The GCAP1 dimer possesses intermolecular hydrophobic contacts involving the side chain atoms of H19, Y22, F73 and V77. The structural model of the dimer was validated by GCAP1 mutations  at the dimer interface that each abolished protein dimerization. Previous studies have shown that each of these mutants either diminished or completely suppressed the ability of GCAP1 to activate the cyclase. These results suggest that GCAP1 dimerization may affect compartmentalization of GCAP1 in the photoreceptors and/or affect regulation of the cyclase activity.Retinal guanylyl cyclases (RetGCs) in vertebrate photoreceptors are regulated by the guanylyl cyclase activator proteins (GCAP1 and GCAP2). Here, we report EPR double electron-electron resonance (DEER) studies on the most ubiquitous GCAP isoform, GCAP1 and site-directed mutagenesis analysis to determine an atomic resolution structural model of a GCAP1 dimer. Nitroxide spin-label probes were introduced at individual GCAP1 residues: T29C, E57C, E133C, and E154C. The intermolecular distance of each spin-label probe (measured by DEER) defined restraints for calculating the GCAP1 dimeric structure by molecular docking. The DEER-derived structural model of the GCAP1 dimer was similar within the experimental error for both the Mg The atotructure . The cry cyan in  and the rization –30 that rization , 32. Ca2recoverin, neurocarecoverin and GCAPrecoverin that areemoglobin.In the current study, we performed EPR DEER analysis to calculate an atomic level structural model of the GCAP1 dimer. Nitroxide spin-label probes were introduced at four different sites in GCAP1  in E. coli strain, BL21(DE3) as described previously. PurificGCAP1CL), all original Cys residues in GCAP1 were substituted by Thr. Subsequent introduction of single and double Cys residues was done using the GCAP1CL cDNA as a template. The resultant cDNA constructs were ligated into the NcoI/BamHI sites of pET11d vector  for subsequent expression in E. coli as described above.Mutations in cDNA coding for a D6S bovine GCAP1 were introduced using a ‘splicing by overlap extension technique’ as previ2+ (2 mM) or Ca2+ (2 mM) was determined using analytical SEC . The calibration procedure was described by GTP. The resultant [32P]cGMP product was separated by TLC using fluorescently-backed polyethyleneimine cellulose plates (Merck) developed in 0.2 M LiCl, eluted with 2 M LiCl and the radioactivity was counted using ScintiSafe liquid scintillation cocktail containing 20% ethanol.The human RetGC1 cDNA was expressed in HEK293 cells from a modified pRCCMV vector (Invitrogen) using calcium phosphate precipitation for the transfection, and the membrane fraction containing expressed RetGC1 was isolated and assayed for activity as previously described in detail. BrieflyCL(E57C), GCAP1CL(E133C), and GCAP1CL(E154C)) served as input for the docking calculation. Since the DEER intermolecular distance distribution for E57C showed two maxima at 26 and 50 Å, two separate calculations were performed using the following intermolecular distance restraints. For the first calculation  and 3 clusters (asymmetric dimer) as shown in CL. Since Cys29, unlike all other Cys residues in GCAP1, is a part of the RetGC-binding interface[CL reduced affinity of the Cys-less protein for the cyclase compared to the wild type , but the GCAP1CL was still able to strongly activate the cyclase at higher concentrations in the absence of Ca2+ (CL(T29C), GCAP1CL(E57C), GCAP1CL(E133C), and GCAP1CL(E154C)). Each of the single Cys mutants were then individually labeled with a nitroxide spin label, and the intermolecular distance between the attached spin label was measured for each mutant using DEER. The GCAP1 Cys-less mutant and each of the single-Cys mutants (GCAP1CL(T29C), GCAP1CL(E57C), GCAP1CL(E133C), and GCAP1CL(E154C)) were all shown to be functional and capable of activating RetGC in a Ca2+-sensitive manner (50 = 5–14 μM) compared to wild type (EC50 < 2 μM), except for the GCAP1CL(T29C), which activated RetGC1 in a manner indistinguishable from the wild type, because the original Cys29 in the cyclase-binding interface became restored in that mutant.Previous studies demonstrated that GCAP1 forms a dimer in solution, 29. In interface, the ori of Ca2+ . Single e manner ; howeverCL(E57C), GCAP1CL(E133C) and GCAP1CL(E154C)) are shown in CL(E57C) (CL(E133C) (CL(E154C) (Representative DEER data for each nitroxide-labeled GCAP1 single Cys mutant (GCAP1CL(E57C)  was bestCL(E57C)  that hasL(E133C)  was accuL(E133C)  with a mL(E154C)  was fit L(E154C)  with a mCL(T29C) could not be accurately measured by DEER, because of apparent strong dipolar coupling of the nitroxide spin label attached at T29C. Evidence of strong dipolar coupling of the nitroxide at this site is evident in the CW-EPR spectrum of GCAP1CL(T29C) (CL(E57C) (CL(E133C) (CL(E57C), GCAP1CL(E133C), and GCAP1CL(E154C) (CL(T29C) containing an attached spin-label (with the N-O group reduced [CL(T29C) determined a translational diffusion coefficient (D = 2.0 x 10−10 m2/s) consistent with a molar mass of ~100 kDa, which corresponds to a protein tetramer for the spin-labeled GCAP1CL(T29C) in contrast to the dimeric wild type protein [CL(T29C) may be the result of protein tetramerization and/or aggregation that is not seen in the wild type protein dimer. Therefore, the strong dipolar coupling of the nitroxide attached at T29C is possibly an artifact of protein aggregation and will not be included in the dimeric structural model below.The intermolecular distance for GCAP1CLT29C)  that exhCL(E57C)  and GCAPL(E133C)  exhibiteL(E154C) . We hypo reduced ) has con reduced  on spin-C  that eCL(E57C) (CL(E57C). One of the models is a symmetric dimer that has a E57C intermolecular distance matching the long distance component at ~50 Å (The intermolecular distances for each of the spin-labeled GCAP1 mutants measured by DEER  were useCL(E57C)  suggestsCL(E57C)  were anaat ~50 Å . The othat ~50 Å .The GCAP1 symmetric dimer  is stabiThe GCAP1 asymmetric dimer  has both1H-15N HSQC NMR spectrum of 15N-labeled F73E mutant (red peaks in To discriminate the two dimeric GCAP1 structures (symmetric vs asymmetric), we constructed a mutant (F73E) that is predicted to disrupt key intermolecular hydrophobic contacts between F73, V77 and W94 in the symmetric dimer , whereas2+ and/or Mg2+ had no detectable effect on the elution time of wild type GCAP1 or any of the mutants. The elution times of each mutant  corresponded to a molar mass of 24 kDa, demonstrating that each mutant is a monomer in solution. Therefore, mutating each of the key residues at the dimer interface  significantly weakened the dimerization association constant, which validates the structural models of the GCAP1 dimer  being part of both interfaces  may dissociate upon binding to membrane-bound RetGC . Mutatioterfaces . In thisterfaces . The exaterfaces . To distterfaces  will remS1 Fig(PDF)Click here for additional data file.


Molecular docking simulation and site-directed mutagenesis indicated that identified compounds target the substrate-binding site in PRMT5. Treatment of lung cancer cells with identified inhibitors led to inhibition of the symmetrical arginine methylation of SmD3 and histones and the cellular proliferation. Oral administration of the inhibitor demonstrated antitumor activity in a lung tumor xenograft model. Thus, identified PRMT5-specific small-molecule inhibitors would help elucidate the biological roles of PRMT5 and serve as lead compounds for future drug development.Protein arginine methyltransferase 5 (PRMT5) plays critical roles in a wide variety of biological processes, including tumorigenesis. By screening a library of small chemical compounds, we identified eight compounds that selectively inhibit the PRMT5 enzymatic activity, with IC NG,NG-dimethylarginine, whereas type II enzymes catalyze the formation of symmetric NG,NG-dimethylarginine. Type III PRMTs catalyze the formation of NG-monomethylarginine, and type IV PRMTs modify the delta nitrogen atom of the arginine residue. All PRMTs have a common catalytic (methyltransferase) domain consisting of a highly conserved core region of around 310 amino acid residues and subdomains important for binding to AdoMet and to the substrate adenosylmethionine ; and 5 μM AdoMet were incubated in 50 mM Tris-HCl (pH 7.5)–1 mM EGTA–1 mM EDTA at 30°C for 2 h. Reaction mixtures (20 μl) were boiled in 5 μl of 5x SDS sample buffer and separated on a 15% polyacrylamide gel. Gels were fixed for 30 min in 40% methanol–10% acetic acid, incubated in 20 ml of Amplify (Amersham Life Science) for 10 min, dried and exposed to X-ray film at -80°C. IC50 was calculated by linear regression analysis of percent inhibition. The other half (20 μl) of each reaction mixture was spread onto DE81 chromatography paper discs (Whatman). The paper discs were washed 4 times with 50 mM NaHCO3 and dried. The amounts of methylated products were quantified by liquid scintillation.Methylation reactions were performed as previously described with a few modifications ,31. ReacThe program Glide ,33 was u3-AdoMet and various concentrations of SmD3. After a 10-, 20- or 30-min incubation at 30 oC or no incubation, the reaction mixtures were spread onto DE81 chromatography paper discs and the amounts of methylated products were quantified by liquid scintillation. Amounts (counts per minute [cpm]) of methylated products and incubation time (minutes) were plotted and reaction velocity (cpm per minute) was deduced.Different amounts of compound C2 or C5 were present in the final methylation reaction mixtures with 1.2 μg of PRMT5, 5 μM AdoMet, 2 μCi of HWaters 2695 HPLC System and a C18 column  were used. The column was equilibrated with 10% acetonitrile and eluated with a linear gradient from 10% acetonitrile to 100% acetonitrile in 60 min. Compounds were dissolved in 10% acetonitrile and the purchased compounds (ChemBridge) were used as the standards.2 incubator and the cell numbers were counted. Each experiment was performed in triplicate.Lung cancer A549 (ATCC) and PC14 (ATCC) cells were cultured in minimum essential medium (Cellgro) with 10% (v/v) fetal bovine serum (FBS) (HyClone), 2% vitamins, 1% L-glutamine, 1% non-essential amino acids and 1% sodium pyruvate. For the treatment with identified compounds, cells  were seeded in 24-well plates in the medium containing 2% FBS. The fresh medium containing identified compounds was added 24 hours later. Cells were cultured for various times in a CO6) or lung tumors (10–100 mg) were lysated using a glass dounce in 1 ml of 1x Passive Lysis Buffer (Promega) and nuclear pellets were collected by centrifugation at 3,000 rpm for 5 min at 4 oC. The supernatant was discard and the pellets were washed with 1 ml of 1x Passive Lysis Buffer. Nuclear pellets were resuspended in 0.4 ml of the buffer  and incubate for 30 min on ice, and centrifuged at 12,000 rpm for 10 min at 4 oC. The supernatant (histones extract) was transferred to a new tube and histones were precipitated by 20% trichloroacetic acid.Histones were purified as reported previously . BrieflyProtein extracts or histones (20 μg) were subjected to 10% or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively then transferred to Immobilon-P membranes (Millipore). The blots were then probed for 2 hours with the primary antibody at dilutions of 1:500 , 1:1,000  or 1:1,000 . The blots were then incubated with a horseradish peroxide-conjugated secondary antibody  for 1.5 h. Immunoreactive proteins were detected using an enhanced chemiluminescence detection system  per the manufacturer’s instructions. Protein concentrations were determined using the Bradford protein assay reagent (Bio-Rad).Male BALB/c mice (8–10 weeks old) were purchased from Charles River Laboratory and were randomized for compound administration. Chemical compounds were dissolved in 0.1 ml of 0.5% (w/v) carboxymethycellulose (Sigma-Aldrich) plus 0.5% Tween 80 (Fisher Scientific) and administrated by oral gavage at the dosage of 50 mg/kg or 100 mg/kg. Mice were sacrificed and blood samples were collected immediately before (n = 5) and at 2 (n = 5), 4 (n = 5), 6 (n = 5) or 8 (n = 8) h after compound administration. Plasma samples were sent to the Pharmaceutical Development Center at MD Anderson Cancer Center for LC-MS/MS assay to determine compound concentrations.Six-week old nude mice were purchased from the National Cancer Institute and maintained in a barred animal facility. Cell injection into the mouse lung was performed as described previously . Mice wet-test was used to determine whether differences between control and experimented samples were statistically significant. *, p<0.05; **, p<0.01; ***, p<0.001. P values less than 0.05 were considered statistically significant.Data are presented as the means of three independent experiments ± the standard deviation. A 2-tailed unpaired Student in vitro and in vivo [NG-monomethyl and NG-NG-dimethyl arginine and does not recognize unmodified arginine. This antibody was used to establish an enzyme-linked immunosorbent assay (ELISA)-based screen for small molecule inhibitors of arginine methylation of SmD3 by PRMT5. The recombinant SmD3 was immobilized on a 96-well plate and incubated with the human recombinant PRMT5-WDR77 complex. Small molecules were added to individual wells using a 12-channel pipette. The methylation reaction was initiated by addition of the methyl donor (AdoMet). The arginine-methylated SmD3 was detected by the monoclonal antibody ab412 and a peroxidase-conjugated secondary antibody. The screen was performed on 10,000 individual compounds from a diverse collection of synthetic chemicals . We identified eight compounds that dramatically (>90% at the concentration of 20 μM) inhibited methylation of SmD3 by PRMT5 (50 (concentrations required to inhibit enzyme activity by 50%) values of the compounds ranged from 0.1 to 6 μM , PRMT1 and PRMT3, using the radioactive methyltransferase assay. The results are shown in Caenorhabditis elegans PRMT5 [To understand the structural basis of the identified compounds’ inhibition of PRMT5, we used the program Glide to investigate the docking of the compounds to PRMT5. Each compound was individually docked in ns PRMT5 . Dockingns PRMT5 . We notins PRMT5 .C. elegans PRMT5) to Met. The mutated PRMT5 was expressed in bacteria and purified by Ni-nitrilotriacetic acid (Ni-NTA) affinity chromatography  of PRMT5  to analyIt has been demonstrated that PRMT5 and its enzymatic activity are required for growth of lung cancer cells ,27. To tThe obtained 3 compounds  have characteristics of Lipinski’s rule-of-five, which defines four physicochemical parameters found in a set of active drugs which made them into phase II clinical trials . We chosThe oral route of administration is central for delivery of a large number of important drugs in various therapeutic areas and is the most preferred dosage form for cancer treatment . We then3-) and methoxy (CH3O-) groups in C9 are not involved in the interaction with PRMT5. By searching the compound library (www.hit2lead.com/search.asp) we found a compound without the methyl (CH3-) group and with an amino group (-NH2) substituting the methoxy (CH3O-) group of C9 (50 (~1 μM) in the cell growth assay is lower than that (~ 6 μM) in the methylation assay. This may reflect the fact that compound C9 is accumulated (by 5-fold) inside cells . This compound interacts with many of the residues in PRMT5 that involved in peptide binding and form a π-π stacking interaction with Phe327 [In this study, an ELISA-based methylation assay was used to screen a library of 10,000 small chemical compounds. By this approach, we identified eight novel chemical compounds that inhibit PRMT5 but not PRMT1 and PRMT3. More recently, a few studies reported small chemical inhibitors specifically targeting PRMT5. Compounds CMP5 and HLCL-61 specifically inhibited PRMT5 rather than PRMT1, CARM1, or PRMT7 ,47. The  of 6 μM . From se of 6 μM . EPZ0156h Phe327 ,50. The We identified a set of genes whose expression was altered by silencing PRMT5 expression in lung cancer A549 cells . A549 cePRMT5 has been shown to be upregulated in a number of different cancers and plays an essential role in growth of various cancer cells ,28. ConsCollectively, identified compounds are powerful probes that could be used to understand more about the biologic roles of PRMT5 and potentially assist in defining a therapeutic strategy for lung cancer treatment.S1 FigThe chemical compound was dissolved in 5% DMSO-0.5% Tween 80. BALB/c mice (8–10 weeks old) were randomized and administrated the vehicle (0.1 ml) or compound C9 dissolved in the vehicle (0.1 ml) by intraperitoneal (I.P.) injection at the dosage of 25, 50 or 100 mg/kg. Mice were sacrificed and blood samples were collected immediately before  and at 2 (n = 5), 4 (n = 5), 6 (n = 5) and 8 (n = 5) h after compound administration. Plasma samples were sent to the Pharmaceutical Development Center at MD Anderson Cancer Center for LC-MS/MS assay to determine compound concentrations.(TIF)Click here for additional data file.S2 FigoC overnight. Cells were collected and stained with propidium iodide (PI). The cell-cycle distributions were determined by flow cytometry analysis (BD AccuriTM C6 Flow Cyometer).A549 cells were infected with the lentivirus expressing non-target (NT) or PRMT5 shRNA as described previously by us . A549 ce(TIF)Click here for additional data file.S3 FigP<0.001.A549 cells were infected with the lentivirus expressing NT or PRMT5 shRNA. A549 cells were grown in the presence of DMSO or compound C5, C9 or C11 (20 μM) for 2 days. A, Bromodeoxyuridine (BrdU) (BD Biosciences) incorporation assay. Cells were plated on a Chamber slide  and cultured in the presence of 10 μM BrdU for 4 h. The BrdU-positive cells (brown) were detected by immunostaining with the monoclonal anti-BrdU antibody (BD Biosciences) and observed under a microscope. B, Percentage of BrdU-positive cells in control, PRMT5-silencing or compound treated cells. The results represent the means of three independent experiments ± the standard deviation. ***, (TIF)Click here for additional data file.S4 Figp<0.05.Mice body weights were measured before and after the treatment for 7, 14 and 21 days. The results represent the means of body weights of five mice ± the standard deviation. *, (TIF)Click here for additional data file.S5 FigRNAs were isolated from cells cultured in the presence of DMSO, EPZ015666 (Sigma-Aldrich) (50 μM), or C9a (50 μM) for 24 hrs and submitted to RT-RCR analysis of gene expression in cancer cell lines. Relative mRNA levels = mRNA in cells treated with PRMT5-inhibtor/mRNA in DMSo-treated cells.(TIF)Click here for additional data file.S6 FigSelleckchem.com) and compounds C9 and C9a. B, Western blot of protein extracts (10 μg) derived from A549 cells treated (4 h) with DMSO (lane 1) or 20 μM of compound C9 (lanes 2), C9a (lanes 3), Roscovitine (lanes 4), or Polbociclib (lane 5) with anti-Rb pS811 , -Rb pT821 , -Rb  or -actin (Sigma-Aldrich) antibody.A, Chemical structures of CDK inhibitors Roscovitine and Polbociclib ((TIF)Click here for additional data file.S1 DataTM C6 Flow Cyometer).Cells were treated with compounds and the cell-cycle distributions were determined by flow cytometry analysis (BD Accuri(PDF)Click here for additional data file.S1 TableRNAs were isolated from cells and submitted for real-time PCR analysis with gene-specific primers as indicated.(XLSX)Click here for additional data file.


Influenza is a major health threat, and a broadly protective influenza vaccine would be a significant advance. Signal Minus FLU (S-FLU) is a candidate broadly protective influenza vaccine that is limited to a single cycle of replication, which induces a strong cross-reactive T cell response but a minimal Ab response to hemagglutinin after intranasal or aerosol administration. We tested whether an H3N2 S-FLU can protect pigs and ferrets from heterosubtypic H1N1 influenza challenge. Aerosol administration of S-FLU to pigs induced lung tissue-resident memory T cells and reduced lung pathology but not the viral load. In contrast, in ferrets, S-FLU reduced viral replication and aerosol transmission. Our data show that S-FLU has different protective efficacy in pigs and ferrets, and that in the absence of Ab, lung T cell immunity can reduce disease severity without reducing challenge viral replication. Influenza virus infection is a global health threat to livestock and humans, causing substantial mortality. The major obstacle in combating influenza is the rapid evolution of the virus, rendering the host Ab response ineffective. Seasonal influenza virus vaccines are therefore strain specific, do not protect well against drifted viruses from the same hemagglutinin (HA) subtype, and offer no protection against infection with heterologous influenza viruses from different HA subtypes. Furthermore, pandemic influenza can arise at any time, originating from either group 1 or 2 avian influenza A viruses (IAV) and can cause devastating mortality. Therefore, a broadly protective influenza A vaccine (BPIV), which could protect against both group 1 and 2 viruses, would be a great advance in preventing seasonal infection and reducing mortality from pandemic influenza  is a replication-incompetent influenza virus, candidate BPIV, and is limited to a single cycle of replication  through RM), which have been shown to be important in cross-protective immunity against influenza infection  glycoproteins against challenge with an H1N1pdm09 (group 1) virus in both pigs and ferrets.8/ml 50% tissue culture infectious doses (TCID50) (95% CI 1.13 to 2.05 × 108/ml). The internal protein gene segments were from influenza A/Puerto Rico/8/34 (H1N1).The design and production of pdmH1N1 S-FLU [eGFP*/N1Eng195)].H1(Eng/195/2009) has been described previously , 5. We m].H1 vaccine on the influenza A/Ann Arbor/6/60 ca backbone was included as a comparator, and a group of mock-immunized control animals received Leibovitz 15 (L-15) media. Influenza A/California/07/2009 (H1N1pdm09) and A/Switzerland/9715293/2013 (H3N2) viruses were used for challenge infection.The pig isolate of A/swine/England/1353/09pdmH1N1 (1353/09pdmH1N1) was used for challenge infection in pigs. The homologous vaccine consisted of the identical β-propiolactone inactivated 1353/09pdmH1N1 with TS6 adjuvant. TS6 adjuvant was kindly provided by Dr. Catherine Charreyre . It contains an oily phase  and an aqueous phase of monopotassium and disodium phosphate.t test (Prism v7.0). H5 (A/Vietnam/1203/2004) −0.2938  = 18.4% reduction; H7 (A/Netherlands/219/2003) −0.2067  = 13.3% reduction; H3 (A/Victoria/361/2011) −0.04246  = 2.9% reduction. Although the 18.4% reduction in the titer of the H5 S-FLU after nebulization was statistically significant, the minimal effects on the H7 and H3 batches did not reach statistical significance. We regard these small effects of the vibrating mesh nebulizer on infectious titer of S-FLU as not biologically significant.We first established that passage through the Aerogen Solo vibrating mesh nebulizer  did not significantly reduce the titer of S-FLU. The cell supernatant containing S-FLU in viral growth medium (DMEM/0.1% BSA/10 mm HEPES pH 7) was passed through a 0.22 μm filter then aerosolized using the nebulizer, captured, and condensed. The effect of nebulization on the infectious titer of S-FLU was measured on three different batches of S-FLU coated in three different HAs by comparison of quadruplicate measurements of the means of the number of doubling dilutions  giving 50% infection of MDCK-SIAT cells  pre- and postnebulization by an unpaired 9 CID50 in 4 ml viral growth medium) was passed into the impactor and subsequently harvested from each of the impactor stages, which fractionate the aerosol droplets by size. In three replicates, the mean aerodynamic size of the aerosol droplets was 1.953 μm with a geometric SD of 1.795. The fine particle fraction, which is the fraction of the aerosol produced with a droplet size <5 μm, was 92.34%, indicating that the aerosol produced was highly respirable.We then assessed the aerosol droplet size distribution using a cascade impactor  at 15 l/min vacuum flow rate. A known quantity of virus  Act 1986. Both institutes conform to Animal Research: Reporting of In Vivo Experiments guidelines. Eighteen, 5–6-wk-old landrace cross female pigs were obtained from a commercial high-health status herd (average weight of 10 kg at the beginning of the experiment). Pigs were screened for absence of influenza A infection by matrix gene real-time RT-PCR and for Ab-free status by hemagglutination inhibition using four swine influenza virus Ags. Pigs were randomly divided into three groups of six and immunized as follows: 1) control unimmunized, 2) Homologous (1353/09pdmH1N1) vaccine containing 2048 hemagglutination units administered i.m. in 1 ml, and 3) H3N2 S-FLU administered by aerosol delivering ∼1.5 × 10nd mouth  followin6 PFU per pig of 1353/09pdmH1N1. Two milliliters were administered to each nostril using a mucosal atomization device, MAD300 . As the analysis of samples from pigs challenged at days 28 and 30 did not show any significant differences, for simplicity in presentation, the results of the assays carried out on pigs challenged on both days have been amalgamated in all figures.For logistical reasons, two IAV challenges were performed, with half of the animals challenged at 28 d postboost (dpb) and the remainder at 30 dpb. Animals were challenged intranasally with 1.5 × 107 TCID50 in 0.5 ml of A/Switzerland/9715293/2013 S-FLU, 107 TCID50 in 0.5 ml of A/Switzerland/9715293/2013 ca, or 0.5 ml of L-15 21 d apart. The ferrets were challenged with 106 TCID50 in 1 ml of influenza A/California/07/2009 (H1N1pdm09) or A/Switzerland/9715293/2013 (H3N2) virus.Four- to six-month-old female ferrets that were seronegative by hemagglutination inhibition assay to circulating influenza A H1N1pdm09 and H3N2 viruses were purchased from Triple F Farms, Sayre, PA. The ferret experiments were conducted in animal BSL2 laboratories at the National Institutes of Health in compliance with the guidelines of the Institutional Animal Care and Use Committee. Ferrets were lightly anesthetized with isoflurane and immunized intranasally with two doses of 10Animals were humanely killed 5 d postchallenge (dpc). At post mortem, the lungs were removed, and digital photographs were taken of the dorsal and ventral aspects. Macroscopic pathology was scored blind, as previously reported . Five luFour nasal swabs (two per nostril) were taken daily after the challenge. Serum and heparin anticoagulated blood samples were collected at the start of the study (prior to immunization) and at days 7, 14, 21, 28, 35, 42, and 49 after the first immunization. Bronchoalveolar lavage (BAL) and tracheobronchial lymph nodes (TBLN) were processed as previously described . Medial Four ferrets from each group were sacrificed at 2 and 4 dpc, and their lungs and nasal turbinates (NT) were harvested. Harvested tissues were homogenized in L-15 medium at 10% (w/v) for lung or 5% (w/v) for NT samples clarified by centrifugation at 2500 rpm for 10 min.6 TCID50 of A/California/07/2009 virus. Challenged ferrets were placed into the section of the cage closest to the air inlet the day of challenge. One day later, a naive ferret was placed into the cage on the other side of the divider. Environmental conditions inside the laboratory were monitored daily and were consistently 19 ± 0.3°C and 60 ± 2.2% relative humidity. The transmission experiments were conducted in the same room to minimize any effects of caging and airflow differences on aerobiology. The naive ferret was always handled before the infected ferret. Animals were carefully handled during nasal wash collections and husbandry to ensure no direct contact occurred between the ferrets. Nasal washes were collected every other day for 14 d and analyzed for the presence and titer of infectious viruses and expressed as TCID50 per ml. On day 14 postinfection, blood was collected from each animal, and serology was performed by hemagglutination inhibition and microneutralization assays.We performed airborne transmission studies using a caging system previously described . Briefly10 TCID50 per g.Viral titers in nasal swabs and BAL from pigs were determined by plaque assay on MDCK cells as previously described . ClarifiNeutralizing Ab titers in serum were determined as previously described , 166 PFU live MDCK-grown1353/09pdmH1N1, 1 × 105 TCID50 H3N2 S-FLU, medium control, or 10 μg/ml Con A (Sigma-Aldrich). Results were expressed as number of IFN-γ–producing cells per 106 cells after subtraction of the average number of IFN-γ+ cells in medium control wells.Frequencies of IFN-γ–secreting pig PBMC and BAL cells were determined by ELISPOT assay using fresh or cryopreserved cells . Cells w6 PFU live MDCK-grown strain 1353/09pdmH1N1 or 1 × 106 TCID50 H3N2 S-FLU or MDCK mock supernatant as control. GolgiPlug (BD Biosciences) was added for a further 4 h before intracellular cytokine staining. Cells were stained for surface markers with CD3ε-AF647 BB23-8E6-8C8, CD4 clone 74-12-4 PerCpCy5.5, CD8α-FITC 76-2-11 (BD Biosciences), and Near-Infrared Fixable LIVE/DEAD stain (Invitrogen). Cells were permeabilized using Cytofix/Cytoperm (BD Biosciences) before intracellular staining with IFN-γ PE P2G10 (BD Biosciences) and cross-reactive anti-human TNF-α-AF650 Mab11 (BioLegend). Samples were fixed in 1% paraformaldehyde before analysis using an LSRFortessa (BD Biosciences). Data were analyzed using FlowJo v10 (Tree Star).Cryopreserved mononuclear cells from blood, TBLN, BAL, spleen, and lung were thawed and stimulated for 12 h at 37°C with either 1 × 10RM that are not accessible to the CD3 Ab, given i.v. TRM is therefore single labeled with PeCy5.Before sacrifice, three animals from each group were infused i.v. with 10 ml of 3.24 mg/ml purified CD3 Ab (clone PPT3) and sacrificed 10 min later. Lymphocytes were isolated and stained ex vivo with anti-mouse Ig-FITC (SouthernBiotech), which labels the circulating intravascular cells. The cells are washed, and normal mouse serum is then added to block any remaining binding capacity of the anti–Ig-FITC. The cells are then washed again and incubated with CD3 Ab labeled with PeCy5 (Abcam). This will bind unsaturated sites of the circulating cells, which are therefore double labeled, as well as all the sites on the TRM, the i.v. CD3 was detected with goat anti-mouse IgG BV421 (BioLegend) and blocked using normal mouse serum as above. Surface markers used were CD3ε-biotin PPT3 (Abcam), CD4 clone 74-12-4 PerCpCy5.5, CD8α-FITC 76-2-11 , and Near-Infrared Fixable LIVE/DEAD stain (Invitrogen). Biotinylated CD3 was visualized with a streptavidin AF647 (BioLegend). Cells were permeabilized using Cytofix/Cytoperm before intracellular staining with IFN-γ PE P2G10 (BD Biosciences) and cross-reactive anti-human TNF-α-AF650 Mab11 (BioLegend). Samples were fixed in 1% paraformaldehyde before analysis using an LSRFortessa.To allow intracytoplasmic staining of TOne-way or two-way ANOVA with Dunnett posttest for multiple comparisons were used to compare immunized groups to the control group, and analysis was performed using GraphPad Prism 6.Groups of six pigs were immunized twice 3 wk apart with an inactivated virus with adjuvant i.m. (Homologous inactivated) or with H3N2 S-FLU by aerosol (S-FLU). The control group was unimmunized (control). Aerosol immunization was carried out using a purpose-built mask and Aerogen Solo nebulizer that allowed efficient vaccine delivery in <5 min to the sedated animals after we had established that the nebulizer did not affect the titer of the S-FLU vaccines and provided a droplet size appropriate for delivery to the lower respiratory tract . Four weThe pigs immunized with the Homologous inactivated vaccine showed the greatest and statistically significant reduction of challenge virus in the nasal swabs at 1, 2, 3, 4, and 5 dpc . S-FLU dFollowing challenge, the unimmunized animals developed typical gross lesions of influenza virus infection . HistopaAnimals immunized with the Homologous inactivated vaccine showed very few gross pathological lesions. Histologically, only a few lung sections showed mild interstitial pneumonia and necrosis of the bronchial and bronchiolar epithelium. Virus NP immunostaining was restricted to very few inflammatory cells within the interalveolar septa. The S-FLU–immunized animals showed small areas of gross pathology. Histopathology showed mild to moderate interstitial pneumonia, edema, and epithelial necrosis within the bronchi and bronchioles. Few bronchiolar epithelial and inflammatory interstitial cells exhibited NP immunostaining .These results indicated that immunization of pigs with a group 2 H3N2 S-FLU significantly reduced gross and histopathology but did not significantly reduce the viral load in nasal swabs and BAL after heterologous challenge with group 1 H1N1pdm09 virus.We determined the Ab response in pigs using microneutralization assay. Sera from the Homologous inactivated vaccine group had neutralizing Ab, with mean inhibitory titers of 1:2291 at 7 dpb, 1:1166 at 28 dpb, and at 1:1801 at 5 dpc. This indicates that the Homologous inactivated vaccine was successfully delivered and induced anti-1353/09pdmH1N1 neutralizing Abs as expected. Also as expected, no neutralizing Ab to the H1N1 virus was detected in the S-FLU or in the unimmunized controls .6 cells). The response to stimulation with H3N2 S-FLU was minimal (mean 39 SFC in homologous inactivated and 17 SFC in S-FLU group). At 28 dpb, just before the challenge, the response was undetectable in any of the groups. At 5 dpc, the S-FLU–immunized animals showed the strongest response to both 1353/09pdmH1N1 and H3N2 S-FLU stimulation   . The red6 cells for S-FLU) because of the low percentage of T cells in BAL. There was also a high medium control background most likely because of the presence of many activated cells in the BAL  (RM). Lymph nodes were inaccessible to the infused CD3 Ab, as has been shown to be the case by others (RM and intravascular cells in the immunized and control pigs (data not shown). A similar pattern has been observed in more than 20 animals from other studies.Because we have shown that aerosol immunization with H3N2 S-FLU induced a strong local immune response, we wished to establish whether the responding cells were part of the Tr cells) . In conty others . There wRM. Because BAL gives a strong Ag-specific response, we can conclude that S-FLU is inducing lung TRM. Our staining in lung indicates that a proportion of lung T cells (39%) are inaccessible to i.v. CD3 Ab and are therefore also part of the TRM population. The TRM in the three S-FLU–immunized animals treated with i.v. Ab had a higher proportion of Ag-specific cells producing IFN-γ and TNF-α than the lung intravascular population (RM in the Homologous inactivated and control groups is unreliable because there were few responding cells and therefore very few events in the gated populations (data not shown). These results demonstrate that aerosol immunization with H3N2 S-FLU induces a large lung TRM population.As BAL is 90% stained only by the ex vivo CD3PeCy5, whereas the blood and spleen are double labeled, this indicates that most BAL cells are part of the blood-inaccessible pool of Tpulation  followinn = 6 per group) were challenged with intranasally delivered homologous wild-type (wt) influenza A/Switzerland/9715293/2013 (H3N2) and the other half with a heterologous influenza A/California/07/2009 (H1N1pdm09) virus. On days 2 and 4 postchallenge, three ferrets in each group were sacrificed, and virus titers in lungs and NT were assessed. The homologous wt H3N2 virus did not replicate in the lower respiratory tract of mock-immunized (or vaccinated) ferrets (4.95TCID50/g) in the NT at 4 dpc, and both vaccines prevented replication of the challenge virus in the NT (p < 0.05).We next determined the protective efficacy of the same batch of H3 S-FLU in ferrets, using as a positive control live attenuated virus H3N2 ca, as we have previously used similar ca viruses for this purpose in ferrets . We also ferrets , so the 7.8 and 106.5 TCID50/g at 2 and 4 dpc, respectively) and to a modest-to-moderate titer (mean 102.9 and 104.3 TCID50/g at 2 and 4 dpc) in the lungs  in H1N1pdm09 titers in the NT at 2 dpc and further reduction  at 4 dpc, although only the S-FLU group on day 4 was significantly different from the mock-immunized group (p < 0.05). A statistically significant reduction in lung virus titers was observed on day 4 postchallenge but only in animals that had received the H3N2 ca virus vaccine (p = 0.002).The H1N1pdm09 virus that was used for heterosubtypic challenge replicated to a high titer in the NT  21 d after the second dose of vaccine and were placed in transmission cages. The following day, an unvaccinated naive ferret was introduced adjacent to each infected ferret. The experimentally infected and respiratory contact ferrets were followed with nasal washes and serology to determine whether the H1N1pdm09 virus transmits by the respiratory route from experimentally infected to airborne contact ferrets. One ferret each in the mock-immunized and H3N2 S-FLU–vaccinated groups reached their humane endpoints 9 dpc from an intercurrent infection in the animal house. Unfortunately, we were not able to identify the etiology of the intercurrent infection that caused weight loss in ferrets. None of the ferrets were found dead. They were euthanized in accordance with our approved animal study protocol because of weight loss. The virus inoculum was not contaminated with bacteria, and the virus dose was confirmed to be correct.All four of the mock-immunized ferrets were infected with H1N1pdm09 and transmitted to their airborne contacts . InfectiRM in BAL and lung tissues of pigs.There is strong epidemiological evidence for an association between a cross-reactive T cell response and heterologous protection between group 1 and 2 IAV in humans , 23. In In earlier experiments in ferrets and mice, in which there was a complete mismatch between the immunizing H1N1 or H5N1 (group 1) S-FLU and challenge H3N2 and H7N9 (group 2), we observed a significant reduction in replication of the infectious challenge virus  and, altIn other experiments in small animals, the effect of fully heterosubtypic immunization was similar to what we observe in pigs. For example, a single-replication cycle H1N1 (group 1) BPIV based on the partial deletion of the M2 gene  induced RM play a major role in protection against influenza viruses in mice (RM in the BAL and lung tissues have greater protective capacity than circulating memory CD8 T cells (RM are associated with reduced symptoms and viral load in respiratory syncytial virus infection in humans (RM in pigs, as has been shown in mice following i.v. administration of CD3 Ab. We demonstrate that more than 90% of BAL cells are inaccessible to intravascular Ab as well as a proportion of the lung tissue cells. Aerosol immunization with H3N2 S-FLU induces a strong immune response of these cells, which may not reduce viral replication, but may be able to induce a beneficial reduction in local inflammation through the release of immuno-modulating cytokines (It is increasingly evident that local immune responses and particularly lung T in mice , 10, 31. T cells , 32, 33.n humans . To our ytokines –37.In summary, our data show that the same vaccine has different protective efficacy in pigs and ferrets. In the absence of Ab, lung T cell immunity can consistently reduce disease severity but does not always abolish viral replication. We suggest that candidate BPIV should be tested in more than one species. The pig maybe a relevant large animal model because it is a natural host for influenza viruses and has very similar respiratory anatomy to humans.Data Supplement


This report summarizes U.S. influenza activityU.S. World Health Organization (WHO) and National Respiratory and Enteric Virus Surveillance System laboratories, which include both public health and clinical laboratories throughout the United States, contribute to virologic surveillance for influenza.During October 2, 2016–February 4, 2017, clinical laboratories in the United States tested 392,901 respiratory specimens for influenza viruses, 38,244 (9.7%) of which were positive . During Public health laboratories in the United States tested 38,141 respiratory specimens collected during October 2, 2016–February 4, 2017. Among these, 15,781 were positive for influenza , 14,606 Age was reported for 13,306 influenza-positive patients, among whom 1,048 (7.9%) were aged 0–4 years, 4,041 (30.4%) were aged 5–24 years, 4,029 (30.3%) were aged 25–64 years, and 4,188 (31.5%) were aged ≥65 years. Influenza A (H3N2) viruses predominated in each age group, representing a range of 82.3% of influenza-positives in persons aged 0–4 years to 93.6% in persons aged ≥65 years. The largest number of influenza B viruses were reported in persons aged 5–24 years.Two human infections with a novel influenza A virus were reported during October 2, 2016–February 4, 2017. One patient from Iowa with exposure to swine in the week preceding illness was infected with an influenza A (H1N2) variant [(H1N2)v] virus.WHO collaborating laboratories in the United States are requested to submit a subset of influenza-positive respiratory specimens to CDC for further virus characterization. CDC characterizes influenza viruses through one or more laboratory tests, including genomic sequencing, antigenic characterization by hemagglutination inhibition (HI), and neutralization assays. Historically, HI data have been used most commonly to assess the similarity between vaccine viruses and circulating viruses to infer how well the vaccine might work until vaccine effectiveness estimates are available.CDC has genetically characterized 892 viruses (101 influenza A (H1N1)pdm09; 593 influenza A (H3N2); and 198 influenza B viruses) collected from October 1, 2016, through February 4, 2017. The hemagglutinin (HA) gene segment of all influenza A (H1N1)pdm09 viruses analyzed belonged to genetic group 6B.1. Influenza A (H3N2) virus HA gene segments analyzed belonged to genetic groups 3C.2a (567 viruses) or 3C.3a (26 viruses). Genetic group 3C.2a includes an emerging subgroup defined as 3C.2a1. The HA of influenza B/Victoria-lineage viruses all belonged to genetic group V1A. The HA of all influenza B/Yamagata-lineage viruses analyzed belonged to genetic group Y3.During October 1, 2016–February 4, 2017, CDC antigenically characterized 484 influenza viruses (74 influenza A (H1N1)pdm09, 267 influenza A (H3N2), and 143 influenza B viruses). All 74 (100%) influenza A (H1N1)pdm09 viruses were antigenically similar to A/California/7/2009, the recommended influenza A (H1N1) component of the 2016–17 Northern Hemisphere vaccine. Among 267 influenza A (H3N2) viruses, 258 (96.6%) were antigenically similar to the A/Hong Kong/4801/2014–like cell propagated reference virus belonging to genetic group 3C.2a, which is the recommended influenza A (H3N2) component of the 2016–17 Northern Hemisphere vaccine. Seventy (90.9%) of 77 influenza B/Victoria-lineage viruses were antigenically similar to B/Brisbane/60/2008, which is the recommended influenza B component of the 2016–17 Northern Hemisphere trivalent and quadrivalent vaccines. All 66 (100%) influenza B/Yamagata-lineage viruses were antigenically similar to B/Phuket/3073/2013, the recommended influenza B component of the 2016–17 Northern Hemisphere quadrivalent vaccine.The WHO Collaborating Center for Surveillance, Epidemiology, and Control of Influenza at CDC tested 807 influenza virus specimens (94 influenza A (H1N1)pdm09, 519 influenza A (H3N2), and 194 influenza B viruses) collected in the United States from October 1, 2016, through February 4, 2017, for resistance to the influenza neuraminidase inhibitor antiviral medications oseltamivir, zanamivir, and peramivir, drugs currently approved for use against seasonal influenza. All 807 influenza viruses tested were found to be sensitive to all three antiviral medications. An additional 114 influenza A (H3N2) viruses were tested for resistance to oseltamivir and zanamivir, and were found to be sensitive to both antiviral medications.From October 2, 2016, through February 4, 2017, the weekly percentage of outpatient visits for influenza-like illness (ILI)Data collected in ILINet are used to produce a measure of ILI activityInfluenza activity levels reported by state and territorial epidemiologists indicate the geographic spread of influenza viruses. For the week ending February 4, 2017 (week 5), Puerto Rico and 43 states  reported widespread activity.CDC monitors hospitalizations associated with laboratory-confirmed influenza infection in adults and children through the Influenza Hospitalization Surveillance Network (FluSurv-NET),The cumulative hospitalization rate  during October 1, 2016–February 4, 2017 was 13.6 among children aged 0–4 years, 4.8 among children and adolescents aged 5–17 years, 7.3 among adults aged 18–49 years, 23.5 among adults aged 50–64 years, and 113.4 among adults aged ≥65 years. Among all hospitalizations reported during October 1, 2016–February 4, 2017, a total of 6,367 (93.6%) were associated with influenza A virus, 395 (5.8%) with influenza B virus, 21 (0.3%) with influenza A and B virus coinfection, and 21 (0.3%) with influenza virus for which the type was not determined. Among 1,729 patients with influenza A subtype information, 1,701 (98.4%) were infected with influenza A (H3N2) virus and 28 (1.6%) were infected with influenza A (H1N1)pdm09 virus.Complete medical chart abstraction data were available for 796 (11.7%) hospitalized patients with laboratory-confirmed influenza as of February 4, 2017. Among 744 hospitalized adults with complete medical chart abstraction, 703 (94.5%) had at least one underlying medical condition that placed them at high risk for influenza-associated complications. The most commonly reported medical conditions were cardiovascular disease (47.1%), metabolic disorders (39.7%), and obesity (38.3%). Among 52 hospitalized children with complete medical chart abstraction, 28 (53.8%) had at least one underlying medical condition, the most commonly reported being asthma (15.4%), chronic lung disease (15.4%), and neurologic disorder (15.4%). Among 59 hospitalized women of childbearing age (15–44 years), 20 (33.9%) were pregnant.CDC tracks pneumonia and influenza (P&I)-attributed deaths through the National Center for Health Statistics (NCHS) Mortality Reporting System. The percentages of deaths attributed to P&I are released 2 weeks after the week of death to allow for collection of sufficient data to produce a stable P&I mortality percentage. Weekly mortality surveillance data includes a combination of machine coded and manually coded causes of death collected from death certificates. There is a backlog of data requiring manual coding within the NCHS mortality surveillance data. The percentages of deaths attributable to P&I are higher among manually coded records than the more rapidly available machine coded records and might result in initially reported P&I percentages that are lower than percentages calculated from final data. Initiatives continue to reduce and monitor the number of records awaiting manual coding.Based on data from NCHS available on February 9, 2017, 7.9%  of all U.S. deaths occurring during the week ending January 21, 2017 (week 3) were attributed to P&I. This percentage is above the epidemic thresholdAs of February 4, 2017 (week 5), 20 laboratory-confirmed influenza-associated pediatric deaths that occurred during the 2016–17 season were reported to CDC. Of the 20 deaths, nine were associated with an influenza A (H3N2) virus infection, one was associated with an influenza A (H1N1)pdm09 virus infection, five were associated with an influenza A virus infection for which no subtyping was performed, four were associated with an influenza B virus infection, and one was associated with an influenza virus for which the type was not determined. Since influenza-associated pediatric mortality became a nationally notifiable condition in 2004, the total number of influenza-associated pediatric deaths per season has ranged from 37 to 171; excluding the 2009 pandemic, when 358 pediatric deaths were reported to CDC from April 15, 2009, through October 2, 2010.Influenza activity in the United States began to increase in mid-December and remained elevated as of February 4, 2017. During the most recent weeks, decreases in activity have been observed in the Northwest (HHS Region 10), while activity has continued to increase in the remainder of the country. During October 2, 2016–February 4, 2017, influenza A (H3N2) viruses accounted for the majority of circulating influenza viruses, but influenza A (H1N1)pdm09 and influenza B viruses also were identified. Influenza activity has been moderate so far this season, and severity indicators are within the range of what has been observed during previous seasons when influenza A (H3N2) viruses predominated. Elevated influenza activity in parts of the United States is expected for several more weeks.Interim estimates of vaccine effectiveness based on data collected from November 28, 2016, through February 4, 2017, indicate that overall the influenza vaccine has been 48%  effective in preventing influenza-related medical visits across all age groups, and specifically was 43% (CI = 29%–54%) and 73% (CI = 54%–84%) effective in preventing medical visits associated with influenza A (H3N2) and influenza B, respectively  virus infection among cats in an animal shelter in New York City was first reported to public health officials on December 14, 2016. Approximately 350 persons with exposure to infected cats during this outbreak were screened or tested for infection and only one human infection with avian influenza A (H7N2) was identified (https://www.cdc.gov/flu/weekly). Additional information regarding influenza viruses, influenza surveillance, influenza vaccine, influenza antiviral medications, and novel influenza A infections in humans is online (https://www.cdc.gov/flu).Influenza surveillance reports for the United States are posted online weekly  viruses were identified most frequently, but influenza A (H1N1)pdm09 and influenza B viruses were also reported. No antiviral resistance to oseltamivir, zanamivir, or peramivir has been identified among influenza viruses tested to date.Elevated influenza activity in parts of the United States is expected for several more weeks. Influenza vaccination remains the most effective way to prevent influenza illness. Antiviral medications are an important adjunct to vaccination in the treatment and prevention of influenza. Early treatment with neuraminidase inhibitor antiviral medications is recommended for patients with severe, complicated, or progressive influenza illness and those at higher risk for influenza complications, including adults aged ≥65 years.


Salmonella enterica subsp enterica serovar Typhimurium (S. Typhimurium) is a serovar with broad host range. To determine the genomic diversity of S. Typhimurium, we sequenced 39 isolates  representing 14 Repeats Groups (RGs) determined primarily by clustered regularly interspaced short palindromic repeats (CRISPR). Analysis of single nucleotide polymorphisms (SNPs) among the 39 isolates yielded an average of 1,232 SNPs per isolate, ranging from 128 SNPs to 11,339 SNPs relative to the reference strain LT2. Phylogenetic analysis of the 39 isolates together with 66 publicly available genomes divided the 105 isolates into five clades and 19 lineages, with the majority of the isolates belonging to clades I and II. The composition of CRISPR profiles correlated well with the lineages, showing progressive deletion and occasional duplication of spacers. Prophage genes contributed nearly a quarter of the S. Typhimurium accessory genome. Prophage profiles were found to be correlated with lineages and CRISPR profiles. Three new variants of HP2-like P2 prophage, several new variants of P22 prophage and a plasmid-like genomic island StmGI_0323 were found. This study presents evidence of horizontal transfer from other serovars or species and provides a broader understanding of the global genomic diversity of S. Typhimurium. Salmonella enterica serovar Typhimurium is the most common Salmonella serovar causing foodborne infections in Australia and many other countries1. The phenotypic diversity of S. Typhimurium has been traditionally illustrated by the Anderson phage typing scheme with more than 200 phage types defined2. Since then, different molecular markers were used to assess its genetic diversity4. S. Typhimurium is divided by multilocus sequence typing into more than 30 sequence types (STs). ST19 is the most prevalent ST internationally and the majority of the STs belong to the ST19 clonal complex5. By application of whole-genome-sequencing (WGS) and genomic analysis, our earlier study of six Australian S. Typhimurium strains from different phage types and 7 published genomes revealed three genomic clusters6. Hayden et al.7 analysed the genomic diversity of 35 US S. Typhimurium isolates together with 21 public genomes7 and found that the 56 S. Typhimurium strains could be divided into 3 clades and at least 10 lineages.S. Typhimurium strains was mainly due to the accumulation of single nucleotide polymorphisms (SNPs). The S. Typhimurium genome consists of a core genome of around 3,800 genes present in all strains and nearly 1,000 accessory genes which are variably present in one or more strains7. The accessory genome contains mostly genes of prophages and genes of unknown function which have contributed to the genetic diversity of S. Typhimurium4. However, the full depth of genomic diversity of S. Typhimurium remains to be explored.Genome sequencing showed that variation among Salmonella generally possesses two CRISPR loci, which comprise conserved direct repeats separated by unique short sequences typically 32 bp long, called spacers9. Variation in the spacer profiles of CRISPRs has been useful for subtyping Salmonella isolates10. Shariat et al.11 investigated CRISPR array composition in four major serovars, Enteritidis, Typhimurium, Newport and Heidelberg, and demonstrated serovar specificity of CRISPR array composition11. Other studies have also found that CRISPR variation is associated with serotype and sequence types, providing good phylogenetic signals for inferring strain relationships13. Hiley et al.14 performed CRISPR and variable number tandem repeat (VNTR) analyses on a diverse collection of 200 Australian S. Typhimurium isolates and 14 reference strains which separated them into 15 Repeats Groups (RGs)14. This presented an opportunity to apply genomic analysis to selected isolates from these RGs to capture a broad range of genetic diversity of S. Typhimurium.Clustered regularly interspaced short palindromic repeats (CRISPR) belong to a family of unique repeat sequences and are possibly associated with adaptive resistance against invasive genetic elements such as phages. S. Typhimurium genomes including 54 of international origins to provide a global overview of S. Typhimurium diversity. We constructed a core genome phylogeny for all 105 strains and examined the correlation of core genome relationships with CRIPSR array composition and prophage profiles.In this study, we sequenced 39 strains, 37 of which were of Australian origin, representing 14 different RGs and we included in the analysis 66 publicly available et al.14  strains4 and 45 other publicly available genomes , were also included in the analysis . The number of SNPs ranged from 128 to 11,319 relative to the strain LT2 4 and the reports of Hayden et al. 7 and Mather et al. 15. This finding indicated that the current dataset had a strain coverage broad enough to achieve a stable core genome and adding more strains to the analysis would not substantially reduce the core genome size. Interestingly, prophage genes contributed up to 13.3%  of the pan genome and 23.4%  of the accessory genome, while plasmid and other mobile elements took up 13.3%  of the accessory genome. For the rest of the genes in the accessory genome, the majority  encoded hypothetical proteins with currently unknown function.Pan genome analysis showed that there were 8,849 genes, including 3,836 core genes, and 5,013 dispensable genes in S. Typhimurium core genome4. The strains were separated into five clades and 19 lineages , separated by the RG14 lineage, and lineage 13 which contained strains belonging to RG4 and RG7 even though they are distinctly different by both CRISPR and VNTR profiles14  shared similar CRISPR1 profiles with RG1 strains but their CRISPR2 profiles were like those in RG4A , a novel OLF-SE9-10012-like prophage and other outlier prophages. The prophage distribution is illustrated in Fig. Our previous studies have shown that much of the genomic diversity within LT2, Gifsy-1DT104, Gifsy-1DT64, Gifsy-1SL1344, Gifsy-1DT2, Gifsy-1CVM23701 and Gifsy-1DT12614. Gifsy-1LT2 was present in five of the 13 RG2 strains and in four RG11A/15 strains. A variant of Gifsy-1LT2 which has three segments different from LT2 occurred in five of the six RG6 strains. Gifsy-1DT104 was found in RG3, RG4, RG7 and RG8. Gifsy-1DT126 was exclusive to RG11. Gifsy-1DT64 was only found in RG14. Gifsy-1CVM23701 was exclusive to some RG12A strains. Gifsy-1SL1344 was found in RG10 and one strain from RG2. Gifsy-1DT2 was distributed widely in many RGs including RG13, RG12, RG1, RG2 and RG1/4A.The lambdoid prophages include Gifsy-1, Gifsy-2, Gifsy-3 and Fels-1. Gifsy-1 was previously subtyped into seven variants, Gifsy-120. Gifsy-3 was found in SARA6, L1873, 14028 S and VNP20009 with >99% DNA sequence similarity to each other. Fels-1 was only found in LT2.Gifsy-2 was found in all strains except SARA8 and is highly conserved with no variants as reported previouslyDT64 and ST64BDT104. The ST64BDT64 variant was found in all Clade I strains and not in any other clades  while the ST64BDT104 variant was only found in some strains in Clades II and IV. Comparison of these ST64BDT104 prophage sequences in RG2, RG7 and RG6B to prototype ST64BDT104 in DT104 showed that some strains contained ST64BDT104 with varying degrees of coverage (range from 57.4% to 94.5%)  than to the SopEφ in S. Javiana 10721 (AOZA01000057) with 93%/98% (coverage/identity), indicating that it may have come from another serovar. A variant of SP-004 was found only in CVM23701 and DT24 while a variant of 186-type P2 was found in D23580 (BTP5) and DT24.Fels-2 was found in nine of the 13 RG2 strains as well as SARA10 (RG unassigned) and L818 (RG11). RE-2010Haemophilus influenzae22, and a distantly related variant, P2-Hawk, was reported in S. Typhimurium strains in tandem with a P4 prophage17. In our study, we found 14 HP2-like prophages in various insertion sites. Six of these were identified as P2-Hawk and were all in RG13 strains.They were almost identical, differing by no more than five SNPs from each other and always in tandem with the P4 phage. A comparative analysis showed that a core genome of 10 kb was shared by HP2-like P2 prophages, but only 2.6 kb was shared by all the genomes of HP2-like, P2, Fels-2, RE-2010 and SopEΦ (Table HP2 phage (NC_003315) was first found in EΦ Table . PhylogeEΦ Table . One varForty six strains were found to contain a P22-like prophage or one of its variants. The P22-like prophages that are located in the STM0323 site have seven publicly available variants including P22 (NC_002371), SE-1 (DQ003260), SPN9CC (JF900176), ST160 (GU573886), ST104 (AAF75053), ST64T (AY052766) and BTP1 (D23580). We first used PHAST to assign the 46 P22-like prophages into the above categories. P22 (NC_002371) or its variant was found in SARA1, SARA17, and SARA21. There were other variants of P22 (NC_002371) in SARA4, L1861, L1867 (RG10), L1853 (RG4A), SARA10 (RG unassigned) and five strains from RG2  but these were located in a novel insertion site, STM0786. SE-1-like prophages were found in SARA12 and L1850 with 100%/99% and 82%/99% coverage/identity, respectively. SPN9CC or one of its variants was found in 16 strains spread across six RGs. ST104 and one of its variants in DT97 were found in another 11 strains. ST160 was found in three strains, of which L1858 and DT99 each had a variant. Additionally, two strains had ST64T and strain D23580 had the BTP1 prophage.nin gene region in the position of 15268–16480 in phage SPN9CC (JF900176) and was in 13 strains from RG12, RG13 and RG10. The third variant had six unique sequences and was present only in L1864 from RG12B.We analysed the genetic diversity within P22 prophages. A phylogenetic tree of core genome sequences showed that the P22-related SPN9CC-like prophages could be divided into three variants Figure . The firThere was considerable diversity of gene content within the P22-like prophages. To better delineate the gene content variation of various P22-like prophages, we analysed the pan-genome of the 46 P22-like prophages found in this study as well as the genomes of seven publicly available P22 and its variants and obtained a 100 kb pan-genome. The pan-genome consisted of 193 DNA fragments, most of which were only shared by some prophage genomes  had high similarity to prophages in multiple serovars, while a few only had high similarity to one or two Salmonella serovars, including serovars closely related to S. Typhimurium such as S. Heidelberg, and more distantly related serovars like S. Newport and S. Paratyphi A. Twenty-four fragments had similarity to E. coli prophages, while 12 fragments were only found in S. Typhimurium prophages. Thus, there has been a considerable exchange of genetic information among diverging P22-like phage lineages, and the exchange appears to be randomly distributed throughout their genomes.We further explored the evolution of P22-like prophages by comparing their sequences with those from Enterobacteria phage HK106 NC_019768. The P22 variant in SARA10 had 66% coverage/99% identity with P22 (NC_002371) but 99% coverage/99% identity with Paratyphi B ATCC 8759 (AOYE01000028.1). SARA4 which was a RG12D/10A hybrid had another variant with 50% coverage/99% identity with P22 (NC_002371). Another prophage, P22_SARA19, was found in SARA19, DT195, ERR277210 and SARA20 but also with considerable sequence divergence from other P22 variants  as the SPN1S in S. Heidelberg strain 12-4374 (CP012924.1). A OLF-SE9-10012-like prophage of 36 kb was found to be inserted at the Fels-1 insertion site in RG9A strains L847, L1855 and L1862. A Shigella SfV-like prophage of 45 kb, which was inserted at STM4243, was found in RG6B strains DT193 and L1874. φW104 (belonging to the family Podoviridae) was detected in all but one RG8 strain and a variant of φW104 was found in SARA10 and SARA14 (unassigned RG) and DT8 (RG14). A 42 kb SEN34-like prophage (belonging to the family Podoviridae) in the STM2067 insertion site was found in strains L1876 and L1879.Five outlier prophages, SPN1S, OLF-SE9-10012-like prophage, a SfV-like prophage, φW104S. Typhimurium strains were classified into 25 prophage profiles , phages may have acted as a driving force in the evolution of S. Typhimurium, regardless of the mechanisms of phage acquisition, 26.Based on the distribution of ST64B, Gifsy-1, Gifsy-3, Fels-1, P2, P4, P22 and φW104 prophages found in this study, the 105 Klebsiella pneumoniae (Supplementary Table S. Enteritidis OLF-SE9-10012 (CP009091.1) with 70%/99% coverage/identity (Supplementary Table S. Muenchen BAA1674 (AOYT01000011.1), S. bongori N268-08 (CP006608.1), S. Hartford str 2012K-0272 (ARYS01000018) and S. Bovismorbificans 3114 (HF969015.2) as well as in multiple strains of S. Weltevreden and S. Bareilly. Close to the 3’ end of the prophage was the location of a variant form of the sopE gene (AF043239) which had been previously identified as a feature of all RG9A genotypes by PCR14.The SPN1S variant had some additional genes which shared identity with phage SPC32N or the sequence from Salmonella serovars. These genes were inserted into different positions of the SfV-like prophage genome, indicating that multiple genetic exchanges have occurred . A SEN34-like prophage was also found in serovar Weltevreden 1655 (CP014996.1) and Paratyphi B SPB7 (NC_010102). L1879 had a 43.5 kb sequence in the same insertion site that had only 14% coverage with SEN34. The remaining sequence was found in a number of S. Typhimurium strains and in other Salmonella serovars.The SfV-like prophage in two RG6B strains showed considerable divergence from SfV (AF339141) . The artAB genes on Gifsy-1DT104 initially found in epidemic DT104 strains29 were found in other strains in six RGs. Similarly, the seven virulence genes carried by Gifsy-2 were present in most strains. However, sopE and sspH1 carried by a P2 and Gifsy-3 respectively were variably present in two different branches, while nanH carried by Fels-1 was only present in one strain (LT2). Some of these genes play a key role in virulence. Specifically, SopE activates RHO GTPases that lead to modification of cytoskeleton of the host cell for invasion and also induce caspase 1 to provoke inflammation30. SopE can also induce the production of nitrate by host cell so that Salmonella can use nitrate respiration in the gut31, which enhances the survival of S. Typhimurium inside the host and competition with gut microbiota. SseK, a T3SS effector, encoded on ST64B and shown to play a role in the inhibition of NF-κB activation33, was found in five RGs. Some P22 prophages carry gtrABC genes which encode glucosyltransferases that glycosylate the galactose residues of the somatic O-antigen in S. Typhimurium34. Modification of the O-antigen may help evade host induced immunity. The distribution of the gtrABC carrying P22 prophages was random across the genome tree with only 3 RGs fully carrying these prophages. The variable carriage of these prophages and virulence genes suggest that S. Typhimurium strains can significantly differ in their pathogenic potential.thrW tRNA) site was found and named as StmGI_0323. It was present in some strains of RG12B and RG12C including L1848, L1854, L1864 and L1877 as well as L796. In L1864, StmGI_0323 occurred in tandem with a SPN9CC-related P22 prophage. The genomic island was also found in DT8 and L1859 in RG14. StmGI_0323 encodes 14 open reading frames (ORFs) (Table E. coli plasmid sequences (>99% at protein level). It was noteworthy that another unrelated genomic island GQ478253 was inserted at this site in RG6B strains L1874 and DT193.A novel genomic island inserted in the STM0323  was used to locate the regular repeats and the intervening spacer sequences. Results were represented as filled rectangular blocks for ‘spacer present’ or an X for ‘spacer absent’ in the same order as for S. Typhimurium spacers in Table et al.8 (Supplementary Table The CRISPR sequences of 36 strains (strain No. from L1849 to L1883) were determined in a previous studyde novo assembled using the Velvet version 1.0.8 and VelvetOptimiser37. Large scaffolds and short contigs generated by Velvet were aligned to the S. Typhimurium LT2 genome (NC_003197) using progressiveMauve version 2.3.138. RAST was used to annotate the sequences from each NGS genome39. The number of coding sequences in the genomes was predicted based on RAST. For draft genomes, SNP calling was performed by Samtools (version 0.1.19) and followed the previously described criteria6. A custom script was used to determine whether a SNP in the genic region is synonymous SNP (sSNP) or non-synonymous SNP (nsSNP). For the complete genome, SNPs were determined by using the NUCmer program in the MUMmer package version 3.040.Genomic DNA was sequenced using the Illumina Genome Analyzer (Illumina) with 250 bp paired end sequencing. Contigs were 41. The prophages were confirmed by searching for integrase from annotated genomes. We subtyped the ST64B prophages and Gifsy-1 prophages into two and seven variants, respectively. ST64B prophage has two variants: ST64BDT104 and ST64BDT64. The sequence of ST64BDT64 (AY055382) and the sequence of ST64BDT104 obtained from DT104 (NC_022569) were used as reference sequences to confirm the variants in our studied strains. Gifsy-1 prophages were subtyped as either Gifsy-1LT2, Gifsy-1DT104, Gifsy-1DT126, Gifsy-1SL1344, Gifsy-1DT2, Gifsy-1CVM23701 or Gifsy-1DT64 based on the unique sequences among these variants as defined previously14. The core genome content of P2 prophage and HP2-like group of P2 prophage were obtained by analysing common shared regions of P2 prophages and HP2-like P2 prophages using progressiveMauve version 2.3.1, respectively38.The presence of prophages from the sequenced strains was screened using PHASTS. Typhimurium core genome SNPs we defined previously4 and core genome content of P2 prophage identified in this study, phylogenetic trees were constructed using the Minimum Evolution algorithms in MEGA 5.0 for 105 S. Typhimurium genomes and 15 P2 prophage genomes, respectively42. Bootstrap analysis was performed with 1,000 replicates. Based on the presence and absence of DNA segments in the P22 pan-genome, a UPGMA tree was constructed using the web-server DendroUPGMA (http://genomes.urv.cat/UPGMA).Based on The raw sequencing data were submitted to GenBank (NCBI) under the BioProject No. PRJNA355598.Supplementary Tablessupplementary info


PPP1R3B) single nucleotide polymorphisms (SNPs) and serum lipid levels, the risk of coronary artery disease (CAD) and ischemic stroke (IS) in the Chinese populations. This study detected such association in a Southern Chinese Han population.Little is known about the association of the protein phosphatase 1 regulatory subunit 3B gene  in 1704 Han Chinese  were determined by the Snapshot technology.Genotypes of 4 novel P < 0.0014). The rs12785A, rs9949A and rs330910T allele carriers were associated with an increased risk of CAD (P = 0.008–0.004). There was strong linkage disequilibrium among the 4 SNPs in the controls and CAD/IS patients. The T-A-A-G haplotype was associated with a decreased risk of CAD and IS, whereas the A-A-T-A haplotype was associated with an increased risk for IS. Haplotype-environment interactions on the risk of CAD and IS were also observed.The rs12785A and rs9949A allele frequency was higher in both CAD/IS patients than in controls. The rs330910T and rs330915T allele frequency was also higher in CAD patients than in controls. The rs330910T allele carriers in controls had lower serum low-density lipoprotein cholesterol (LDL-C) levels than the rs330910T allele non-carriers  contains supplementary material, which is available to authorized users. The mortality and morbidity of coronary artery disease (CAD) and ischemic stroke (IS) remain very high in different districts –3. AlthoPPP1R3B) is located on chromosome 8p. The gene which encoding a regulatory subunit of protein phosphatase 1 is involved in modulation of glycogen synthesis in liver and skeletal muscle . Table S2. Association between the PPP1R3B genotypes and BMI or blood pressure in controls. Table S3. Linkage disequilibrium analysis of the 4 PPP1R3B SNPs. Table S4. Haplotype analysis of the 4 PPP1R3B SNPs. Figure S1. Annotation of SNPs in UCSC. Non-coding (ncRNA) and untranslated region (3’UTR) are colored blue. (DOC 392 kb)


Cohnella sp., [Coh00831  and Coh01133 (KP335160: 3678 bp)] were identified. The genes were heterologously expressed in E. coli to both determine their type and further characterize their properties. The isolated DNA was PCR amplified with gene specific primers and cloned in pET28a, and the recombinant proteins were expressed in E. coli BL21 (DE3). The temperatures and pH optima of purified recombinants Coh 01133 and Coh 00831 enzymes were 70°C and 8, and 60°C and 6, respectively. These enzymes are stable more than 90% in 60°C and 50°C for 90 min respectively. The major reactions released sugars which could be fractionated by HPLC analysis, from soluble starch were mainly maltose (G2), maltotriose (G3) and maltotetraose (G4). The enzymes hydrolyzed pullulan to maltotriose (G3) only. Enzyme activities for both proteins were improved in the availability of Mn2+, Ba2+, Ca2+, and Mg2+ and reduced in the presence of Fe2+, Li2+, Na2+, Triton X100 and urea. Moreover, Co2+, K+, and Cu2+ had a negative effect only on Coh 01133 enzyme.Some industries require newer, more efficient recombinant enzymes to accelerate their ongoing biochemical reactions in harsh environments with less replenishment. Thus, the search for native enzymes from extremophiles that are suitable for use under industrial conditions is a permanent challenge for R & D departments. Here and toward such discoveries, two sequences homologous to amylopullulanases  from an endogenous  Costs associated with food and beverage enzymes are expected to reach $1.7 billion US dollars by 2018 , 4. PullCohnella sp. A01 toward their identification and biochemical characterization were heterologously expressed in E. coli in the search for industrial enzymes. The enzyme features were compared with some previously known orthologs, and phylogenetic analysis were carried out to define their relatedness to either GH13 or GH57.Here, two amylopullulanases from an endogenous Taq DNA polymerase, BamH I, Nhe I, Nde I, Sal I, T4 DNA ligase, and IPTG were purchased from Thermo Scientific™. Strains including E. coli DH5α and BL21 (DE3), pET-28a vector and Ni-NTA resin were purchased from Invitrogen . Ampicillin (A0166-5G), kanamycin (K1377-5G), agarose (A9539-50G), pullulan (91335), and starch (S2630) were acquired from Sigma . The DNA extraction kit was obtained from BioNEER . HPLC-grade water and acetonitrile gradient grade for liquid chromatography were purchased from Merck. Other materials were purchased from Merck  and Sigma . Cohnella sp. A01 was obtained from sampled water of a shrimp pond at Choebdeh  and further characterized by microbiological and 16s rRNA sequencing (Accession No. JN208862.1) ) as solvent at a flow rate of 1 ml per min and column temperature of 20°C.2, CaCl2, KCl, NiCl2, LiCl2, FeCl2, MnCl2, ZnCl2, BaCl2, CuCl2, CoCl2, and NaCl), detergents , 2 and 5 mM EDTA as chelator, and 10 mM iodoacetic acid as reducing agent on the recombinant enzymes activities were analyzed. Enzymes (50 μl) at their pH optima were added to mixtures of 50 μl 1% pullulan and each of above-mentioned chemicals. The reactions were incubated at optimum temperatures for 30 min.The effects of 5 mM metal cations  pullulan. The gel was washed with 2.5% Triton X100 and incubated for 1 h with the proper buffer providing optimum pH for each enzyme, 50 mM either sodium acetate (pH = 6.0) or sodium phosphate (pH = 8.0), at 4°C. The gels were incubated at 60°C for 1 h; then Lugol’s reagent was added to the gel, and enzyme activity was photographed.http://www.cazy.org) proteins, representative members of the amylopullulanases and α-amylases families, were collected and run through MEGA5. The four conserved motifs that have been reported to be the main scaffolds for α-amylase family of enzymes at positions of: I: 316-AADGSV-S, II: 383-EVRLDKV, III: 408-YEAWR and IV: 507-VDGTDK in Coh 00831 (KP335161 GenBank accession no.) amino acid sequence and I: 567–GTSPLVDLSGNA, II: 670- SPALTDLA, III: 729- VEVNG and IV: 816- TDGEG in Coh 01133 (KP335160 GenBank accession no.) amino acid sequence were evident   of the enzymes have been summarized in Table C in cat value of the Coh 01133 enzyme toward pullulan substrate was smaller compared with Coh 00831 enzyme. In contrast, Coh 00831 enzyme had a lower kcat value for soluble starch compared with those of Coh 01133 enzyme , maltotriose (G3) and maltotetraose (G4) and hydrolyzed pullulan to maltotriose (G3) demonstrating that the enzymes show typical pullulanase activity. Neither enzyme produced glucose (G1)  .2+, Ba2+, Ca2+, and Mg2+ improved the activity of both enzymes, while Fe2+, Li2+, Na2+, Triton X100, and urea caused sharp reductions in enzyme activity. More specifically, Co2+, Cu2+, and K+ in Coh 01133 amylopullulanase reduced enzyme activity greatly. EDTA reduced the activities of both enzymes, specifically at 5 mM. In contrast, iodoacetamide had no major effect on enzyme activity , from shrimp ponds in southern parts of Iran. Following genome sequencing (data not yet published), two amylopullulanase genes, Coh 00831 gene and Coh 01133 gene, were PCR amplified and used for heterologous protein expression and further biochemical characterizations. The enzymes demonstrated themselves to be thermophilic as expected from the niche from which the bacterium was isolated. The Km values for the two recombinant enzymes were the lowest for pullulan and starch when compared with previously reported homologues from other bacteria . The recombinant enzyme had an optimum temperature of 60°C, higher than a few previously reported bacterial amylopullulanases  shown in Table C in kcat and kcat/Km values were greater for starch than pullulan, indicating that starch is a better substrate for the enzyme  and Coh 01133, (~127 kDa), were PCR amplified and heterologously expressed in E. coli. Recombinant enzymes were assayed against pullulan and starch and demonstrated greater affinity toward starch. Thus, both enzymes have the potential to be used for liquefaction in the starch industry. The effects of metal divalent cations were studied, and Ca2+, Mg2+, Mn2+, and Ba2+ were demonstrated to improve enzyme activity by more than 50%.Two amylopullulanse genes from an endogenous bacterium belonging to GH57, namely S1 FileTable A in S1 File: Bacterial α-amylases and amylopullulanases used for multiple sequence alignment and phylogenetic tree. Table B in S1 File: The % identity of proteins from this study with other Amylopullulanase. Table C in S1 File: Molecular weight, pH optimum, temperature optimum and Kinetic parameters of some amylopullulanases from GH13 and GH57. Figure A in S1 File: Chromatogram of Ni-NTA sepharose column (c1), chromatogram of ion exchange chromatography (c2)(PPTX)Click here for additional data file.


G9a using short-hairpin RNA and pharmacological interference. Knockdown of G9a reduced H3K9me2 levels and impaired both HCC cell growth and sphere formation. However, transforming growth factor β1-induced epithelial mesenchymal transition (EMT) was not suppressed by G9a knockdown. Combined analyses of chromatin immunoprecipitation followed by sequencing and RNA-sequencing led to successful identification of 96 candidate epigenetic targets of G9a. Pharmacological inhibition of G9a by BIX-01294 resulted in both cell growth inhibition and induction of apoptosis in HCC cells. Intraperitoneal administration of BIX-01294 suppressed the growth of xenograft tumors generated by implantation of HCC cells in non-obese diabetic/severe combined immunodeficient mice. Immunohistochemical analyses revealed high levels of G9a and H3K9me2 in 36 (66.7%) and 35 (64.8%) primary HCC tissues, respectively. G9a expression levels were significantly positively correlated with H3K9me2 levels in tumor tissues. In contrast, in non-tumor tissues, G9a and H3K9me2 were only observed in biliary epithelial cells and periportal hepatocytes. In conclusion, G9a inhibition impairs anchorage-dependent and -independent cell growth, but not EMT in HCC cells. Our data indicate that pharmacological interference of G9a might be a novel epigenetic approach for the treatment of HCC.Histone H3 lysine 9 dimethylation (H3K9me2) is mainly regulated by the histone lysine methyltransferase G9a and is associated with the repression of transcription. However, both the role of G9a and the significance of H3K9me2 in hepatocellular carcinoma (HCC) cells remain unclear. In this study, we conducted loss-of-function assay of  Epigenetics is defined as a mechanism that regulates gene expression without changes in the underlying DNA sequence . EpigeneAberrant epigenetic modifications are frequently observed in a wide range of cancers and are considered to play a central role in carcinogenesis and cancer progression . G9a oveG9a in culture. Candidate targets of G9a were investigated by chromatin immunoprecipitation followed by sequencing (ChIP-seq) and RNA-sequencing (RNA-seq). In addition, pharmacological disruption of G9a was conducted in culture as well as in vivo xenograft models. Finally, G9a expression levels and H3K9me2 levels in primary HCC surgical samples were determined by immunohistochemical analyses.In the present study, we performed loss-of-function analyses of G9a using lentivirus-mediated knockdown of We first examined the basal expression of G9a and H3K9me2 levels in a number of different HCC cell lines, namely Huh1, Huh7, PLC/PRF/5, and Huh6 cells -positive cell sorting. A lentiviral vector expressing the shRNA targeted against luciferase (Luc) was used as a control. Two different shRNAs (sh-G9a-1 and sh-G9a-2) were able to suppress G9a protein expression as well as reduce H3K9me2 levels  in several cancers such as breast cancer and head and neck squamous cell carcinoma , 13. Intn Figure  and 2B. n Figure . Howevers Figure . Immunocs Figure . Concords Figure . In addih7 cells .G9a knockdown HCC cells. miR200a and 200b expression levels showed no significant changes after G9a knockdown   Figure . The numG9a knockdown. RNA-seq profiling identified 371 upregulated (> 2-fold expression) and 141 downregulated (< 0.5-fold expression) genes. Combined examination of RNA-seq profiling and ChIP-seq data led to successful identification of 96 candidate targets for G9a whose expression was de-repressed (> 2-fold expression) accompanied by a decrease in H3K9me2 levels (< 0.5-fold expression) after G9a knockdown  mice. Following implantation of 2 × 106 Huh1 or Huh7 cells into NOD/SCID mice, intraperitoneal administration of BIX-01294 (10 mg/Kg) to recipient mice was started immediately. As a result, tumor growth was suppressed by BIX-01294 treatment in both Huh1 and Huh7 implanted mice  and 35 (64.8%) of 54 HCC samples were classified as H3K9me2low and H3K9me2high, respectively. Importantly, the number of G9alowH3K9me2low and G9ahighH3K9me2high tumors were found to be 10 (18.5%) and 27 (50.0%), respectively .To begin to understand the possible relevance of G9a to primary HCC, we conducted immunohistochemical analyses to examine G9a expression and H3K9me2 levels in 54 primary HCC tissues and corresponding non-tumor tissues. Tumor samples showed varying degrees of G9a expression and varying levels of H3K9me2 Figure . In conty Figure . Fisher'G9a is a member of the SET domain containing Suv39 family and catalyzes the dimethylation of histone H3K9 . SubsequG9a knockdown decreased H3K9me2 levels, but not H3K9me3 levels. In line with previous reports, G9a knockdown interfered with cell proliferation and tumorigenicity in HCC cells. Recently, inhibition of G9a has been shown to induce not only apoptosis, but also autophagic cell death via the suppression of mTOR pathway [To gain insights into the role of G9a in HCC cells, we performed loss-of-function assays in cultured cells.  pathway , 23. Aut pathway . FurtherIn vitro assays showed that G9a depletion failed to cancel TGF-β1-induced EMT in Huh7 and PLC/PRF/5 cells, which have an epithelial cell morphology. However, co-IP assays on Huh7 cells showed an interaction between G9a and SNAIL. These findings suggest that the role of G9a in regulating EMT differs with the cancer types. Further analyses would be necessary to explore the subject.EMT is a process in which epithelial cells lose their characteristics while acquiring the properties of mesenchymal cells. There is increasing evidence that EMT is deeply involved in the initiation of metastasis for cancer progression . Recent p21WAF1/CIP1, a cyclin-dependent kinase inhibitor, plays an important role in cell cycle arrest [p21WAF1/CIP1 is silenced by histone modifications such as histone methylation and deacetylation in HCC cells. Tissue inhibitor of metalloproteinase 3 (TIMP3) inhibits tumor development via cell cycle arrest and senescence in carcinogen-induced HCC mouse model [G9a knockdown might contribute to cell growth inhibition in vitro and in vivo experiments. In addition, differentiation marker genes such as cytokeratin 19 (CK19) and several CYP enzymes were de-repressed after G9a knockdown. Given that anchorage-independent growth ability (sphere formation capability) reflects the stemness feature of tumor cells [G9a knockdown cells.In this study, combined analyses of ChIP-seq and RNA-seq successfully identified 96 candidate epigenetic targets of G9a. Among these, e arrest . We preve arrest . Taking se model . Restoreor cells , it is pIn vitro assays revealed that both cell proliferation and sphere formation of HCC cells were significantly impaired by BIX-01294 treatment in a dose-dependent manner. Subsequent xenograft transplantation experiments showed that BIX-01294 suppressed the onset and growth of subcutaneous tumors. Taken together, BIX-01294 appears to exhibit therapeutic value for the treatment of HCC.Recently, a number of HKMT inhibitors have been developed as potential therapeutic cancer agents . Among tHypoxia is a common characteristic of many solid tumors including HCC . IntratuG9a knockdown and pharmacological ablation of G9a reduced H3K9me2 levels and inhibited cell proliferation and tumorigenicity. However, it appeared that G9a exhibited a minimal effect on EMT in HCC cells. Considering that a significant relationship was observed between G9a expression and H3K9me2 levels in primary HCC samples, G9a might be an important epigenetic target in HCC treatment. Further analyses are necessary to elucidate the epigenetic regulatory mechanisms of G9a in HCC cells.In conclusion, we have successfully demonstrated that both Human HCC cell lines were obtained from the Health Science Research Resources Bank . For the sphere formation assay, 1,000 cells were plated onto ultra-low attachment 6-well plates . The number of spheres (> 100 μm in diameter) was counted following 14 days of culture. For the pharmacological inhibition of G9a, BIX-01294  was used at final concentrations of 1 μM or 5 μM. For the induction of EMT, human TGF-β1  was used at a final concentration of 10 μg/mL.G9a  and luciferase(Luc) were constructed. Recombinant lentiviruses were produced as described elsewhere [Lentiviral vectors (CS-H1-shRNA-EF-1a-EGFP) expressing short hairpin RNA (shRNA) that targets the human lsewhere . The celHCC cell proliferation was assessed using trypan blue staining after 48, 96 and 144 hours in culture. Cell migration was assessed by the scratch-wound assay.G9a-knockdown cells were selected by cell sorting for EGFP expression. These cells and BIX-01294-treated HCC cells were subjected to Western blot analysis using anti-G9a , anti-tubulin , anti-histone H3K9me2 and anti-histone H3  antibodies. Endogenous SNAIL was immunoprecipitated and subjected to Western blotting with rabbit anti-SNAIL  and mouse anti-G9a  antibodies.Cells were fixed with 2% paraformaldehyde and blocked with normal goat serum. Cells were then stained with anti-G9a , anti-E-cadherin , anti-caspase 3 , and anti-H3K9me3 (Millipore) antibodies, followed by incubation with Alexa 555- or Alexa 488-conjugated immunoglobulin G (IgG)  antibodies. Cells were coverslipped using a mounting medium that contained 4′,6-diamidino-2-phenylindole dihydrochloride .Quantitative real-time PCR was performed with an ABI PRISM 7300 Sequence Detection System  using the Universal Probe Library System  according to the manufacturer's directions. The sequences of primers are listed in RNA-Seq was performed as previously described . In brie7 Huh7 cells were subjected to immunoprecipited using anti-H3K9me2  antibodies. To evaluate the histone modification H3K9me2 in each gene, normalized tag numbers in the region from 2 kb upstream to 2 kb downstream of the transcription start site (TSS) were separated by reads per million (RPM) mapped reads of the corresponding input. The RPM values of the sequenced read were calculated using BEDTools to visualize with Integrative Genomics Viewer (http://www.broadinstitute.org/igv).ChIP sequencing (ChIP-Seq) was also performed as previously described . In brie6 HCC cells stably expressing shRNA against G9a or luciferase were implanted into the subcutaneous space on the right and left sides of the backs of recipient NOD/SCID mice, respectively. In the BIX-01294 treatment model, a total of 2 × 106 Huh1 or Huh7 cells were implanted into the subcutaneous space on the backs of NOD/SCID mice. BIX-01294 (10 mg/Kg) was administered intraperitoneally 3 times a week. Tumor formation and growth were observed weekly. Subcutaneous tumors were also subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry with anti-H3K9me2, anti-CASP3, and anti-Ki67  antibodies. For the analyses of xenograft tumors, small pieces of tumor were digested in DMEM containing 5 mg/mL collagenase type II (Roche). The cell suspension was centrifuged on Ficoll  to remove dead cells and debris. The harvested cells were subjected to sphere formation assays. All experiments were performed in accordance with the institutional guidelines for the use of laboratory animals.NOD/SCID mice  were bred and maintained in accordance with our institutional guidelines for the use of laboratory animals. A total of 2 × 10low or H3K9me2low (< 50% of tumor cells) and G9ahigh or H3K9me2high (≥ 50% of tumor cells).A total of 54 tumor and non-tumor liver tissue pairs were subjected to histological examination. All patients provided informed consent. They included 41 men and 13 women whose average age was 67.6 years (range: 27–84 years). Paraffin embedded sections of tumors and surrounding non-tumor tissues were examined by H&E staining and immunohistochemistry with anti-G9a and anti-H3K9me2 antibodies. Based on the percentage of cells that strongly expressed these markers, HCC tissues were divided into two groups: G9aU test or Chi-square test. Fisher's exact test was used to assess the relationship between H3K9me2 levels and G9a expression. P values less than 0.05 were considered significant.Data are presented as the mean ± standard error of the mean (SEM). Statistical differences between 2 groups were analyzed by either Mann-Whitney Data of RNA-seq and ChIP-seq were deposited in DNA Data Bank of Japan (DRA005376).