Influenza NS1 protein is the main viral protein counteracting host innate immune responses, allowing the virus to efficiently replicate in interferon (IFN)-competent systems. In this study, we analyzed NS1 protein variability within influenza A (IAV) H3N2 viruses infecting humans during the 2012-2013 season. We also evaluated the impact of the mutations on the ability of NS1 proteins to inhibit host innate immune responses and general gene expression. Surprisingly, a previously unidentified mutation in the double-stranded RNA (dsRNA)-binding domain (I64T) decreased NS1-mediated general inhibition of host protein synthesis by decreasing its interaction with cleavage and polyadenylation specificity factor 30 (CPSF30), leading to increased innate immune responses after viral infection. Notably, a recombinant A/Puerto Rico/8/34 H1N1 virus encoding the H3N2 NS1-T64 protein was highly attenuated in mice, most likely because of its ability to induce higher antiviral IFN responses at early times after infection and because this virus is highly sensitive to the IFN-induced antiviral state. Interestingly, using peripheral blood mononuclear cells (PBMCs) collected at the acute visit (2 to 3 days after infection), we show that the subject infected with the NS1-T64 attenuated virus has diminished responses to interferon and to interferon induction, suggesting why this subject could be infected with this highly IFN-sensitive virus. These data demonstrate the importance of influenza virus surveillance in identifying new mutations in the NS1 protein, affecting its ability to inhibit innate immune responses and, as a consequence, the pathogenicity of the virus.IMPORTANCE Influenza A and B viruses are one of the most common causes of respiratory infections in humans, causing 1 billion infections and between 300,000 and 500,000 deaths annually. Influenza virus surveillance to identify new mutations in the NS1 protein affecting innate immune responses and, as a consequence, the pathogenicity of the circulating viruses is highly relevant. Here, we analyzed amino acid variability in the NS1 proteins from human seasonal viruses and the effect of the mutations in innate immune responses and virus pathogenesis. A previously unidentified mutation in the dsRNA-binding domain decreased NS1-mediated general inhibition of host protein synthesis and the interaction of the protein with CPSF30. This mutation led to increased innate immune responses after viral infection, augmented IFN sensitivity, and virus attenuation in mice. Interestingly, using PBMCs, the subject infected with the virus encoding the attenuating mutation induced decreased antiviral responses, suggesting why this subject could be infected with this virus. Orthomyxoviridae family responsible for seasonal epidemics and occasional pandemics, being one of the most common causes of respiratory infections in humans (Influenza A (IAV) and B (IBV) viruses are members of the n humans . IAVs arn humans . Despiten humans .The defense mechanisms provided by the innate immune system restrict influenza virus replication . Virus-s3–Influenza virus NS1 protein allows the virus to replicate efficiently by suppressing the host innate immune responses by a variety of mechanisms  activation through sequestration of this RNA helicase and its activating ligand –14 or th–16–in vitro and in vivo, the sensitivity of the virus to the antiviral effect of type I IFN, and virus pathogenesis in mice. This is, to our knowledge, the first time that a mutation in the dsRNA binding domain of IAV NS1 affects CPSF30 binding and host protein expression, including the antiviral action of type I IFN. Moreover, a virus containing the I64T mutation resulted in an attenuated phenotype in mice. Furthermore, PBMCs isolated from the subject infected with the H3N2 IAV encoding the T64-NS1 protein showed evidence of a defect in the induction of innate antiviral responses. This deficiency in the ability to mount an effective antiviral response may account for the ability of the IFN-deficient virus to efficiently replicate in this and possibly other similar subjects.While much is known about the anti-type I IFN function of NS1 proteins derived from avian and some pandemic IAVs , 22, 25,2 using Dulbecco's modified Eagle's medium  supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 50 μg/ml gentamicin (Gibco).Human embryonic kidney , human lung epithelial carcinoma , African green monkey kidney epithelial , and canine kidney epithelial  cells were grown at 37°C in air enriched with 5% COPolymerase II expression pCAGGS plasmids  containiTo introduce mutations E26K, I64T, and R224K into the NS1 protein of 1918 NS1, overlapping PCRs were performed using the primers described in Plasmids encoding NS1 variants under the control of the phage T7 polymerase were obtained by subcloning NS1 from the pCAGGS-HA NH2 plasmids into pcDNA3 plasmid using EcoRI and XbaI restriction enzymes. pcDNA3 plasmids encoding PR8 and 1918 NS1 proteins have been described previously .The NS1 genes of IAV H3N2 85 and 87 were cloned in the previously described pDZ plasmid encoding nonoverlapping NS1 and NEP open reading frames (ORFs) (pDZ-NSs) . NS1 ORF4 cells/well) were infected with 10-fold serial dilutions of tissue culture supernatants (TCS). At 8 h postinfection (hpi), cells were fixed and permeabilized using 4% formaldehyde, 0.5% Triton X-100 in phosphate-buffered saline (PBS) for 20 min at room temperature. After washing with PBS, cells were incubated in blocking solution  for 1 h at room temperature, washed with PBS, and incubated with the IAV NP monoclonal antibody (MAb) NR 4282 (BEI Resources) diluted in 1% BSA for 2 h at 37°C. After washing with PBS, cells were incubated with a fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG secondary antibody (Dako) diluted 1:1,000 in 1% BSA for 1 h at 37°C. IAV NP-positive cells were visualized and enumerated to determine the virus titer (FFU per milliliter) using a fluorescence microscope. Viral infections were performed in the presence of 1 μg/ml of tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma).IAV were titrated by immunofocus assay (fluorescent focus units [FFU] per milliliter) in MDCK cells. Briefly, confluent plates of MDCK cells  were infected with 10-fold serial dilutions for 1 h at room temperature, overlaid with agar, and incubated at 37°C. Viral plaques were visualized and counted 1 day postinfection (dpi) using crystal violet.Vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP)  was titrCocultures (1:1) of 293T and MDCK cells in 6-well plates were cotransfected in suspension with 1 μg of the seven ambisense wild-type (WT) plasmids  of influenza A/Puerto Rico/8/34 H1N1  plus the ambisense split NS plasmids (pDZ-NSs) encoding the NS1 proteins of IAV 85 or 87 using DNA-IN   were infected in duplicate at low multiplicity of infection . After 1 h of virus adsorption at room temperature, cells were washed and overlaid with DMEM containing 0.3% BSA, antibiotics, and TPCK-treated trypsin (1 μg/ml for MDCK cells and 0.25 μg/ml for A549 cells). In the experiments to measure IFN sensitivity, the medium also contained 2,000 U/ml of universal IFN-α (Axxora). At the indicated times postinfection, TCS were collected and viral titers were determined by immunofocus assay (FFU/ml) as described above.To determine virus growth rates 4 cells/well) of human 293T cells were cotransfected  with 100 ng/well of the pCAGGS-HA NH2 NS1 plasmids (or the empty plasmid as a control), 50 ng/well of plasmids expressing Firefly luciferase (Fluc) under the control of IFN-β (pIFNB Fluc), or the IFN-stimulated response element (ISRE) (pISRE-Fluc) promoters (4 cells/well in triplicate) were treated with the UV-inactivated TCS for 24 h and then infected  with VSV-GFP for 16 h  infection, triplicate wells  constitutively expressing GFP and Fluc reporter genes under the control of the IFN-β promoter (MDCK IFN-β-GFP-/IFN-β-Fluc)   were moc-β-Fluc) . Fresh M-β-Fluc) . The GFPP value of <0.05 and a fold change of >1.5 were considered in the analysis. All sequence reads that did not align to the canFam3.1 genome were used as input to the Trinity assembler for each sample individually. The resulting contigs from each assembly were then compared (BLASTN 2.2.29+) against an influenza A/New York/392/2004 (H3N2) (PRJNA15622) reference to identify contigs derived from IAV. All of the ORFs of IAV genes were sequenced. To analyze influenza virus single-nucleotide polymorphisms (SNPs), bwa-0.7.12, samtools-1.3.1, and bcftools-1.3 software programs were used.MDCK cells were mock infected or infected at an MOI of 0.01 with viruses obtained from subjects 65 and 85. At 16 hpi, total RNAs were extracted using an RNeasy minikit (Qiagen) and further purified using an oligo(dT) column to obtain the cellular and viral mRNAs by following the manufacturer's recommendations. The mRNAs were subjected to deep sequencing at the University of Rochester Genomics Research Center. Sequence reads were cleaned according to a rigorous preprocessing workflow using Trimmomatic version 0.32 before mapping them to the canFam3.1 reference genome using SHRiMP2.2.3. Cufflinks2.0.2 was then used in conjunction with the canFam3.1 annotated genes to perform differential expression analysis. Differentially expressed genes were classified according to Gene Ontology (GO) annotation using the DAVID software . Only geCanis familiaris (dog) genes . Quantification was achieved using the 2CT−ΔΔ method  with viruses obtained from nasal washes/swabs from subjects 65 and 85, isolated from MDCK cells . At 24 hT method .4 cells/well in triplicate) were transiently cotransfected, using DNA-IN, with 200 ng/well of pCAGGS-HA NH2 NS1 expression plasmids, or the empty plasmid as a control, together with 50 ng/well of pCAGGS plasmids expressing GFP , containing Laemmli sample buffer (Bio-Rad) and β-mercaptoethanol and then boiled for 5 min before running 10% SDS-PAGE gels. Proteins were transferred to nitrocellulose membranes (Bio-Rad) and detected by Western blotting. Primary rabbit anti-HA (NS1) and anti-FLAG (CPSF30) polyclonal antibodies diluted 1:1,000 , MAb anti-actin diluted 1:2,000 , and MAb anti-ISG15 diluted 1:200  were used, followed by incubation with a 1:1,000 dilution of goat anti-rabbit and goat anti-mouse IgG antibodies conjugated to horseradish peroxidase (Sigma-Aldrich). Proteins were detected by chemiluminescence using the SuperSignal West Femto maximum-sensitivity substrate (Thermo Scientific) by following the manufacturer's recommendations.4 cells/well) were cotransfected with 100 ng/well of the pCAGGS-HA NH2 NS1 plasmids or the empty plasmid as a control, with 50 ng/well of plasmids expressing Fluc under the control of the IFN-β (pIFNB-Fluc) or the ISRE (pISRE-Fluc) promoters  were transiently transfected with 200 ng/well of the pCAGGS-HA NH2 NS1 plasmids, or the empty plasmid as control, using DNA-IN by following the manufacturer's instructions. At 24 hpt, cells were transfected with 100 ng of polyinosinic-poly(C)  using DNA-IN or were treated with 250 U/ml of universal IFN-α. Sixteen hours after poly(I·C) or IFN-α treatment, cells were infected  with VSV-GFP  by following the manufacturer's recommendations. Human 293T cells  were transiently transfected with 8,000 ng of a pCAGGS plasmid expressing a FLAG-tagged version of the human CPSF30 (in vitro-synthesized NS1 proteins and 20 μl of an anti-FLAG affinity resin (Sigma-Aldrich). After extensive washing, precipitated proteins were dissociated from the resin using disruption buffer and analyzed by Western blotting as described above using rabbit anti-HA (NS1)- and anti-FLAG (CPSF30)-specific polyclonal antibodies.HA-tagged NS1 variants were synthesized n CPSF30 . At 30 hin vitro using pcDNA3 plasmids and the TNT7 transcription/translation kit (Promega). The mixture was washed 3 times with TBS buffer containing 1 mM EDTA and 0.05% Tween 20, and the bound proteins were eluted in loading buffer at 95°C over 5 min. The eluted proteins were analyzed by Western blotting using a rabbit anti-HA-specific polyclonal antibody, as previously described.To prepare poly(I·C)-conjugated agarose beads, poly(C)-conjugated agarose beads (Sigma) containing approximately 62.5 μg of poly(C) were washed five times with TBS buffer . The beads then were resuspended in buffer containing 50 mM Tris and 50 mM NaCl and incubated overnight with 500 μg of inosinic acid (Sigma). The beads were washed twice with TBS, resuspended in TBS buffer containing 1 mM EDTA and 0.5% Triton X-100, and incubated at 4°C for 2 h with the HA-tagged NS1 variants synthesized Guide for the Care and Use of Laboratory Animals of the National Research Council (n = 5) and mortality . Mice showing more than 25% loss of body weight were considered to have reached the experimental endpoint and were humanely euthanized.Female 6-week-old C57BL/6 mice were purchased from the Jackson Laboratory and maintained in the animal care facility at the University of Rochester in a pathogen-free environment. All animal protocols were approved by the University of Rochester Committee of Animal Resources and complied with the recommendations in the  Council . Mice wen = 3) were sacrificed and lungs were extracted and homogenized. Virus titers were determined by immunofocus assay (FFU per milliliter) as indicated above. Geometric mean titers (GMTs) were determined and statistical analyses (Mann-Whitney test) were performed using GraphPad Prism software. Levels of IFN-β induction were analyzed in mouse lungs at 1, 3, and 5 dpi. To that end, mice (n = 3) were sacrificed and lungs were extracted and incubated in RNAlater (Ambion) at 4°C for 24 h prior to freezing at −80°C. Lungs were homogenized in lysis buffer using a gentleMACS dissociator (Miltenyi Biotec), and total RNA was extracted using an RNeasy minikit (Qiagen). RT reactions were performed at 37°C for 2 h using the high-capacity cDNA transcription kit and random hexamer oligonucleotides to generate cDNAs. qPCRs were performed using a TaqMan gene expression assay (Applied Biosystems) specific for the IFN-β murine gene (Mm00439552_s1). Quantification was achieved using the 2CT−ΔΔ method . Individuals reporting influenza-like illness  were asked to visit the Vaccine Research Unit (VRU) at the University of Rochester for nasal and nasopharyngeal swabbing and blood sampling. Starting with nasopharyngeal swabs, MDCK cells were inoculated and observed for cytopathic effect at 3 dpi. Supernatants then were collected and used to generate virus stocks in MDCK cells . The stuKJ942684.1]) and NEP-3′-RS . The cDNAs were amplified by PCR using Pfx polymerase (Life Technologies) and the same VS and RS primers. Amplified PCR products were used for Sanger sequencing (Genewiz).RNA was obtained from 300 μl of patient nasal washes/swabs using the QIAamp viral RNA extraction kit (Qiagen) according to the manufacturer's instructions. RT reactions were performed during 2 h at 37°C using the high-capacity cDNA reverse transcription kit (Applied Biosystems) and the primers NS1-NCR-5′-VS  in RPMI medium containing 10% fetal bovine serum and 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 50 μg/ml gentamicin. The following day, PBMCs were treated with 2,000 U/ml of IFN-α or infected  with r85. At 21 h after treatment or infection, PBMCs were collected and total RNA was extracted using an RNeasy minikit (Qiagen). RT reactions were performed at 37°C for 2 h using the high-capacity cDNA transcription kit (Applied Biosystems) and random hexamer oligonucleotides to generate cDNAs. qPCRs were performed using TaqMan gene expression assays (Applied Biosystems) specific for human IFN-β, IFIT2, and interleukin-29 (IL-29) (IFN-λ3) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes  or a TaqMan assay specific for the influenza virus M gene . Quantification was achieved using the 2CT−ΔΔ method  were thawed and rested overnight . For this reason, and to simplify the results, only the transcriptome from the 65 virus-infected cells will be shown and compared to gene expression in mock- and 85 virus-infected cells. A total of 234 and 637 genes were differentially expressed in cells infected with 65 and 85 viruses, respectively, compared to mock-infected cells , and 79 genes were differentially expressed in cells infected with 65 and 85 viruses (see Table S3). The genes differentially expressed in cells infected with the two viruses were clustered according to their main biological function using DAVID software . However, influenza virus NS1 is the main viral protein that suppresses host innate immune responses allowing the virus to replicate efficiently . However, as these genes have not been implicated to date in the antagonism of innate immune responses after influenza virus infection, these data suggested that the differences in innate immune responses are not due to coinfections with other influenza viruses.KU715844, KU715845, KU715848, KU715849, KU715851, KU715853, KU715854, KU715855, KU715856, KU715857, KU715859, and KU715860). Clinical isolates from patients 54FUR0056, 54FUR0058, 54FUR0064, 54FUR0065, 54FUR0068, 54FUR0072, 54FUR0073, 54FUR0090, and 54FUR0091 (referred to here by the last two numbers in their identifiers) encoded the same NS1 sequence. However, IAV infecting patient 55 presented an E26K change, while the virus infecting patient 87 presented E26K and R224K changes. Finally, the virus from patient 85 presented E26K, I64T, and R224K changes (To further assess variability in the NS1 protein of IAV H3N2 viruses circulating within the 2012-2013 season in the Rochester (NY) area, the NS1 protein sequences of 10 additional clinical samples from the same 2012-2013 season were obtained   or IFN-αTo further evaluate the effect of mutations E26K, I64T, and R224K on the ability of NS1 to inhibit host innate immune responses and gene expression, these mutations were introduced in the 1918 NS1 background . To analTo further analyze the effect of NS1 I64T mutation on the antagonism of innate immune responses, human A549 cells were transiently transfected with plasmids expressing 1918-NS1-I64 (WT protein), 1918-NS1-T64, and PR8 NS1. At 24 hpt, cells were mock treated or treated with poly(I·C)  or IFN-αin vitro-transcribed and -translated NS1 proteins from patients 85 and 87 (differing only at I64T position), or with PR8 NS1 as control, and with agarose beads conjugated with an anti-FLAG antibody  or with PR8 NS1 as a control. We also included agarose beads conjugated to poly(C) to demonstrate the specific interaction of NS1 with poly(I·C). As expected, all NS1 proteins were pulled down using poly(I·C) agarose beads but not with the poly(C) agarose beads, suggesting that the pulldown was specific  were incubated with the specific . No signspecific , suggestspecific , were lospecific .in vivo (in vivo (7 FFU/ml at the peak of infection (48 hpi). However, in A549 cells, r85 was attenuated compared to r87 at all tested times postinfection. Moreover, r85 viral titers were approximately 5- to 10-fold lower than those for r87 . Th. Thin vi. Th. Thi to better overcome the antiviral state induced by type I IFN compared to r85 (T64), MDCK cells were infected  with r85 and r87 viruses. After viral adsorption, cells were left untreated (control) or were treated with 2,000 U/ml of universal type I IFN-α, and virus titers were determined at 24 and 48 hpi. After IFN-α treatment, compared to type I IFN-untreated cells, r85 viral titers were decreased more than 20-fold (∼ 95%) at both 24 and 48 hpi. In contrast, r87 viral titers were only decreased by 6- and 4-fold (∼85 and 75%) at 24 and 48 hpi, respectively . These dTo test whether I64T mutation diminishes the NS1 protein's ability to counteract host innate immune responses in the context of virus infection, MDCK cells constitutively expressing GFP and Fluc reporter genes under the control of the IFN-β promoter (MDCK IFN-β GFP-CAT/IFN-β Fluc)  were mocNDV infection is highly affected by previous antiviral states induced in the cells . Therefoin vivo. To that end, mice (n = 5) were infected intranasally (i.n.) with 50 FFU of r85 or r87 or were mock infected. Morbidity (body weight loss) and mortality  were monitored for 14 days  were infected (i.n.) with 50 FFU of r85 and r87, and virus titers in lungs extracted at 1, 3, and 5 days postinfection (dpi) were evaluated (3 FFU/ml) were detected in 2 out of 3 mice infected with virus r87, and no infectious virus was detected in mice infected with virus r85. High viral titers (>105 FFU/ml) were detected in the lungs of mice infected with IAV r87 at 3 and 5 dpi, while low titers (<102 FFU/ml) were detected in the lungs of 2 out of 3 mice at 3 dpi and no virus was detected at 5 dpi in mice infected with IAV r85  were analyzed. Interestingly, from 6,980 sequences, most of them  contain I64 (99.9%), 2 contain T64 (0.03%), 1 contains L64 (0.015%), and 1 contains V64 (0.015%) . These data suggest, as shown in the manuscript, that amino acid I64 is relevant for the NS1 function(s), as other amino acids at this position are present only in very minor frequencies.To analyze whether the low frequency of the I64T mutation also applies to other influenza subtypes, such as human H1N1, human 2009 pandemic H1N1, human H5, human H7, avian H1N1, avian H3N2, swine H1N1, swine 2009 pandemic H1N1, and swine H3N2, NS1 protein sequences from the Influenza Research Database were further analyzed . As observed for human H3N2 viruses, the frequencies of amino acids other than I64 were very low (<0.12%) in all analyzed sequences.We have shown that the virus containing NS1-T64 is impaired in antagonizing innate antiviral responses and is highly attenuated, at least in mice. Given these results and the observation that H3N2 IAVs with an NS1-T64 mutation are very rare in the human population, it was surprising that a virus with this mutation was found in a human subject reporting flu symptoms . To analyze whether subject 85, infected with the virus containing the T64-NS1 protein, presented a defect in IFN responses, PBMCs from this subject and subjects 87, 64, and 68 (collected during the 2012-2013 season), as well as 21, 23, and 44 (collected during the 2010-2011 season) as controls, were treated with IFN-α  or infecin vivo, probably because this virus is highly sensitive to IFN responses. Furthermore, we show that the patient infected with this virus presents a defect in the induction of type I IFN responses, probably explaining why this subject was infected with the highly sensitive IFN virus and developed flu symptoms.Influenza virus NS1 protein counteracts host innate immune responses, allowing the virus to replicate efficiently even in IFN-competent systems , 9, 38. Some reports have shown variability in the NS1 proteins from seasonal IAVs –45, and –16–By blocking gene expression in infected cells, the NS1 protein suppresses the expression of type I IFN and type I IFN-stimulated genes, most of which display antiviral activity , 18, 47.20–Influenza virus NS1 protein inhibits host gene expression and, therefore, type I IFN and IFN-stimulated genes by binding and inhibiting the cellular factor CPSF30 –18. To a–in vitro and in vivo. However, r85 grew to lower titers than r87 in the human A549 cells  were rescued. To this end, we used a virus containing a split NS segment . Interes49 cells . Similar49 cells . Our res49 cells  and 9D a49 cells . In agre in mice , 40.It was surprising that subject 85 was infected and reporting flu-like symptoms with a virus encoding the T64-NS1 protein , as we have shown that this mutation is quite rare in the human population (0.03%), most probably because this virus is highly sensitive to IFN responses and is highly attenuated, at least in mice . PBMCs fin vivo. In addition, screening viral sequences in patients infected with viruses bearing mutations that impact IFN antagonism might be a valid strategy to identify patients with possible innate immune defects.Altogether, analyzing the NS1 sequences from influenza viruses infecting subjects and the effect of mutations on NS1 protein functions represents a feasible strategy to identify NS1 mutations affecting innate immune responses and pathogenicity


OA also improved bone properties, Ca balance, and exerted modulatory effects on renal CYP27B1 and CYP24A1 expressions in aged normal female Sprague–Dawley rats. In addition, OA significantly increased renal CYP27B1 mRNA and promoter activity, and suppressed CYP24A1 mRNA and protein expressions in human proximal tubule HKC-8 cells. OA exerted bone protective effects in mature OVX mice and aged female rats. This action on bone might be, at least in part, associated with its effects on Ca and vitamin D metabolism. The present findings suggest that OA is a potential drug candidate for the management of postmenopausal osteoporosis.Oleanolic acid (OA) is a triterpenoid with reported bone anti-resorption activities. The present study aimed to characterize its bone protective effects in vivo and to study its effects on vitamin D metabolism, both in vivo and in vitro. OA significantly increased bone mineral density, improved micro-architectural properties, reduced urinary Ca excretion, increased 1,25(OH) The worldwide estimated osteoporosis-related fracture rate is around 40% in aged women and about 13% in men  and estrsitivity . Vitaminsitivity . Howeversitivity ,9. In adsitivity . Moreovesitivity ,12,13,14Oleanolic acid (3β-hydroxyolean-12-en-28-oic acid)  is a bonFructus Ligustri Lucidi (FLL), an OA-rich Chinese herb, could regulate bone turnover markers and enhance calcium balance in mature OVX rats [Our group was the first to report that OVX rats . Indeed,OVX rats  of FLL. OVX rats  and agedOVX rats ,29. The OVX rats ,31. ReceOVX rats ,33. Thes3 2D3) is essential for normal bone mineralization and calcium homeostasis in the body [2D3 is tightly controlled by the biosynthetic enzyme, 25-hydroxyvitamin D 1-α-hydroxylase (CYP27B1), as well as the catalytic enzyme, 25-hydroxyvitamin D 24-hydroxylase (CYP24A1) [2D3 itself [2D3 [2D3 by inducing renal CYP27B1 activity in rat renal proximal tubule cells [2D3 and alter vitamin D metabolism.1α,25-dihydroxyvitamin Dthe body . The cirCYP24A1) . CYP27B1CYP24A1)  and is tCYP24A1) , 1,25 cells were determined. The results provide evidence of the osteoprotective effects of OA in OVX mice and aged rats.n = 9), OVX + vehicle , OVX + 17β-oestradiol , OVX + OAL , and OVX + OAH . The animals were pair-fed with 3 g, the minimum daily average food intake, phytoestrogen-free AIN-93M rodent diet . The 17β-Oestradiol (E2) was from Sigma–Aldrich  and OA (purity > 98%) was from Shanghai Winherb Medical Technology Co. . The body weights of the animals were monitored on a weekly basis. One day before sacrifice, mice were individually housed in metabolic cages for urine collection. The mice were then sacrificed by cardiac stick exsanguinations under anaesthesia. Serum was prepared in aliquots and stored at −80 °C for biochemical measurements. The uterine index was calculated from the wet weight of uterus over the body weight. The left tibia and intact lumbar vertebra were collected, with soft tissue cleared, wrapped in saline-soaked gauze and stored at −20 °C for micro-computed tomography (μCT) analysis.Forty-two four-month-old C57BL/6J mice were purchased from The Chinese University of Hong Kong. Mice were randomly subjected to either sham-operated or ovariectomy (OVX) and were orally administrated by daily gavage for 6 weeks, as follows: sham + vehicle : control diets with oral administration of OA (25 mg/kg/day) or its vehicle by daily gavage, and a high calcium diet  served as a positive control group. All animals were pair-fed with 15 g/day of a control diet with water supply ad libitum for 12 weeks. Diets were purchased from Harlan Teklad . Before sacrifice, rats were individually placed into metabolic cages for 24 h urine and feces collection. Upon sacrifice, blood, duodenal mucosa, and cortex of the left kidney were collected. Serum in aliquots and tissue samples were stored at −80 °C for further analysis. The left tibia, femur, and the intact lumbar vertebra were collected with soft tissues cleaned, wrapped by phosphate buffered saline (PBS)-soaked gauze, and stored at −20 °C for micro-CT scanning and bone calcium content measurement.Twenty-four retired breeder Sprague–Dawley (SD) rats, aged nine-months-old, were purchased from Beijing Vital River Laboratories . The retired breeders were raised to thirteen-months-old to establish the aged rat model. Before the treatment regimen, rats were pair-fed with a normal calcium diet  for five days as acclimation. Aged female rats were randomly assigned to three groups  and ASESC No.: 130602 (rat study)).2D3 level was determined using a mouse 1,25-dihydroxyvitamin D3 2D3) ELISA kit  for OVX mice and a 1,25-(OH)2-Vitamin D ELISA Kit  for aged rats. Rat serum PTH level was determined by Rat Bio Active Intact PTH ELISA Kit . Urinary calcium, phosphorous, and DPD were corrected by urinary creatinine (Cr) levels, which were determined by picric acid methods, using commercial kits .The concentrations of calcium and phosphorous in serum and urine samples were measured by standard colorimetric methods, using commercial kits, following the manufacturers’ instructions . Mice serum osteocalcin (OCN) level was measured by a mouse osteocalcin ELISA kit . Mice urinary deoxypyridinoline (DPD) level was measured using a METRATM DPD EIA kit . Serum 1,25(OH)3 and 21 μm3, respectively, by the μCT system . Scans were performed at a medium resolution and using energy of 70 kVp, intensity of 114 μA, with an integration time of 300 ms. Distal femurs and proximal tibias were scanned in 100 slices for OVX mice and 210 slices for aged rats, from the metaphyseal growth plates. The volume of interest was contoured from 50 or 100 serials of slices (corresponding to a 0.525 mm region and 2.1 mm region), starting from the disappearance of the condyle, for evaluation. For the lumbar vertebra (L4), 100 slices for OVX mice and 150 slices centered in L4 for aged rats were scanned and evaluated. Morphometric parameters included bone mineral density , bone volume/tissue volume , trabecular number , trabecular thickness , trabecular separation , and connectivity density  were calculated using a three-dimensional direct model with a constant threshold of 300.The left tibias, left femurs and fourth lumbar vertebrae (L4) of OVX mice and aged rats were scanned at voxel sizes of 10.5 μmThe left tibia of both OVX mice and aged rats were first dried at 110 °C in muffle furnace for 12 h and then incinerated at 800 °C for 20 h. The ash weight was recorded. 100 mg of bone ash was weighed and dissolved in 2 mL of 37% HCl and diluted by Milli-Q water for atomization. The bone calcium content was determined by atomic absorption spectrophotometer .The calcium content in 24 h fecal samples from aged rats was determined by the same method as for bone calcium, described previously . The cal2–95% air atmosphere, at 37 °C. Forskolin  and 1,25(OH)2D3  were applied as positive controls for determining CYP27B1 and CYP24A1 expressions, respectively. For assessment of promoter activities, mRNA, and protein expressions, the medium was changed to a chemically–defined, serum-free medium containing the following supplements for 24 h before drug treatment: insulin (5 μg/mL), transferrin (5 μg/L), Na2SeO3 (5 ng/mL), tri-iodothyronine (0.37 nmol/L), epidermal growth factor (2.5 ng/mL), and hydrocortisone (1 nmol/L).Human proximal kidney tubule cells (HKC-8) were a kind gift from Dr. Racusen of Johns Hopkins University . Cells w® Reagent . Total RNA (4 μg) was reverse-transcribed by M-MLV Reverse Transcriptase  following the manufacturer’s instructions. Real-time PCR was performed in a total of 20 μL of reaction mixture, containing 10 μL of SYBR Green Mastermix  and 0.5 μL of cDNA product by 7900HT Fast Real-Time PCR System  using the two-step program: initial denaturation at 95 °C for 10 min, 40 cycles of denaturation at 95 °C for 15 s and 60 °C for 1 min. The sequences of primers for target genes and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are listed in RNA from animal tissues and HKC-8 cells was isolated using TRIzol2, 10% glycerol, 1% Nonidet P-40) supplemented with protease inhibitors: 1 mM PMSF, 2 μg/mL aprotinin, 2 μg/mL leupeptin, 10 mM NaF, and 1 mM sodium orthovanadate . Protein concentrations were determined using the Bradford protein assay . The proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transblotted to polyvinylidene difluoride (PVDF) membranes  and probed with the primary antibodies: rabbit anti-CYP27B1 , rabbit anti-CYP27B1 , or mouse anti-β actin , followed by IgG-HRP-conjugated secondary antibodies anti-rabbit  or anti-mouse . Membranes were incubated with enhanced chemiluminescence (ECL) substrate  for 5 min. The bound antibodies were visualized and quantified by Lumi-Imager . The signal intensity of the bands was presented as Biochemical Light Units (BLU).Cortexes of kidneys and HKC-8 cells were homogenized and lysed in Nonidet P-40 Lysis Buffer , 1,25D (10−8 M) or OA (10−8–10−5 M) for 24 h. A dual luciferase reporter assay was conducted using reagents from Promega. The promoter activities of CYP27 or CYP24 were normalized to pRL-TK. Data were presented as relative luciferase units (RLU).HKC-8 cells were transiently transfected with CYP27- or CYP24-promoter construct. The CYP27-promoter construct, a reporter plasmid containing a full-length insert of 1576 bp CYP27B1 promoter region, was a kind gift from Dr. Farzana Perwad from University of California, San Francisco . CYP24-pp-values less than 0.05 were considered to be statistically significant.All data obtained from the experiments were presented in the form of mean ± SEM (standard error of mean). The differences between groups of data were analyzed by GraphPad Prism Version 6.00 . The significances between different groups of means were evaluated by one-way analysis of variance (ANOVA). Post-test analysis of multiple comparisons was carried by Tukey’s test at a confidence level of 95%. p < 0.001 vs. sham), and treatment of mice with 17β-oestradiol (E2) and both dosages of OA significantly suppressed OVX-induced body weight . Unlike E2, OA treatment in low and high dose groups did not induce a uterus index in OVX mice (p < 0.05 vs. OVX) and with OA . Treatment of OVX mice with E2 and OA at both dosages significantly increased trabecular BMD at all three bone sites. In addition, the OVX-induced bone micro-architecture deterioration in mice was effectively prevented by the administration of E2 (vs. OVX) and both dosages of OA treatments (vs. OVX), as revealed by the significant improvements in BV/TV and Conn.D, and decrease in Tb.Sp at all three sites of bone in OVX mice. Treatment of OVX mice with OA also increased Tb.N at the distal femur and L4, and increased Tb.Th at the proximal tibia and distal femur  and urinary deoxypyridinoline (DPD) levels were significantly increased in OVX mice (vs. Sham). Treatment of OVX mice with E2, but not OA, significantly suppressed OVX-induced serum OCN levels and urinary DPD levels vs. OVX, . Bone asal femur .p < 0.001 vs. OVX, p < 0.05 vs. OVX, p < 0.05 vs. OVX, The regulatory effects of OA on renal vitamin D metabolic enzymes were then investigated. E2 and OA significantly induced renal CYP27B1 mRNA expression in OVX mice (28k (CaBP28k) and duodenal calbindin D9k (CaBP9k) were studied. Both E2 and OA treatments were found to significantly induce the mRNA expression of renal calcium channel TRPV5 , renal calbindin Dp < 0.001 vs. NCD) and serum 1,25(OH)2D3 levels (p < 0.001 vs. NCD) but did not alter urinary calcium and serum PTH levels in aged rats. In contrast, treatment of aged rats with OA did not change urinary phosphorous levels but effectively reduced urinary Ca/Cr levels in aged rats (p < 0.05 vs. NCD). However, OA treatment did not alter serum 1,25(OH)2D3 levels nor serum PTH levels in aged rats , nor OA, altered serum calcium and phosphorous levels in aged rats. HCD treatment significantly decreased urinary phosphorous excretion (ged rats .p < 0.001 vs. NCD) and fecal calcium loss (p < 0.01 vs. NCD) and enhanced calcium balance (p < 0.05 vs. NCD) in aged rats. Administration of OA to aged rats significantly enhanced net calcium balance by two-fold (p < 0.05 vs. NCD) to a comparable level with that of the HCD group.p < 0.05 vs. NCD, p < 0.01 vs. NCD, p < 0.05 vs. NCD, OA significantly induced bone calcium content in the tibia of aged rats to a comparable level with that of the HCD group OA increased BMD and improved bone microarchitecture at three bone sites  in both OVX mice and aged rats. Our results agree with previous reports in which a quinoxaline derivative of OA was shown to increase the BMD in femurs of OVX mice , and 3-m2D3 and PTH.OA behaves like E2, which exerts bone protective actions in an OVX model. E2 as well as OA, at 50 and 100 mg/kg/body weights, was shown to improve BMD and bone properties at three bone sites of OVX mice. However, unlike E2, OA did not suppress the OVX-induced increases in serum OCN and urinary DPD levels in mice. Thus, the bone protective effects exerted by OA in OVX mice might be different from E2 and mediated by distinct mechanisms of actions. In addition, OA increased BMD and improved bone properties in an aged rat model. Such protective actions of OA might be mediated by its reported bone anti-resorption activities ,18,19,232D3 [2D3 levels were also increased in OVX mice, it is unclear if the induction of renal TRPV5 mRNA by OA is mediated via its E2-like actions or via its actions on 1,25(OH)2D3. Indeed, unlike E2, OA did not increase duodenal CaBP9K mRNA in OVX mice, suggesting that its actions on Ca transport protein expression might be distinct from estrogen. Using an in silico target identification tool [2D3 level.OA mimicked E2 in suppressing urinary calcium loss and inducing renal TRPV5 mRNA expression in OVX mice. However, renal TRPV5 expression could be independently induced by 1,25(OH)2D3  and estr2D3  in OVX mion tool , OA was ion tool . However2D3 in aged rats with excess dietary Ca intake [2D3 in aged rats, a vitamin D-independent mechanism might be involved in this action. As the activities and protein expression of the calcium transport proteins were not been measured in the present study, future studies will be needed to characterize the effects of OA as well as its mechanism of actions involved in regulation of intestinal and renal Ca transport in aged rats.OA mimicked HCD in improving calcium balance in aged female rats. The improvement in Ca balance by OA was associated with an increase in duodenal TRPV6 mRNA expression in aged female rats. Thus, apart from the direct effects on bone, our results indicated that OA might protect bone through enhancing calcium balance in aged rats, which, in turn, via its actions in regard to increasing calcium entry across brush border membranes . Howevera intake . However2D3 levels and renal CYP27B1 mRNA expression in OVX mice following treatment for 6 weeks. The actions of OA on vitamin D metabolism in OVX mice appeared to be different from those of E2. The present study as well as others [2D3 levels in OVX mice. In addition, E2 could induce CYP27B1 and suppress CYP24A1 expression in our study using mice and in other studies using avian species [2D3 levels nor renal CYP27B1 expression in aged female rats following treatment for 3 months. This observation agrees with those reported for aged rats in which the induction of renal CYP27B1 by stimuli, such as PTH, were blunted [2D3 production [2D3 levels, future study will be needed to study the mechanism of OA on regulating the local production of 1,25(OH)2D3 in bone and duodenum in aged rats.Our results are the first to report that OA at 100 mg/kg/day could significantly increase serum 1,25(OH)s others ,57 showe species  models.  blunted . In contoduction ,61. As O2D3 to further induce, rather than inhibit, CYP24A1 promoter activity. The discrepancies between the responses of endogenous CYP24A1 expression and the transfected CYP24A1 promoter activities could be accounted for by the composition of the promoter region in the plasmid constructs. Indeed, the promoter employed in the present study is a partial promoter with a 5′-flanking region and partial exon 1 of CYP24A1 promoter which contains a vitamin D receptor (VDR) responsive region [OA significantly induced CYP27B1 and mRNA and stimulated CYP27B1 promoter activity in a dose-dependent manner, suggesting that OA regulated CYP27B1 expression transcriptionally. The induction of OA on CYP27B1 mRNA and promoter activity were of similar levels as those induced by PTH and forskolin in HKC-8, as reported by previous studies ,63. In ae region ,47, and 2D3 via its actions on extra-renal CYP27B1 in bone and duodenum in both animal models. Future studies of the actions of OA on intestinal Ca absorption in OVX animal models as well as the proteins and activities of intestinal and renal Ca-transporters would have provided more evidence on the mechanisms involved. One limitation of our study was that we only measured the renal CYP27B1 and CYP24A1 expression levels and mRNA expressions of the Ca-transporter in the intestine and kidney. Although we observed beneficial effects of OA on bone, Ca balance, and modulatory effects on vitamin D metabolism, the molecular targets and mechanisms involved in OA regulating Ca and vitamin D metabolism remain unclear. Further studies will be needed to investigate if OA could alter local production of 1,25(OH)This study demonstrates that OA exerts osteoprotective effects via multiple in vivo targets. Apart from the reported effects of OA on bone cells ,18,19, t


INK4a; 3) induced by TGFbeta and suppressed by RAS/MAPK(Erk) signaling (the latter explaining the lack of L1CAM induction in RAS-induced senescence); and 4) induced upon downregulation of growth-associated gene ANT2, growth in low-glucose medium or inhibition of the mevalonate pathway. These data indicate that L1CAM is controlled by a number of cell growth- and metabolism-related pathways during SC development. Functionally, SC with enhanced surface L1CAM showed increased adhesion to extracellular matrix and migrated faster. Our results provide mechanistic insights into senescence of human cells, with implications for future senolytic strategies.Aging involves tissue accumulation of senescent cells (SC) whose elimination through senolytic approaches may evoke organismal rejuvenation. SC also contribute to aging-associated pathologies including cancer, hence it is imperative to better identify and target SC. Here, we aimed to identify new cell-surface proteins differentially expressed on human SC. Besides previously reported proteins enriched on SC, we identified 78 proteins enriched and 73 proteins underrepresented in replicatively senescent BJ fibroblasts, including L1CAM, whose expression is normally restricted to the neural system and kidneys. L1CAM was: 1) induced in premature forms of cellular senescence triggered chemically and by gamma-radiation, but not in Ras-induced senescence; 2) induced upon inhibition of cyclin-dependent kinases by p16 Human population is aging rapidly, raising serious global health and societal concerns, at the same time highlighting the urgent need to better understand the process of aging at the molecular, cellular and organismal levels. Recent research has revealed aging-associated accumulation in diverse tissues of the so-called senescent cells (SC) which, along with their secreted products, appear to causally contribute to aging ,2. From in vivo. Indeed, there is accumulating evidence that SC contribute to pathogenesis of several aging-associated degenerative diseases including atherosclerosis 6, 0.12 mM K4Fe[CN]6 × 3 H2O, 1 mM MgCl2 in PBS, pH 6.0) at 37°C and 5% CO2 for 4 to 24 hours (depending on the cell type) according to Dimri et al.  × 100. The size of the area was measured using ImageJ Software; velocity was analyzed with tracking plug-in.BJ cells sorted according to the L1CAM surface level were serum-starved for 24 hours and then seeded into collagen-containing Matrigel inserted in the column  according to the manufacturer’s protocol. Migration directed towards serum-containing medium was monitored with time-lapse Leica Microscope DMI6000 for 8 hours. Cell tracking was performed using ImageJ Software. For estimation of velocity and distance of the tracked cells, Chemotaxis and Migration Tool Software (Ibidi) was used. To evaluate random migration, the same procedure was used except that BJ cells were stained with antibody against L1CAM prior to seeding into Matrigel .2O), or rat tail collagen 1 (2 μg/ml in H2O) at 37°C at 5% CO2 for 1 hour. After washing with 0.1% BSA dissolved in DMEM (WB), the wells were blocked with blocking buffer (0.5% BSA in DMEM) at 37°C at 5% CO2 for 1 hour. After subsequent washing with WB, the plate was chilled on ice for 5 minutes, cell suspensions were seeded into the wells and left to adhere in a CO2 incubator at 37°C for 30 minutes. Next, the plate was shaken at 200 rpm for 10 to 15 seconds and washed three times with WB. Cells were then fixed with 4% formaldehyde at room temperature for 15 minutes, washed with WB and stained with crystal violet (5 mg/ml in 2% ethanol) for 10 minutes. The plate was subsequently washed several times with H2O to get rid of the remains of crystal violet. After drying up the plates, 2% SDS (in H2O) was added and incubated at room temperature for 30 minutes. Optical density at 550 nm was estimated by a spectrophotometer .A 96-well cell culture plate was coated with laminin-1 , fibronectin (20 μg/ml in HAgeI and EcoRI sites in the pLKO.1 vector. A non-target shRNA plasmid was obtained from Sigma (cat. No. SHC016). Lentiviral particles were produced by co-transfection with pMD2.G and psPAX2 (a kind gift from Didier Trono) in HEK293T cells as described previously [siRNAs targeting L1CAM  were introduced into the cells using Lipofectamine™ RNAiMAX . Nonsense siRNA sequences  were used as a negative control. shRNAs targeting L1CAM were introduced into the cells using a lentiviral vector. The shRNA vector targeting L1CAM was obtained by insertion of double-stranded oligo: 5'-CCGGCAGCAAGAGACATATCCACAACTCGAGTTGTGGATATGTCTCTTGCTGTTTTT-3' into the RhoA activity was measured using G-LISA Activation Assay . The procedure was performed according to manufacturer’s protocol.Confluent cell monolayer was scratched by pipette tip. After 4 hours, cells were stained (live) with the L1CAM antibody, then fixed, permeabilized and stained with antibody against a marker of MTOC, γ-tubulin. For determination of cell polarization, the position of MTOC was measured relatively to perpendicular lines (90°) positioned toward the scratch line . The nucSupplementary FileSupplementary Table 1Supplementary Video 1Supplementary Video 2


Listeria monocytogenes causes severe disease mainly in the vulnerable populations of the young, old, pregnant, and immunocompromised. Here, we present the genome sequence of L. monocytogenes H34, a serotype 1/2b, lineage I, sequence type 489 (ST489) strain, isolated from a neonatal sepsis case in Uruguay. The foodborne pathogen  L. monocytogenes strain H34 was isolated from blood and lung cultures from a newborn with sepsis.In August 2016, 10 human cases of listeriosis were observed in Uruguay. As part of a subcluster consisting of 2 strains with similar pulsed-field gel electrophoresis (PFGE) patterns (ApaI and AscI), http://bigsdb.pasteur.fr/) identified H34 as a serotype 1/2b, lineage I, sequence type 489 (ST489), clonal complex 489 (CC489) strain. While lineage I, serotype 1/2b strains are commonly associated with human clinical cases  and 7-gene multilocus sequence typing (MLST)  cells in a PacBio RSII device (Pacific Biosciences). A de novo assembly was performed with the HGAP_3 algorithm . The DNA was sheared to 10 kb in size and size-selected, and PacBio libraries were prepared using C4-P6 sequencing chemistry . A total of 4.5 × 10http://rast.nmpdr.org) determined the lab strain Scott A to be the closest relative to H34.The H34 genome is 2,892,474 bp in size, with a G+C content of 38.0%, and it carries no plasmids. PGAP annotated 2,823 coding sequences and 89 RNAs, and RAST (Listeria pathogenicity islands 1 (LIPI-1)  a deletion in the actA coding sequence (CDS) and (ii) several single nucleotide polymorphisms within the phospholipase C (plcB) CDS and its 5′ untranslated region (UTR).The H34 sequence was further analyzed with a focus on the virulence genes. All genes associated with the (LIPI-1)  were preactA CDS has a 105-nucleotide (nt) in-frame deletion in the P-region of the ActA protein. While the P-region is not required for actin polymerization, it has a role in determining the length of the actin tails and the speed of bacterial motility .The H34 motility , and itsmotility . Within plcB CDS, leading to either a nonfunctional or shortened PlcB protein (85 amino acids), and H34 contains the same mutations in the 5′ UTR of plcB, as described for strain H4 by Jiang et al. (There are several nonsense mutations within the first 246 bp of the  comK CDS.The H34 genome contains 2 incomplete prophages, as determined by Phaster ; none ofCP020774.The complete H34 genome has been deposited in the NCBI GenBank under accession number


D) of 4.06 × 10−9 M. By diversifying the V-regions of the lead mAbs and selecting for cross reactivity we generated five mAbs that bound each of the seven subtypes. Three scFv binding non-overlapping epitopes were converted to IgG that had KD for the different BoNT/F subtypes ranging from 2.2×10−8 M to 1.47×10−12 pM. An equimolar combination of the mAbs was able to potently neutralize BoNT/F1, F2, F4 and F7 in the mouse neutralization assay. The mAbs have potential utility as diagnostics capable of recognizing the known BoNT/F subtypes and could be developed as antitoxins to prevent and treat type F botulism.Human botulism is primarily caused by botulinum neurotoxin (BoNT) serotypes A, B and E, with around 1% caused by serotype F (BoNT/F). BoNT/F comprises at least seven different subtypes with the amino acid sequence difference between subtypes as high as 36%. The sequence differences present a significant challenge for generating monoclonal antibodies (mAbs) that can bind, detect and neutralize all BoNT/F subtypes. We used repertoire cloning of immune mouse antibody variable (V) regions and yeast display to generate a panel of 33 lead single chain Fv (scFv) mAbs that bound one or more BoNT/F subtypes with a median equilibrium dissociation constant (K N) and a receptor-binding domain (HC). There are at least seven distinct BoNT serotypes (A-H) [Botulism occurs in infants and adults and is caused by botulinum neurotoxin (BoNT), the most poisonous substance known. ,2. Botules (A-H) –5, defines (A-H) ,7. For ses (A-H) –10. Subtes (A-H) .2 whose seven serotype-specific components have short serum half-lives (7.5–34.2 hours), which preclude its effective use for prophylaxis and may predispose to relapse of botulism after treatment [Four BoNT serotypes  cause virtually all human botulism. BoNTs are also classified as Category A biothreat agents, HHS Tier 1 select agents/toxins, one of the seven highest-risk threat agents for bioterrorism . The onlreatment . As an areatment  are beinreatment . These mreatment ,24.For this work, we sought to generate a panel of mAbs that bound most, or all, of the highly disparate (up to 36% different at the amino acid level) BoNT/F sub-serotypes for the purpose of generating a therapeutic antitoxin.The USAMRIID Institutional Animal Care and Use Committee approved the animal care and use protocol to conduct the animal studies reported here. Research was conducted under an IACUC approved protocol in compliance with the Animal Welfare Act, PHS Policy, and other Federal statutes and regulations relating to animals and experiments involving animals. The facility where this research was conducted is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care, International (AAALAC/I) and adheres to principles stated in the Guide for the Care and Use of Laboratory Animals, National Research Council, 2011. The specific national regulations and guidelines to which this animal care and use protocol adheres are the following: (1) 7 USC, Sections 2131–2159, Chapter 54 “Animal Welfare Act”, and  Health Research Extension Act of 1985, Public Law 99–158 “Animals in Research” and the Public Health Service Policy in Humane Care and Use of Laboratory Animals; (3) Biosafety in Microbiological and Biomedical Laboratories, 5th Edition, NIH, Human and Health Services Publication (CDC) 21–112; (4) Army Regulation 40–33 “The Care and Use of Animals in DOD Research, Development, Test and Evaluation or Training Programs” and (5) DOD Instruction 3216.01 “Use of Animals in DOD Programs”. DOD uses the “The Guide for the Care and Use of Laboratory Animals”, 8th Edition, Institute for Laboratory Animal Research, National Research Council, as a guideline for evaluation and accreditation of program and it is based on the actual national regulations and guidelines for animal care and use programs. The animals used in this study were euthanized using carbon dioxide gas following the AVMA Guidelines on Euthanasia prior to spleen removal.The University of California, San Francisco (UCSF) Institutional Review Board approved the human use protocol used for the studies described here. Human donors were laboratory workers being immunized to work with BoNT who were recruited via an informational letter and who signed informed consent.Saccharomyces cerevisiae strain EBY100 was used for library construction and BoNT/F domain display. Escherichia coli DH5α was used for subcloning and preparation of plasmid DNA, while the strain BL21 was used for BoNT/F fragment expression. Chinese hamster ovary (CHO) cells were used for immunoglobulin G (IgG) expression. Yeast Peptone dextrose (YPD) medium was used for growth of EBY100 cells. Selective growth dextrose casamino acids media (SD-CAA) was used for selection of pYD4-transformed EBY100 as previously described [escribed  and seleN) and F1-7 C-terminal portion of the heavy chain (HC) were amplified from synthetic BoNT/F1-7 genes. All the cDNAs were subcloned into the plasmid pET28b with a hexahistidine (His) tag  or a maltose binding protein (MBP) tag (F2-6HC). The fragments were expressed in the E. coli strain BL21 and purified as described [C fragments were purified using the agarose Dextrin Sepharose High Performance , eluted into PBS with 10mM maltose. The His-tagged fragments were purified with a Ni-NTA agarose . The recombinant proteins were dialyzed with PBS buffer overnight to remove imidazole or maltose prior to use. IgG were generated by subcloning VH and Vk genes into a mammalian expression vector with human heavy and kappa light chain constant regions, establishment of stable CHO cell lines by transfection and purification of IgG by Protein G chromatography as previously reported [The cDNA of 14 BoNT/F fragments including F1-7 N-terminal portion of the light chain  BoNT/F1, BoNT/Bf2, and BoNT/F6; 2.5 x 103 LD50 BoNT/Af4; 0.5 x 103 LD50 BoNT/F7). BoNT/F5 boosts were done with nontoxic BoNT/F5 LC-HN. For BoNT/F LC-HN immunization, 0.25–2.5 μg/mouse of antigen were subcutaneously injected with adjuvant on Days 1,14 and 28, 0.25–2.5 ug/mouse and 0.5–5.0 μg/mouse of antigen was intramuscularly injected with adjuvant; at day 42, proteins were intravenously injected in PBS. Antibody titers after the third vaccination ranged from 0.2–10 x 10−5. Mice were euthanized and spleens were removed 3 to 5 days after their final vaccinations and processed to extract mRNA for scFv library construction.For the BoNT/F toxin immunization, mice were vaccinated three times at two week intervals  with 10 μg/mouse of catalytically inactive BoNT/ACEF HC. Mice boosted with BoNT/F5 were also vaccinated with BoNT/F5 LC. Mice were boosted twice with 1 μg active toxin  or from spleens of immunized mice. cDNA synthesis, Vescribed . LibraryMonoclonal BoNT/F antibodies were isolated as described ,26 by usD) of yeast-displayed scFv was measured by flow cytometry, as previously described [N binders) or mAbs 6F8 and 6F3 (for HC binders) as secondary antibodies. Briefly, 2x106 yeast were incubated in serially diluted BoNT/F toxin or fragments for 1 hour at room temperature, then with a detection antibody and a R-phycoerythrin (PE)-conjugated goat anti-human or anti-mouse antibody together with Alexa Fluor® 647-labeled SV5 tag antibody. Mean fluorescence intensity (MFI) was measured by flow cytometry as previously described [The equilibrium dissociation constant  or BoNT/F1, F2, F3, F4, F5, F6, F7 HC individually at room temperature for 1 hour. All subsequent washing and staining steps were performed at 4°C using ice-cold FACS buffer as above. Yeast-displayed scFvs were incubated for 30 minutes with 2 μg/mL of a mixture of Alexa-647 conjugated IgGs 6F5.1 and 6F11 (for LC-HN binders) or mAbs 6F3 and 6F8 (for HC binders) as the secondary antibody for binding detection, together with an Alexa-488 labeled anti-SV5 IgG for detection of scFv expression. Finally, the yeasts were washed using cold FACS buffer and BoNT/F subtype binding was measured by flow cytometry.The yeast-displayed scFv were incubated with 100nM BoNT/F1, F2, F3, F5, F6, F7 LC-HH gene of the scFv was PCR-amplified with primers with gap tails and gel purified. The plasmids containing a pYD4-VK repertoire [H gene was transformed into EBY100 together with the linearized pYD4-VK plasmids using LiAC as described previously [™ DNA polymerase , in which 0.05mM MnCl2 was applied to introduce random mutation. The cDNA with mutations was gel purified and transformed into EBY 100 together with NcoI/NotI digested pYD4 plasmid by LiAC method as described previously [The antibodies that potentially bound all seven subtypes of BoNT/F were affinity matured by chain shuffling, followed by error prone PCR mediated random mutagenesis. For the chain shuffling libraries, the Vpertoire  were lineviously . For theeviously . The libD values were measured and the colonies with highest affinity to each BoNT/F subtype were selected.In order to isolate the colonies that had improved affinity to all of the BoNT/F subtypes, each library was sorted at least five times; each subsequent round of sorting used a different BoNT/F subtype, with the antigen concentration decreased from 100nM to 100pM from the first to last round of sorting. After the final round of sorting, the collected yeast clones were grown on SD-CAA plates followed by plasmid isolation and DNA sequencing. KN and HC domains were PCR amplified from the seven synthesized BoNT/F genes. Gel purified cDNA fragments were digested with NcoI/NotI and cloned into the plasmid pYD4. The recombinant plasmids were then used to transform EBY100 by LiAC, which were grown on SD-CAA plate for 72 hours. Individual colonies were picked, grown in SD-CAA medium and were induced with SG-CAA medium at 18°C for 48 hours to express the BoNT/F domain on the yeast surface. Yeast colonies were incubated with 2 μg/ml one of the BoNT/F IgGs at room temperature for 1 hour, followed by PE-conjugated goat anti-mouse IgG together with Alexa-488 labeled anti-SV5 IgG at 4°C for 30 minutes. Yeast clones were washed and assayed by flow cytometry.cDNA encoding each subtype of the BoNT/F1 LC, HN and FC. In order to compare the structure of BoNT/F subtypes, the amino acid sequence of BoNT/F1 was aligned with the other subtypes using the online tool “MultAlin”, , [http://espript.ibcp.fr) [BoNT/F1 was modeled on BoNT/A crystal structure (pdb ID: 2NZ9) using the software UCSF-Chimera . The modltalin), . The aliibcp.fr) . which tibcp.fr) . The difThe mouse neutralization assay was performed as described previously . BrieflyThe BoNT/F subtypes differ from each other by 1.1% to 36.1% at the amino acid level . BoNT/F1C) or translocation domain (HN) fused to the light chain (LC) (LC-HN) of sub-serotypes F1, F2, F3, F4, F5, F6 and F7 were designed and cloned into plasmid pET28b to contain His tags or MBP tags. His-tagged BoNT/F1 and BoNT/F7 HC and MBP-tagged BoNT/F2, 3, 4, 5, 6 HC were expressed in the cytoplasm and could be purified , which showed improved affinity to BoNT/F1 but low affinity to other subtypes. Then V gene of 4E17.2 (6F5) was randomly mutated by error prone PCR and sorted using extremely low BoNT/F1 holotoxin concentrations (100pM), which yielded the scFv 6F5.1 which has a 3.75 to >700 fold increase in affinity for the BoNT/F subtypes with the KD ranging from 0.32 nM for BoNT/F4 to 2.97nM for BoNT/F7  and LC-HN were subcloned into the plasmid pYD4 and displayed on the surface of EBY100. The yeast-displayed domains were incubated with Alexa-647 conjugated IgG and Alexa-488 labeled antiSV5 IgG and detected by flow cytometry. The results demonstrated that 6F5.1, 6F11, 6F12, and 6F13 bound BoNT/F LC-HN and BoNT/F HN, indicating that they bound on HN domain, and that 6F8 and 6F15 bound BoNT/F HC . A broad range of specific activity of the different BoNT/F subtypes  (50) of BoNT/F1 and 1 μg of the mAb combination protected 8/10 mice, indicating a median effective dose (ED50) of less than 1 μg (50) challenge dose of BoNT occurred at a mAb dose of 20 μg, with an ED50 of less than 10 μg. The mAb combination was least potent for BoNT/F2, with an ED50 of approximately 50 μg, likely reflecting the lower affinity of mAb 6F13 for this subtype (22 nM) compared to the other BoNT/F subtypes  . Five mihan 1 μg . For BoN50 of BoNT/F1. In contrast, 50ug of HBAT was needed to neutralize 6600 LD50 of BoNT/F1. This difference would be expected given that only a fraction of polyclonal antitoxins bind the BoNT F1. For BoNT/F2, F4 and F7, the mAb combination also demonstrated much potent neutralizing capacity than HBAT, with the ED50 of <50ug for BoNT/F2, < 5.0ug for BoNT/F4 and < 10ug for BoNT/F7. By comparison, the range of ED50s of the equine antitoxin for the four BoNT subtypes were more than 100 fold lower than the mAb combination; the lowest ED50 was for BoNT/F7, with 300ug of HBAT rescuing 9 of 10 mice with 344 LD50 of BoNT/F7 challenge but without showing any protection if mice exposed to 11000 LD50 of BoNT/F7 , since 1.0ug of mAbs neutralized 20,000LD BoNT/F7 .Using repertoire cloning, a panel of murine scFv antibody fragments were generated that bound one or more of the BoNT/F subtypes. Only one of these lead scFv bound all seven BoNT/F subtypes, reflective of the fact that the subtypes differ by up to 36% at the amino acid level . Using yin vivo reducing the risk of reintoxication [The mAbs reported here have potential diagnostic and therapeutic utility due to their subtype cross reactivity. The ability to bind all seven BoNT/F subtypes allows sensitive detection of all known BoNT/F’s and increases the probability that yet-to-be discovered subtypes will also be detected ,36. Therxication .S1 FigA. Cartoon of botulinum neurotoxin primary structure. B. SDS-PAGE analysis of BoNT/F fragments expressed from E. coli BL21 (DE3). The upper panel shows the expression of BoNT/F LC-HN fragments fused with a 6 x His tag (~110Kd). C. Expression of BoNT/F HC fragments fused with a 6 x His tag  or fused to a maltose binding protein (MBP) tag .(TIF)Click here for additional data file.S2 FigN fragments  or were incubated with BoNT/F HC fragments (6F8 and 28H4) and then an Alexa-647 labeled secondary IgG was used for binding detection. An Alexa-488 labeled anti-SV5 IgG was used to detect scFv mAb expression on yeasts. Binding to BoNT/F4 was measured using crude BoNT/F4 culture supernatant due to the absence of recombinant domains.Yeast displayed scFv were incubated with BoNT/F LC-H(TIF)Click here for additional data file.S3 FigYeast displayed BoNT/F1 LC, HN, LC-HN or HC was incubated with Alexa-647 labeled IgG and Alexa-488 labeled anti-SV5 IgG.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


In 2014, a sentinel chicken surveillance for avian influenza viruses was conducted in aquatic bird habitat near Wuxi City, Jiangsu Province, China. Two H7N2, one H5N6, and two H9N2 viruses were isolated. Sequence analysis revealed that the H7N2 virus is a novel reassortant of H7N9 and H9N2 viruses and H5N6 virus is a reassortant of H5N1 clade 2.3.4 and H6N6 viruses. Substitutions V186 and L226 (H3 numbering) in the hemagglutinin (HA) gene protein was found in two H7N2 viruses but not in the H5N6 virus. Two A138 and A160 mutations were identified in the HA gene protein of all three viruses but a P128 mutation was only observed in the H5N6 virus. A deletion of 3 and 11 amino acids in the neuraminidase stalk region was found in two H7N2 and H5N6 viruses, respectively. Moreover, a mutation of N31 in M2 protein was observed in both two H7N2 viruses. High similarity of these isolated viruses to viruses previously identified among poultry and humans, suggests that peridomestic aquatic birds may play a role in sustaining novel virus transmission. Therefore, continued surveillance is needed to monitor these avian influenza viruses in wild bird and domestic poultry that may pose a threat to poultry and human health. Since the first emergence of avian influenza A(H7N9) virus in early 2013  white leghorn chickens  were placed at sentinel surveillance site. A high rate of females to males (4:1) was chosen to avoid fighting among roosters. Before the sentinel chickens were placed into the surveillance site, cloacal and throat swabs of each chicken were collected, pooled, and screened for influenza A virus using a real-time RT-PCR assay which targeted the matrix gene  containing 50% glycerol, penicillin (2000 U/ml), gentamicin (250 mg/ml), polymixin B (2000 U/ml), nystatin (500 U/ml), ofloxacin HCl (60 mg/ml), and sulfamethoxazole (200 mg/ ml) and were kept at 4°C for ~4 h until they were transported to the laboratory, where they were stored at −80°C for virus isolation within 2 days.The chicken handling and sampling was performed in accordance with experimental animal administration and the ethics committee of Academy of Military of Medical Sciences. The protocol was approved by the biosafety committee of Beijing institute of Microbiology and Epidemiology.All of swab samples were inoculated into the allantoic cavities of 9-day-old SPF embryonated chicken eggs  for virus isolation. Allantoic fluid were harvested after incubation at 37°C for 72 h or immediately after embryos died, and tested for hemagglutination (HA) activity with horse red blood cell and real time RT-PCR to detect the presence of influenza A virus. All virus isolation procedures were conducted in a biosafety level 3 facility. The whole viral genome of isolated viruses were sequenced by using Ion Torrent PGM sequencing technology .2 incubator for 1 h for virus absorption. Then covered with 1% agar containing MEM, penicillin (1000 unit/ml), streptomycin (1000 μg/ml), fungizone (2.5 μg/ml), MEM vitamin solution, 0.5% bovine serum albumin, and 2 μg/ml trypsin. After 2 days of incubation at 37°C in a CO2 incubator and stained with 0.025% neutral red containing 1% agarose, 42 and 53 plaques for #4315 and #6395 were initially picked for further purification and resuspended in 500 μl of infection medium. The subtypes of all resuspended virus stocks were verified using real time RT-PCR analysis (rRT-PCR), and virus stocks showing a single subtype were further purified in SPF chicken embryos. After three serial rounds of purification, the H7N2 and H9N2 virus isolates were successfully separated and their genome were fully sequenced. The sequences data generated in this study were deposited in Global Initiative on Sharing All Influenza Data . We used the maximum likelihood method to generate phylogenetic trees using MEGA version 6.0.6 (www.megasoftware.net/) with a bootstrapping resampling process (1000 replications) to assess the robustness of individual nodes of the phylogeny.Two swab samples, #4315 and #6395, that contained mixed H7 and H9 subtypes were subjected to the standard plaque purification  of 1623 samples  were positive for HA activity after virus isolation. Subtype determination, using rRT-PCR analysis targeting the H5, H7, and H9 HA genes, showed that specimens #4315 and #6395 were positive for all H7 and H9 genes, and specimen #7393 was only positive for H5 gene. To confirm these results, three HA-positive allantoic fluid specimens were subjected to sequencing on Ion Torrent PGM, and results consist with rRT-PCR that both specimens #4315 and #6395 that contained influenza A H7, H9, and N2 gene segments suggesting a coinfection of H7 and H9 subtypes, while specimens #7393 only presented with H5 and N6 type of influenza A virus. We then attempted to isolate individual H7 and H9 viruses from these two samples using a plaque purification. Fortunately, we were able to segregate the expected subtypes, H7N2 and H9N2 from two co-infected samples. Thus, totally five strains were isolated and designated as A/chicken/Wuxi/SC4315/2014 (H7N2), A/chicken/Wuxi/SC6395/2014 (H7N2), A/chicken/Wuxi/SC4315/2014 (H9N2), A/chicken/Wuxi/SC6395/2014 (H9N2), and A/chicken/Wuxi/SC7393/2014 (H5N6).To characterize H7N2 viruses, molecular and phylogenetic analysis were conducted. The HA gene segments of the two H7N2 viruses most closely matched those of H7N9 viruses isolated from human and poultry in China in 2013 with an up to 99.6% nucleotide sequence identity  clade 2.3.4.4 from Guangdong Province, China. The virus likely originated from highly pathogenic H5 avian influenza viruses with N1, N2, and N8 neuriminidases detected in poultry in China since 2010 and from viruses detected in Vietnam in 2014  were closely related to those of H9N2 viruses that were isolated from poultry in China, providing the evidence that H7N9 viruses continue to evolve and reassort with H9N2 viruses in poultry in China. Molecular analysis showed that the substitutions at A138, A160, V186, and L226 (H3 numbering) in HA protein were observed, which is proposed to enhance binding capacity of human like receptors and mammalian adaption. A deletion of three amino acids in the viral NA stalk has been observed. It has been suggested that amino acid deletion within the stalk domain may be associated with transmission of influenza from wild birds to domestic poultry  virus that belong to clade 2.3.4.4 from a sentinel chicken. However, any death bird was not observed in study site during period of surveillance although H5N6 viruses have been detected in fatal wild birds (Yu et al., The study had several limitations. Firstly, samples from wild birds were not collected in the study area due to the restriction of regulations. Secondly, environmental samples were not collected to test the presence of avian influenza viruses. Thirdly, the low virus isolation rate could be due to the possible low prevalence of avian influenza viruses and low viral load in samples.In conclusion, our results suggest transmission of avian influenza viruses from wild birds to poultry. Hence, public health officials must consider methods to disrupt peridomestic novel avian influenza virus transmission in these aquatic bird environments.M-JM, TZ, Y-HQ, S-HC, G-LW, M-NW, YH, G-YM, L-QF, G-CG, BL, and Y-GT conducted the study and analyzed data, M-JM, S-HC, TZ, G-LW, M-NW, YH, and G-YM processed samples and virus isolation., M-JM and WC conceived and designed the study and wrote the manuscript.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.


Our current view of DNA methylation processes is strongly moving: First, even if it was generally admitted that DNMT3A and DNMT3B are associated with de novo methylation and DNMT1 is associated with inheritance DNA methylation, these distinctions are now not so clear. Secondly, since one decade, many partners of DNMTs have been involved in both the regulation of DNA methylation activity and DNMT recruitment on DNA. The high diversity of interactions and the combination of these interactions let us to subclass the different DNMT-including complexes. For example, the DNMT3L/DNMT3A complex is mainly related to de novo DNA methylation in embryonic states, whereas the DNMT1/PCNA/UHRF1 complex is required for maintaining global DNA methylation following DNA replication. On the opposite to these unspecific DNA methylation machineries , some recently identified DNMT-including complexes are recruited on specific DNA sequences. The coexistence of both types of DNA methylation (un/specific) suggests a close cooperation and an orchestration between these systems to maintain genome and epigenome integrities. Deregulation of these systems can lead to pathologic disorders. Asp [DNA methylation, occurring in CpGs motifs, is the reaction catalyzing the covalent transfer of a methyl group from S-andenosyl methionine (SAM) to the fifth carbon of cytosines (C). DNA methylation is involved in numerous biological events , and it concerns about 70–80% of CpGs in mammalian DNA. DNA methylation, which is generally observed in a condensed chromatin and associates with transcriptional gene silencing when it occurs in promoters, is processed by two distinct mechanisms: (i) the inheritance DNA methylation that allows the maintenance of DNA methylation marks on the new strand using the parental methylated strand as a matrix, following DNA replication and (ii) de novo DNA methylation which occurs on both strands independently of DNA replication. De novo methylation happens predominantly during embryogenesis and is further maintained by the DNA methylation inheritance machinery after DNA replication and cell division. DNA methylation is processed by a family of enzymes, the DNA methyl transferases (DNMTs), which are divided in three classes: DNMT1, DNMT2, and the DNMT3A/3B/3L. The DNMT2 functions have been poorly investigated: This enzyme may methylate the consensus sequence TTNCGGAR but DNMT2 is probably mainly involved in the methylation of C38 of tRNADNMT1, a large protein of 1616 amino acids (aa) which mainly catalyzes DNA methylation inheritance activity, is composed of a large regulator N-terminal region (1000 aa) and a small catalytic C-terminal region. In this last region, 10 catalytic domains are essential for the interaction with the SAM. C-ter and N-ter regions are linked by 12 repeats of KG di-peptides. The N-ter region is composed of (i) a binding protein domain able to interact with a large panel of proteins , (ii) a De novo DNA methylation activity, catalyzed by DNMT3A and DNMT3B, is essential during embryonic development or gametogenesis but is also frequently associated to aberrant gene repression in many pathologies  . The strSince 10 years, our knowledge on the roles of DNMTs in DNA methylation has highly raised. More and more partners of each DNMT have been reported, and our view of DNA methylation machineries is currently moving, let us understand that (i) crosstalks exist between de novo and maintaining DNA methylation machineries and (ii) DNA methylation can be mediated by different DNMT-including complexes and some of them are not associated with specific DNA sequences while other complexes may target the methylation in a specific loci. The different kinds of DNMT-including complexes involved in DNA methylation are summarized in Fig. Mutations in the RFTS domain of DNMT1 dramatically reduced its activity. Moreover, this RFTS domain is probably involved in the allosteric activation of DNMT1. Indeed, stable DNMT1 homodimers are linked by a hydrophobic interaction requiring the RFTS domain of each DNMT1. Although monomeric DNMT1 presented a 2- to 50-fold increase activity in the presence of hemi-methylated DNA, compared to methylated DNA, many different partners could also be involved in the recruitment of DNMT1 on hemi-methylated DNA –12. A stThe concomitant presence of several repressive marks has often been observed on silenced promoters. Indeed, the presence of 5mC is often correlated with histone deacetylation, suggesting that DNA methylation and histone regulation machineries thinly cooperate. Maintaining of DNA methylation in heterochromatin required DNMT1/HDAC1 interaction and the deacetylation of histones . HDAC2 aAlthough most of the unspecific inheritance DNA methylation activity is probably processed by the DNMT1/PCNA/UHRF1/HDAC/G9a complex, additional DNMT1-including complexes have been reported.MAGEA10) [Following DNA replication, some nucleosomes are deposited on the new replicated strand. Kinetics of histone modifications and DNA methylation are highly related but are dependent of both, the local DNA sequence and the nature of DNMTs/HDACs/HMTs complexes recruited. Although, in vitro, the DNA methylation of heterochromatin can be catalyzed by free DNMT1 on mononucleosomes, the interaction of DNMT1 with the ATP-dependent nucleosome remodeler SNF2H (SNF2 homolog) strongly increased DNMT1 recruitment on these nucleosomes . Some HMMAGEA10) .CFP1 (CysxxCys finger protein 1), which presents a high affinity for unmethylated DNA, has been shown to interact with DNMT1  . CFP1−/−MBDs (methyl-CpG-binding domain protein) are organized in a family of five proteins  known to interact with methylated DNA and are also involved in the recruitment of DNMT1. Indeed, the MBD2/MBD3 heterodimer which preferentially interact with hemi-methylated DNA could also recruit DNMT1, during late S phase, in replication loci . MeCP2 wDMAP1 (DNMT-associated protein1) is a transcriptional co-repressor also involved in DNA methylation inheritance. DNMT1/DMAP1 interaction (via aa 12–105 of DNMT1) was involved in both early (euchromatin) and late S phase (heterochromatin) of DNA replication and in the recruitment of PCNA . The DNMIn spite of the absence of a clear affinity for a particular DNA sequence, each DNMT1-including complex could be associated to a different or a partially redundant DNA methylation profile. Indeed, a specific peptide-mediated disruption of DNMT1/PCNA, DNMT1/HDAC1, DNMT1/DNMT3B, or DNMT1/HP1 interactions promoted global DNA hypomethylation in astrocytes and increased tumor growth , 56, 57.As already seen above, DNA methylation deregulation is associated with tumorigenesis. An association of DNMT1 with two distinct viral oncoproteins has been reported. Both DNMT1/E1A (in adenovirus) and DNMT1/E7 (in papillomavirus) interactions increased inheritance DNA methylation. Although mechanisms governing this phenomenon are still unclear, it has been proposed that viral oncoproteins might, as already described for DNMT3L, promote DNMT1 DNA binding and SAM recruitment .miRNA-21 (microRNA) and miRNA-148a that control the DNMT1 gene expression [miRNA-29b inhibited the expression of SP1  and consequently decreased DNMT1 expression [A deregulation of inheritance methylation activity was reported in many pathologies. Indeed, in lupus patients, a decrease of DNMT1 was mediated by the overexpression of pression . In acutpression . In spitpression . S127 anpression . On the pression , 64. An pression . Moreovepression .IGF2 and XIST) and a progressive global DNA hypomethylation [In spite of their predominant role in de novo methylation, DNMT3A and DNMT3B are also involved in maintaining DNA methylation . Cooperahylation , 71. Jeohylation .CASP8 promoter in glioma cells [In cells, maintaining and de novo DNA methylation activities are not compartmentalized and evident crosstalks between these machineries have been underlined. Indeed, in colorectal cancer cells, invalidation of DNMT1 or DNMT3B had minor effects on global DNA methylation while double invalidation reduced to more than 90% the 5mC content , 74. Morma cells , 76.DNMT3B gene induce a specific hypomethylation of heterochromatin satellite-2 sequences leading to the ICF  syndrome. Indeed, de novo DNA methylation of CpG-rich sequences  could be easily catalyzed by a processive enzyme such as DNMT3B , whereas DNMT3A is a distributive enzyme [Mutations in the e enzyme . Contrare enzyme , 79. More enzyme .Alu sequences, in the promoters of imprinted genes and in CpG-rich regions with CpG spaced from 8 to 10 pb [The absence of methylation of H3K4 (H3K4me0) also controls the DNMT3 recruitment on chromatin during gametogenesis and embryogenesis. Moreover, the identification of H3K4me0 by DNMT3L (via PHD domain) could also promote in a DNMT3L/DNMT3A/3B interactions manner, the recruitment of DNMT3A and DNMT3B on DNA. Crystal structure of DNMT3L/DNMT3A complexes revealed that these proteins associated in dimers or tetramers (1–2 DNMT3A linked via their C-ter region to the C-ter region of 1–2 DNMT3L) –85. Thesto 10 pb , 89. Aboto 10 pb –92.As seen above for unspecific maintaining DNA methylation, de novo methylation also frequently requires cooperation between DNMTs and chromatin remodelers . e.g., iB-GLOBIN gene), suggesting that de novo methylation activity is also closely related to histone methylation [DNMT3A could also directly read the H3K36me3 and H4R3me2 marks to complete gene repression in a DNA methylation manner  an initial addition of the H3K9me2/3 mark by G9a, (ii) the identification of this mark by HP1, and (iii) the silencing was then completed by the recruitment of DNMT3A and DNMT3B for DNA methylation [RASSAF1A gene required (i) addition of H3K9me3 marks on the promoter by the SETDB1/HDAC1 complex, (ii) recruitment of DNMT3A (via DNMT3A/SETBD1 and DNMT3A/HDAC1 interactions), and (iii) DNA methylation. The formation of the BRG1/G9a/DNMT3A complex was induced following stress induction in mice and responsible for the repression of the motor MYH6 gene and cardiac dysfunctions [Similar mechanisms were also reported in euchromatin: as an example, the kinetic of hylation , 94, 106hylation , e.g., tunctions .The direct methylation of the murine DNMT3A (K44me2) by G9a or by GLP (G9a-like protein) could also be recognized by MMP8 (M-phase phosphoprotein 8). The DNMT3A/MPP8/G9a(or GLP) silencing complex was predominantly recruited close to H3K9me marks .UHRF1, the essential component of the unspecific maintaining DNA methylation activity catalyzed by the DNMT1/PCNA/UHRF1/G9a complex, is also able to interact with DNMT3A and DNMT3B independently of the presence of DNMT1 . These iD4Z4 subtelomeric repeat was dependent of DNMT1 and not DNMT3B [Although DNMT1 is predominantly involved in maintaining DNA methylation and that its affinity for hemi-methylated DNA is 2- to 50-fold more important than for unmethylated DNA, large high-resolution sequencing of repetitive elements or of single copy genes revealed a role of DNMT1 in de novo methylation . Indeed,t DNMT3B . Among tt DNMT3B . Recruitt DNMT3B . All of CG(T/G/A)(C/A/T)(A/T/C), (T/A/C)(A/T)(T/G/A)CG(T/G/C)G(G/C/A), and (A/C)(C/G/A)(A/G)CGT(C/G)(A/G). Others reported that CANAGCTG and CCGG(A/T)NC(C/G)C sequences were more frequently found in methylated genes, following overexpression of DNMT3A and DNMT3, respectively [As seen above, de novo methylation is crucially related to many pathologies. In cancer cells, a paradoxal global DNA hypomethylation is frequently concomitant with both local hypo and hypermethylations of genes. These defects in DNA methylation could induce TSG silencing or resistance to cells death inducers . In glioectively . HoweverThe mechanisms involved in DNA repair, following DNA breakages, abasic sites formation or inappropriate matching, have been intensively investigated. However, the question of how epigenetic marks are repaired is still unclear. Indeed, 5mC are mutational hotspots, as the non-correction of a 5mC deamination lead to the incorporation of T:G mismatches in DNA. Even if 5mC deamination is properly corrected by a C, loss of methylation can potentially upregulate the expression of the concerned genes. The association of DNMTs with DNA repair machineries may also occasionally provoke de novo methylation and aberrant gene silencing. Indeed, DNMT1 could interact with the MMR (mismatch repair), the major DNA repair complex. DNMT1 recruitment on DNA break was independent of DNA replication and S phase, but was still mediated by PCNA . Moreovemethyl-CpG-binding domain protein 4) and TDG (G/T mismatch-specific thymidine DNA glycosylase), two enzymes involved in the (base excision repair) BER complex [PS2 promoter [DNMT3A or DNMT3B was also involved in DNA methylation following DNA repair, via a direct association with MBD4  leading to the recruitment of DNMT1 and the methylation of FAS promoter [In many cancers, the activation of the oncogene promoter .HOX genes expression by (i) inducing PRC2-mediated ubiquitinylation of H2AK119, (ii) inducing the PRC1-mediated H3K27me3 mark, (iii) a direct interaction of BMI1  with DMAP1 leading to the recruitment of DNMT1 and the silencing of particular genes [HOX genes [MYT1 promoter was dependent of interactions between DNMT3A or DNMT3B (via their PHD domain) and PRC components, and the recruitment of the complex on the MYT promoter was performed in an EZH2-dependent manner. Interestingly, in these cells, the EZH2-dependent recruitment of DNMT3A was associated with H3K27me3 but not always with DNA methylation suggesting new roles for DNMT3A on gene repression independently of de novo methylation [The polycomb group (PcG) system is composed of four interdependent multi-protein repressor complexes (PRC1 and 2/3/4) involved in the regulation of homeotic genes during development and chromatin remodeling in stem cells. Since some particular areas of DNA are controlled by PcG and are generally highly methylated, it has been suggested that PcG complexes are connected to DNA methylation machineries, to silence specific loci. PRC1–4 multi-protein complexes sequentially inhibit ar genes , 128. A OX genes , 130. DNOX genes . In ES chylation .OCT-4 gene which occurs during development required (i) the local G9a-mediated H3K9 methylation and (ii) the recruitment of MBD2, MBD3, and GCNF/DNMT3A/DNMT3B complex on specific RAREs boxes specifically recognized by the repressor GCNF (germ cell nuclear factor) [OCT-4 promoter and interacted with G9a also favored a direct interaction of G9a with DNMT3A and DNMT3B (via ANK domain of G9a) [The presence of transcriptional repressors in DNMT-including complexes may explain some specific targeting of DNMTs. Indeed, the complete silencing of the  factor) , 134. Mo of G9a) .RP58 response elements although local DNA methylation was not observed [IL-4 or IGFBP-3) and required direct interactions of HBX with both DNMT3A and HDAC1 [Interaction of DNMT3A with the transcriptional repressor RP58 also promoted the recruitment of DNMT3A on observed . Additiond HDAC1 , 137.The specific silencing of gene coding for rRNA (rDNA) requires both DNA methylation and chromatin remodeling and is orchestrated by a putative complex including TIP5 (TTF-I interacting protein 5), SNF2h, HDCA1, DNMT1, and DNMT3B. Interaction of TIP-5 with H4K16ac marks on rDNA was required for the recruitment of HDAC1, DNMT1, and SNF2h and for the consecutive local deacetylation and DNA methylation in these promoters , 139.Roles of TFs in DNA methylation are still unclear. However, in regard to recent data of literature, it appeared that DNMTs are able to “use” a TF as a co-repressor.SLIT2 promoter was mainly processed by the canonical DNMT1/PCNA/UHRF1 complex, but methylation of specific CpGs localized in or near a SP1 box, within the promoter, was methylated by the DNMT1/SP1 complex  by catalyzing both DNA methylation and histone deacetylation, on specific DNA loci recognized by P53. Repressive capacities of DNMT1/P53-including complexes are also controlled by additional regulators. Indeed, following DNA damage, DNMT1/P53-mediated silencing of the SURVIVIN gene was dependent of NBS1 (Nijmegen breakage syndrome)/DNMT1 interaction [CDC25C required SP1 (specificity protein-1)/P53 interaction, allowing the recruitment of DNMT1 close to SP1 and P53 response elements [ERα (estrogen receptor: ESR1) silencing in MDA-MB-468BC cells, suggesting that abnormal TFs may mediate specific DNA methylation in cancers [Activation of the TF P53 increased the expression of a large group of genes while surprisingly, a fraction of genes with a P53 box where repressed . A ternaeraction . Likewiselements . P53/mSI cancers .IL-3) [Modified TFs  or an increase expression of a particular TF may contribute to generate new TF/DNMTs interactions or to favor pre-existing interactions in pathologic tissues and therefore to induce specific DNA methylation. Indeed, the t(q22.q22) translocation, which was frequently observed in acute myeloid leukemia, induces the formation of the chimerical protein RUNX1  -MTG8  which mimics an oncogenic TF. In normal cells, RUNX1 bound to the enhancer sequence TGT/CGGT whereas MTG8 is a transcriptional repressor able to interact with other co-repressors. Direct or indirect DNMT1/RUNX1-MTG8 interaction was observed in a complex including the co-repressors HDACs, mSIN3a and N-Cor and all components synergistically silenced some specific genes  , 147.PTPN6 gene was mediated by the recruitment of DNMT1/STAT3(phosphorylated)/HDAC1 complex on STAT3 boxes n PTPN6 promoter [The repression of the promoter . Moreovepromoter .During development, many genes are thinly and kinetically regulated. One of the TF, controlling this timing is HESX1 (HESX homeobox 1), which mediates the repression of HESX1-target genes by both recruiting co-repressors (such as TLE1 or N-Cor) and by specific DNA methylation in a HESX1/DNMT1 interaction manner.RASSAF1A promoter and its methylation in lymphoblastic leukemia [The target of DNMT1-including complexes on specific TF-response elements can also be mediated by indirect interactions with TFs. Indeed, the presence of the transcriptional repressor DAXX lead to the silencing of ReIB target genes which are normally activated by this TF. This DNA methylation repression is mainly mediated by the indirect recruitment of DNMT1 via the DAXX/DNMT1 and DAXX/RelB interactions and is completed by the further recruitment of HDAC2 on these promoters . Similarleukemia .p21) although the existence of P53/DNMT3A-mediated de novo methylation was not clearly demonstrated [p16(INK4a). Silencing of the target genes was then completed by histone modifications mediated by the PU.1/mSIN3a/HDAC/MeCp2 complex [miRNA-124-3 promoter [c-MYC box in CCND1 promoter, in a MIZ1-dependent manner. On the opposite, in the absence of MYZ1, c-MYC recruitment on c-MYC response element induced the transcriptional activation of CCND1 [RARβ2 promoter induced the recruitment of DNMT1 and DNMT3A by direct interactions [TFs are also capable of inducing the specific recruitment of DNMT3A and DNMT3B in DNA. DNMT3A can interact with P53 (via its C-ter) leading to the repression of P53-regulated genes  by inducing LANA/DNMT3A/E2F interaction-mediated de novo methylation [The E2F family members regulate the expression of many genes during cell cycle by interacting to ilencing , 160. Th S phase . The DNMR-a gene , 46. Morhylation .TRAF1 by the indirect recruitment of DNMT3A and DNMT3B via the complex DNMT3L/NFKBp65.Little is currently known about the specificity of each DNMT for one or another protein partner. Although some redundancy may explain that invalidation of one DNMT could sometimes be balanced by another one (P53 can interact with both DNMT1 and DNMT3A), the analysis of cell-validated or in vitro putative TF/DNMT interactions revealed that some interactions are specific to one DNMT , 156. AsNY-ESO1 gene in glioma and mesothelioma cells required the sequential recruitment of three independent complexes: (i) HDAC1/mSIN3a/NCOR complex which deacetylates the promoter, (ii) DNMT3B/HDAC1/EGR1 complex which induces a local DNA methylation and increases histone deacetylation, and (iii) DNMT1/PCNA/UHRF1/G9a complex which maintains DNA methylation and introduces the H3K9me2 repressive mark [Epigenetic silencing appears thinly regulated and orchestrated and frequently requires the presence of several DNMT-including complexes, polycomb proteins and HDACs, interacting together in a specific kinetic. A list of the main repressors directly or indirectly interacting with DNMTs is proposed in Table ive mark . The queive mark . A secon


P = 0.015). Stratified analysis showed that Sox2 expression correlated with shorter lifespan only in patients with cardiac gastric cancers (P = 0.002) or stage I or II gastric cancers (P = 0.002); but not in patients with non-cardiac cancers (P = 0.556) or stage III or IV gastric cancers (P = 0.121). Analysis on a database cohort validated the correlation between Sox2 expression and poor prognosis in stage II cancer. Also, expression of Sox2 was associated with lymphnode metastasis in patients with cardiac gastric cancer (P = 0.037). A multivariate analysis revealed that Sox2 was an independent prognostic factor in cardiac gastric cancer. Our results indicate that predictive value of Sox2 in gastric cancer is associated with cardiac cancer location and with early cancer stages (I and II).Cancer stem cells (CSCs) are thought to be the "root" of cancer. Although stemness-related factors ALDH1A1 and Sox2 have been used as markers to identify gastric CSCs, the expression pattern and significance of these factors in gastric cancer have not been sufficiently demonstrated. In this study, the expressions of ALDH1A1 and Sox2 were detected by immunohistochemistry in 122 gastric cancer specimens. And the correlation between Sox2 or ALDH1A1 expression and clinicopathological parameters and overall survival data were analyzed. The positive rate of ALDH1A1 expression was 60%, but there was no significant difference between survival rates of ALDH1A1-positive and ALDH1A1-negative patients. Sox2 was expressed in 42% of specimens and was associated with poor prognosis of patients ( Gastric cancer is one of the most common malignancies worldwide, especially in Eastern Asia, Central and Eastern Europe, and South America. CompletRecently, the cancer stem cell (CSC) theory has become a highlight of the cancer research field. CSCs have been identified not only in leukemia, but also in solid tumors, including gastric cancer –6. CSCs Gastric cancer is a heterogeneous disease that is often reported as a single entity. Topographically, gastric cancer can be classified into cardiac gastric cancer and non-cardiac gastric cancer. In thisA total of 122 Gastric adenocarcinoma tissues were collected from 2010 to 2013 at PLA Army General Hospital . No preoperative radiotherapy or chemotherapy was performed before surgery for the patients; patients were monitored every three to six months. There were 100 male patients and 22 female patients. The mean age is 63 years . Tumor stage was classified according to the 7th Union International Cancer Control (UICC) TNM staging system. To analyze for stratification based on the tumor location, gastric cancers that meet Siewert type II and III, according to Siewert classification, were cl2O2, and then was subjected to antigen retrieval. The slides were incubated with mouse anti-human ALDH1A1  or rabbit anti-human Sox2  at 4 Covernight. The horseradish peroxidase (HRP) labeled secondary antibody was added for 30 minutes at room temperature. At last, Reactions were revealed with 3, 3′-diaminobenzidine . Slides were then counterstained with hematoxylin, dehydrated with alcohol and xylene, and mounted on cover slips. The Immunohistochemistry results were interpreted as follows [Immunohistochemistry was performed as described. Briefly follows , 19–20: P value < 0.05 in univariate analysis were used in the multivariate regression. P values<0.05 were considered statistically significant.All data were analyzed with SPSS13.0 statistical software . The Pearson Chi-square test and Fisher’s exact test were used to evaluate comparisons of clinicopathological characteristics. Kaplan–Meier survival plots and log-rank statistics were used to compare the survival rates of patients with follow-up data. Multivariate analyses were performed using the Cox proportional hazards regression model. Variables with P = 0.375), sex(P = 0.701), Lauren classification(P = 0.623), cardiac cancer(P = 0.199), T stage(P = 0.430), N stage(P = 0.166), TNM stage (P = 0.894) or ALDH1A1 expression(P = 0.192) in gastric cancer (P = 0.006). No correlation was found between ALDH1A1 expression in gastric cancer and age (P = 0.194), sex(P = 0.688), Lauren classification(P = 0.856), T stage(P = 0.050), N stage(P = 0.390), or TNM stage(P = 0.118) (Sox2 was found located in nuclei and ALDH1A1 was stained in cell plasma . Forty-tc cancer . Sixty pc cancer . The exp= 0.118) .P = 0.015). In addition, similar survival rates were observed regardless of ALDH1A1 positivity (P = 0.172) while patients with negative ALDH1A1 and Sox2 expression has higher survival rate than patients with ALDH1A1 and/or Sox2 positive expression  to analyze the relationship between ALDH1A1 and Sox2 mRNA levels and survival of gastric cancer patients; we found that high ALDH1A1 mRNA levels were associated with better survival, while, high Sox2 mRNA levels were associated with poor prognosis  and Fifty-three percent (62/116) were deceased at this time point. Kaplan–Meier survival curves showed that patients with Sox2 negative tumors had better prognoses compared to those with Sox2 positive tumors . There were no associations found between Sox2 expression and age, sex, Lauren classification, invasion depth, and TNM stage in cardiac gastric cancers  can be divided to three types according to Siewert classification. The SieSox2 is a transcription factor that plays an important role in fetal development and in cancer biology–37. Sox2ALDH1 is a predominant isoform of aldehyde dehydrogenase, which participates in the metabolism of a wide variety of aliphatic and aromatic aldehydes–44. The This study is limited owing to its small cohort size. Predictive survival in relation to Sox2 expression could not be addressed with respect to each TNM stage.In conclusion, we found that positive Sox2 expression was associated with worse survival in patients with cardiac gastric cancer and earlier cancer stages (Stages I and II). Tumor location and stage may be important factors involved in gastric cancer heterogeneity, and should be considered in future studies.S1 Fig(TIFF)Click here for additional data file.S2 Fig(A) ALDH1A1 (212224_at); (B) Sox2 (228038_at).(TIFF)Click here for additional data file.S3 Fig(TIFF)Click here for additional data file.S4 Fig(A) Overall survival of patients in the Stage I and II group and in the Stage III and IV group. (B) Overall survival of patients in the cardiac gastric cancer and in non-cardiac cancer groups.(TIFF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.