee hospital. At examination, swelling of upper and lower lids, more prominent on lower part was seen . Proptos3/mm3, neutrophil:32%, lymphocyte 50%, monocyte 14%, eosinophil 4%, hemoglobin 10.4 g/dL, mean corpuscular volume (MCV) 67.1fl, mean corpuscular hemoglobin (MCH) 18.6 pg, platelet count 152000/mm3, ESR 25 mm/1sthour, C. reactive protein 6, blood sugar 85 mg/dl. Total cerebrospinal fluid (CSF) cell count was 10 including 2 white blood cell . The sugar and protein of CSF were 69 and 22 mg/dl, respectively. Gram staining of CSF did not show any organism. Other rare disorders that present with proptosis such as Wegener granulomatosis, leukemia, Burkitt lymphoma and histiocytosis were investigated. Peripheral antineutrophil cytoplasmic antibodies (P-ANCA) and C-Anti-neutrophil cytoplasmic antibodies (C-ANCA) were negative and bone marrow aspiration and trephine biopsy revealed normocellular marrow with no evidence of metastasis. Immunohistochemical study for S100, CD30, CD20, CD3 and BCL-2, myeloperoxidase was negative in histocyte-like cells.Laboratory investigation revealed: white cell count 18.2×10Computer tomography scan of the orbits demonstrated partly cystic solid occupying lesion in the medial aspect of left orbit with compression of the optic nerve and irregularity of the wall with central necrosis detected .1, IgG2, IgG3, IgG4, IgA, C3, C4, CD+3 ( T-cell), CD+4, (helper T-cell), CD+8(cytotoxic T-cell), CD+16, CD+4/ CD+8 ratio, B-cell, and Natural killer cell. HIV test, blood and sinus culture results were negative.Anterior orbitotomy and orbital mass biopsy of the left eye were done, no evidence of pus was detected and only necrotic tissue was seen. Four pieces of creamy-white, rubbery tissue was removed and sent to pathology and mycology labs. The pathology result was reported as vague aggregates of histiocytes with perivascular lymphocyte. Some areas of necrosis with hyphen elements were detected. In the mycology lab, direct smear of tissue with potassium hydroxide showed non-septated hyphae  and afteSurgical exenteration of the left eye was done and amphotericin B (1 mg/kg) and clindamycin (30 mg/kg) daily were started for him. He was discharged after about 4 weeks and treatment was extended with itraconazole (5 mg/kg/day) for 6 months. Two-year follow up examination revealed no evidence of recurrence.The ethics committee of Clinical Microbiology Research Center, Shiraz University of Medical Sciences has reviewed and approved the study regarding the patient written consents before participating in the study.Zygomycetes has coincided with an increase in the incidence of Mucorals  . Standar