Fusarium poae strain SX63, and molecularly characterized. FpV2 and FpV3, with respective genome sequences of 9518 and 9419 base pairs (bps), are both predicted to contain two discontinuous open reading frames (ORFs), ORF1 and ORF2. A hypothetical polypeptide (P1) and a RNA-dependent RNA polymerase (RdRp) are encoded by ORF1 and ORF2, respectively. Phytoreo_S7 domain (pfam07236) homologs were detected downstream of the RdRp domain  of the ORF2-coded proteins of both FpV2 and FpV3. The same shifty heptamers (GGAAAAC) were both found immediately before the stop codon UAG of ORF1 in FpV2 and FpV3, which could mediate programmed –1 ribosomal frameshifting (–1 PRF). Phylogenetic analysis based on RdRp sequences clearly place FpV2 and FpV3 in a taxonomically unassigned dsRNA mycovirus group. Together, with a comparison of genome organization, a new taxonomic family termed Fusagraviridae is proposed to be created to include FpV2- and FpV3-related dsRNA mycoviruses, within which FpV2 and FpV3 would represent two distinct virus species.Two novel double-stranded RNA (dsRNA) mycoviruses, termed Fusarium poae dsRNA virus 2 (FpV2) and Fusarium poae dsRNA virus 3 (FpV3), were isolated from the plant pathogenic fungus,  Hypoviridae, Narnaviridae, Alphaflexiviridae, Gammaflexiviridae, Endornaviridae, and Barnaviridae), five consist of dsRNA genomes , and the other two families hold RNA reverse-transcribing genomes (Metaviridae and Pseudoviridae) [Mycoviruses, or fungal viruses, selectively infect fungi and are widespread in all major taxonomic fungal groups . Most myviridae) . In addiviridae) ,7,8,9.Megabirnaviridae, Hypoviridae, Partitiviridae, Narnaviridae, and Reoviridae, or the unassigned negative-strand ssRNA and ssDNA mycoviruses, can cause clearly abnormal symptoms in the host, such as reducing mycelial growth, decreasing production of spores and/or sclerotia, suppressing biosynthesis of secondary metabolites, and attenuating aggressiveness or virulence [Hypoviridae family is a classic example and has been reported as a promising biocontrol agent for combating chestnut blight [In most cases, mycovirus infection causes little or inconspicuous symptoms in their host ; neverthirulence ,10,11,12t blight . In addiFusarium species. These filamentous fungi include important plant pathogens [Fusarium spp. also produce mycotoxins that may have a negative impact on public health [F. poae but low in other Fusarium species. Among 55 F. poae isolates collected from wheat in different geographical regions in the world, all contained dsRNAs and encapsidated virus-like particles [F. poae isolate, they were stable after repeated subculturing [F. poae isolates containing dsRNAs displayed any morphological changes, which demonstrates that the mycoviruses do not greatly harm the host. One of these mycoviruses, obtained from F. poae isolate A-11, was later identified as Fusarium poae virus 1 (FpV1), a member of the genus Partitivirus—which was the only report of the complete genome sequence of mycovirus from F. poae [The hosts of dsRNA mycoviruses contain many athogens  that cauc health . The frearticles . In addiulturing . None of F. poae .Fusarium poae strain SX63. We recommend that FpV2 and FpV3, together with the previously-reported SsNsV-L, BcRV1, FgV3, MpRV2, FvV1, and FvV2, are placed in a group, for which a new family Fusagraviridae is proposed.In this study, we present the sequence and molecular characteristics of two novel relative dsRNA mycoviruses, termed Fusarium poae dsRNA virus 2 (FpV2) and Fusarium poae dsRNA virus 3 (FpV3), both isolated from identical Fusarium spp., was identified as F. poae by PCR amplification of the translation elongation factor (EF-1) fragment [ca. 9 and 2.5 kb, respectively  with a partial consensus sequence of the provisional large tegument protein UL36 , which is conserved in the large tegument protein in the family herpesviridae : ORF1 and ORF2 on their genomic plus strand, with long 5′-UTRs of 1029 and 1190 bp, and relatively short 3′-UTRs of 48 and 55 bp, respectively . The 5′-sviridae . NeverthThe 3′-proximal ORF2 of FpV2 and FpV3  potentially encode 1361 and 1354 aa proteins with predicted molecular masses of 152.6 and 149.4 kDa, respectively. These proteins have a low percentage of sequence similarity (28%–44%) to the putative RNA-dependent RNA polymerases (RdRps) of the same nine unclassified viruses mentioned above, as well as two other unclassified dsRNA viruses found in a BLASTP search: Diplodia scrobiculata RNA virus 1 (DsRV1)  and PhytTotiviridae, four members of the family Chrysoviridae, two members of the genus Phlegivirus (suggested) [Megabirnaviridae and Partitiviridae were subjected to phylogenetic analysis. It was showed that FpV2 and FpV3 form a clear, well-supported taxonomic cluster together with SsNsV-L, BcRV1, FgV3, MpRV2, FvV2, FvV1, PgV2, DsRV1, PMeV, and PiRV3 in the unclassified dsRNA viruses. This clade is closely relevant with the clades for Phlegivirus, Megabirnaviridae, Chrysoviridae, and Totiviridae. However, it is distantly related to the clade for Partitiviridae. Results also showed that within the unassigned clade, FpV2 is placed between the FvV1 and PgV2 branches, and FpV3 is placed between the FgV3 and MpRV2 branches. The putative RdRp encoded by FpV2 shares the highest sequence identity with that of MpRV2 (36%) and PgV2 (36%), respectively, while RdRp encoded by FpV3 shares the highest sequence identity with that of FgV3 (44%), SsNsV-L (44%), and GaTV2 (44%), respectively (A phylogenetic tree was constructed using the neighbor-joining (NJ) method based on the RdRp sequences of FpV2, FpV3, and 33 selected RNA viruses A. Togethggested) , the unaectively .Furthermore, phylogenetic analysis based on the viral P1 protein clearly showed that the unassigned dsRNA virus clade includes more than two groups, with one of them grouping FpV2, FpV3, SsNsV-L, BcRV1, FgV3, MpRV2, FvV1, and FvV2 B, supporet al. [Chrysoviridae and Endornaviridae through multiple horizontal gene transfer (HGT) events among diverse virus groups. The function of this domain in these non-phytoreoviruses is not known. Interestingly, the S7 domain is located downstream of the RdRp domain of ten members of the closely-related 12 unclassified viruses, including FpV2, FpV3, SsNsV-L, FgV3, BcRV1, MpRV2, FvV1, FvV2, DsRV1, and PgV2. However, the S7 domain is located upstream of the RdRp domain in Endornaviruses and Chrysoviruses [The Phytoreo_S7 domain is characteristic of a protein family composed of several phytoreovirus S7 proteins that are viral core proteins with the activities of binding nucleic acid. The Phytoreo_S7 domain pfam07236) were detected by CDD searches only in SsNsV-L  and FgV3 were detet al.  revealedoviruses . A similEscherichia coli expression system [Programmed –1 ribosomal frameshifting is a common translational recoding mechanism in RNA viruses as same as in other organisms . When –1n system . Thus, fMegabirnaviridae, Chrysoviridae, and Totiviridae  containing FpV2, FpV3, SsNsV-L, BcRV1, FgV3, MpRV2, FvV1, and FvV2 viruses. The proposed family can be readily distinguished from other known mycovirus families on account of the size of their monopartite genomes (8112~9518 bp), the genomic structure with putative –1 PRF translational recoding mechanism, a long 5′-UTR (865–1310 bp) and a relatively short 3′-UTR (7–131 bp), and the arrangement of S7 and RdRp domains   and FgV3 , SMC   in MpRV2, which proteins have the ability of binding DNA and acting in organizing and segregating chromosomes for partition; SIS (Sugar ISomerase)  in SsNsV-L, which exist in many phosphosugar isomerases and phosphosugar binding proteins, and in proteins that regulate the expression of genes referred to synthesis of phosphosugars; flhF (flagellar biosynthesis regulator FlhF)  and PHA03418   in FvV1  and 26.46%–43.27% (RdRp), respectively, among the members of the family or RdRp) ; Second, in FvV1 ; Third, aviridae . These dFusarium poae strain SX63 was isolated from the disease glumes of wheat infected with Fusarium spp. collected in the Shanxi province of China. The F. poae strain SX63 was routinely cultured on potato dextrose agar (PDA) at 25 °C in the dark. Mycelial plugs were stored in 25% glycerol at −80 °C. Small mycelial agar plugs were grown on top of cellophane membranes overlaid on PDA plates for three days at 25 °C in the dark. The mycelium was subsequently harvested for dsRNA extraction.The harvested mycelial mass was ground in liquid nitrogen to a fine powder; the dsRNA was then extracted from the fine powder, following the CF-11 cellulose chromatography method previously described  . Total n® HS DNA Polymerase  on a Thermal Cycler  according to the manufacturer’s instructions. The products were fractionated on 1.0% agarose gel and agarose gel-purified with a gel extraction kit . The resulting products were cloned into the PMD18-T vector  and transformed into Trans 5α Chemically Competent Cells . Positive clones were selected and sequenced by the Sanger method at the Beijing Genomics Institute .cDNA synthesis and molecular cloning of dsRNA were performed as previously described ,36. The ® High Fidelity (HiFi) PCR SuperMix  for specific PCR amplification. PCR products were purified, cloned, and sequenced as previously described.dsRNA-specific primers were designed for RT-PCR amplifications based on the obtained sequences in order to identify the gap sequences between different clones. RT-PCR amplifications were performed as previously described ,36. In bet al. [et al. [The 5′- and 3′-terminal sequences of dsRNA were determined to obtain the complete nucleotide sequences by a 3′ RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RLM-RACE) protocol, as described by Xie et al.  and Chib [et al. . All ampThe whole nucleotide sequences of dsRNA were completed and identified by overlapping of cDNA clones, and each base was ascertained by sequencing at least four independent clones.The resulting nucleotide sequences were assembled and analyzed using DNAMAN Software . The translations of ORFs were also conducted using DNAMAN software, and potential ORFs were found by using the NCBI ORF Finder tool . ProteinPhylogenetic trees were estimated using the NJ method on the aligned amino acid sequences. The deduced amino acid sequences of the polyproteins or conserved domains were aligned using DNAMAN 6.0 Software with default parameters . The NJ The complete genomic sequences of dsRNA1 and dsRNA2 isolated from the strain SX63 have been deposited in the GenBank database with accession Nos. KU728180 and KU728181, respectively.Hypoviridae, Narnaviridae, Alphaflexiviridae, Gammaflexiviridae, Endornaviridae, Barnaviridae, Reoviridae, Partitiviridae, Chrysoviridae, Totiviridae, Megabirnaviridae, Metaviridae, and Pseudoviridae [F. poae strain SX63. A comprehensive comparison of genome organization and a powerful phylogenetic analysis led us to recommend the creation of a novel family designated Fusagraviridae to include above mentioned FpV2- and FpV3-related dsRNA mycoviruses . The identification of FpV2 and FpV3 inevitably supports the suggestion of the assignation of a new virus family, in which FpV2 and FpV3 represent two distinct virus species.At present, mycoviruses are classified into 13 families, including oviridae . In addioviridae ,7,8,9. I


SCID mice and significantly inhibited by cabozantinib, as was tumor phospho-Met content. These studies indicate the prevalence and functionality of the HGF/Met signaling pathway in UC cells, suggest that paracrine HGF may contribute to UC tumor growth and progression, and that support further preclinical investigation of Met inhibitors for the treatment of UC is warranted.There is mounting evidence of oncogenic hepatocyte growth factor (HGF)/Met signaling in urothelial carcinoma (UC) of the bladder. The effects of three kinase inhibitors, cabozantinib, crizotinib and EMD1214063, on HGF-driven signaling and cell growth, invasion and tumorigenicity were analyzed in cultured UC cell lines. SW780 xenograft growth in SCID and human HGF knock-in SCID (hHGF/SCID) mice treated with cabozantinib or vehicle, as well as tumor levels of Met and pMet, were also determined. Met content was robust in most UC-derived cell lines. Basal pMet content and effector activation state in quiescent cells were low, but significantly enhanced by added HGF, as were cell invasion, proliferation and anchorage independent growth. These HGF-driven effects were reversed by Met inhibitor treatment. Tumor xenograft growth was significantly higher in hHGF/SCID mice  In 2013, 72,570 new cases of bladder cancer (BCa) and 15,210 bladder cancer-related deaths were estimated in the U.S. alone . These cin vivo [FGFR3 (13%), ERBB2 (6%), FGFR1 (6%) and MET (2%) [et al. [MET or HGF genes in 3% and 2%, respectively, of cases in the Nature Genetics 2013 dataset (n = 99) [n = 131) [n = 201). Together these studies identified genetic aberrations in HGF and MET at an average combined frequency of 10% in 528 BCa cases.The cell surface receptor tyrosine kinase (RTK) for hepatocyte growth factor (HGF), known as Met, is widely present in cells of epithelial origin. HGF/Met signaling is frequently implicated in cancer, driving tumor invasiveness and metastasis . Evidencin vivo ,10,11. Ain vivo . The RTKMET (2%) . The RASMET (2%) . A prelio [FGFR3 %, ERBB2 MET (2%)  further  [et al. , as well(n = 99) , in 10% n = 131) , and in While several Met kinase inhibitors are in preclinical and clinical development, there use for the treatment of BCa has been limited. Here, we focused on three small molecule Met inhibitors, crizotinib, cabozantinib and EMD1214063. Crizotinib is an orally bioavailable small molecule inhibitor of Met and anaplastic lymphoma kinase (ALK), which has been US FDA approved for the treatment of ALK-positive non-small cell lung cancer . CrizotiAccurately selecting therapies that will be effective against a specific tumor is the ultimate goal of clinicians treating BCa. In a prior study we found that levels of soluble Met ectodomain (sMet), which are readily measured in the urine of bladder cancer patients and those with no evidence of cancer, could distinguish patients with BCa from those without, and patients with or without muscle-invasive BCa, suggesting the potential utility of urinary sMet as a BCa biomarker for surveillance following initial treatment . In paralow), intermediate (Metint) or high (Methigh) Met content  for multiple human tumor derived cell lines  and 12 UC derived cell lines as measured by electrochemiluminescent two-site immunoassay are listed in  content A. This o50 and IC90 values, respectively; 50 and IC90 values, respectively; None of the UC derived cell lines produced detectable levels of HGF, as determined by two-site electrochemiluminescent immunoassay (200 femtomole limit of detection), suggesting that autocrine HGF/Met signaling is rare in UC of the bladder. Treatment with exogenous HGF (1 nM) induced significant Met kinase activation (pMet/Met SI ratio) in all UC derived cell lines within 20 min, which was suppressed by the small molecule Met kinase inhibitors crizotinib at 5 and 10 nM,  and MAPK pathways downstream of Met mediates HGF-driven cell survival, migration, invasion and proliferation. HGF stimulated both Akt and Erk phosphorylation in all UC derived cell lines . Treatme TCC-SUP A, left, nd SW780 A, right d HT1376 C. Treatm TCC-SUP B, top, t5, SW780 B, bottom5, SW780 C, left, ines J82 B, bottomines J82 C. These low lines RT4, T24M2, T24M3 and TCC-SUP, the Metint lines UMUC5 and SW780, and the Methigh line J82 Aveo). Growth of SW780 cell xenografts in SCID mice was significantly slower than in hHGF/SCID mice (3 (~day 18), significantly blocked, and later reversed, SW780 tumor growth in both SCID  overexpression of ET genes ,14, and ET genes . EpithelET genes . ReinforET genes . HGF proET genes . A priorET genes . FinallyET genes . These sMET can confer resistance to crizotinib and other Met kinase inhibitors [MET gene mutation in the four TCGA bladder cancer datasets (11/569 cases or 1.9% combined), the small molecule Met inhibitors crizotininb, cabozantinib and EMD1214063 uniformly and potently suppressed Met and Met-effector activation, and HGF-driven invasion, growth and soft agar colony formation in all UC cell lines tested. Our cell-based assay results do not reveal a relationship between Met abundance and biological response to HGF, or between Met abundance and drug suppression of HGF-induced responses, in the 12 UC-derived cell lines studied. In this regard BCa is not similar to certain other cancers, such as gastric and lung adenocarcinomas, where frequent MET gene amplification leads to ligand-independent activation and enhanced sensitivity to Met inhibition [Mutations in hibitors . Consisthibition  are suggSubcutaneously implanted SW780 cells grew significantly faster in hHGF/SCID mice relative to SCID mice, revealing the ability of paracrine delivered HGF to drive UC tumor growth. Cabozantinib fully suppressed SW780 xenograft growth in mice, and prevented the elevated tumor pMet observed in vehicle-treated hHGF knock-in mice. Additional studies will be needed to discern meaningful differences in efficacy among these agents against UC tumor growth, particularly whether the broader target spectrum of cabozantinib and, to a lesser degree, crizotinib, is potentially advantageous, and whether drug effects on the host, such as on tumor-stroma interactions and the tumor vasculature, also contribute to anti-tumor efficacy.Tissue culture media and supplements were obtained from Invitrogen . Antibodies against pMet (1234/1235), pErk, tErk, pAkt, and tAkt were obtained from Cell Signaling Technology . Anti-Met (C-28) was obtained from Santa Cruz Biotechnology . Purified recombinant human HGF was obtained from ProSpec . Crizotinib, cabozantinib and EMD1214063 were obtained from the Open Chemical Repository Collection, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.SW480, SW620, CRL 7636, CRL 7637, HTB 125, MCF7, 786-0, ACHN, PC3, UMUC3, UMUC5, H596, A549, MKN45, RT-4, TCC-SUP, T24, HT1197, SW780, J82, 5637 and HT1376 cells were obtained from ATCC . PC3M was obtained from MD Anderson Cancer Center, University of Texas . T24M2 and T24M3 cell lines were derived from metastases of T24 xenografts in mice. Cells were cultured in DMEM medium containing 10% FBS and antibiotic-antimycotic. Cells were grown in 5% CO2 at 37 °C.Cells were washed with cold PBS, extracted in Laemmli buffer, sonicated, heated for 5 min at 95 °C prior to SDS-PAGE and electrophoretic transfer to nitrocellulose. Membranes were blocked with 5% milk in TBST  for 1 h at 25 °C then incubated 16 h at 4 °C with primary antibody in TBST/0.5% milk. Membranes were washed three times with TBST, incubated with horseradish peroxidase-labeled secondary antibody for 1 h at 25 °C, washed for 3 h with TBS prior to ECL detection . Phospho- and total Met content in Triton X-100 cell extracts were analyzed by electrochemiluminescent two site immunoassay using a SectorImager 2400 plate reader  as described . The asshttp://imagej.nih.gov/ij/, 1997–2012). For proliferation assays, cells were plated in triplicate at a density of 5 × 104 cells/35-mm dish in defined medium. HGF ± crizotinib or cabozantinib were added on days 1, 2, and 4. Cells were trypsinized and counted using a hemocytometer on days 3 or 6.Invasion assays were performed using 8 um pore size BD Bioscience BioCoat Matrigel Invasion chambers  according to the manufacturer’s instruction. Images were captured by light photomicrography and quantitation was performed using Image J software V1.47  in phenol-red free DMEM was added to 96 well plates. Cells in 0.3% agarose in phenol-red free DMEM were added on top of the base layer. Cells were fed with DMEM with or without HGF and crizotinib or cabozantinib every other day. After one week, MTT was added to quantify the viable colonies by absorbance using a Victor plate reader .http://www.informatics.jax.org) were acquired from The Jackson Laboratory. SW780 cells (3 × 106 per site) suspended in matrigel were injected subcutaneously into both flanks of 5 mice per treatment group, and tumor volumes were calculated from caliper measurements. When tumor volume reached 150 mm3, mice were treated with either vehicle or cabozantinib once daily by oral gavage. Tumors excised from mice were disrupted in non-ionic detergent containing buffer, lysates were cleared by centrifugation, and analyzed using two-site immunoassays for Met and pMet [Experiments with SCID and hHGF/SCID mice were performed in accordance with National Institutes of Health Guidelines for Care and Use of Laboratory Animals using protocols approved by the Institutional Animal Care and Use Committee of the Center for Cancer Research, National Cancer Institute. hHGF/SCID mice (HGF/SCID: Rideout WM 3rd Mouse Genome Database (MGD) at the Mouse Genome Informatics website, The Jackson Laboratory (Bar Harbor, MN, USA; and pMet .p = 0.05 was the threshold for statistical significance.Significant differences between two groups were determined by One Way ANOVA with post hoc comparison of all groups using GraphPad Prism 5 software, where Met protein content in 12 UC-derived cell lines ranged from 12 ng/mg to 133 ng/mg total protein, comparable to that of several other well-studied tumor-derived cell lines from other prevalent cancers. HGF production was conspicuously absent, but exogenous HGF stimulated the activation of Met and known downstream effectors, and significantly enhanced UC cell invasion, proliferation and anchorage-independent growth. Concomitant treatment with cabozantinib, crizotinib or EMD1214063 effectively suppressed these HGF-driven effects. Cabozantinib also significantly inhibited HGF-driven tumor xenograft growth and Met activation in SCID and, more dramatically, in hHGF/SCID mice. Our results suggest that further studies are warranted to fully define the prevalence and oncogenic impact of HGF/Met signaling in bladder cancer, and to identify reliable and practical biomarkers of pathway activation for patient selection and predicting response to treatment with HGF/Met targeted therapeutics.


Crude odds ratios (ORs) for PD in the quartiles of serum 25(OH)D were 1 (reference), 0.710 , 0.631 , and 0.483 , respectively. Crude ORs for PD in quartiles of sunlight exposure were 1 (reference), 0.809 , 0.623  and 0.533 , respectively. A significant positive correlation between serum 25(OH)D and sunlight exposure was found, but serum 25(OH)D was not correlated with daily vitamin D intake. This study indicates that lower levels of serum 25(OH)D and sunlight exposure are significantly associated with an increased risk for PD.Circulating vitamin D has previously been found to be lower in patients with Parkinson’s disease (PD), while the effects of sunlight exposure have not yet been fully investigated. Therefore, we evaluated the associations between serum vitamin D, vitamin D intake, sunlight exposure, and newly-diagnosed PD patients in a Chinese population. This case-control study measured serum 25-hydroxyvitamin D (25(OH)D) levels and sunlight exposure in 201 patients with newly-diagnosed PD and 199 controls without neurodegenerative diseases. Data on vitamin D intake and sunlight exposure were obtained using a self-report questionnaire. Multivariable logistic regressions were employed to evaluate the associations between serum 25(OH)D levels, sunlight exposure, and PD. Adjustments were made for sex, age, smoking, alcohol use, education, BMI, and vitamin D intake. There were significantly lower levels of serum 25(OH)D (20.6 ± 6.5 ng/mL), daily vitamin D intake (8.3 ± 3.7 g/day), and sunlight exposure (9.7 ± 4.1 h/week) in patients with PD compared to healthy controls ( Parkinson’s disease (PD) is a common progressive neurodegenerative disorder mostly occurring in elderly humans. Although the pathogenesis of PD is unclear, environmental factors, including some vitamins, have been suggested to be linked with the development of PD ,3,4,5,6.Previously, vitamin D was considered to be only related to bone metabolism, osteoporosis, and falling. However, vitamin D has recently emerged as a critical factor in the pathogenesis of chronic diseases and progressive neurodegenerative disorders ,7,8. AddLow circulating vitamin D occurs in populations of all ages and geographic latitudes. Sources of vitamin D include exposure to sunlight, diet, and vitamin D supplements. However, neither diet alone, nor the current recommendation of 800 to 1200 international units of vitamin D supplementation may be enough to provide adequate levels of circulating 25(OH)D. Moreover, 20% of vitamin D is obtained from food , whereas 80% is obtained from ultraviolet radiation inducing skin synthesis as a product of skin 7-dehydrocholesterol trans-formations . PreviouA total of 201 newly-diagnosed cases of PD were recruited from the clinic of the Department of Neurology, Yongchuan Hospital of Chongqing Medical University, Chongqing, China, while 199 age- and sex-matched healthy controls were recruited from three community hospitals during the winter of 1 November 2014 and 1 January 2015 in Chongqing, China. PD was diagnosed by neurologists according to the UK PD Society Brain Bank clinical diagnostic criteria. Participants were excluded if they fulfilled any of the following exclusion criteria: presence of any other neurodegenerative disease, thyroid dysfunction, and recent modification of lifestyles or dietary patterns. All participants completed a questionnaire-based interview and received a physical examination. Interviewers, doctors, and nurses were trained in administering the questionnaire and physical examination prior to study commencement. All participants were informed about the details of the study and signed a consent form. The study protocol was approved by the local ethics committee.Educational status, sunlight exposure habit, smoking, drinking, and medical history of all participants were obtained by face-to-face questionnaires. Dietary intake was measured by the validated semi-quantitative food-frequency questionnaire . RequireAfter fasting for 10 h, samples of blood were obtained from all participants, centrifuged at 2500 g for 15 min for serum, and stored at −80 °C. Serum 25(OH)D concentrations were detected by ACQUITY Ultra Performance Liquid Chromatography . Inter- and intra-assay coefficients of 25(OH)D at 20 ng/mL were 4.56% and 3.81%, respectively. Serum 25(OH)D concentration levels of less than 20 ng/mL were considered as vitamin D deficiency; levels between 20 and 30 ng/mL were considered as vitamin D insufficiency; and levels over 30 ng/mL were considered as vitamin D sufficiency [p < 0.05 was considered statistically significant.Clinical variables were expressed as mean ± standard deviation (SD) or percentages as appropriate. The chi-square test was employed to assess the differences in proportions. Associations between serum 25(OH)D, sunlight exposure and PD development were analyzed by multiple variable logistic regression analysis by adjusting for the covariates of age, sex, BMI, smoking, alcohol use, and vitamin D intake. The correlations between serum 25(OH)D and sunlight exposure were analyzed by multiple linear regression analysis. A value of As serum 25(OH)D was not correlated with vitamin D intake, vitamin D intake was analyzed as a covariate in the logistic regression . 25(OH)Dp < 0.05).A significant positive correlation between serum 25(OH)D and sunlight exposure was observed, but serum 25(OH)D was not correlated with daily vitamin D intake . Table 3In this case-control study, we found that levels of serum 25(OH)D and sunlight exposure were lower in patients with PD and were inversely associated with the risk for PD. Several studies have indicated lower levels of serum vitamin D in PD ,9 but laThe present study supports the hypothesis that lower serum vitamin D could be a risk factor for PD. Although it is unclear how vitamin D could protect against PD, vitamin D has been found necessary for the regulation of some neurodegenerative processes, such as neurotrophin, inducible nitric oxide synthase, glutathione and monoamine synthesis, and apoptosis. In addition, vitamin D has multiple beneficial effects on the nervous system, such as anti-oxidative stress, calcium regulation of neurons, immune system modulation, nerve conduction strength, and detoxification . Additioet al. reported that men working outdoors have a lower risk of PD [In order to reach conclusive results, future studies investigating the effects of sunlight exposure on serum 25(OH)D need to include the exposure time as a measurement. We found a significant inverse association between sunlight exposure and PD. In line with our study, Kenborg sk of PD . Howeversk of PD . Meanwhisk of PD ,22,23, aSome limitations of the present study need to be considered. First, the design of a case-control study could not provide causal effects of serum vitamin D on PD development. Second, the vitamin D status could not be fully represented due to absent measurements of serum 1,25(OH)2D3. In addition, the study took place in winter and no measurements were taken during summer. Although serum vitamin D is relatively stable over time , the posIn sum, our study found that lower levels of serum 25(OH)D and sunlight exposure are significantly associated with a higher risk for PD. Further cohort and vitamin D supplementation studies are needed to confirm this finding.


Fragaria vesca (2n = 2x = 14), the woodland strawberry, is a perennial herbaceous plant with a small sequenced genome (240 Mb). It is commonly used as a genetic model plant for the Fragaria genus and the Rosaceae family. Fruit skin color is one of the most important traits for both the commercial and esthetic value of strawberry. Anthocyanins are the most prominent pigments in strawberry that bring red, pink, white, and yellow hues to the fruits in which they accumulate. In this study, we conducted a de novo assembly of the fruit transcriptome of woodland strawberry and compared the gene expression profiles with yellow  and red  fruits. De novo assembly yielded 75,426 unigenes, 21.3% of which were longer than 1,000 bp. Among the high-quality unique sequences, 45,387 (60.2%) had at least one significant match to an existing gene model. A total of 595 genes, representing 0.79% of total unigenes, were differentially expressed in YW and RG. Among them, 224 genes were up-regulated and 371 genes were down-regulated in the fruit of YW. Particularly, some flavonoid biosynthetic pathway genes, including C4H, CHS, CHI, F3H, DFR and ANS, as well as some transcription factors (TFs), including MYB (putative MYB86 and MYB39), WDR and MADS, were down-regulated in YW fruit, concurrent with a reduction in anthocyanin accumulation in the yellow pigment phenotype, whereas a putative transcription repressor MYB1R was up-regulated in YW fruit. The altered expression levels of the genes encoding flavonoid biosynthetic enzymes and TFs were confirmed by quantitative RT-PCR. Our study provides important insights into the molecular mechanisms underlying the yellow pigment phenotype in F. vesca. Fragaria vesca, commonly called woodland strawberry, is emerging as an advantageous alternative system for the cultivated octoploids as well as the Rosaceae family due to its small genome size (240 Mb), diploidy (2n = 2x = 14), small herbaceous stature, ease of propagation, short reproductive cycle, and facile transformation , and detailed information is presented in Some flavonoid pathway genes were down-regulated in the fruits of YW compared with the fruits of RG in both biological replicates, including the genes encoding C4H [EC 1.14.13.11], CHS [EC 2.3.1.74], CHI [EC 5.5.1.6], F3H [EC 1.14.11.9], DFR [EC 1.1.1.219] and ANS [EC 1.14.11.19] . The gen2(YW/RG) = -4.36] in both biological replicates. Some other TFs were down-regulated in YW, including two putative MYB TFs (annotated as MYB86 and MYB39), two putative MADS TFs (annotated as AGL11-like and AGL15-like gene), one putative WD-repeat protein (WDR) TFs (2 (YW/RG) = 3.95].To identify regulatory factors that potentially controlled flavonoid biosynthesis, candidate transcription factors were chosen from the transcriptome data. We initially focused on MWB (MYB-bHLH-WD40) complex proteins, which were key factors in the regulation of primary and secondary metabolism . InteresWDR) TFs . NoticeaWDR) TFs , annotatTo confirm the results of the Illumina RNA-Seq analysis, the expression levels of six flavonoid pathway genes  and the differently expressed TFs  were tested in RG and YW by qRT-PCR using the same samples prepared for RNA-Seq. According to the qRT-PCR data , the traF. vesca was sequenced in 2010. This provided an invaluable resource for studying the molecular mechanisms influencing strawberry development. When a reference genome is available, the sequencing reads are aligned primarily by mapping on to the sequenced reference genome. However, although reference-based approaches is a robust and relatively precise way of characterizing transcript sequences, this method remains problems by its inability to account for un-sequenced genome or structural alterations within mRNAs, such as spliced variants, and also does not solve the problem of hypervariable sequences and private genes [de novo assembly strategy. De novo assembly can reconstruct short sequences of transcripts into entire sequences of transcriptomes, identify all of the expressed genes, separate isoforms and quantify transcript expression levels, which do not depend on the genome [F. vesca, RG and YW, corresponding to 75,426 unigenes.The genome of te genes ,27,28. Me genome . In thisc locus (yellow fruit color) in F. vesca, and the results showed that the c locus were tightly linked with the F3H gene [F. vesca. RNAi silencing of F3H in strawberry fruits also exhibited that the anthocyanin content was greatly reduced and flavonol was also decreased [F. vesca and F. ×ananassa was reflected in the expression of F3’H and DFR1, and F3’H deficient lines displayed white or pale pigmentation phenotype [F. vesca in our RNA-Seq data. The co-down-regulation of many structural genes in YW indicates that yellow fruit phenotype is unlikely a mutation of one specific flavonoid pathway gene and multiple genes or transcription factors interacting with each other may account for the yellow coloration of woodland strawberry fruit.Genome-wide transcriptome analysis of the red and yellow strawberry fruit using RNA-Seq technology revealed significant down-regulation of a number of flavonoid pathway genes, both early biosynthetic genes C4H, CHS as well as CHI and late biosynthetic genes ANS and DFR  concurreF3H gene , which secreased . Recentlhenotype . In thisFvMYB10 in ‘Alpine’ strawberry F. vesca resulted in plants with elevated leaves, petioles, stigmas and fruit anthocyanin concentrations; while the mature fruit of FvMYB10 RNAi lines showed white fruit skin and white flesh [FaMYB1 was described as a transcriptional repressor in regulating the biosynthesis of anthocyanins and flavonols in strawberry [FvbHLH33, which is a potential bHLH partner for FvMYB10, did not affect the anthocyanin pathway when knocked down using an RNAi construct [FaTTG1 gene caused enhanced anthocyanin accumulation in strawberry fruit off plant [The structural genes of plant flavonoid biosynthetic pathway are largely regulated at transcriptional level. The R2R3-MYB TFs played a key role in the regulation of the flavonoid pathway in most plant species ,36. R2R3te flesh . FaMYB1 rawberry . FvbHLH3onstruct . Suppresff plant .FvMYB10, FvbHLH33 and FaTTG1) were not involved in DEGs. Unexpectedly, MYB1 gene was significant down-regulated in the fruits of YW, and it was previous reported that the high level of transcripts of FcMYB1 was detected in white Chilean strawberry [FvMYB10 from YW and RG were sequenced and a missense mutation was found in FvMYB10 due to a G to C base substitution at the 35th nucleotide of the cDNA sequence of YW, resulting in an amino acid Trp to Ser change  in tobacco flowers induced a decrease in anthocyanin accumulation [ABS/TT16 from Arabidopsis is a member of Bs MADS subfamily GGM13-like gene, which was initially found in the control of flavonoid biosynthesis in the yellow seed coat [IbMADS10) from sweet potato (Ipomoea batatas L.) was reported to be related to the red pigmentation [IbMADS10 gene in Arabidopsis showed high accumulation of the anthocyanin pigments [SQUAMOSA-class MADS transcription factor, VmTDR4, resulted in substantial reduction in anthocyanin levels in ripe bilberry (Vaccinium myrtillus) fruits [PyMADS18 and putatively involved in anthocyanin biosynthesis was found in pear (Pyrus communis L.) [In addition, a number of other TFs were differentially expressed in RG and YW, including two putative MYB TFs (annotated as MYB86 and MYB39), two putative MADS TFs (annotated as AGL11-like and AGL15-like), one putative WDR TF and one R3 single-repeat MYB TF (annotated as MYB1R). Among these TFs, MYB86 and MYB39 have not been proved to be involved in flavonoid biosynthesis until now. WDR is more likely to enhance gene activation rather than a direct regulatory function because it commonly has no obvious catalytic activity ,41. MYB1 Mimulus ,42,43. Omulation . MADS ismulation ,45,46. Reed coat . A new Mentation . Over-expigments . Silenci) fruits . A gene unis L.) . Therefode novo assembly of the fruit transcriptomes of two botanical forms of F. vesca contrasting in fruit pigmentation. Our data revealed significant down-regulation of certain flavonoid biosynthetic genes, including C4H, CHS, CHI, F3H, DFR and ANS, concomitant with the pigment-deficient phenotype. In addition, we have identified some transcription factors including MYB, WDR and MADS that potentially control flavonoid biosynthesis.In summary, our results showed the use of RNA-Seq technology to perform a S1 Fig(EPS)Click here for additional data file.S1 File(RAR)Click here for additional data file.S2 File(DOC)Click here for additional data file.S1 Table(XLS)Click here for additional data file.S2 Table(XLS)Click here for additional data file.


Dehalobacter strains (strain CF and strain DCA) were assembled from the metagenome of an anaerobic enrichment culture that reductively dechlorinates chloroform (CF), 1,1,1-trichloroethane  and 1,1-dichloroethane . The 3.1 Mbp genomes of strain CF  and strain DCA  each contain 17 putative reductive dehalogenase homologous (rdh) genes. These two genomes were systematically compared to three other available organohalide-respiring Dehalobacter genomes , and to the genomes of Dehalococcoides mccartyi strain 195 and Desulfitobacterium hafniense strain Y51. This analysis compared 42 different metabolic and physiological categories. The genomes of strains CF and DCA share 90% overall average nucleotide identity and >99.8% identity over a 2.9 Mbp alignment that excludes large insertions, indicating that these genomes differentiated from a close common ancestor. This differentiation was likely driven by selection pressures around two orthologous reductive dehalogenase genes, cfrA and dcrA, that code for the enzymes that reduce CF or 1,1,1-TCA and 1,1-DCA. The many reductive dehalogenase genes found in the five Dehalobacter genomes cluster into two small conserved regions and were often associated with Crp/Fnr transcriptional regulators. Specialization is on-going on a strain-specific basis, as some strains but not others have lost essential genes in the Wood-Ljungdahl (strain E1) and corrinoid biosynthesis pathways (strains E1 and PER-K23). The gene encoding phosphoserine phosphatase, which catalyzes the last step of serine biosynthesis, is missing from all five Dehalobacter genomes, yet D. restrictus can grow without serine, suggesting an alternative or unrecognized biosynthesis route exists. In contrast to D. mccartyi, a complete heme biosynthesis pathway is present in the five Dehalobacter genomes. This pathway corresponds to a newly described alternative heme biosynthesis route first identified in Archaea. This analysis of organohalide-respiring Firmicutes and Chloroflexi reveals profound evolutionary differences despite very similar niche-specific metabolism and function.The genomes of two closely related  Chloroflexi, Firmicutes, and Proteobacteria  to chloroethane (CA) via 1,1-dichloroethane , and also dechlorinates chloroform (CF) to dichloromethane , CfrA, while the second population dechlorinates 1,1-DCA to CA using a distinct RDase, DcrA. We have now isolated strain CF and strain DCA corresponding to the two assembled genomes  and G+C content (44–45%) and comprise ~2900 coding sequences . Sequence insertions within 16S rRNA genes have been reported in some Desulfitobacterium strains  and only two differ from their orthologs in strain DCA. The first, DCF50_p1199, differs from its ortholog in strain DCA, DHBDCA_p1132, by 25 nucleotides out of 1542 corresponding to six amino acids and is not closely related to any other rdhA gene of known function. These two orthologous genes were not found expressed in the ACT-3 culture , is the gene encoding the RDase CfrA. Its ortholog in strain DCA is the dcrA gene (DHBDCA_p1180) encoding DcrA. CfrA and DcrA differ by 22 amino acids. The gene cfrA resides in a cluster , and cfrK   annotated as thiamin biosynthesis lipoprotein ApbE, which might be involved in the maturation of iron-sulfur clusters  and strain DCA (3.06 Mb) are remarkably similar to each other, sharing 90% average nucleotide identity based on a Mauve , thus the positive selection appears driven by the presence of these chlorinated substrates. Since 1,1,1-TCA was the original contaminant at the site, and 1,1-DCA is not known to be naturally occurring, the original strain likely dechlorinated at least 1,1,1-TCA or perhaps both 1,1,1-TCA and 1,1-DCA, although other scenarios are possible, including evolution during laboratory enrichment.Sixty-seven (67) of the 70 SNPs that distinguish strains CF and DCA in the region between s Figure . This isDesulfitobacterium sp. strain PR ; however, it encodes an RDase that, unlike CfrA and DcrA, dechlorinates all three substrates, CF, 1,1,1-TCA and 1,1-DCA  regions associated with hot recombination sites including some tRNA genes and the tmRNA gene ssrA  cluster into two small regions designated cluster 1 and cluster 2  share an amino acid sequence identity of 98.2% and a nucleotide identity of 97.5%. Moreover, similar rdhA genes are often close to each other in the genome within the two rdhA clusters  in a genome, they are hot genome rearrangement sites via intra-genomic homologous recombination.Transposition events (TEs) are known to have positive, neutral and negative effects on the host , and many strain-specific transposition events were identified when aligning the genomes of strains CF and DCA. In the genome of strain CF, 27 distinct ISs were identified, 14 of which exist in more than one copy in the CF genome; an extreme case is that of IS (DCF50_p264) that exists in nine copies in the genome. While strains CF and DCA share all ISs, their copy numbers vary in some instances, revealing many strain-specific transposition events that have happened since the differentiation of the two strains  transposition has played an important role in shaping Dehalobacter genomes including recA, recFOR and ruvABC  a sBC Table  and (ii)BC Table . FinallyBC Table , would rBC Table . SequencrdhA clusters, major sequence variations between Dehalobacter strains were observed in three other regions of these genomes, labeled regions A, B, and C in Figure Dehalobacter genomes .Beyond the two Dehalobacter genomes, many genes involved in type IV pilus assembly were identified  and B2 (Strain DCA) have a low G+C content, and poor sequence conservation was found between all five Dehalobacter genomes, indicating a hypervariable region. Region D  as well as the obligate organohalide-respiring genus Dehalococcoides. Relevant gene expression information is also included based on a recent proteomic study of Dehalobacter restrictus strain PER-K23 , aldehyde dehydrogenase (DCF50_p2406), 6-phosphofructokinase (DCF50_p872), pyruvate kinase (DCF50_p653), pyruvate formate lyase (DCF50_p2283 and p2284), and formate hydrogen lyase (DCF50_p760 to p766). This suggests that Dehalobacter have the potential to ferment or use electron donors other than H2 or formate. All of these genes have orthologs in D. hafniense strain Y51, and most Desulfitobacterium strains are capable of pyruvate fermentation. Other than rdhA genes, no genes for other electron–accepting reactions were identified in any of the Dehalobacter genomes. Only putative cytochrome b genes were found. Thus, considering the presence of 86 rdhA genes in these five genomes, and lack of fermentative growth, these Dehalobacter strains appear specialized for organohalide respiration  to produce pyruvate using pyruvate-flavodoxin oxidoreductase (Dcf50_ p269 or p2740). Heterotrophic CO2 fixation is supported by some 14CO2 fixation experiments with strain PER-K23 .Many anaerobes have an incomplete tricarboxylic acid cycle (TCA cycle), which mainly serves anabolic purposes. For example, Dehalobacter genomes possess complete biosynthesis pathways for all amino acids except serine, and the enzymes involved in these pathways were detected by proteomic analysis in strain PER-K23 , the enzyme that catalyzes the last step in serine biosynthesis, is missing from all five Dehalobacter genomes , the gene encoding L-threonine aldolase (EC 4.1.2.5), which converts L-threonine to glycine, is also not present in the genomes of strains CF and DCA . An alternative salvage mechanism must operate in Dehalobacter cells since the growth of strain PER-K23 only requires arginine, histidine, and threonine but not cysteine, glycine, or serine , a multifunction enzyme . Then, the newly described alternative pathway likely catalyzes the steps from siroheme via iron-coproporphyrin III to heme, as was recently demonstrated in the methanogenic archaeon Methanosarcina barkeri  is a cofactor of cytochrome b, a subunit of hup-type Ni,Fe-hydrogenases found in all five es Table . Howeveres Table , which sDehalobacter genomes. These two formate dehydrogenases were found expressed in strain PER-K23 . Strain PER-K23 has a peptidoglycan-containing cell wall  encodes a complete nitrogen fixation operon  and associated histidine kinase  or sensor (PAS) domains  with Taxon Object ID of 2510065016  signaling domains  for Pfam protein domains associated with signal transduction and gene regulatory activity as defined by the MIST2 database  or IMG (img.jgi.doe.gov) for use with the pfam_scan.pl script (Finn et al., ST assemblies the Dehalobacter genomes. ST and PW annotate the Dehalobacter genomes. EE, ST, PW, and SH analyze the data. ST, PW, FL, and EE write the paper.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.


Preclinical and clinical data suggest that combination of a BRAF and a MEK inhibitor  may increase the efficacy over BRAFi monotherapy in BRAF-mutant metastatic melanoma and that addition of a MEKi may overcome or delay resistance to BRAFi.This phase Ib/II study is evaluating the combination of LGX818, a potent, selective BRAF inhibitor, and MEK162, a selective MEK1/2 inhibitor, in patients with BRAF-mutant tumors (NCT01543698). The objectives for the phase Ib portion of the study were determination of the maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D) for daily (qd) LGX818 + twice daily (bid) MEK162. Dose escalation was guided by a Bayesian logistic regression model with overdose control.At the time of data cut-off , 30 patients were enrolled  and treated at the following dose levels (DLs) with LGX818 qd + MEK162 bid, respectively: 50 mg + 45 mg, 100 mg + 45 mg, 200 mg + 45 mg, 400 mg + 45 mg, 450 mg + 45 mg, and 600 mg + 45 mg. One dose limiting toxicity, grade 3 alanine aminotransferase elevation, was reported at the 600 mg + 45 mg DL. The MTD was not yet determined and two RP2Ds have been declared: 600 mg + 45 mg and 450 mg + 45 mg. Common adverse events  suspected to be treatment related were nausea, diarrhea, fatigue, visual impairment, and headache. No events of fever, hyperkeratosis, or squamous cell carcinoma were observed. Among patients evaluable for response, complete response was observed in 1/9 (11%) BRAFi-naïve melanoma patients and partial responses were observed in 7/9 (78%) BRAFi-naive melanoma patients, 3/14 (21%) BRAFi-pretreated melanoma patients, and 2/3 (67%) thyroid cancer patients.Preliminary data indicate that the combination of LGX818 + MEK162 is active and demonstrates no substantial increases in adverse events compared to single-agent therapies. The absence of fever, hyperkeratosis, and squamous cell carcinoma suggest a distinct safety profile for this BRAFi/MEKi combination. Enrollment continues in the phase II portion of this study where the efficacy of LGX818 600 mg qd + MEK162 45 mg bid is being assessed.


Macrophages are found across all vertebrate species, reside in virtually all animal tissues, and play critical roles in host protection and homeostasis. Various mechanisms determine and regulate the highly plastic functional phenotypes of macrophages, including antimicrobial host defenses , and resolution and repair functions . The study of inflammatory macrophages in immune defense of teleosts has garnered much attention, and antimicrobial mechanisms of these cells have been extensively studied in various fish models. Intriguingly, both similarities and differences have been documented for the regulation of lower vertebrate macrophage antimicrobial defenses, as compared to what has been described in mammals. Advances in our understanding of the teleost macrophage M2 phenotypes likewise suggest functional conservation through similar and distinct regulatory strategies, compared to their mammalian counterparts. In this review, we discuss the current understanding of the molecular mechanisms governing teleost macrophage functional heterogeneity, including monopoetic development, classical macrophage inflammatory and antimicrobial responses as well as alternative macrophage polarization towards tissues repair and resolution of inflammation. Macrophage lineage cells present a remarkably versatile array of functional specializations across vertebrates. As resident cells in virtually all tissues, macrophages aid in maintaining homeostatic environments, and upon infection, are typically one of the first cell types to encounter intruding pathogens, where they orchestrate appropriate immune responses. Our understanding of macrophage biology has greatly expanded since the first description of starfish larvae phagocytes in 1882 by Élie Metchnikoff, who was later awarded the Nobel Prize for his contribution to cellular immunity in 1908 [Much of our understanding of macrophage biology comes from research in mammalian models, where distinct macrophage subsets of have been characterized, including classically activated cells by interferons (IFN) and tumor necrosis factor alpha (TNFα) (M1); alternatively activated cells by IL-4 and IL-13 M2a); macrophages activated by immune complexes or apoptotic cells (M2b); and regulatory macrophages, deactivated by IL-10, TGF-β, or glucocorticoids (M2c), which culminate in the various effector subtypes, broadly described as having a “kill” or “heal” response  ,27,28,29From a phylogenetic perspective, it is notable that birds and mammals possess a single, alternatively spliced CSF-1 gene that produces both membrane-bound and secretory forms of this cytokine ,34. IntrThe mammalian macrophage lineage cell CSF-1R gene expression increases progressively with cell development, with committed myeloid precursors possessing the lowest transcript levels of this receptor, monocytes and immature macrophages expressing significantly greater levels of the CSF-1R, and mature macrophages exhibiting still greater levels of the CSF-1R transcript . MoreoveMacrophage activation was first coined by Mackaness in the 1960s after describing antimicrobial activity of mouse macrophages to secondary infection with intracellular bacteria . Later, In fish, type II interferons have been well characterized ,57. IFNγEscherichia coli challenge is uncompromised, whereas knock-down of both IFNγs results in significant increases in infection-induced mortalities [Yersinia ruckeri infections, while knock-down of both IFNs further compounds embryo mortalities. Presumably, IFNγ and IFNγrel induce both overlapping and distinct antimicrobial mechanisms.Interestingly, certain fish species possess at least two distinct type II IFNs, termed IFNγ and IFNγ-related (IFNγrel). Recombinant IFNγrel has been shown to activate macrophage reactive intermediate production  in goldftalities . NotablyWe have documented that the recombinant forms of the goldfish (rg) IFNγ and rgIFNγrel have very different effects on kidney macrophage functions . While rin vivo and other in vitro fish models. Notably, the zebrafish IFNγrel has recently been shown to elicit more robust pro-inflammatory gene expression than IFNγ in larvae microinjected with respective IFN expression constructs [Further confirmation of this functional dichotomy between the fish type II IFNs is warranted using nstructs . InteresIn comparison to other vertebrates that possess a single IFNγ receptor 1 (IFNGR1), it is particularly notable that certain teleosts possess two distinct IFN gamma-receptor binding chains (IFNGR1-1 and IFNGR1-2) that confer distinct but poorly understood interactions with the IFNγ and IFNγrel molecules of these respective species ,48,63. TE. coli inoculation, where TNFα expression occurred in the presence of bacteria, thus underlining the microbial activation and M1 polarization of macrophages [Akin to many fish cytokines, multiple isoforms of the key inflammatory cytokine, tumor necrosis factor alpha (TNFα), have been documented across numerous fish species ,82,83,84rophages . TNFα3 eGranulocyte macrophage colony-stimulating factor (GM-CSF) has been added to the list of M1 stimuli in mammals . GM-CSF i.e., neutrophils and monocytes), and influencing Th1 polarization, which in turn amplifies classical activation [Stimulation and polarization of macrophages to an M1 subtype results in the production of large amounts of TNF, IL-1β, IL-6, IL-12 IL-15, and IL-23, for further activation of proximal cell types, aiding chemotaxis of inflammatory leukocytes , hydroxyl radical (OH·), and hyperchlorous acid (HOCl), which efficiently kill microorganisms [et al., 2008). The NADPH oxidase complex is made up of six individual subunits that are segregated in resting cells, including the cytosolic components p40phox (phagosome oxidase), p47phox, p67phox, and a guanosine triphosphatase (GTPase) Rac 1 or Rac 2 migrates to membrane-associated subunits gp91phox  and p22phox [Edwardsiella tarda, a natural pathogen of various economically important fish and the etiological agent of edwardsiellosis, has several strategies to combat ROI production of teleost macrophages, reviewed by [The destruction of internalized microorganisms is key to the innate immune response. It is well established that fish phagocytes, including macrophages, generate reactive oxygen intermediates as an antimicrobial defense, similar to mammalian phagocytes. During the macrophage respiratory burst, an assembly of multicomponent enzyme nicotinamide adenine dinucleotide phosphate (NADPH) subunits at the plasma membrane results in the transfer of electrons from NADPH to molecular oxygen, resulting in the superoxide anion . Upon forganisms ,94 , which additionally serves potent antiparasitic/antimicrobial functions [Akin to superoxide production, the inducible nitric oxide (NO) system of teleost macrophages is well conserved compared to those described in mammals. Classically activated macrophages are distinguished by the expression of inducible nitric oxide synthase (iNOS/NOS2) that catalyzes the conversion of compound ,113. Simunctions ,115. Indunctions .Aeromonas salmonocida, Renibacterium salmoninarum, Yersinia ruckeri [Mycobacterium marinum [The fish iNOS has been characterized with marked similarity to the mammalian enzyme counterpart, possessing the putative binding sites for heme, tetrahydrobiopterin, calmodulin, flavine mononucleotide, flavine adenine dinucleotide, and NADPH, suggesting cofactor conservation and function . First i ruckeri ,124,125, marinum ,127,128.Following pathogen uptake, the phagosome undergoes various maturation steps that are an important process to destroy the internalized microbe, which culminates in phagolysosome formation. Both M1- and M2-type macrophages rapidly form phagolysosomes in order to degrade internalized material. Interestingly, recent reports of M1-stimulated macrophages show relatively neutral pH following the internalization of zymosan, in contrast with M2-driven macrophages that rapidly acidify their phagosomes . This isSalmonella, Leishmania, and Mycobacteria [2+ into the phagosome in RAW264.7, a NRAMP1-deficient cell line, shows iron transport into phagosomes, which may aid in catalyzing ROS and limiting bacterial growth [Solute carrier 11 member 1, Slc11a1 , is a divalent metal ion transporter, present in the late endosomes and lysosomes of professional phagocytes . Transcrbacteria . Althougl growth . Contrarl growth ,137, leal growth .NRAMP1 has been cloned in a number of fish species, including carp , fugu 1, channelV. anguillarum [E. ictaluri [M. marinum [Teleost fish appear to possess multiple Slc11 orthologs per genome including two fugu Slc11a genes  and threuillarum , E. ictaictaluri , and M.  marinum ,147.Mycobacteria and Salmonella, and ferroportin-deficient mice are more susceptible to intracellular pathogens [The first identification of ferroportin  was in zebrafish  and miceathogens . The conathogens .V. anguillarum infections. Interestingly, the zebrafish mutant weissherbst is a hyperchromatic blood mutant with a mutation in functional ferroportin [Ferroportin has been identified in zebrafish  and turbroportin , which mIndoleamine 2,3-dioxygenase (IDO), or tryptophan 2,3 dioxygenase (IDO), is a macrophage enzyme that catalyzes the degradation of tryptophan, and is up-regulated in IFNγ-stimulated macrophages . M1 macrBony fish IDO orthologs appear to possess very low tryptophan-degradative enzymatic efficiencies compared to the mammalian IDOs , suggestM. marinum significantly up-regulate their proto-IDO gene expression, where live M. marinum induces substantially greater proto-IDO transcript levels than the heat-killed bacteria [M. marinum survival in macrophages, and may indicate that this fish proto-IDO serves to dampen immune cell bystander responses, akin to its mammalian counterpart. A summary of mammalian and teleost M1 macrophage function is depicted in Notably, goldfish macrophages infected with bacteria . This suM2 macrophages are generally considered as being antagonists to M1 macrophages, having “anti-inflammatory” or “pro-healing” functions. In mammals, the best characterized M2 stimuli are IL-4 and IL-13 , produced by Th2 cells, eosinophils, basophils, NKT cells, and macrophages themselves . The recTo date, at least two genes have been identified in fish that share homology with both the mammalian IL-4 and IL-13 cytokines (IL-4/13A and IL-4/13B) , althougl-arginine. Whereas iNOS of M1 macrophages converts l-arginine to l-citruline and NO, arginase, a putative marker of M2 macrophages, converts l-arginine to l-ornithine and urea [l-ornithine, which is a precursor for polyamines and proline components of collagen that are important for tissue repair [A major bifurcation of M1 and M2 function involves the metabolism of and urea ,179,180.e repair . Interese repair .NG-hydroxy-l-arginine, suggesting an evolutionary conservation of polarized macrophages in lower vertebrates [Mammals possess two arginase isoforms including arginase-1, located in the cytosol and induced by IL-4 and IL-13 , and argtebrates . Indeed,tebrates ,10,120. tebrates . For exctebrates ,188.Trypanoplasma borreli immune complex formation generated distinctly activated carp macrophage subsets, promoting the resolution of inflammation and parasite clearance [Repeated stimulation of M1-like macrophages by inflammatory stimuli has been documented to result in the adoption of M2-like macrophage phenotypes and unresponsiveness to subsequent inflammatory cues. For example, mammalian M2b or type-2 macrophages are generated in response to immune complexes (IC) and Gram-negative bacteria lipopolysaccharides (LPS), resulting in lower IL-12 and higher IL-10 production . This isDeactivation of macrophages by glucocorticoids (GCs) and IL-10 has also been described to culminate in the unique M2c macrophage activation state, otherwise referred to as “regulatory macrophages”. The diffusion of GCs across the plasma membrane leads to interaction with the glucocorticoid receptor (GCR), which results in nuclear translocation and direct transcriptional up-regulation of some immune genes and down-regulation of others, resulting in macrophage phenotypes and transcriptional profiles distinct from those observed following IL-4 macrophage stimulation ,191. Foret al., 2011b) [In mammals, IL-10 signals through IL10R1 and IL10R2, leading to activation of STAT3 and inhibition of pro-inflammatory cytokine expression and, thus, Th1 and M1 functions . IL-10 i, 2011b) , demonstAkin to their mammalian counterparts, macrophages of teleost fish exhibit a plethora of functional roles including those pertaining to homeostasis, as well as host immune defenses, and are largely governed by their respective tissue niches and microenvironments. Perturbations in these homeostatic environments by pathogens or injury polarize resident macrophage populations aptly towards the appropriate functionalities, with the sum of the cell types altering the physiology of the hosts towards an M1 inflammatory type, or the M2 resolution and repair types. Although these broad functional states have been demonstrated in fish macrophages, further research into the molecular regulation by both mammalian orthologs and novel fish-specific molecules will further the understanding of teleost macrophage functional regulation. Undoubtedly, the growing genetic resources of teleost animal models combined with transcriptomic/proteonomic/metabolomic technologies and live-imaging techniques of differentially activated teleost macrophages will be integral in deciphering the intricacies of macrophage functional regulation in teleosts.


ALDH18A1, a gene encoding the mitochondrial enzyme Δ1-pyrroline-5-carboxylate synthase (P5CS). It is a rare disorder with only six pathogenic mutations and 10 affected individuals from five families previously described in the literature. Here we report the identification of novel compound heterozygous missense mutations in two affected siblings from a Lebanese family by whole-exome sequencing. The mutations alter a conserved C-terminal domain of the encoded protein and reduce protein stability as determined through Western blot analysis of patient fibroblasts. Patient fibroblasts exhibit a lipid droplet phenotype similar to that recently reported in Warburg Micro syndrome, a disorder with similar features but hitherto unrelated cellular etiology.Autosomal recessive cutis laxa type 3A is caused by mutations in  ELN (MIM 130160), FBLN5 (MIM 604580), EFEMP2 (MIM 604633), or LTBP4 (MIM 604710) which encode secreted proteins that interact with the extracellular matrix (ECM) which encodes a component of a Golgi-localized H+-ATPase , which encodes a RAB6-interacting Golgin  and ARCL3B (MIM 614438) is caused by mutations in PYCR1 (MIM 179035). These genes encode mitochondrial enzymes involved in proline synthesis, Δ1-pyrroline-5-carboxylate synthase (P5CS) and pyrroline-5-carboxylate reductase 1, respectively  and GO as well as ARCL3B  comprises a group of distinct but clinically overlapping disorders within which inelastic wrinkled and redundant skin is a common feature , height 77.5 cm (5th percentile), and head circumference 42.3 cm (<3rd percentile). He had a small and receding forehead, a flat occiput, deeply set eyes, cupped, simple, large ears, a hooked nose with a prominent columella, short philtrum, long, thin fingers with some ulnar deviation. External genitalia showed bilateral ascended testes. Neurological examination revealed a hypotonic boy, unable to support himself in sitting, decreased muscle bulk, and increased deep tendon reflexes. Ophthalmological examination revealed the presence of a bilateral aphakia. Abdominal ultrasonography and echocardiography did not show any abnormalities. A brain MRI was performed and revealed thinning of the corpus callosum with enlargement of the subarachnoid space anteriorly within the hemispheric fissure, lateral and 3rd ventricles were prominent in size without evidence for hydrocephaly, cerebellar hypoplasia, mildly delayed myelination, a mega cisterna magna, small optic chiasm, and abnormal lenses within the globes. Auditory evoked potential was normal. A total body x-ray revealed a short left first metacarpal, and a bilateral coxa valga. Upon several examinations, the clinical course was unremarkable except that he had severe developmental delay. At the last follow-up, he was 11 years old. He could not sit unaided and was speechless. Head circumference was 43 cm, height 104 cm, and weight 12 kg . In addition, to the known features, he had deep set eyes, prominent cheeks, hypoplastic alae nasi, very thin skin with visible fine vessels, and multiple joint contractures.Patient 2 was first evaluated by us at the age of 1 year. At birth, his weight was 2600 g, and his length 49 cm. His clinical course was identical to that of his affected brother except that he did not have cataracts, and had a normal skin appearance at birth. At examination, he was 5 years and 9 months. His weight was 11 kg, height 92.5 cm, and head circumference 39.7 cm (<3rd percentile). Physical examination showed the same dysmorphic features present in his brother with the addition of camptodactyly to the left fingers. Abdominal ultrasonography did not show any abnormalities. Metabolic screening was carried out for both patients and no anomalies found. Serum and urine amino acids, urine organic acids, very long chain fatty acid (VLCFA), phytanic acid, electrolytes, aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatine kinase (CK), carnitine, acylcarnitine, acylglycine and tests for congenital disorders of glycosylation were all normal.ALDH18A1, c.2246G>A, p.Arg749Gln and c.2294G>A, p.Arg765Gln. These mutations were confirmed by Sanger sequencing and were present in both affected siblings. The c.2246G>A, p.Arg749Gln mutation was transmitted by the father and the c.2294G>A, p.Arg765Gln mutation by the mother  deficient in Tbc1d20, there is a significant increase in LD size when cells are treated with oleate for 18 or 24 h to induce LD formation  cells as compared to two control fibroblast cell lines following both 18 and 24 h oleate treatment  are associated with Troyer syndrome, a complex form of hereditary spastic paraplegia (MIM 275900) characterized by short stature and cognitive deficits in addition to progressive ascending spasticity. SPG20 has been found to associate with both LDs and mitochondria, and SPG20 deficiency has been linked to LD enlargement and accumulation (Lu et al. ALDH18A1, or indeed, ARCL resulting from mutations in other genes.Together, the above data identify two novel pathogenic mutations in


Based on patch-clamp studies, the channels play crucial roles in facilitating the calcium influx necessary to trigger lymphocyte activation and proliferation. Using selective channel inhibitors in experimental animal models, in vivo studies then revealed the clinically relevant relationship between the channel expression and the pathogenesis of autoimmune diseases. In renal diseases, in which “chronic inflammation” or “the overstimulation of cellular immunity” is responsible for the pathogenesis, the overexpression of Kv1.3-channels in lymphocytes promotes their cellular proliferation and thus contributes to the progression of tubulointerstitial fibrosis. We recently demonstrated that benidipine, a potent dihydropyridine calcium channel blocker, which also strongly and persistently inhibits the lymphocyte Kv1.3-channel currents, suppressed the proliferation of kidney lymphocytes and actually ameliorated the progression of renal fibrosis. Based on the recent in vitro evidence that revealed the pharmacological properties of the channels, the most recent studies have revealed novel therapeutic implications of targeting the lymphocyte Kv1.3-channels for the treatment of renal diseases.Delayed rectifier K Previouferation –11. Sincferation  and seruferation , 14, Kv1feration , 15  [ in situ in the cortical interstitium, and thus dramatically increased their numbers [Recently, Liu et al. demonstrated that CD4on (UUO) . In the on (UUO) . In this numbers , 34.+-channels (Kv), Ca2+-activated K+-channels (KCa), Ca2+ release-activated Ca2+-channels (CRAC), Mg2+-inhibited Ca2+-permeable current (MIC) channels, and swelling-activated Cl− channels (Clswell) [ in vitro studies. Patch-clamp studies revealed that these channels generate the K+-diffusion potential across the plasma membranes and thus play a role in regulating the resting membrane potential and controlling the cell volume [2+ influx [2+ signaling pathway necessary to trigger the lymphocyte activation and proliferation.A variety of ion channels that are expressed in T lymphocytes include voltage-dependent KClswell) . Among tClswell) , 2. In pClswell) , 38, 39.l volume , 41. The+ influx , 37. Con+-channels in lymphocytes was increased in such patients and the activity of the channels was strongly associated with the severity of renal dysfunction. In experimental animal models of renal diseases, such as renal allograft rejection [Peripheral lymphocytes are activated and the serum cytokine levels are known to be elevated in patients with end-stage renal disease –14. Baseejection  and glomejection , immunosejection , 10, theIn previous studies, the overexpression of Kv1.3-channnels was demonstrated in cells under various pathologic conditions, such as cancer , 44, isc in vitro studies, CCBs, including nifedipine, verapamil, and diltiazem, repress the migration of leukocytes and inhibit their proliferation [β, tumor necrosis factor α (TNF-α), and interferon γ (IFN-γ) [In addition to their cardiovascular effects on hypertension and ischemic heart disease , CCBs arferation , 58. Rec (IFN-γ) , 60. Acc (IFN-γ) , 62, exe (IFN-γ) , 64. In  (IFN-γ) , benidip (IFN-γ) , 60, the (IFN-γ) . in situ proliferation in advanced CRF rat kidneys [In our recent study, the overexpression of Kv1.3-channels in kidney lymphocytes was strongly associated with their kidneys , and the kidneys , since b kidneys . Previou kidneys  and angi kidneys , for theIn advanced CRF rat kidneys with benidipine treatment, the size of the cortical interstitium, which included the areas of fibrosis, edema, and the inflammatory leukocyte infiltration, was smaller . In thes2+-activated K+-channels (KCa3.1) for renal fibrosis, since fibroblasts overexpressed the channels under a pathologic condition [ in vitro studies, these drugs exerted immunomodulatory properties besides their anti-inflammatory, antimicrobial, and anticholesterol effects [22, and PAP-1, actually repressed the immune response in lymphocytes [+ reabsorption. Therefore, the channel inhibition may also affect such tubular functions and the urinary K+ secretion, which was actually demonstrated in previous studies using isolated collecting ducts [Grgic et al. demonstrated the therapeutic efficacy of blocking the intermediate-conductance Caondition . In a seondition . As an eondition , 34. In ondition , macroliondition , 21 (Tab effects , 71–75.  effects  and sincphocytes , 76–78 (phocytes , the ampphocytes , the usephocytes , 82. In phocytes  and predphocytes . In thesng ducts . Howeverng ducts , 85, 86,In a physiological condition, Kv1.3-channels expressed in T lymphocytes play crucial roles in the initiation of the immune response. In renal diseases, such as CKD, acute glomerulonephritis, and renal allograft rejection, the channels contribute to the pathogenesis of the diseases. In rat kidneys with advanced CRF, the overexpression of the channels in lymphocytes facilitated the progression of tubulointerstitial fibrosis by promoting lymphocyte proliferation, suggesting that the channel could be a potent therapeutic target for advanced-stage CKD. Benidipine, one of the most commonly used CCBs, which also strongly and persistently inhibits the lymphocyte Kv1.3-channel currents, suppressed the proliferation of kidney lymphocytes and actually ameliorated the progression of renal fibrosis. Since other drugs, such as NSAIDs, macrolide antibiotics, and statins, also effectively suppress the channel currents in lymphocytes, they may be useful for treating or preventing renal diseases.“Chronic inflammatory diseases” are a category of diseases, in which “chronic inflammation” or “the overstimulation of cellular immunity” is responsible for the pathogenesis . Besides


Shigella flexneri serotype 2 variant  was isolated in 2008 and first reported in China in 2013. In the present study, epidemiological surveillance from 2003 to 2013 in China suggested that this serotype first appeared in Guangxi in 2003; it then emerged in Shanghai and Xinjiang in 2004 and in Henan in 2008. Of the 1813 S. flexneri isolates, 58 S. flexneri serotype 2 variant strains were identified. Serotype 2 variant has emerged as a prominent serotype in recent years, with 2a (32.6%), X variant (25.2%), 1a (9.4%), X (6.3%), 2b (5.4%), and 1b (3.6%). According to phenotypic and genotypic analysis, the serotype 2 variant originated from 2a to 2b. A higher antibiotic resistance rate was observed between 2009 and 2013 than that between 2003 and 2008. Among 22 cephalosporin-resistant isolates, blaTEM-1, blaOXA-1, blaCTX-3, blaCTX-14, and blaCTX-79 were detected. Among 22 fluoroquinolone-resistant isolates, a Ser80Ile mutation in parC was present in all of the isolates. Moreover, 21 isolates had three gyrA point mutations  and one isolate had two gyrA point mutations (Ser83Leu and His211Tyr). The prevalence of His211Tyr in the fluoroquinolone-resistant isolates is concerning, and the mutation was first reported in China. Besides, 22 isolates harbored the aac(6′)-Ib-cr gene, and two isolates harbored qnrS1. In view of the increased epidemic frequency and multidrug-resistant strain emergence, continuous surveillance will be needed to understand the actual disease burden and provide guidance for shigellosis. Shigella species, is recognized as a major public health burden and continues to be an important cause of diarrheal diseases. Worldwide, Shigella episodes were estimated to be 164.7 million annually, of which, 163.2 million were in developing countries, resulting in 1.1 million deaths  and S. sonnei (32.7%) were two major causative species  based on the combinations of antigenic determinants present on the O antigen of the cell envelope lipopolysaccharide  was first reported by Qiu et al. in 2013  were routinely grown in a 37°C incubator in Luria–Bertani agar plates  and monoclonal antibody reagents .Fresh stool samples from diarrhea patients with clinically suspected dysentery were collected in sentinel hospitals. Samples were cultured for http://www.shigatox.net/ecmlst/cgi-bin/index. The PCR amplification conditions were as follows: 94°C for 5 min; 33 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min; and 72°C for 10 min with Ex Taq DNA polymerase . The PCR amplicons of 15 housekeeping genes were sequenced, and sequences were edited using SeqMan 7.0. Then, the sequences were uploaded to the EcMLST website for comparison, which allowed us to determine the sequence type.MLST of these isolates was carried out using the protocols described at Escherichia coli (ATCC 25922) strain was used as the quality control strain.The antimicrobial MICs of 21 antimicrobials including ceftazidime (CAZ), ceftriaxone (CRO), cefepime (FEP), cefoperazone (CFP), cefazolin (CFZ), cefoxitin (FOX), imipenem (IPM), nitrofurantoin (NIT), piperacillin (PIP), ampicillin (AMP), ticarcillin (TIC), tetracycline (TE), tobramycin (TO), gentamicin (GEN), amikacin (AK), aztreonam (ATM), chloramphenicol (C), ticarcillin/clavulanic acid (TIM), levofloxacin (LEV), norfloxacin (NOR), and trimethoprim/sulfamethoxazole (SXT) were determined by broth microdilution using a 96-well microtiter plate  according to the manufacturer's instructions, and MIC values follow Clinical and Laboratory Standards Institute recommendations. An S. flexneri were analyzed by PFGE according to the standard protocol for S. flexneri developed by the Centers for Disease Control and Prevention of the USA. Salmonella enterica serotype Braenderup H9812 was digested with XbaI and used as molecular weight standard. Slices of agarose plugs were digested with NotI  at 37°C for 3 h. Electrophoresis was carried out in 1% agarose SeaKem Gold gel  with the CHEF Mapper system (Bio-Rad) with the following run parameters: 6 V/cm and a linear increase in switching times from 2.16 to 54.17 s over a period of 20 h. The interpretation of the PFGE patterns was performed with BioNumerics software version 6.0 . A tree indicating relative genetic similarity was constructed based on the unweighted pair group method of averages and a position tolerance of 1.2%.All isolates of blaSHV, blaTEM, blaOXA, and blaCTX−M. The variable regions of class 1 integrons and class 2 integrons were also amplified with primers listed in Table aadA1, aadA2, aadA5, and cmlA1, together with the forward primer hep58, were used to amplify the gene cassettes of class 1 integron-positive strains. The forward primer hep74 and reverse primer hep51 were used to amplify the gene cassettes of class 2 integron-positive strains. To understand the underlying mechanism conferring resistance to quinolones, plasmid-mediated quinolone resistance determinants (PMQRs) such as qnrA, qnrB, qnrD, qnrS, and aac(6′)-Ib-cr and quinolone resistance–determining regions (QRDRs) of the DNA gyrase (gyrA) and topoisomerase IV (parC) genes were amplified , followed by ticarcillin , tetracycline , chloramphenicol , trimethoprim/sulfamethoxazole , norfloxacin , cefazolin , ceftriaxone , cefoperazone , piperacillin , tobramycin , gentamicin , ticarcillin/clavulanic acid , cefoxitin , nitrofurantoin , aztreonam , and levofloxacin . None of the isolates were resistant to cefepime, imipenem, ceftazidime, and amikacin. In addition, strains intermediately resistant to ceftazidime, cefoperazone, levofloxacin, norfloxacin, piperacillin, tobramycin, gentamicin, aztreonam, and ticarcillin/clavulanic acid were also observed , and only one strain from Shanghai was not. All of the isolates had 26 antibiotic-resistance profiles, dominated by resistance to TE/TIC/AMP/C , followed by TE/TIC/AMP/C/SXT , TE/TIC/AMP/C/SXT/NOR , and TE/TIC/AMP/C/NOR   formed a single cluster (containing 2a and 2b isolates). This result suggested that the serotype 2 variant strains were closely related to serotype 2a and 2b. Besides, the 58 S. flexneri serotype 2 variant isolates generated 45 PFGE patterns  from Xinjiang and strain HN08096 from Henan had the gyrA mutation of Asp87Asn, while the rest of the strains except strain HN09103 had the gyrA mutation of Asp87Gly. Only two species of PMQR determinants [qnrS and aac(6′)-Ib-cr] were identified. All of the isolates harbored the aac(6′)-Ib-cr gene, and only two isolates from Shanghai harbored qnrS. Sequencing analysis determined that the two qnrS genes were qnrS1 -Ib-cr]  and parC (Ser80Ile). Furthermore, different amino acid substitutions at the same position may result in different quinolone susceptibility levels -Ib-cr gene is identified in many Enterobacteriaceae and is responsible for low-level resistance to fluoroquinolones -Ib-cr–positive S. flexneri 2a strain was first isolated in 1998 -Ib-cr–positive, suggesting that the aac(6′)-Ib-cr gene had been present in China for many years.Fluoroquinolone resistance in ls Ruiz, . The HisS. flexneri to escape protective host immune responses, which may drive the emergence of novel and atypical serotypes under pressure (Allison and Verma, Serotype conversion is a major mechanism for In summary, studies on the serotype 2 variant have become very important owing to the increased epidemic frequency and the prevalence of MDR strains. In view of the fact that this serotype was prevalent in several provinces in China, it is likely to provoke a major crisis because MDR clones can and may have already spread to other countries. Therefore, continuous surveillance will be needed to determine the distribution and resistance development of this serotype so as to understand the actual disease burden and provide guidance for the clinical treatment of shigellosis.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.


Arabidopsis and rice, few reports have examined the function of JAZ proteins in maize. In this report, we examined the phylogenetic relationship and expression pattern of JAZ family genes in maize. In addition, a tassel and endosperm-specific JAZ gene, ZmJAZ14, was identified using microarray data analysis and real-time RT-PCR, and its expression was induced by polyethylene glycol (PEG), jasmonate (JA), abscisic acid (ABA), and gibberellins (GAs). ZmJAZ14 was shown to be localized in the nucleus and possessed no transcriptional activating activity, suggesting that it functions as a transcriptional regulator. We found that overexpression of ZmJAZ14 in Arabidopsis enhanced plant tolerance to JA and ABA treatment, as well as PEG stress, while it promoted growth under GA stimulus. Moreover, ZmJAZ14 interacted with a subset of transcription factors in Arabidopsis, and the accumulation of several marker genes involved in JA, ABA, and GA signaling pathways were altered in the overexpression lines. These results suggest that ZmJAZ14 may serve as a hub for the cross talk among the JA, ABA, and GA signaling pathways. Our results can be used to further characterize the function of JAZ family proteins in maize, and the gene cloned in this study may serve as a candidate for drought tolerance and growth promotion regulation in maize.Jasmonate (JA) is an important signaling molecule involved in the regulation of many physiological and stress-related processes in plants. Jasmonate ZIM-domain (JAZ) proteins have been implicated in regulating JA signaling pathways and the cross talk between various phytohormones. Maize is not only an important cereal crop, but also a model plant for monocotyledon studies. Although many JAZ proteins have been characterized in  Plant hormones are implicated in many aspects of plant growth and development, as well as in the response to environmental cues. Both positive and negative regulators are involved in hormonal signal-transduction pathways to mediate various pathways in response to the same stimulus. Therefore, cross talk between various signaling pathways is essential to balance developmental and stress responses. Jasmonates (JAs), a class of fatty acid-derived hormone molecules, are important signaling molecules involved in the regulation of many physiological and stress-related processes in plants, including root growth, senescence, fruit ripening, wounding, water deficit, and pathogen attack –8.Recently, the underlying mechanism of JA signaling networks during stress responses and development including cooperation between various hormone responses were addressed . Jasmonamyb21 mutant showed reduced fertility, and myb24 exacerbated this phenotype, while overexpression of MYB21 partially restores the sterility phenotype of coi1 [JAZ proteins belong to the TIFY family, which constitutes several plant-specific groups of proteins with various functions . The TIF of coi1 . These r of coi1 . Repress of coi1 . These sArabidopsis, grape, rice, and maize [Arabidopsis [Glycine soja [JAZ genes in maize.JAZ proteins are found in many plant species, and the number of isoforms within this family is variable among different species. Twelve, 11, 15, and 23 genes encoding JAZ family proteins have been reported in nd maize , respectnd maize ,29, but bidopsis ,30,31, rbidopsis ,32, tobabidopsis , and Glyine soja , few funZmJAZ family genes. ZmJAZ14 was identified as a tassel and endosperm-specific JAZ gene and its coding protein is located in the nucleus, indicating that it may function as a transcriptional regulator. The expression of ZmJAZ14 in response to various phytohormone stimuli and abiotic stresses was investigated using quantitative reverse transcription-PCR (qRT-PCR). In addition, we found that overexpression of ZmJAZ14 in Arabidopsis enhanced plant tolerance to JA and ABA treatments, as well as polyethylene glycol (PEG) stress, and promoted growth under GA stimulus. Moreover, ZmJAZ14 interacted with a subset of TFs in Arabidopsis, and the expression levels of marker genes involved in JA, ABA, and GA signaling pathways were altered. Taken together, these results supported the essential role of ZmJAZ14 as a regulator of cross talk among the JA, ABA, and GA signaling pathways.In this report, we provide information on the phylogeny and expression pattern of Zea mays L.) inbred line B73 were grown in the experimental station of Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Hebei province, China. For expression analysis of ZmJAZ14 and ZmJAZ4, root, stalk, leaf, and apex samples were collected from the flare opening stage, silks and tassels were collected at the flowing period, as well as embryos and endosperms were sampled on 10, 15, 20, and 25 days after pollination. For phytohormone and abiotic stress treatments, the maize seedlings with three leaves were used. The seedlings were planted in vermiculite and transplanted into water 2 h before treatments to balance the variable conditions. The seedlings were then subjected to water (control), 250 mM NaCl, 20% PEG, 100 μM JA, 100 μM ABA, 100 μM GA, and 100 μM SA. Samples of shoots and roots were separated and taken as three biological replicates at 0, 0.5, 1, 3, 6, 9, and 24 h after treatment.The plants of maize  seeds were chilled at 4°C for 3 days. Seeds were then sterilized by 75% ethanol containing 0.02% Tween 20 for 5 min and washed with distilled water four times. Seeds were then sown on 0.5× MS medium and transferred to a culture room with a 16 h/8 h light/dark photoperiod at 22°C.Arabidopsis lines, after 7 days of growth on 0.5× MS medium, the seedlings were transferred to 0.5× MS solid medium with or without 10 mM NaHCO3, 15% PEG, 25 μM JA, 10 μM GA, and 1 μM ABA. The phenotypes were observed 7 days after transplantation and were vertically grown in a chamber. WT and transgenic lines were grown on the same plate to control for plate-to-plate variation. The lengths of roots were measured using ImageJ software. Statistical calculations were performed using SPSS software.To examine phytohormones and abiotic responses of transgenic http://plntfdb.bio.uni-potsdam.de/v3.0). The deduced protein sequences of ZmTIFY proteins were aligned using ClustalX 2.0.5 program. The phylogenetic tree was constructed using the neighbor-joining method in MEGA 4.0 software. The ZmTIFY proteins and their accession numbers used for phylogenetic tree construction are as follows: ZmJAZ1 (GRMZM2G343157), ZmJAZ2 (GRMZM2G445634), ZmJAZ3 (GRMZM2G117513), ZmJAZ4 (GRMZM2G024680), ZmJAZ5 (GRMZM2G145412), ZmJAZ6 (GRMZM2G145458), ZmJAZ7 (GRMZM2G382794), ZmJAZ8 (GRMZM2G086920), ZmJAZ9 (GRMZM2G145407), ZmJAZ10 (GRMZM2G171830), ZmJAZ11 (GRMZM2G005954), ZmJAZ12 (GRMZM2G101769), ZmJAZ13 (GRMZM2G151519), ZmJAZ14 (GRMZM2G064775), ZmJAZ15 (GRMZM2G173596), ZmJAZ16 (GRMZM2G338829), ZmJAZ17 (GRMZM2G126507), ZmJAZ18 (GRMZM2G116614), ZmJAZ19 (GRMZM2G066020), ZmJAZ20 (GRMZM2G089736), ZmJAZ21 (GRMZM2G036351), ZmJAZ22 (GRMZM2G036288), ZmJAZ23 (GRMZM2G143402).The TIFY family proteins were identified in maize by searching the TF database , and the self-activation tests were surveyed on selective medium lacking Leu, Trp, Ade, and His (QDO). The assay was performed in duplicates and a similar result was observed.To examine the self-activation of ZmJAZ14 in yeast, the coding region of EcoRV and BamHI and introduced into the EcoRI-BamHI site (the EcoRI site was blunted by T4-DNA polymerase) of pCAMBIA-3301 to generate the binary vector pCAMBIA-UBI. To construct the overexpression vector pCAMBIA-UBI-ZmJAZ14, the coding region of ZmJAZ14 was amplified with the primers JAZ14-U and JAZ14-L, and the resulting fragment was cloned into the BamHI-BstEII site of pCAMBIA-UBI to generate pCAMBIA-UBI-ZmJAZ14. This construct was transformed into Arabidopsis using the floral dip method. The transgenic plants were screened on 0.5× MS medium containing 20 mg/L phosphinothricin. The homozygous lines were generated by self-fertilization.The maize ubiquitin promoter was amplified using the primers UBI-U and UBI-L. The PCR fragment was digested with ZmJAZ14, ZmJAZ4, ZmJAZ12 and ZmJAZ20 were applied  was used as an internal control. qRT-PCR was performed using ABI 7500 system , as described previously [RNA was extracted using Trizol reagent  following the manufacturer’s instructions and then treated with RNase-free DNase I  to remove genomic DNA. First strand cDNA was synthesized using M-MLV reverse transcriptase . For the spatial and temporal expression pattern in various maize tissues and the expression profiles under phytohormone and abiotic stimuli, the gene specific primers for  applied , and theeviously . The PCRArabidopsis, a GAL-4 based yeast two-hybrid system was used. The TFs in Arabidopsis were amplified using primers listed in ZmJAZ14. The interaction tests were surveyed as described in previous transcriptional activation analyses.To examine the interaction between ZmJAZ14 and TFs in ZmJAZ14, MYC2, MYC3, MYB21 and NINJA were amplified with gene specific primers (The coding region of  primers  and clonThe maize plants (inbred line B73) were grown in the experimental station of Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Hebei province, China. Since the maize inbred line B73 is a common non-transgenic maize variety and the field studies did not involve endangered or protected species, we had permission from the Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Hebei province, China to conduct this study.http://plntfdb.bio.uni-potsdam.de/v3.0). ZmJAZ proteins were divided into four groups based on phylogenetic analysis  and WT lines grown in Murashige & Skoog (MS) medium alone, the growth trend differed between OE and WT lines when phytohormones and 15% PEG were added to MS medium. The transgenic plants grew better and formed relatively more lateral roots than the WT under 25 μM JA, 10 μM GA, and 1 μM ABA treatment. In addition, the OE lines had better PEG stress resistance than the WT, as the root growth of OE plants was significantly better than that of WT when grown in MS with 15% PEG , VSP2 (Vegetative storage protein) and NADP-ME  for JA, Em1 and Em6 (group I late embryogenesis abundant genes) for ABA, and AtGA20ox1 (GA 20-oxidase enzyme) for GA. The results showed that the expression levels of PDF1.2, VSP2, NADP-ME, Em1 and Em6 were significantly down-regulated in the OE lines, whereas the accumulation of AtGA20ox1 significantly increased , and their expression patterns were determined by analyzing the microarray data. In addition, the majority of ZmJAZs were present as sister pairs and share high similarity between paralogs, and the duplication of ZmJAZ genes may be associated with the chromosomal block duplications [ZmJAZ4 and ZmJAZ14 both accumulated specifically in developing endosperms. This result indicated that ZmJAZ4 and ZmJAZ14 may function redundantly and play essential roles in the maturation of endosperm.Maize is not only an important commercial crop, but also a model plant for genetics and evolutionary studies. The kernel quality and stress resistance during reproductive and grain-filling stages are the major factors that influence corn yield. Therefore, identifying functional genes essential for growth and stress-resistance regulation at the seed developing stages is useful for breeding maize varieties with high and stable yield. Recently, many genes have been characterized that play important roles during maize development. JAZ proteins constitute a major TF family in plants and play essential roles in regulating growth and development. Since the identification of JAZ proteins in bidopsis ,13, grapbidopsis , and ricbidopsis , whereasbidopsis , bZIP [4bidopsis , and MADbidopsis  familiesications . ZmJAZ4 The nuclear localization of ZmJAZ14 may contribute to increase our understanding of ZmJAZ14. The nuclear localization signal (NLS) of JAZ proteins had been reported to be ambiguous, though many studies have demonstrated that JAZ is localized in the nucleus and functions as a transcriptional regulator . NotablyIt is also found that ZmJAZ14 does not possess transcriptional activation ability . AlthougZmJAZ14 gene identified many cis-elements required for meristem, seed, and endosperm-specific expression. In addition, ABA-responsive elements were found in the promoter region  [ZmJAZ14 expression in maize seedlings. In shoots, this gene was induced by salt, PEG, GA, and ABA and salt, JA, GA, and ABA in roots. In contrast, the transcript level of ZmJAZ14 tended to be down-regulated both in shoots and roots under SA treatment, showing antagonism between the defense hormones SA and JA. Notably, the induction of ZmJAZ14 by JA was delayed as compared to other hormones, and its induction level by JA was relatively weaker than those by NaCl, PEG, ABA, and GA in shoots  , ethylenid (NAA) , woundinid (NAA) , herbivoid (NAA) , salt, aid (NAA) . Hormonen shoots . This reZmJAZ14 was specifically expressed in tassel and endosperm, its accumulation was induced under various hormone and stress treatments in both shoots and roots. In addition, the up-regulation of ZmJAZ14 transcripts associated with the endosperm development may be attributed to the increasing level of ABA content in seeds. These results suggested that the function of ZmJAZ14 may not only restrict to tassel and endosperm development, but also in many aspects of growth and stress responses. Moreover, overexpressing ZmJAZ14 Arabidopsis lines showed a stronger tolerance toward 15% PEG as well as an enhanced response to GA treatment than the WT, while they exhibited a reduced response to JA and ABA stimuli.The physiological functions of many JAZ proteins have been determined using reverse genetic analysis ,32,48. AZmJAZ14 in JA signaling, the root lengths were measured in the OE and WT plants under 50 μm JA treatment Click here for additional data file.S2 FigZmJAZ4 was normalized with ZmActin1. The qRT-PCR data were analyzed according to the 2-ΔΔCt method and the error bars indicate standard deviations.The relative expression level of (TIF)Click here for additional data file.S3 FigZmJAZ12 amd ZmJAZ20 was normalized with the ZmActin1 gene. The qRT-PCR data were analyzed according to the 2-ΔΔCt method and the error bars indicate standard deviations.The maize seedlings were treated with water (control), 250 mM NaCl, 20% PEG, 100 μM JA, 100 μM ABA, 100 μM GA, and 100 μM SA. The shoots and roots were separated and collected. Relative mRNA abundance of (TIF)Click here for additional data file.S1 TableA word document contains primer sequences used in gene cloning, vector construction and qRT-PCR.(DOCX)Click here for additional data file.S2 Tablecis-elements in the 2 kb region before the start codon of ZmJAZ14 were analyzed by Plantcare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).The putative (DOCX)Click here for additional data file.


Malignant pleural mesothelioma (MPM) is an aggressive, locally invasive, cancer elicited by asbestos exposure and almost invariably a fatal diagnosis. To date, we are one of the leading laboratory that compared microRNA expression profiles in MPM and normal mesothelium samples in order to identify dysregulated microRNAs with functional roles in mesothelioma. We interrogated a significant collection of MPM tumors and normal pleural samples in our biobank in search for novel therapeutic targets.Utilizing mRNA-microRNA correlations based on differential gene expression using Gene Set Enrichment Analysis (GSEA), we systematically combined publicly available gene expression datasets with our own MPM data in order to identify candidate targets for MPM therapy.P = 0.022) and miR-20a-5p (P = 0.026) was clearly associated with epithelioid histology. We interrogated the predicted targets of these differentially expressed microRNA families in MPM cell lines, and identified KCa1.1, a calcium-activated potassium channel subunit alpha 1 encoded by the KCNMA1 gene, as a target of miR-17-5p. KCa1.1 was overexpressed in MPM cells compared to the  mesothelial line MeT-5A, and was also upregulated in patient tumor samples compared to normal mesothelium. Transfection of MPM cells with a miR-17-5p mimic or KCNMA1-specific siRNAs reduced mRNA expression of KCa1.1 and inhibited MPM cell migration. Similarly, treatment with paxilline, a small molecule inhibitor of KCa1.1, resulted in suppression of MPM cell migration.We identified enrichment of target binding sites for the miR-17 and miR-30 families in both MPM tumors and cell lines. RT-qPCR revealed that members of both families were significantly downregulated in MPM tumors and cell lines. Interestingly, lower expression of miR-17-5p  contains supplementary material, which is available to authorized users. Malignant pleural mesothelioma (MPM) is an aggressive tumor occurring in the lining of the lungs, induced by exposure to asbestos. MPM has poor prognosis, and palliative chemotherapy is often the only treatment modality that can be offered , 2. SincMMP14 ; CRI1, EP300 interacting inhibitor of differentiation 1, ; FFPE, formalin-fixed paraffin embedded; GSEA, Gene Set Enrichment Analysis; GSN, gelsolin, ; KCa1.1, calcium-activated potassium channel 1, subunit alpha 1 protein; KCNMA1, calcium-activated potassium channel 1, subunit alpha 1 gene ; KEGG, Kyoto Encyclopedia of Genes and Genomes; MMP14, matrix metallopeptidase 14 (membrane inserted), ; MPM, Malignant Pleural Mesothelioma; NME2, NME/NM23 nucleoside diphosphate kinase 2, ; PDGFC, platelet derived growth factor C,