H9N2 influenza A viruses (IAV) are considered low pathogenic avian influenza viruses (LPAIV). These viruses are endemic in poultry in many countries in Asia, the Middle East and parts of Africa. Several cases of H9N2-associated infections in humans as well as in pigs have led the World Health Organization (WHO) to include these viruses among those with pandemic potential. To date, the processes and mechanisms associated with H9N2 IAV adaptation to mammals are poorly understood. The non-structural protein 1 (NS1) from IAV is a virulence factor that counteracts the innate immune responses. Here, we evaluated the ability of the NS1 protein from A/quail/Hong Kong/G1/97 (HK/97) H9N2 to inhibit host immune responses. We found that HK/97 NS1 protein counteracted interferon (IFN) responses but was not able to inhibit host gene expression in human or avian cells. In contrast, the NS1 protein from earlier H9N2 IAV strains, including the first H9N2 A/turkey/Wisconsin/1/1966 (WI/66), were able to inhibit both IFN and host gene expression. Using chimeric constructs between WI/66 and HK/97 NS1 proteins, we identified the region and amino acid residues involved in inhibition of host gene expression. Amino acid substitutions L103F, I106M, P114S, G125D and N139D in HK/97 NS1 resulted in binding to the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30) and, in consequence, inhibition of host gene expression. Notably, changes in the same amino acid residues resulted in the lack of inhibition of host gene expression by WI/66 NS1. Importantly, our results identified a new combination of amino acids required for NS1 binding to CPSF30 and inhibition of host gene expression. These results also confirm previous studies demonstrating strain specific differences in the ability of NS1 proteins to inhibit host gene expression. Orthomyxoviridae family and are classified in subtypes based on the antigenic features of the two surface glycoproteins present on the viral envelop: hemagglutinin (HA) and neuraminidase (NA) (Influenza A viruses (IAV) are members of the ase (NA) . To datease (NA) , 2013. Aase (NA) .Based on the World Organization for Animal health , avian-origin influenza viruses are classified in low pathogenic (LPAIV) and high pathogenic (HPAIV) avian influenza viruses, depending on the severity of the disease that they induce in poultry . LPAIV sHost innate immune responses play an important role in the defense against viral infections, including influenza . The defIn this study, we analyzed the ability to inhibit host gene and innate immune responses of the NS1 protein from A/quail/Hong Kong/G1/1997 (HK/97) H9N2, a prototype strain of the predominant G1-like lineage . We founL-glutamine, 2 mM) at 37 and 39°C, respectively, with 5% CO2.Human embryonic kidney 293T, HEK293T , human lung epithelial carcinoma A549 (ATCC CCL-185) and UMNSAH/DF-1 chicken embryo fibroblast, DF-1 (ATCC CRL-12203) cells were grown in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (FBS) and 1% PSG , A/duck/Hong Kong/702/1979 (HK/79), A/quail/Hong Kong/A28945/1988 (HK/88), A/quail/Hong Kong/G1/1997 (HK/97) and A/guinea fowl/Hong Kong/WF10/1999 (HK/99) were generated using standard molecular biology techniques as previously described . Polymer5cells/well, triplicates) were transiently co-transfected in suspension, using Lipofectamine2000 (Invitrogen), with 1 μg/well of pCAGGS-HA-NH2 NS1 protein expression plasmids, or an empty pCAGGS-HA-NH2 as internal control, together with 25 ng/well of pCAGGS plasmids expressing Gaussia luciferase (Gluc)   and the e (Gluc)  and plac5 cells/well, triplicates) were transiently co-transfected, using a calcium phosphate-based mammalian transfection kit (Stratagene), with 2 μg/well of the pCAGGS-HA-NH2 NS1-expressing plasmids, or empty pCAGGS-HA-NH2 plasmid as control, together with 0.25 μg/well of plasmids expressing Firefly Luciferase (FFluc) under the control of the IFN-β (pIFN-β-FFluc) or the ISRE (pISRE-FFluc) promoters  promoters, HEK293T cells  containing 0.1% Tween 20 (PBS-Tween) and incubated overnight at 4°C with rabbit anti-HA (Sigma) or anti-FLAG (Sigma) polyclonal antibodies (pAb) to detect expression of NS1 and CPSF30, respectively. A monoclonal antibody (mAb) specific for actin (Sigma) was used as an internal loading control. To detect the primary antibodies, anti-rabbit (pAb) or anti-mouse (mAb) IgG antibodies conjugated to horseradish peroxidase  were used. Proteins were detected by chemiluminescence using the Super Signal West Femto maximum-sensitivity substrate (Thermo Scientific) following the manufacturer’s recommendations and photographed using a Kodak Image Station.6 cells/well) were transiently transfected with 2 μg/well of a pCAGGS plasmid expressing a FLAG-tagged human CPSF30 (in vitro from the pcDNA3 plasmids using the TNT7 transcription/translation kit (Promega), following manufacturer’s recommendations. Cleared cell lysates expressing the FLAG-tagged CPSF30 were incubated overnight at 4°C with the in vitro synthesized HA-tagged NS1 proteins and 30 μl of an anti-FLAG affinity resin (Sigma-Aldrich). After washing three times in Tris-Buffered Saline (TBS) containing 0.1% Tween-20, precipitated proteins were dissociated from the resin using Laemmli buffer and high temperature (95°C) and analyzed by Western blot as described above using rabbit anti-HA(NS1) and anti-FLAG (CPSF30) specific pAbs (Sigma).For production of human CPSF30, HEK293T cells  cells were transiently transfected with 1 μg/well of the respective pCAGGS expression plasmids containing an N-terminal HA-tagged NS1 constructs, using lipofectamine 2000 (LPF2000) (Invitrogen). At 24 hpt, cells were fixed with 4% (vol/vol) formaldehyde diluted in 1X PBS, permeabilized with 0.1% (vol/vol) Triton X-100, and blocked using 2.5% bovine serum albumin (BSA) diluted in 1X PBS. After blocking, transfected cells were incubated with a polyclonal antibody (pAb) against the HA epitope tag  and probed with a FITC-conjugated donkey anti-rabbit secondary antibody (Dako). Staining with 4′,6-diamindino-2-phenylindole (DAPI) was used to visualize cell nuclei. Protein expression was observed under a Leica fluorescent microscope. Microscope images were colored using Adobe Photoshop CS4 (v11.0) software.To evaluate the subcellular localization of NS1 proteins, human A549 cells or DF-1 chicken embryo fibroblast  H9N2 IAV could block host gene expression, human HEK293T or avian DF-1 cells were co-transfected with expression plasmids encoding GFP and Gluc together with a plasmid encoding the NS1 gene of HK/97 Figure . Cells tNext, we explored whether the NS1 proteins from different H9N2 strains would share the same phenotype displayed by the NS1 from the HK/97 strain Figure  and GlucIn order to evaluate which region(s) in the NS1 of H9N2 IAV is (are) responsible for influencing host gene expression, we constructed chimeras between the NS1 proteins from HK/97 (unable to inhibit host gene expression) and WI/66 (able to inhibit host gene expression) Figure . To evalBased on the results obtained with our chimeric H9N2 NS1 constructs Figure  we made Our results with individual mutants Figure  suggesteWe then evaluated the ability of each combination of amino acids in WI/66 NS1 Figure  to blockTo assess whether the same residues responsible for the loss of inhibition of host gene expression in WI/66 NS1 could restore the ability of HK/97 NS1 to inhibit host gene expression, we introduce individually the same amino acid changes in HK/97 NS1 Figure  and evalin vitro transcribed and translated HK/97 NS1 WT, HK/97 NS1 13 (L103F/I106M/P114S/G125D/N139D) and WI/66 NS1 WT fused to an HA-tag   bound toIn order to evaluate the ability of HK/97and WI/66 NS1 proteins and their respective HK 13 (L103F/I106M/P114S/G125D/N139D) and WI 13 (F103L/M106I/S114P/D125G/D139N) mutants to counteract IFN and ISG responses, HEK293T cells were co-transfected with pCAGGS plasmids expressing HK/97 WT, HK 13, WI/66 WT and WI 13 NS1 proteins together with reporter plasmids expressing Firefly luciferase (FFluc) under the control of the IFN-β Figure  or ISRE H9N2 IAV have been detected in domestic and wild avian species around the world . H9N2 IAThe ability of NS1 protein to counteract host innate immune responses and to allow viral replication in IFN-competent organism is common for IAV . One mecIn this study, we evaluated the ability of the NS1 protein from different H9N2 IAV isolates to inhibit host gene expression. We showed that HK/97 H9N2 NS1 protein, a reference virus for the G-1 lineage and one of the lineages more distributed in Asia , is unabThe NS1 protein from canine (CIV)  and equiin vivo studies in birds as well as in mammals with H9N2 IAV encoding NS1 proteins that lack/present the ability to counteract host gene expression will help to elucidate their role in viral pathogenicity, transmission and host adaptation.In summary, our study contributes in the current knowledge of the mechanism used for IAV to counteract host gene expression and identified a new combination of amino acids required for NS1 binding to CPSF30 and inhibition of host gene expression. Likewise, our studies further demonstrate the presence of strain specific differences in the ability of IAV NS1 proteins to inhibit host gene expression. Moreover, our results also confirm that inhibition of host gene expression by IAV NS1 is important for adaptation of IAV into mammals. Future LR, AN, and LM-S conceived and planned the experiments, contributed to the interpretation of the results, and wrote the manuscript. LR and AN carried out the experiments. All authors provided critical feedback and helped shape the research, analysis, and 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.


Scientific Reports 10.1038/s41598-018-33108-4, published online 02 October 2018Correction to: In Figure 5B, the XPS spectra are incorrect. The correct Figure 5 appears below as Fig. In addition, the Acknowledgements section in this Article contains errors.“Dr. Zafar Khan Ghouri gratefully acknowledges the support of Chemical Engineering Program, Texas A&M University at Qatar and Central laboratories Unit, Qatar University.”should read:“Dr. Zafar Khan Ghouri and Dr. Ahmed Abdala would like to acknowledge the financial support from Texas A&M University at Qatar and Qatar Foundation.”


In addition, it is possible that the antigen processed during natural LCMV infection is an 11-mer peptide (KAVYNFATCGI) rather than the widely used 9-mer. Although previous affinity measurements using purified proteins for these antigen variants revealed minimal differences, we applied highly sensitive two dimensional (2D) biophysical based techniques to further dissect TCR interaction with these closely related GP33 variants. The kinetic analyses of affinity provided by the 2D micropipette adhesion frequency assay (2D-MP) and bond lifetime under force analyzed using a biomembrane force probe (BFP) revealed significant differences between 41M, 41C and the 11-mer 41CGI antigen. We found a hierarchy in 2D affinity as 41M peptide displayed augmented TCR 2D affinity compared to 41C and 41CGI. These differences were also maintained in the presence of CD8 coreceptor and when analysis of total TCR:pMHC and CD8:pMHC bonds were considered. Moreover, the three ligands displayed dramatic differences in the bond lifetimes generated under force, in particular the 41CGI variant with the lowest 2D affinity demonstrated a 15-fold synergistic contribution of the CD8 coreceptor to overall bond lifetime. Our analyses emphasize the sensitivity of single cell and single bond 2D kinetic measurements in distinguishing between related agonist peptides.The LCMV GP33 CD8 epitope has long been one of the most widely used antigens in viral immunology. Of note, almost all of the  CD8+ cytotoxic T lymphocytes (CTLs), which recognize peptides presented by major histocompatibility complex (MHC) class I molecules, are critical for the antigen specific clearance of viral infections , 2. All A major question that persists is how T cells can simultaneously possess a high level of specificity coupled with extreme sensitivity for as few as a single pMHC molecule , 17. Thib MHC class I epitopes (in C57BL/6 mice); GP33−41, GP276−286, and NP396−404  (41C) and the 11 amino acid long GP33−43 (KAVYNFATCGI) (41CGI) (Lymphocytic choriomeningitis virus (LCMV) is one of the best characterized viral model systems in mice –26. LCMVP396−404 , 33. Botb response have been analyzed with the more stable APL. Because of these changes in the peptide antigen used for the LCMV response, we sought to determine how the three immunodominant GP33 epitopes alter the 2D kinetics of TCR recognition of the respective antigens and the CD8 T cell functional response. In addition to the TCR:pMHC interaction we wanted to investigate the contribution of CD8 coreceptor to the TCR signaling. When blocked or in the absence of CD8 coreceptor, CD8+ T cells require longer and more substantial TCR engagement  9-mer 41M, was created by introducing a single methionine at the carboxy terminal end replacing the cysteine at position 41, , 34, 35.gagement , 39. It gagement . FurtherC57BL/6 (B6) mice were purchased from the National Cancer Institute (NCI) and Charles River. P14 (Thy 1.2) TCR transgenic mice were housed and bred at the University of Utah. All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee at the University of Utah. C57BL6 Thy 1.1 congenic mice used as hosts for adoptive transfers were purchased from NCI. All mice were between 8 and 12 weeks of age.Lymphocytic choriomeningitis virus (LCMV) Armstrong were kindly provided by Dr. Matthew Williams (University of Utah) and was made as described .GP33-41M (KAVYNFATM), GP33-41C (KAVYNFATC) and cognate GP33−43 (KAVYNFATCGI) peptides were synthesized at the University of Utah on the Prelude X peptide synthesizer (Protein Technologies).5 PFU of Armstrong by intraperitoneal (i.p.) injection 24 h after adoptive cell transfer.Naïve CD8+ T cells were isolated from spleens of P14 (Thy 1.2) transgenic mice using MACS CD8+ T cell magnetic separation kit (Miltenyi Biotec) and intravenously transferred to congenic C57BL/6 (Thy1.1) hosts. Mice were infected with 2 × 105pfu Armstrong and sacrificed at 8 days post infection. Spleens were harvested and stimulated with either KAVYNFATM (41M), KAVYNFATC (41C) or KAVYNFATCGI (41CGI) for 1 h and then intracellular cytokine staining was performed.8–12 weeks old C57BL/6 mice were injected i.p. with 2 × 10b GP33 specific CD8+ T cells was measured using the previously characterized 2D-MP (33−41M (KAVYNFATM), GP33−41C (KAVYNFATC) or GP33−43 (KAVYNFATCGI) monomers. For relative 2D affinity measurements of TCR:pMHC interaction, monomers with the Db D227K mutation were used abrogating CD8 binding to MHC or without the Db D227K mutation measuring normalized adhesion bonds of the TCR:pMHC:CD8 trimolecular interaction. All monomers were obtained from the NIH Tetramer Core Facility. In both sets of experiments the adhesion frequency between a single T cell and a ligand coated RBC aspirated on opposing pipettes was observed using an inverted microscope. An electronically controlled piezoelectric actuator repeated a T cell contact and separation cycle with the pMHC coated RBC 50 times while keeping contact area (Ac) and time (t) constant. Upon retraction of the T cell, adhesion (binding of TCR:pMHC) was observed as a distention of the RBC membrane, allowing for quantification of the adhesion frequency (Pa) at equilibrium. Surface pMHC (ml) and TCR (mr) densities were determined by flow cytometry using an anti-TCRβ PE antibody  and an anti-H2Db antibody  both at saturating concentrations along with BD QuantiBRITE PE beads for standardization (BD Biosciences). The calculation of molecules per area was determined by dividing the number of TCR and pMHC per cell by the respective surface areas. The relative 2D affinities were calculated using the following equation: AcKa = –ln [1-Pa(1)]/mrml. Normalized adhesion frequency was calculated using the equation [–ln(1-Pa(s))/ml (pMHC)]. Geometric means of all measured single cell affinities and normalized adhesion bonds are reported ± SEM. The centerpiece of our micropipette system is an Olympus IX71 inverted microscope equipped with fluorescence and a 100X oil immersion phase contrast and suspended on a TMC CleanBench vibration isolation table. The micropipettes are mounted onto the stage by adapters made by Narishige and are controlled by both fine and coarse micromanipulators. One micropipette is attached to the piezoelectric actuator via a Physik Instrumente P-840.1 piezo amplifier control module that is controlled by LabVIEW software on the imaging workstation. Micropipettes are held by Narishige HI-7 injection holders affixed by the heads of Narishige UT-2 universal joints (with joint removed) on custom mounts. Aspiration pressure of the micropipettes is maintained by Kontes water columns on height adjustable Velmex Unislide height adjusters. Micropipettes are produced on a Sutter Instruments P-1,000 pipette puller and finished using a Narishige MF-900 microforge.The relative 2D affinity of naïve P14 H2Ded 2D-MP , 43–47. 33−41M (KAVYNFATM), GP33−41C (KAVYNFATC) or GP33−43 (KAVYNFATCGI) monomers and placed on the apex of an RBC that was aspirated onto a micropipette. This bead served as a force probe. The position of the edge of the bead was tracked by a high-resolution camera  with < 3 nm displacement precision. The T cell of interest was brought into contact with the glass bead, then retracted a set distance and held by the computer-controlled piezoelectric actuator. The retraction and hold-phase generated a force on the TCR:pMHC bond, which can be altered based on the distance the T cell is retracted. The camera then recorded the time it took for the T cell to disengage the glass bead, which was visualized as the RBC retracted and the bead returned to its starting position. Repeated cycles (known as force-clamp cycles) can be carried out at a single force in order to generate an average bond lifetime between the TCR and pMHC complex. For an optimal response to antigen, the bond lifetime increases with increasing force before reaching a peak bond lifetime, which is typical of a catch bond physiology. By varying the force and measuring the bond lifetimes one can determine what type of bond occurred. Bond lifetimes were analyzed as described (Bond lifetime measurements under force were captured using the biomembrane force probe Assay (BFP). Procedures for coupling pMHC to glass beads have been described previously , 48. In escribed  using a escribed . The BFPSplenocytes isolated from infected mice were plated at 2 × 106 cells per well in a 96-well plate and cytokine production was tested in response to either 41M, 41C or 41CGI peptides. Tested concentrations ranged from 1 uM to.03 nM in various dilutions. Cells were incubated with peptide for 1 h at 37° Celsius in R10 media and washed before Brefeldin A was added  and the cells incubated for another 4 h. R10 media was composed of RPMI 1640 (Mediatech), 10% heat inactivated FBS (Hyclone), 10 mM HEPES buffer (Mediatech), 2 mM L-glutamine (Mediatech), 50 μM 2-mercaptoethanol (2ME) (Sigma), and 100 μg/ml gentamicin (Mediatech). Additional samples were also cultured without peptide as a negative control and stimulation with a PMA  ionomycin  combination was used as a positive control. Cells were then washed and stained with surface antibodies in the dark for 30 min on ice in FACS staining buffer composed of phosphate buffered saline (PBS) (Mediatech), 0.1% bovine serum albumin (BSA) (Fisher Scientific), and 0.05% sodium azide (Sigma). Surface markers were stained with anti-CD90.2 FITC , anti-CD44 PerCP Cy5.5 , anti-CD3 Brilliant Violet 605  anti-CD4 Brilliant Violet 711  and anti-CD8 Brilliant Violet 785 . Using Tonbo bioscience Fix/Perm kit, cells were fixed and permeabilized as per manufacturer's protocol. Intracellular antibody staining with anti-IFNγ APC-Cy7 , anti-TNFα PE-Cy7 , anti-IL-2 APC , anti-Nur77 PE  and anti-IRF4 eFluor 450  antibodies was performed as per manufacturers' protocol in a permeabilization buffer for 30 min on ice. Cells were washed and kept on ice before being run on the LSRFortessa X-20 cell analyzer (Beckton Dickson). All flow cytometry data were analyzed using FlowJo software (Treestar).*P > 0.05, **P > 0.01, ***P > 0.001, and ****P > 0.0001.Statistical significance of measured values was determined by Ordinary one-way ANOVA and Tukey's multiple comparison test using the Prism Software (GraphPad). Statistical significance indicated as ns = no significance, D values of 17 μM and 45 μM (b monomer (D227K mutation of the MHC I α3 domain that abolishes CD8 binding)  3D affinity data has shown the APL 41M and 41C to have similar 3D affinities, with Knd 45 μM , 9.1 μM,nd 45 μM  and 2.3 nd 45 μM . More rend 45 μM  and the nd 45 μM , 22. Thend 45 μM , 53–55 mbinding) , 56–59.  Figures . Further Figures  demonstrb) (b-CD8 binding) to quantify the number of TCR:pMHC and CD8 to MHC bonds (2 respectively)  , 46 but b) . One canHC bonds . Similar) Figure . Using t) Figure . Additio) Figure . These dPreviously, using optical tweezer technology, it has been demonstrated that T cells generate piconewton (pN) force in response to agonist pMHC , 61. TheHere, we apply force to the TCR:pMHC bond with and without the contribution of CD8 for all three pMHC variants Figures . In eachLiu et al. have revealed CD8 coreceptor to be critical for T cell mediated force generation and cell spreading . TherefoUsing the BFP assay, we and others have shown that the bond lifetime of a given TCR:pMHC pair changes when the applied force is varied , 9–11. Iex vivo with each variant peptide. The orphan nuclear hormone receptor, Nur77, has been shown to be rapidly upregulated in T cells stimulated with antigen via the TCR, but not by inflammatory stimuli  cells also produced the most IFNγ Figures  and IL-2ex vivo with our peptide variants. Similar to our observation with monoclonal P14 cells, more polyclonal CD8 T cells exhibited increased frequencies of Nur77 expression upon stimulation with 41M as compared to 41C and 41CGI , the 9-mer 41C was six times more efficient than 41CGI at inducing upregulation of H-2Db. However, in competition experiments performed on the T-2Db cell line, the 11-mer 41CGI was six times more efficient at competing for pMHC than the 9-mer 41C  and the 11-mer (41CGI) are the immunodominant GP33 epitopes. In RMA-S MHC stability assays  and the 41M mutation with ranges from 2 to 45uM –52. At tThe crystal structures of several of the GP33 pMHC complexes have been reported with no major differences that explain our 2D kinetic findings . In geneex vivo with our three peptides, we demonstrated that 41M stimulated P14 CD8 T cells give a stronger signal than 41C as measured by Nur77 upregulation. Additionally, both 41M and 41C stimulated cells induce significantly stronger signals than 41CGI stimulated cells. While Nur77 is often used as a readout of signal strength and indicative of functionality (While 2D kinetics are not impacted by peptide loading as they are single molecule interactions, it is important to note that functional differences might be impacted by peptide loading. However Gairin et al. show that, based on binding, different assays give varying results on whether 41C or 41CGI is most stable . Our 2D ionality , 81, theionality , 83 but ionality .Although T cell responses are aggregations of the TCR bond lifetimes, bond lifetime under force is a single molecule measurement independent of peptide loading and is a critical parameter in determining T cell functionality , 10, 22.Unlike the OT-I APL system where often the question is how 2D affinity differences elicit changes in the generation of effector and memory T cell populations, here the question is how the use of GP33 variants might change the perception of the GP33 epitope immunodominance within the CD8 response to LCMV. Our data clearly show that 41M elicits higher 2D affinity, increased number of total bonds with CD8 and longer bond lifetime under force as well as a more robust CD8 T cell response than both 41C and 41CGI. Super agonists are ligands for the TCR that stimulate the T cells more than the processed antigenic peptide . By our EK and BE conceived the project and wrote the manuscript. EK and BL performed BFP experiments. EK and RA performed 2D-MP experiments. EK and JJ performed intracellular flow cytometry experiments. EK analyzed all data. BL and RA discussed findings and RA edited 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.


Beta-blocker treatment (0.5 g/L propranolol given in the drinking water) maintained a low T3 state in MI animals, dampening both BAT D2 activity (44% reduction) and serum T3 (66% reduction in serum T3) compared to that of the non-treated MI group 12 weeks after surgery (P<0.05). Propranolol improved cardiac function (assessed by echocardiogram) in the MI group compared to the non-treated MI group by 40 and 57%, 1 and 12 weeks after treatment, respectively (P<0.05). Our data suggested that the beta-adrenergic pathway may contribute to BAT D2 hyperactivity and T3 normalization after MI in rats. Propranolol treatment maintained low T3 state and improved cardiac function additionally.Considering the recognized role of thyroid hormones on the cardiovascular system during health and disease, we hypothesized that type 2 deiodinase (D2) activity, the main activation pathway of thyroxine (T4)-to-triiodothyronine (T3), could be an important site to modulate thyroid hormone status, which would then constitute a possible target for β-adrenergic blocking agents in a myocardial infarction (MI) model induced by left coronary occlusion in rats. Despite a sustained and dramatic fall in serum T4 concentrations (60–70%), the serum T3 concentration fell only transiently in the first week post-infarction (53%) and returned to control levels at 8 and 12 weeks after surgery compared to the Sham group (P<0.05). Brown adipose tissue (BAT) D2 activity (fmol T4·min Ischemic heart disease remains the leading cause of death worldwide . OverallMost symptoms resemble a hypothyroid state, i.e., chronic fatigue, mood disorders, swelling, skin disorders, dyslipidemia, and decreased metabolic rate. Cardiovascular repercussions include decreased heart rate and cardiac contractility, as well as mild increases in peripheral vascular resistance. However, thyroid hormone replacement therapy has shown some benefits in patients with ischemic heart disease and recent evidence has suggested that low T3 status could be a necessary allostatic response to reduce the metabolic rate of ischemic heart. Indeed, either systemic or cardiac hypothyroidism elicited by HF can be partially associated with the induction of ectopic cardiac type 3 deiodinase (D3) activity, the main inactivating pathway of TH . These fAlthough high expression levels of ectopic cardiac D3 activity have been initially proposed to explain hypothyroidism following MI, the mechanism whereby T3 can be normalized after MI remains unclear. Because type-1 deiodinase (D1) activity remains low throughout 12 weeks of MI, the type 2 deiodinase (D2) pathway has become the potential mechanism underlying, at least in part, the progressive re-establishment of serum T3 after MI . Both D1It has been demonstrated that D2 activity can be modulated by several factors, including sympathetic stimulation . BecauseMale Wistar rats (200–250 g) were maintained in cages (4 per cage) in a room under controlled temperature (24±2°C) and lighting (lights on from 6:00 to 18:00 h), with free access to food and water. Animal handling and experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health  and the Institutional Committee of Ethics and Animal Welfare (CEAW number: 23083.004836/20120-58).®, Brazil), a skin incision was performed at the left parasternal level, followed by dissection of the pectoralis major and minor muscles. The incision was made between the 4 or 5th left intercostal spaces, through which the heart was externalized. Left coronary artery was located and ligated with a 6-0 silk suture, as close as possible to its origin on the aorta. The heart was then quickly placed in its original anatomical position. Sham-operated group (Sham) was subjected to the same surgical procedure as the infarcted group but without left anterior coronary artery occlusion. Prophylactic doses of veterinary antibiotic  and analgesic flunixin meglumine  were given.MI was induced following the procedure previously described and modified by our group . After aElectrocardiogram (ECG) was registered one day after MI and echocardiogram (ECHO) evaluations were performed 1, 6, and 12 weeks after surgery to assess cardiac function. Echocardiograph color-system  equipped with a 10 MHz electronic-phased-array transducer was used. Images were obtained from the left parasternal and apical windows. Short-axis 2-dimensional views of the left ventricle (LV) were taken at the level of papillary muscles to obtain the M-mode recordings. Systolic function was expressed by the ejection fraction , calculated by the Simpson's method, after the LV volume calculation: systolic and diastolic LV long axis were measured on long-axis view, and systolic and diastolic LV short axis, traced at the level of papillary muscle, were measured on transversal view.Pathological analysis was performed as previously described . Heart wSerum T3 and T4 were determined by specific Coated-Tube RIA kits . All procedures were performed following the recommendations of the respective kit.125I] T4 , 20 mM dithiothreitol, and 1 mM propylthiouracil (PTU) in 100 mM potassium phosphate buffer, pH 6.9, containing 1 mM EDTA.Pituitary gland or BAT (40 mg) were homogenized with 0.5 mL (pituitary glands) or 1 mL (BAT) of 0.1 M sodium phosphate buffer containing 1 mM EDTA, 0.25 M sucrose, and 10 mM dithiothreitol, pH 6.9. Each homogenate sample (100 μL containing 150 μg protein for BAT samples and 50 μg for pituitary samples) was incubated for 3 h at 37°C with 1 nM [g for 3 min at 4°C, and an aliquot of the supernatant (360 μL) was collected to measure 125I liberated during deiodination reaction. Protein concentration was measured by the Bradford method after incubation of homogenates with NaOH (2.5 N). Enzyme activity is reported as femtomoles of T4 deiodinated per min per mg of protein.Total reaction volume was 300 μL. The reaction was stopped at 4°C in ice-bath followed by addition of 200 μL fetal bovine serum  and 100 μL trichloroacetic acid , and vigorous agitation. The samples were centrifuged at 8000 Wistar rats underwent MI  or sham operation  and serial blood sample collections  through the jugular vein to measure serum T3 and T4 concentrations. ECHO exams were performed on the first and twelfth week post-surgery, immediately before blood collection. At the end of 1, 8, and 12 weeks after the surgical procedure, the animals were randomly euthanized by decapitation after sedation (n=4-10/time/group) and the tissues (BAT and pituitary gland) were collected for D2 activity measurement.Wistar rats underwent MI (n=32) or Sham (n=28) procedures and were randomly assigned to four groups: rats treated daily with propranolol hydrochloride (Sigma Aldrich), a β-adrenergic antagonist in drinking water (0.50 g/L): Sham+Prop (n=14) and MI+Prop (n=16) or vehicle: Sham+water (n=14) and MI+water (n=16), during 1 (n=7–8/group) or 12 (n=7–8/group) weeks after surgery. At the end of each protocol, all groups underwent ECHO analyses and were euthanized the next day. Blood and BAT samples were collected for T4 and T3 serum measurement by radioimmunoassay and D2 activity assessments, respectively.t-test or one-way ANOVA, followed by Bonferroni multiple comparison post-test, using GraphPad Prism® 4 . Pearson's product moment correlation was used to assess the relationship between the D2 activity and the T3/T4 ratio in each period, and a Student’s t-test for paired data was used to assess the significant differences. Differences were considered statistically significant for P<0.05.Data are reported as means±SE. Statistical analyses were performed by unpaired Student’s ECG and ECHO validated the efficiency of the MI model. As expected, MI was characterized by rightward deviation of the frontal QRS axis (ÂQRS), Q wave was observed in L1, and QRS index (I-QRS) decreased . Animalsvs Sham group at 8 and 12 weeks after surgery (Despite sustained decrease of serum T4 concentrations (approximately 60-70%) of MI compared to the Sham group since the first week post-surgery , serum T surgery . T3/T4 rvs Sham group, with no differences in the first week (P>0.05). D2 activity progressively increased only in infarcted rats , suggesting that cardiac adrenergic stimulation was blunted. ECHO analysis  demonstrPathology data  showed tvs MI+Water at both times (Propranolol reduced the infarct size at the first week post-MI (P<0.05) but not 12 weeks post-MI (P>0.05) as observed in MI+Propranolol Serum T3 levels observed in MI-animals in this Phase  reproducAs expected, BAT D2 activity increased in MI+Water compared to the Sham+Water group at 12 weeks <0.01. MIThis study provides experimental evidence on the role played by BAT D2 activity over TH economy during the progression of HF following MI. BAT D2 activity progressively increased after MI, and it was followed by reestablishment of circulating T3 levels, independently of T4 levels. The increased T4 to T3 conversion could correspond to an important mechanism responsible for serum T3 normalization observed at the late phase of MI, for previous evidence has suggested that D2 might also be important for serum T3 supply, besides its crucial role in intracellular T3 levels ,14,24. TAs expected, cardiac function was significantly impaired early after coronary ligation, as evidenced by decreased EF at week 1, and this condition persisted towards the following weeks. Circulating TH levels were found decreased after 1 week in MI group, corroborating previous evidence on the development of hypothyroidism after ischemic heart disease. However, while T4 levels remained decreased during the following weeks, circulating T3 levels progressively increased to reach control levels at week 8, resulting in increased T3/T4 ratio. These findings indicated that T3 levels were normalized. In normal conditions, 50% of the intracellular T3 comes from deiodination processes induced by D2 , and a dIn keeping with this, BAT D2 activity progressively increased after MI, and it was positively correlated with the T3/T4 ratio. The progressive increase in T4 to T3 conversion was expected, in as much as increased D2 activity can be associated with the longer half-life of D2 protein and increased Dio2 gene transcription . Howeverper se, increased BAT D2 activity could also be induced by the higher sympathetic activity towards BAT. Indeed, early stage HF-induced hypothyroidism was not significantly affected by propranolol, though serum T3 normalization in the late stage of cardiac disease was prevented, corroborating the hypothesis that T4 to T3 deiodination was decreased by such pharmacological intervention. These data suggest that the β-adrenergic pathway plays a crucial role on BAT D2 hyperactivity and serum T3 normalization at late stages of HF pathophysiological progression. Future studies employing surgical or pharmacological sympathectomy will be needed to further address this hypothesis.D2 activity can be modulated by several factors, such as hypothyroidism, α- and β-adrenergic activation, and bile acids ,34. SinAnother potential mechanism to explain serum T3 normalization lies in D3 activity. If high cardiac D3 activity is associated with low serum thyroid hormones in the early phase of HF , T3 normBeta-adrenergic blockers therapy has long been demonstrated to improve survival in patients with HF . CardiacIn conclusion, our results suggest that the normalization of serum T3 concentrations after MI in rats can be induced by a progressive increase of D2 activity in BAT, which was potentially induced by the beta-adrenergic pathway post-MI in rats. When this pathway was blocked by propranolol, the low thyroid hormone state persisted, and cardiac function was improved additionally, suggesting a new beneficial effect of beta-blockers in a HF model.


Asfarviridae family that harbours the B-cell lymphoma 2 or Bcl-2 homolog A179L. A179L has been shown to bind to a range of cell death-inducing host proteins, including pro-apoptotic Bcl-2 proteins as well as the autophagy regulator Beclin. Here we report the crystal structure of A179L bound to the Beclin BH3 motif. A179L engages Beclin using the same canonical ligand-binding groove that is utilized to bind to pro-apoptotic Bcl-2 proteins. The mode of binding of Beclin to A179L mirrors that of Beclin binding to human Bcl-2 and Bcl-xL as well as murine γ-herpesvirus 68. The introduction of bulky hydrophobic residues into the A179L ligand-binding groove via site-directed mutagenesis ablates binding of Beclin to A179L, leading to a loss of the ability of A179L to modulate autophagosome formation in Vero cells during starvation. Our findings provide a mechanistic understanding for the potent autophagy inhibitory activity of A179L and serve as a platform for more detailed investigations into the role of autophagy during ASFV infection.Subversion of programmed cell death-based host defence systems is a prominent feature of infections by large DNA viruses. African swine fever virus (ASFV) is a large DNA virus and sole member of the  L, Bcl-w, Mcl-1, A1 and Bcl-b [The inhibition of programmed cell death pathways in infected host cells is a widely used strategy employed by many large DNA viruses. Successful inhibition is often achieved by the use of virus encoded homologs of the B-cell lymphoma-2 (Bcl-2) family of proteins, which are crucial arbiters of intrinsic or mitochondrial initiated apoptosis . Bcl-2 fnd Bcl-b , althougnd Bcl-b ,5. Pro-and Bcl-b ,7. The Bnd Bcl-b .Iridoviridae, grouper iridovirus encodes pro-survival GIV66 [Viruses utilize a diverse set of molecular strategies to inhibit premature host cell apoptosis. These include direct caspase inhibition and inhibition of the intrinsic apoptosis . For exaal GIV66 ,36.Asfarviridae, composed of large double stranded DNA viruses [African swine fever virus (ASFV) is the sole member of the family  viruses . ASFV is viruses . ASFV wa viruses ,40 that  viruses .In cellular assays, A179L is a potent inhibitor of apoptosis, is essential to protecting both Hela cells as well as insect cells against apoptosis  and is l−1  for 20 min and resultant cell pellets were resuspended in 50 mL lysis buffer A ). The cells were lysed with sonication . The lysate was transferred into SS34 tubes for further centrifugation at 16,000 rev min−1  for 20 min. The supernatant was loaded onto a 5 mL His Trap HP, 5 mL column  equilibrated with buffer A. After sample application, the column was washed using 100 ml of buffer B  and 25 mM of imidazole). Bound protein was then eluted with buffer C  and 300 mM of imidazole) and dialysed overnight into buffer A at 4 °C. The target protein was then concentrated using a centrifugal concentrator with 30 kDa molecular weight cut-off (Amicon® Ultra 15) to a final volume of 1 mL. Concentrated A179L was subjected to size-exclusion chromatography using a Superdex S200 10/300 column mounted on an ÄKTApure system  equilibrated in 25 mM Tris pH 7.5, 150 mM NaCl with 5 mM DTT , where it eluted as a single peak. Using SDS–PAGE analysis, the final sample was determined to be 95% pure. Eluted protein was concentrated using a centrifugal concentrator with a 30 kDa molecular weight cut-off (Amicon ® Ultra 15) to a final concentration of 21 mg/mL.Synthetic codon-optimized cDNA encoding A179L (Uniprot Accession number P42485) was cloned into the bacterial expression vector pMAL c4x-1-M(RBS) using SacI at the 5’ end and EcoRI at the 3’ (Genscript). Recombinant A179LΔC31, a construct with the 31 C-terminal residues truncated, was expressed in BL21 DE3 Codon Plus RIPL cells using the auto-induction method  for 24 hSynthetic cDNA encoding for a codon-optimized double (V73Y/G89Y) mutant of A179L was cloned into the pGEX-6P-3 vector (Genscript). Expression and purification of wildtype A179L as well as A179L V73Y/G89Y was performed as previously described .Sus scrofa Beclin peptide used was: DGGTMENLSRRLKVTGDLFDIMSGQT . All other peptides employed were described previously [Binding affinities were measured using a MicroCal iTC200 system  at 25 °C as previously described . The BH3eviously .−1 using the sitting-drop method at 20 °C in 0.2 M ammonium sulfate, 0.1 M Bis-Tris pH 5.5 and 25% w/v PEG 3350. The crystals were flash-cooled at −173 °C using 30% (w/v) glucose as cryo-protectant. Native diffraction data were collected at the Australian Synchrotron MX2 beamline using an EIGER 16M detector at a wavelength of 0.9537 Å and an oscillation range of 0.1° per frame. Data integration and scaling was performed using XDS [work/Rfree of 21.4/25.5 with 96.4% of residues in the favoured region of the Ramachandran plot and no outliers. Details of the data-collection and refinement statistics are summarized in A complex of A179L with Beclin BH3 peptide was prepared as previously described . MBP-A17sing XDS  and AIMLsing XDS . The strsing XDS . The strsing XDS . A179L:BRecombinant, replication deficient human adenovirus 5 (rAd) encoding full-length wildtype A179L and A179L V73Y/G89Y tagged with an N-terminal HA tag were generated using standard techniques. Vero cells were transduced with rAd, incubated for 21 h and then incubated for another 3 h in complete media or in Earles balanced salt solution to induce starvation. Cells were then fixed with methanol and stained with anti-HA  and anti-LC3B . Images were captured using a Leicia SP8 confocal microscopy and the number of LC3 puncta per cell determined for 30 cells per condition using Imaris 9.2.1. Statistical analysis was performed in MiniTab (version 18) using analysis of variance (ANOVA) plus Tukey multiple comparison test to determine statistical differences between groups.L [To understand the structural basis for A179L inhibition of autophagy, we determined crystal structures of A179L bound to a peptide spanning the Beclin BH3 motif . PreviouL .The Beclin BH3 peptide binds into a surface groove formed by helices α2-5 of A179L B. A supeTo examine the effect of mutations in the A179L binding groove on autophagy regulation, we generated an A179L V73Y/G89Y double mutant. Circular dichroism spectroscopy indicates that the A179L V73Y/G89Y double mutant maintains a strongly alpha helical fold identical to wildtype A179L . IsotherStructural and functional homologs of Bcl-2 are used by numerous large DNA viruses to subvert programmed cell death-based host defence systems. The majority of these Bcl-2 family members subvert apoptosis ; howeverL [L:Beclin BH3 [The ability to bind the BH3 motif of Beclin has previously been reported for a number of endogenous cellular Bcl-2 proteins, including Bcl-2  and Bcl-L , as wellL  from KSHL ,60 as weL . For thrclin BH3  and M11:L and M11 complexes with Beclin, including the hallmark ionic interaction of BH3 motifs with Bcl-2, which is found between the Beclin BH3 D119 and an Arg from Bcl-2, which is further supported by an additional hydrogen bond from Beclin D119 to a conserved Asn in Bcl-2 (L complexes, whereas in the M11:Beclin complex the recipient acidic residue is a Ser that forms a hydrogen bond with K117 instead. The unique A179L interaction with Beclin is a hydrogen bond between A179L Y46 with the main chain of Beclin L114, with the equivalent residue to Y46 in Bcl-2, Bcl-xL and M11 a Phe. The high level of similarity between the A179L, Bcl-2 and Bcl-xL complexes with Beclin is also reflected in the affinities of these interactions, which are comparable with A179L binding Beclin with a KD of 1.9 μM, with the corresponding KD for Bcl-2 being 1.7–8.0 μM [L being 1.1–2.3 μM [D of 40 nM and a weaker one of 1.1 μM being reported [Interestingly, all major side chain mediated ionic interactions and hydrogen bonds observed in the A179L:Beclin complex are recapitulated in Bcl-2, Bcl-xin Bcl-2 . An addi7–8.0 μM ,57 and f1–2.3 μM ,56. ConfAutophagy is an intracellular bulk degradation pathway that is conserved in eukaryotic cells and in the last decade has been implicated in a range of cellular processes. Autophagy plays an important role in the innate and adaptive immune response to infection and can directly degrade invading pathogens ,63. HoweIntroduction of the large hydrophobic residue Tyr in two locations within the binding groove of A179L ablates the ability of A179L to bind to Beclin and its ability to inhibit the formation of autophagosomes during starvation. A previously reported mutant of A179L, A179L G89A, was shown to no longer bind promiscuously to BH3 motifs from all major pro-apoptotic Bcl-2 proteins and Beclin, and instead only bound Puma BH3 , indicatIn summary, we report the structural basis for A179L-mediated inhibition of autophagy by determining the crystal structure of A179L bound to the Beclin BH3 motif. Furthermore, we show that disruption of Beclin binding via targeted mutations in the A179L ligand-binding grove ablates the ability of A179L to suppress autophagosome formation. These findings provide a mechanistic platform for more detailed investigations into the role of A179L during autophagy inhibition and to delineate the relative contributions that A179L-mediated suppression of apoptosis and autophagy signaling makes during ASFV infection and viral persistence.


Escherichia coli (NMEC) strains of Sequence Type complex (STc) 95 in France.To describe the temporal dynamics, molecular characterization, clinical and ex vivo virulence of emerging O1:K1 neonatal meningitis Dictyostelium discoideum amoeba model.The national reference center collected NMEC strains and performed whole genome sequencing (WGS) of O1:K1 STc95 NMEC strains for phylogenetic and virulence genes content analysis. Data on the clinical and biological features of patients were also collected. Ex vivo virulence was assessed using the S88:H7 and O18:H7 serotypes. However, the model showed differences between several subclones, although they harbor the same known virulence determinants.Among 250 NMEC strains collected between 1998 and 2015, 38 belonged to O1:K1 STc95. This clonal complex was the most frequently collected after 2004, representing up to 25% of NMEC strains in France. Phylogenetic analysis demonstrated that most (74%) belonged to a cluster designated D-1, characterized by the adhesin FimH30. There is no clinical data to suggest that this cluster is more pathogenic than its counterparts, although it is highly predominant and harbors a large repertoire of extraintestinal virulence factors, including a pS88-like plasmid. Ex vivo virulence model showed that this cluster was generally less virulent than STc95 reference strains of O45The emerging clonal group O1:K1 STc95 of NMEC strains is mainly composed of a cluster with many virulence factors but of only moderate virulence. Whether its emergence is due to its ability to colonize the gut thanks to FimH30 or pS88-like plasmid remains to be determined.The online version of this article (10.1186/s12866-018-1376-4) contains supplementary material, which is available to authorized users. Escherichia coli is a commensal of the gastrointestinal tract of vertebrates, including humans [E. coli is the most frequent bacteria involved in preterm meningitis and the second most frequent in newborn meningitis, causing a high rate of mortality or sequelae [g humans . It is ag humans . In partsequelae , 4.E. coli strains responsible for neonatal meningitis belong to Sequence Type complex 95 (STc95) in the Warwick Multi-Locus Sequence Typing (MLST) scheme [S88:K1:H7 [S88:K1:H7, has highlighted the presence of a 134 kb-plasmid, called pS88, encoding three iron capture systems  and other putative extraintestinal virulence genes. The important role of this plasmid in the experimental pathogenicity of S88 has been demonstrated [Sequence types (ST) are grouped into clonal complexes by their similarity to a central allelic profile (genotype). Most 88:K1:H7 –8. ComplE. coli meningitis [E. coli isolates were sent to the national reference center (NRC) and studied. The highly pathogenic phylogenetic subgroup STc95 represented 56% of the 141 strains analyzed and among them, serogroup O1 (27.7%) was the most frequently identified during this period relative to serogroups O18 (19.1%) and O45S88 (11.3%).Between 2001 and 2013, a French prospective national survey collected data for 325 children hospitalized with Dictyostelium discoideum amoeba model as previously used to evaluate E. coli extraintestinal pathogenic strains [Whole genome sequencing (WGS) of a large collection of strains belonging to STc95 was recently performed [ strains .S88, by determining their respective rates between 1998 and 2015 . All these STs differ from ST95 by one allele and are therefore considered to be part of STc95   and O18:K1 (UTI89) reference strains, FimH54 and FimH18, respectively.FimH is an adhesin of type I that is involved in the adhesion of endothelial cells preceding the invasion of cerebral spinal fluid (CSF) . The disblaTEM-1 except one strain harboring blaTEM-84. Alleles of blaTEM-1 were blaTEM-1-A (n = 1), blaTEM-1-B (n = 8) and blaTEM-1-C (n = 7). Allele blaTEM-1-B was associated with genes encoding resistance to streptomycin (strAB) and sulphonamide (sul2 and dfrA5) in all cases but one. Antimicrobial resistance was not associated with a particular WGS subgroup or with pS88-associated genes. Thirteen isolates were devoid of any antimicrobial resistance gene.No extended spectrum beta-lactamase or carbapenemase was found among these meningitis isolates. Seventeen strains (45%) produced a penicillinase, all harboring p = 0.02)  than the subclone containing strains S136, S368, S384, and S386 (mean score 0.65).Grazing scores of the amoeba virulence assay are not shown [see Additional file We tested a representative strain of cluster D-1, S172, cured of its plasmid, and a transconjugant of J53 in our model to determine whether plasmid pS88-like may play a role in the virulence of O1:K1 D-1 strains in our model. The grazing score was clearly affected, with a loss of virulence for S172△pS172, while J53pS172 gained virulence [see Additional file E. coli lineages that causes human extraintestinal infections. The extraintestinal virulence of STc95 E. coli is exemplified by their ability to cause neonatal meningitis. In France, this group represents 56% of the neonatal meningitis strains collected by the national reference center [S88:K1). During the last 18 years, there has been a change in the relative frequency of major serotypes in STc95 NMEC, with a significant increase of O1:K1:H7 strains that were characterized by WGS.The clonal complex STc95 is one of the major n = 34 versus n = 4). These two subgroups were present in all continents studied  with a similar repartition  that contain O1:H7 strains, described by Gordon et al. , strainsectively . Subgroun = 28), D-2 (n = 4), and D-3 (n = 2). The closely related clusters D-2 and D-3 are minor relative to cluster D-1 and carry a different ST (ST421) and a different FimH (92) or no FimH gene (D-3). Cluster D-1, which carries FimH30, may represent a group of strains with a high capacity to induce neonatal meningitis. It also carries genetic determinants characteristic of the extraintestinal virulence plasmid pS88, which are absent from clusters D-2 and D-3, and rarely present in subgroup A. This plasmid may be key to the virulence of this group, as shown for the recently described clone O45S88:K1:H7 [S88:K1:H7, S88, which carries the pS88 plasmid, appears to be closely related to D-2 and D-3 strains. Thus, it is possible that clone O45S88:K1:H7 was derived from D-2/D-3 strains, after switching its O antigen gene cluster and acquiring the pS88 plasmid.Phylogenetic analysis allowed us to distinguish three clusters within subgroup D, called D-1 n = 2, D-2 .Another notable difference between the O1:K1 clusters is the adhesin FimH. It binds specifically to D-mannose residues attached to the surface of glycoproteins that line vaginal, perineal, and bladder cells, as well as enterocytes . FimH is tropism . It also tropism . HoweverE. coli ST131 O25b:H4, found throughout the world, carry FimH30 [E. coli isolates, although the specific role of FimH30 was not assessed [The strains of NMEC O1:K1:H7 described here, carry mostly the fimH30 allele, whereas the other main serotypes responsible for meningitis carry the FimH54 (O45:K1:H7) and FimH18 (O18:K1:H7) alleles. Of note, most strains of the multi-resistant epidemic clonal group of assessed . It is pblaTEM-1 except for one strain), resistance to streptomycin, tetracyclin and sulphonamid and about one third of strains were devoid of any acquired resistance mechanism. We found no association between resistance and a particular WGS-subgroup or with the presence of pS88 virulence factors, suggesting that virulence and resistance genes are harbored by different plasmids or genomic islands such as Integrative and Conjugative Elements (ICE).Antimicrobial resistance was limited to the production of penicillinase , characterized by the presence of the pS88-like plasmid. Clinical data and an ex vivo model failed to show higher pathogenicity of strains belonging to this major cluster D-1. Whether its high prevalence results from a high capacity to colonize the gut, due to adhesion through the FimH30 allele and/or the presence of the pS88-like plasmid, are yet to be determined.This work describes the recent dominance of the meningitis-causing E. coli strains responsible for neonatal meningitis in France were collected by the E. coli national reference center (NRC). All strains were subjected to serogroup and svg PCR [E. coli of STc95 were identified among these isolates and included in the study for WGS.Between January 1, 1998 and December 31, 2015, 250 Clinical and biological data  were collected.E. coli isolates were sequenced. The Nextera XT kit (Illumina) was used to prepare libraries. Sequencing was performed on a MiSeq instrument using reagent kit V3 600 cycles (Illumina technology). Sequences were analyzed using the Center of Genomic Epidemiology (CGE) website (www.genomicepidemiology.org). Genes were annotated using Rast® software (version 2.0), and the sequences aligned and compared using BioEdit® Sequence Alignment Editor (V7.2.5).The whole genomes of the 38 The SPAdes assembler was used to construct assemblies. Contigs < 500 bp were removed. The quality of the de novo assemblies was estimated using standard metrics [see Additional file The CGE website was used to confirm the ST of the strains using the MLST tool . In silihttps://cge.cbs.dtu.dk/services/CSIPhylogeny/). CFT073 strain (O6:K5:H1) was used as outgroup in order to restrict our analysis to core genome SNPs. A phylogenetic tree, based on concatenated alignment of the 19,547 SNPs identified, was constructed by the Neighbor-Joining method available on MEGA software (version 3.1) with 100 bootstrap replicates [SNP (single nucleotide polymorphism) calling was performed with CSI Phylogeny (version 1.4) available on CGE web site  strains O1:K1 was assessed by their resistance to phagocytosis in an amoeba ex vivo model as described by Adiba et al. [E. coli strains [Additional file S88:K1) [E. coli REL606 was used as a positive control for phagocytosis and the pathogenic strain E. coli 536 as a negative control [The virulence of neonatal meningitis a et al. . We asse S88:K1) , 8. E. c control . We also3, 104, 105 cells) for each bacterial population. Each strain was tested in triplicate. Results were interpreted in terms of D. discoideum grazing. Strains not able to resist amoebic phagocytosis were considered to be grazing sensitive (GS) (and thus less virulent) and strains resistant to amoebic phagocytosis, grazing resistant (GR) (and thus more virulent). A grazing score was calculated for each strain, corresponding to the mean of each series of replicates: 0 for GR and 1 for GS.The previously described amoebic phagocytosis protocol  was usedp value < 0.05 was considered statistically significant.Categorical variables were compared using the Fischer exact-test. Continuous variables were expressed as the mean ± SD and compared using the Mann-Whitney test. A Additional file 1:E. coli meningitis strains; description: grazing scores for representative strains. (DOCX 15 kb)ex vivo amoeba model outcomes for several representatives of O1:K1 STc95 Additional file 2:Primers of serotype PCR; description: list of primers and sequences used for serotype PCR. (DOCX 17 kb)Additional file 3:E. coli isolates. (DOCX 22 kb)Sequencing data information; description: N50, N75, Minimum contig Length, Maximum contig Length, Average contig Length, Total contig length, Mean coverage, Number of reads, Number of contigs for all O1:K1 Additional file 4:166 genes searched by local blast analysis using the NCBI blast tool; description: list of genes searched in the strains. (DOCX 15 kb)


Shin et al. identify PIP4K2A as a putative tumor suppressor in glioblastoma using an in vivo RNAi screen system. PIP4K2A competes with PTEN to negatively regulate PI3K signaling via p85/p110 component degradation in vitro and in vivo, and its expression is significantly down-regulated in GBM patients. (GBM) is the most malignant brain tumor with profound genomic alterations. Tumor suppressor genes regulate multiple signaling networks that restrict cellular proliferation and present barriers to malignant transformation. While bona fide tumor suppressors such as PTEN and TP53 often undergo inactivation due to mutations, there are several genes for which genomic deletion is the primary route for tumor progression. To functionally identify putative tumor suppressors in GBM, we employed in vivo RNAi screening using patient-derived xenograft models. Here, we identified PIP4K2A, whose functional role and clinical relevance remain unexplored in GBM. We discovered that PIP4K2A negatively regulates phosphoinositide 3-kinase (PI3K) signaling via p85/p110 component degradation in PTEN-deficient GBMs and specifically targets p85 for proteasome-mediated degradation. Overexpression of PIP4K2A suppressed cellular and clonogenic growth in vitro and impeded tumor growth in vivo. Our results unravel a novel tumor-suppressive role of PIP4K2A for the first time and support the feasibility of combining oncogenomics with in vivo RNAi screen.Glioblastoma   Glioblastoma  is the most lethal primary brain tumor with standard-of-care therapies providing only partial palliation . DespiteIn the majority of cancer patients, including those with GBM, malignancies develop due to abnormalities in the structure and orientation of oncogenes and/or tumor suppressor genes. In part, functional loss of tumor suppressor genes is a prevalent event in tumor initiation and progression. Inactivation of tumor suppressors results from genetic alterations, including genomic mutations, allelic deletions, structure variations, as well as epigenetic silencing due to DNA methylation . Indeed,3), the product of activated phosphatidylinositol 3-kinase (PI3K). PI3K consists of p110 catalytic and p85α regulatory subunits that are often aberrantly activated in response to receptor tyrosine kinases. The PI3K–Akt oncogenic pathway provides proliferative and antiapoptotic signals and is frequently dysregulated in various tumor classes (Phosphatase and tensin homologue (PTEN) is a tumor-suppressor protein that is often deactivated due to genomic deletion and/or mutation across a wide range of human cancers. PTEN-dependent signaling dysregulation is frequently observed in GBM, with mutation occurring in between 5% and 40% of all GBM cases and loss of heterozygosity in 60% to 80% of all cases . PTEN de classes . In respCDKN2A/B, PTEN, and RB1, is frequently observed across a broad range of cancer types, including GBM. As genomic deletion is one of the key elements behind malignant transformation, we sought to determine putative tumor suppressor genes using genomic and transcriptome data of GBM patient specimens (n = 228) that are available from the Rembrandt database. The vast majority of the candidate genes were located on chromosome 10, as loss of heterozygosity on chromosome 10 is a common genetic event during GBM progression. We selected five to seven individual shRNA clones targeting each candidate gene from Cold Spring Harbor shRNA libraries and generated them in a pooled format. By integrating each tumor cell with an individual unique shRNA, we reasoned that a subset of population would outgrow the rest due to selective growth advantage. After a thorough optimization process, we generated orthotopic xenograft models that were injected with shRNA pool-transduced GBM cells  and found that PIP4K2A expression was significantly higher in NPCs compared to the GBMs as well  of 88 GBM tissue specimens and their adjacent non-neoplastic tissues to determine PIP4K2A protein expression levels. Quantification of immunohistochemical analyses using the TMA system revealed significant differences between matched neoplastic and non-neoplastic tissues as PIP4K2A protein level was much higher in the non-neoplastic regions compared with its corresponding tumor sections, consistent with our previous results (As  results . CollectPIP4K2A expression levels. Transduction with lentivirus expressing PIP4K2A wild-type (PIP4K2A) significantly inhibited cellular viability and cellular growth in PIP4K2A-transduced GBM cells compared to the control groups .To explore the functional role of PIP4K2A in GBM, we first investigated its ability to regulate cellular proliferation. Patient-derived GBM cells were used to evaluate PIP4K2A’s potential tumor-suppressive role because they exhibit minimal Cancer stem/initiating cells possess an innate ability to initiate tumor growth in vivo. Although some cancers may not directly follow the cancer stem/initiating cell architecture, multiple studies have shown that GBMs harbor a subset of highly tumorigenic, stem-like cells called glioma stem cells GSCs; . They haClonogenic growth in the form of neurospheres is an in vitro indicator of GSC self-renewal capability . To assePIP4K2A in tumor growth in vivo, we performed in vivo growth competition assays in which control (GFP-labeled) and PIP4K2A-transduced tumor (RFP-labeled) cells were equally mixed at a 1:1 ratio and co-injected into mouse brains  and S6K phosphorylation, a downstream target of AKT . On the 3 in the cell membrane. The interaction between p85 and p110 is integral to the stability of p110  and helical domains (E542K and E545K) of p110α  exhibited minimal p85 and p110 protein expression levels, while PIP4K2Alow tumors  showed significantly higher levels of p85 and p110 . It belongs to the class II PIPK family, also known as the phosphatidylinositol-5-phosphate 4-kinase family, and the major function of these proteins is to phosphorylate at position four of the inositol ring to generate a new lipid messenger, the phosphatidylinositol-4,5-bisphosphate . PtdIns4,5P2 plays a vital role in phosphoinositide signaling, regulating several essential cellular processes, including vesicle transport, cellular proliferation, adhesion, apoptosis, and nuclear events . A full Down-regulation of PIP4K2A attenuates growth of acute leukemia cells but not of primary normal human hematopoietic stem and progenitor cells. Whether tumor suppression is dependent only on the loss of PIP4K activity remains unclear . FurtherN251S . These results suggest that kinase activity of PIP4K2A is not essential in initiating p85/p110 PI3K complex degradation in GBM.There are three mammalian isoforms of PtdIns5P 4-kinases type II: α, β, and γ . PIP4K2AN251S  and PIP4PTEN mutation or deletion, often through the complete loss of its locus on chromosome 10q, is found largely in GBM patients  and p110 family catalytic subunits . The interaction between p85 and p110 is critical for the stability of p110 . Moreovepatients . Further systems . We founlativity . We suspIn the present study, we demonstrate in vivo RNAi screen as a powerful tool to identify and validate putative tumor suppressors in GBM. The PI3K signaling pathway is frequently dysregulated in GBM. Given the profound and unprecedented effects of PIP4K2A on tumor growth inhibition in vitro and in vivo through regulating PI3K signaling via p85/p110 component degradation, we highlight the newly discovered role of PIP4K2A in GBM.shRNAs were obtained from Open Biosystems as pGIPZ lentiviral vectors. The overall shRNA barcode-screening procedure was performed as described previously . For theAfter receiving informed consent, GBM specimens were obtained from patients undergoing surgery at the Samsung Medical Center in accordance with the Samsung Medical Center Institutional Review Board. This work was performed in compliance with all relevant ethical regulations for research using human specimens. Patient-derived primary GBM cells and normal NPCs were cultured as previously described . For sphLentiviral vectors expressing shRNAs for PIP4K2A, PI3K (p85), and Cbl were purchased from Sigma-Aldrich: PIP4K2A , p85 , and Cbl . The following shRNAs were used throughout the experiments: PIP4K2A , p85 , and Cbl . To generate the recombinant lentivirus that expresses PIP4K2A wild-type and PI3KC-E545K mutant, a cloning package including entry (pCR8/GW/TOPO Vector) and lentiviral destination (pLenti6/V5-Dest) vectors were used (Invitrogen) and validated by sequencing and immunoblot analysis. As V5 epitope consists of 15 amino acids, and its molecular mass is measured at ∼5 kD, exogenous PIP4K2A protein shows higher molecular mass compared with the endogenous PIP4K2A. For viral production, 293T cells were cotransfected with a lentiviral expression vector and packaging plasmid (psPAX2 and pCMV-VSVG) using CalPhos Mammalian Transfection Kit (Clontech). Virus-containing supernatants were collected and concentrated by ultracentrifugation. The titer of each lentivirus was determined by serial dilution.5 per mouse) were injected into the brains of mice by stereotactic intracranial injection . Mice were sacrificed either when 25% body weight loss or neurological symptoms  were observed.All mouse experiments were performed according to the guidelines of the Animal Use and Care Committees at the Samsung Medical Center and Association for Assessment and Accreditation of Laboratory Animal Care–accredited guidelines . 6-wk-ol5 GBM cells were mixed with 5 µl HBSS buffer and injected intracranially into the striatum of adult nude mice by using a stereotactic device (Kopf Instruments). Mice with tumor formation were sacrificed for primary culture. Primary tumors were harvested, minced, and incubated in CDD1 for 10 min. Dissociated cells were filtered through 40-µm mesh and then processed for FACS analysis using FACS Calibur flow cytometry (BD Biosciences). The expression levels of GFP and RFP were determined using the FlowJo program.For in vivo growth competition assays, GBM cells were labeled with either GFP or RFP using lentiviral infection. After checking fluorescence signals through FACS analysis, GFP-labeled GBMs were infected with control lentivirus (empty control vector), and RFP-labeled GBMs were infected with PIP4K2A-expressing lentivirus. GBM samples were dissociated into single cells using Accutase, and 103 cells per well, and each sample was plated and incubated in quintuplicate for 6 d. Cell proliferation was detected with the Ez-Cytox according to the manufacturer’s instructions. Optical density values were measured by using a microplate reader at an absorbance of 450 nm. For a direct cell count, the number of viable cells was counted by the trypan blue dye exclusion assay with a hemocytometer.Cell proliferation was measured using the EZ-Cytox cell viability kit (DAEIL Lab) according to the manufacturer’s protocol. GBM cells were transduced with either control lentivirus (empty control vector) or PIP4K2A-expressing lentivirus. PIP4K2A and control cells were plated onto 96-well plates at 10http://bioinf.wehi.edu.au/software/elda/).For LDAs, cells were transduced with either control lentivirus (empty vector) or PIP4K2A-expressing lentivirus. PIP4K2A or control infected cells were plated in 96-well plates at a range of 1–100 cells per well. After 1–2 wk, the number of wells without spheres was counted. At the time of quantification, each well was examined for the formation of tumorspheres. The LDA clonogenic index is calculated as the inverse of the x-intercept of the regression between the number of wells without spheres (y axis) and the number of cells seeded at a range of 1–100 cells per well x axis; . Stem ceFor analysis of PIP4K2A, PI3K (p85), and PI3K (p110) by immunohistochemistry, TMA containing neoplastic and matching non-neoplastic tissues was used . Brain tThe following antibodies were used for immunoblotting: PIP4K2A 1:1,000 , p85 1:1,000 , p110α 1:1,000 , p110β 1:1,000 , pAKT 1:1,000 , AKT 1:1,000 , pS6K 1:1,000 , S6K 1:1,000 , Cbl 1:1,000 , PTEN 1:1,000 , and β-actin 1:5,000 . The following antibodies were used for immunohistochemistry or immunofluorescence: PIP4K2A 1:50 , pAKT 1:200 , p85 1:100 , and p110 1:100 .Cells and tissues were fixed in 4% PFA for 20 min. After blocking and permeabilizing with 0.3% Triton X-100 and 10% goat or donkey serum in PBS for 1 h, samples were probed with the following primary antibodies for overnight at 4°C: PIP4K2A, p85, and pAKT. Appropriate fluorescence-tagged secondary antibodies (Invitrogen) and 4,6-diamidino-2-phenylindole dihydrochloride (Roche) were used for visualization. Microscopy was done with confocal microscope imaging.Protein co-IP in GBM cells was performed essentially as previously reported . GBM celt test was used to determine statistical significance. P values <0.05 were considered significant.All data were expressed as means ± SD or ± SEM from at least three independent experiments. Quantification in immunostaining analyses was performed by using ImageJ software , and results were presented as the percentage of pixels mean area. For the animal survival studies, P values were determined by log-rank test. Student’s PIP4K2A and PTEN expressions and Kaplan–Meier survival analysis of GBM patients based on PIP4K2A expression. Fig. S2 shows the effects of overexpression and shRNA-mediated knockdown of PIP4K2A on cellular growth and PIP4K2A, p85, and pAKT protein expression levels. Fig. S3 shows effects of PIP4K2A or PTEN overexpression with or without MG132 treatment on PIP4K2A, PTEN, p85, pAKT, or ubiquitin protein expression levels or binding activities in GBMs or NPCs. Fig. S4 shows PIP4K2A mRNA expression levels in breast carcinoma versus normal breast tissues (left) and lung adenocarcinoma versus normal lung tissues (right). Fig. S5 shows the effects of PIP4K2A kinase mutants on PIP4K2A and p85 protein expression levels and cellular growth.Fig. S1 shows correlation between


Schizophrenia is a severe mental disorder with an unclear pathophysiology. Increased expression of the immune gene C4 has been linked to a greater risk of developing schizophrenia; however, it is not known whether C4 plays a causative role in this brain disorder. Using confocal imaging and whole-cell electrophysiology, we demonstrate that overexpression of C4 in mouse prefrontal cortex neurons leads to perturbations in dendritic spine development and hypoconnectivity, which mirror neuropathologies found in schizophrenia patients. We find evidence that microglia-mediated synaptic engulfment is enhanced with increased expression of C4. We also show that C4-dependent circuit dysfunction in the frontal cortex leads to decreased social interactions in juvenile and adult mice. These results demonstrate that increased expression of the schizophrenia-associated gene C4 causes aberrant circuit wiring in the developing prefrontal cortex and leads to deficits in juvenile and adult social behavior, suggesting that altered C4 expression contributes directly to schizophrenia pathogenesis. Elevated expression of the gene encoding complement C4 is associated with an enhanced risk of schizophrenia, but the mechanism underlying this link is unclear. This study shows that overexpression of the C4 gene in mice leads to mis-wiring of the prefrontal cortex and deficits in social interactions. Schizophrenia (SCZ) is a neurodevelopmental disorder characterized by disruptions in brain connectivity that lead to an array of symptoms including psychosis and deficits in cognition and social interactions ,2. CurreAmong the many clinical traits of SCZ are deficits in social cognition such as social-cue perception and emotion regulation, which suggest dysfunction of the prefrontal cortex (PFC) . MoreoveC4, which is located in the MHC locus, is associated with SCZ such that specific structural variants and mutations that cause increased expression of C4 confer greater risk for this brain disorder . To. ToC4b kdy areas . This alt test, ****p < 0.0001). Furthermore, we found that 99% of cells expressing GFP had higher amounts of mC4 mRNA relative to the mean transcript levels of nearby untransfected cells  cells. Therefore, we generated an antibody that detected both mC4 and hC4 in HEK cells that were transfected with C4 . Using tWe were unable to detect either endogenous or exogenous C4 in histological sections using our antibody or other commercially available antibodies. Therefore, we created a C4-GFP fusion protein construct (pCAG-C4-GFP) to visualize exogenous C4 protein expression without the need for antibodies. We confirmed C4-GFP expression in HEK cells using western blot . In bothSince we found C4 in PSD fractions, we wondered whether increased expression of C4 in developing neurons led to structural alterations in dendritic spines. We used IUE to produce control (pCAG-GFP) or C4 overexpression conditions . Since early postnatal development is an important period for synaptic maturation and refinement of cortical circuitry , we measp < 0.0001). Whereas spine density in mC4- or hC4-overexpressing neurons was similar to that of control conditions at P7 and P14, apical tuft dendritic spine density was approximately 30% lower at P21  and an increase in the interevent interval (IEI) . Increas10-90 or Decaytau between control and mC4-transfected neurons , suggesting that there could be mC4-dependent changes in the location of inhibitory synapses. Taken together, our data suggest that increased expression of mC4 alters the developmental wiring of cortical neurons by decreasing their excitatory synaptic drive.Further analysis of postsynaptic current kinetics revealed no differences in mEPSC Rise neurons . Althoug neurons , it signm) in control and mC4-overexpressing neurons  with microglia (Iba1) or microglial lysosomes (CD68) . We obseSince increased expression of mC4 led to specific spine loss in L1 apical tufts with no changes in basal dendrites , we testAdditionally, we used expansion microscopy (ExM), a biological sample–preparation procedure that allows for nanoscale imaging with standard confocal microscopes  S9 Fig)S9 Fig), Using ExM, we observed clear examples of PSD-95 colocalized with Iba1 , suggestTo determine whether overexpression of mC4 in the frontal cortex is sufficient to cause deficits in early (P18) social behaviors, we administered a task that allowed us to measure sensorimotor abilities of mice and maternal–pup social interactions . For theTo test sensorimotor abilities , we transferred P18 pups to an arena that contained fresh bedding in two neutral corners and nesting material from the animal’s home cage in the corner opposite to the starting corner, and mice were free to explore the arena for 3 min . SimilarTo test maternal–pup social interactions , we placed P18 pups in an arena that contained nest bedding, an empty wire mesh cup, and a wire mesh cup containing the pup’s dam, and the behavior of each pup was monitored for 5 min . We founN = 22 control mice, N = 20 mC4 mice). We first tested adult mice in a novel-object task to assess whether interest in a novel object was affected by C4 overexpression  in opposing corners, and the behavior of each mouse was monitored for 5 min . We founWe showed here that overexpression of C4 in the mPFC can perturb circuit development. We demonstrated that, during normal early postnatal development, excitatory neurons in the mouse mPFC express low levels of C4. Moreover, overexpression of C4 in these cells led to spine dysgenesis that was evident through a transient decrease in filopodia and medium-sized spine density during the third week of postnatal development. We also showed that C4-dependent spine abnormalities were accompanied by a decrease in excitatory synaptic drive onto pyramidal cells in the mPFC. C4 overexpression caused microglia to engulf more PSD-95 from transfected neuron’s apical dendrites in L1, where spine loss was observed. Lastly, increased expression of C4 in the frontal cortex was sufficient to reduce social interactions in juvenile (P18) and adult (P60–70) mice. Our findings implicate C4 in shaping the developmental trajectory of cortical circuits and provide a causal link between increased C4 levels and PFC dysfunction. These data show for the first time, to our knowledge, that C4-driven circuit dysfunction in the PFC drives cellular and behavioral phenotypes observed in SCZ rather than merely being a consequence of disease and/or pharmacological treatment.Our morphological characterization of L2/3 mPFC neurons revealed that C4-mediated connectivity deficits are both circuit and spine-type specific. That is, overexpression of C4 caused a decrease in spine density of apical but not basal dendrites of L2/3 neurons. These data provide evidence that C4-driven synaptic alterations in the mPFC are cortical layer and/or input specific. Interestingly, previous data have shown that exposure to repeated stress in adolescent rats can reduce connectivity in superficial L1 of the mPFC , suggestWhen we further investigated which spine types were affected by C4 overexpression, we found a specific loss of filopodia and medium-sized spines, whereas the density of mature mushroom spines were intact. This is in line with previous observations that complement-mediated pruning is activity dependent . Large mMajor contributors to synaptogenesis are dendritic filopodia, which are thin, specialized postsynaptic structures that orchestrate synapse formation by dynamically sampling potential presynaptic partners, thus optimizing circuit wiring ,53. It iIn our experiments, we did not observe a significant difference in apical tuft protrusion density between adult (P60) control and C4 conditions. However, in human SCZ postmortem tissue, spine loss of PFC cortical L3 neurons persists into adulthood . NotablyOur electrophysiological data strengthen our morphological findings and provide additional insight into the functional abnormalities caused by C4 overexpression. Previous studies have shown that microglia are able to trogocytose—or “nibble” and phagocytose—small volumes of neurons and synapses . It is pIn addition to being the brain’s resident macrophage, microglia can influence brain development by playing roles in synapse formation, elimination, and maintenance ,41,59. OSocial deficits in individuals with SCZ are an early feature of this disease, and poorer social functioning is associated with worse functional outcome in adults with SCZ ,6. TherePrevious work has shown that mice deficient for various complement proteins, including C3R, C1q, C3, and C4, have a reduced pruning of synaptic terminals in the visual system during development ,24,25. SAlthough polygenetic mechanisms underlie SCZ , we provAll experimental protocols were conducted according to the National Institutes of Health (NIH) guidelines for animal research and were approved by the Boston University Institutional Animal Care and Use Committee .C4b KO mice in a C57BL/6 genetic background  and wild-type C57BL/6 mice of both sexes at age P30 were used . Males and females were used for all experiments, and no differences were found between sexes.All mice were group housed on a 12-hr light and dark cycle with the lights on at 7 AM and off at 7 PM and with food and water ad libitum. Offspring were housed with dams until weaning at P21. For spine density and morphology, microglia engulfment, electrophysiology, and behavior experiments, wild-type CD-1 mice of both sexes from age 7 to 70 d were used . For in situ hybridization, C4b  and hC4A  were subcloned  into the pCAG backbone to produce pCAG-mC4 and pCAG-hC4A, respectively. Endogenous PSD-95 was fluorescently labeled in vivo using PSD95-FingR (EF1a-PSD95.FingR-RFP) ). (TIF)Click here for additional data file.S7 FigN = 19 ROIs . mC4: N = 17 ROIs . One-way ANOVA with Bonferroni’s multiple comparisons. p = 0.998. (B) Microglia lysosomal areas, as measured by area of microglia positive for CD68, were not different between conditions. Area of MG CD68+ (%) = area of microglial CD68+ / total microglia area. Control: N = 26 ROIs (from 5 mice including 345 microglia). mC4: N = 26 ROIs (from 5 mice including 319 microglia). t test. p = 0.19. Mean ± SEM. For underlying data, see https://osf.io/7em3s/?view_only=0e7ffde4ebd344dc83af83b5a605c451. mC4, mouse C4; mPFC, medial prefrontal cortex; ROI, region of interest.(A) Microglia (“MG”) density in superficial layers of the mPFC was not affected by mC4 overexpression. Control: (TIF)Click here for additional data file.S8 Figt test. p < 0.0001. (C) Zoomed images from image in (A) showing boxed regions in L1 (yellow inset) and L4 (red inset). White arrowheads: PSD95-FingR puncta. Blue: DAPI (cell nuclei). Scale bar = 5 μm. For underlying data, see https://osf.io/7em3s/?view_only=0e7ffde4ebd344dc83af83b5a605c451. L, layer; P, postnatal day; PSD, postsynaptic density.(A) Representative confocal image (60X) showing cytoarchitecture  and labeling of endogenous PSD-95 by FingR  in P21 coronal sections. Scale bar = 25 μm. (B) PSD95-FingR labeling pattern was consistent with endogenous location of synaptic PSD-95 in L2/3 pyramidal neurons. Mean normalized fluorescent intensity  as a function of distance from pia (μm). Dark blue line: mean. Light blue shade: SEM. Bar graph: mean normalized fluorescent intensity (y-axis) is greater in L1 (yellow inset) than in L4 (red inset). (TIF)Click here for additional data file.S9 Figp < 0.0001. (F) Graph showing the short diameter of each microglia before (blue) and after (red) expansion and after post-scaling (purple). One-way ANOVA with Tukey’s. ****p < 0.0001. (E-F) The average scaling factor was approximately 2.78 . N = 86 microglia (45 control microglia and 41 mC4 microglia). Post-scaled = post / scaling factor (2.78). Inset image of microglia shows the long (E) and short (F) diameter of soma (white line with arrows). The coefficient of variation between pre-expansion, post-expansion, and post-expansion scaled microglia sizes was 30.24%, 34.35%, and 29.22%, respectively. The variance between the pre-expansion and post-expansion scaled sizes was compared with an F-test, and no difference was found between these populations . Mean ± SEM. For underlying data, see https://osf.io/7em3s/?view_only=0e7ffde4ebd344dc83af83b5a605c451. ExM, expansion microscopy; Iba, ionized calcium binding adaptor molecule 1; PFC, prefrontal cortex.(A) Representative photograph of a coronal PFC brain section before (left) and after (right) expansion. Scale bar = 1 cm. (B) Representative confocal image (40X) of a lysosome (CD68+) before (left) and after (right) expansion. Scale bar = 1 μm. (C) ExM revealed greater detail of lysosome morphology. Mean gray value line intensity scan (for dotted lines shown in B). Blue line: pre-expansion. Yellow line: post-expansion. (D) Representative confocal image (40X) of an Iba+ microglia before (left) and after (right) expansion. Scale bar = 5 μm. (E) Graph showing the long diameter of each microglia before (blue) and after (red) expansion and after post-scaling (purple). One-way ANOVA with Tukey’s. ****(TIF)Click here for additional data file.S10 Fig(A-B) Representative confocal images (40X) of a single expanded microglia from control (A) and mC4 (B) conditions, respectively. The columns in (A) and (B) show different z-planes of a single microglia in each of the columns that contain lysosomes that were either positive (+) or negative (−) for PSD-95. These images show that mC4 condition microglia contained a greater number of lysosomes positive for PSD-95. The first row shows microglia (Iba1) with a silhouette drawn in white. The second row shows lysosomes (CD68) within the microglia shown in the top panel. The bottom two rows are a zoomed region (yellow inset from row 2) showing lysosomes (third row) and PSD-95 (fourth row) with a silhouette of the lysosome drawn in white. White arrowheads show PSD-95 within lysosomes. Scale bar (A) = 1 μm. Scale bar (B) = 1.63 μm. ExM, expansion microscopy; Iba1, ionized calcium binding adaptor molecule 1; mC4, mouse C4; PSD, postsynaptic density.(TIF)Click here for additional data file.S11 FigN = 21 mC4 mice. (B) Total number of GFP+ cells for juvenile mice for control and mC4. N = 36 mice (15 control and 21 mC4 mice). (C) Percentage of GFP+ cells per area in adult mC4 mice. N = 20 mC4 mice. (D) Total number of GFP-positive cells per area for adult mice (P60) for control and mC4. N = 42 mice (22 control and 20 mC4). (E) Representative sections showing rostro-caudal extent of transfections in the frontal cortex. Images in left panels are zoomed areas from the right panels (red square). Black numbers: Bregma coordinates. Left panel scale bar = 0.5 mm. Right panel scale bar = 1 mm. Mean ± SEM. For underlying data, see https://osf.io/7em3s/?view_only=0e7ffde4ebd344dc83af83b5a605c451. ACC, anterior cingulate cortex; AI, anterior insular cortex; AO, anterior olfactory nucleus; Cpu, caudate-putamen; Fr3, frontal cortex area 3; FrA, frontal association cortex; GFP, green fluorescent protein; IL, infralimbic cortex; L, layer; LO, lateral orbitofrontal cortex; M1, primary motor cortex; M2, supplementary motor cortex; mC4, mouse C4; MO, medial orbitofrontal cortex; P, postnatal day; Pir, piriform cortex; PL, prelimbic cortex; S1, primary somatosensory cortex; VO, ventral orbitofrontal cortex.(A) Percentage of GFP+ cells per area in juvenile (P18) mC4 mice. (TIF)Click here for additional data file.S12 Figt test with Welch’s correction. *p < 0.05. (B) Average time per each grooming occurrence was longer for mC4 mice compared to controls in the MI1 task. t test with Welch’s correction. **p < 0.01. (A-B) N = 36 mice (15 control and 21 mC4). Mean ± SEM. For underlying data, see https://osf.io/7em3s/?view_only=0e7ffde4ebd344dc83af83b5a605c451. M2, supplementary motor cortex; mC4, mouse C4; MI1, maternal interaction 1.(A) mC4 mice had a greater number of grooming occurrences during the MI1 task. (TIF)Click here for additional data file.S13 Figt test. p = 0.2141. (B) Control and mC4 mice traveled similar total distances in the OF task. t test. p = 0.1986. (C) Control and mC4 mice spent a similar amount of time exploring the open arms of the EZM task. t test. p = 0.0651. (A-C) N = 42 mice (22 control and 20 mC4 mice). Mean ± SEM. For underlying data, see https://osf.io/7em3s/?view_only=0e7ffde4ebd344dc83af83b5a605c451. EZM, elevated-zero maze; mC4, mouse C4; OF, open field; P, postnatal day.(A) Control and mC4 mice (P60) had a similar average velocity in the OF task. (TIF)Click here for additional data file.


Highly pathogenic avian influenza (HPAI) A(H5N1) viruses have been circulating since 2003 in Indonesia, with major impacts on poultry health, severe economic losses, and 168 fatal laboratory-confirmed human cases. We performed phylogenetic analysis on 39 full-genome H5N1 virus samples collected during outbreaks among poultry in 2015–2016 in West Java and compared them with recently published sequences from Indonesia. Phylogenetic analysis revealed that the hemagglutinin gene of all samples belonged to 2 genetic groups in clade 2.3.2.1c. We also observed these groups for the neuraminidase, nucleoprotein, polymerase, and polymerase basic 1 genes. Matrix, nonstructural protein, and polymerase basic 2 genes of some HPAI were most closely related to clade 2.1.3 instead of clade 2.3.2.1c, and a polymerase basic 2 gene was most closely related to Eurasian low pathogenicity avian influenza. Our results detected a total of 13 reassortment types among HPAI in Indonesia, mostly in backyard chickens in Indramayu. Highly pathogenic avian influenza viruses (HPAI) continue to be a major global problem for both animal and human health. Since the first outbreak of HPAI A(H5N1) in Guangdong, China, in 1996, these viruses have caused outbreaks in various species of birds globally. HPAI H5N1 is endemic in multiple countries and causes a major impact on poultry health and severe economic losses. In addition, >860 laboratory-confirmed human cases of HPAI H5N1 have been reported to the World Health Organization (WHO). In Indonesia, 200 laboratory-confirmed human cases of avian influenza A(H5N1) have been reported, with a case-fatality rate of 84%, which is higher than the current global case-fatality rate of 53% . The specimens were kept chilled and shipped by overnight courier to the 2 collaborating veterinary laboratories, Disease Investigation Center (DIC) Subang and West Java Animal Health Laboratory Cikole.The samples were collected after detection of clinical signs in or increased mortality of birds. The criteria for increased mortality was set at >5% of the population in birds vaccinated against H5N1 and 10% in those unvaccinated for 2 consecutive days. When the criteria were met, oropharyngeal and cloacal samples were collected from 5 sick birds and pooled into viral transport medium containing brain–heart infusion broth and antimicrobial drugs according to European Union instructions (http://www.roche.com), and transported to the Eijkman Institute for Molecular Biology in Jakarta for Sanger sequencing. Two additional HPAI H5N1–positive samples, collected in 2016 and obtained from the Animal Health Laboratory (AHL) Cikole of West Java, were also inactivated and included in this study for Sanger sequencing.We tested the collected samples in veterinary laboratories using a national protocol for influenza A screening developed from a real-time reverse transcription PCR (RT-PCR) targeting the matrix gene. Specimens with a cycle threshold value <30 were inactivated using binding buffer of High Pure Viral RNA kit  with Uni12 primer (http://www.takarabio.com) or Toyobo KOD FX Neo  if the genomes were not successfully amplified using the Takara product.At the Eijkman Institute, we rescreened the specimens and extracted RNA in accordance with the protocol of the manufacturer and synthesized cDNA by Invitrogen Super Script III First-Strand Synthesis SuperMix  for PCR products for which gel separation was necessary, and subsequently sequenced them using a BigDye Terminator v3.1 Cycle Sequencing Kit in an ABI 3130 Genetic Analyzer (both from Thermo Fisher).The primers used were primarily designed by Wageningen Bioveterinary Research. We obtained additional primer sequences from the Australian Animal Health Laboratory and from scientific literature (http://www.dnastar.com) and aligned them by using MUSCLE (https://www.fludb.org) and confirmed results through further phylogenetic analysis . We included in the analysis sequences obtained from viruses detected during other recent outbreaks in Indonesia (2014–2016). These viruses had been collected via passive outbreak surveillance by the Disease Investigation Centres (DIC) in Medan, Sumatra; Wates, Central Java; and Denpasar, Bali, under the Directorate General for Livestock and Animal Health Services and the Indonesian Ministry of Agriculture (DGLAHS-MoA). Viruses were submitted by DIC Wates of DGLAHS-MoA to GenBank, and then downloaded to GISAID    Table 1.t-test to estimate the significance of the comparison between the 2 averages of antigenic distances.We used deduced HA amino acid sequences to calculate estimated antigenic distances of viruses based on 27 aa residues in HA, as described previously  and neuraminidase (NA) nucleotide and deduced amino acid sequence data confirmed that viruses in our samples were HPAI H5N1 with polybasic cleavage motif (Q-R-E-R-R-R-K-R-G-L-F) and (Q-R-E-K-R-R-K-R-G-L-F). Phylogenetic analysis showed that the HA genes of the HPAI H5N1 viruses in our study samples all belong to clade 2.3.2.1c. In-depth analysis revealed that Indonesia 2015–2016 HPAI H5N1 clade 2.3.2.1c has evolved into 2 putative new subgroups, A and B. The APD between the 2 subgroups within clade 2.3.2.1c was >1.5% (3.3% ± 0.4%); the bootstrap value was >60%; and the APDs within the 2 groups within clade 2.3.2.1c were <1.5% (0.9% ± 0.1% for subgroup A and 1.9% ± 0.2% for subgroup B). One sample collected by DIC Medan in 2016 from Sumatra Island was identified as clade 2.1.3.2a  Figure 1We observed the evolution of clade 2.3.2.1c of Indonesia 2015–2016 HPAI H5N1 viruses into putative new subgroups (A and B) for the polymerase basic 1 (PB1), polymerase (PA), nucleoprotein (NP), and neuraminidase (NA) genes, as became apparent from comparing respective phylogenetic trees of these genes . In all cases, the distance difference between subgroup A or B and the 3 seed vaccine strains was significant (p<0.05)  H5 Evolution Working Group  was observed mostly in West Java, whereas another subgroup (B) was seen in diverse regions of Indonesia . AdditioWe identified reassortment events in West Java, mostly in backyard chickens in Indramayu. The high poultry density, the presence of different poultry types, and the frequent contacts between poultry farms and between domestic poultry and wild birds may have led to reassortment in West Java , Korea , Japan (H1N1), and Mongolia (H7N1); viruses that until recently had not been detected in Indonesia  bird markets, and keeping poultry separated from wild birds will help to prevent introduction, adaptation, and reassortment of LPAI viruses to a possibly novel zoonotic HPAI virus as currently observed in China and other countries  viruses circulating in Indonesia, 2015–2016.Additional information about reassortments among avian influenza A(H5N1) viruses circulating in Indonesia, 2015–2016.


Streptococcus agalactiae is a major pathogen and is the leading cause of neonatal infections in industrialized countries. The diversity of strains isolated from two pregnant women was investigated. Here, we present the draft genome sequences of strains W8A2, W8A6, W10E2, and W10F3, obtained in order to ascertain their phylogenetic affiliation. Streptococcus agalactiae is a major pathogen and is the leading cause of neonatal infections in industrialized countries. The diversity of strains isolated from two pregnant women was investigated. Here, we present the draft genome sequences of strains W8A2, W8A6, W10E2, and W10F3, obtained in order to ascertain their phylogenetic affiliation. Streptococcus agalactiae, or group B Streptococcus (GBS), is a major pathogen in humans and is the leading cause of neonatal infections in industrialized countries  using a CRISPR typing approach . For twoGBS isolates grew in Todd-Hewitt (TH) broth (BD Biosciences). The genomic DNA was isolated using phenol-chloroform extraction and precipitated with 1 M sodium chloride and 2 volumes of ice-cold 100% ethanol. Whole-genome sequencing and library construction were performed by GenoScreen . Libraries from purified genomic DNA were prepared for Illumina sequencing with a Nextera XT sample prep kit  according to the supplier’s recommendations. Sequencing was performed using an Illumina HiSeq platform in paired ends of 250 bp. At least 2.8 million reads were obtained for each strain, for a mean coverage of more than 339× for each based on a 2.1-Mb genome size .A quality check of sequencing data was performed using FastQC v0.11.5 , and triAll of the strains were assembled into fewer than 80 contigs  with a total assembly size of 2.1 Mb. About 2,000 coding genes were annotated . The metrics for each sample are shown in http://phaster.ca/, remnant phages were discovered on the 4 genomes, and an intact prophage sequence (79% identity with Streptococcus prophage 315.2 [GenBank accession no. NC_004585.1]) was found on W10F3 isolates (contig 4).Whole-genome sequencing (WGS) analysis confirmed the presence and structure of CRISPR-Cas genes previously described for these isolates . Using Pcyl operon, containing 12 genes, including the cylE gene (cyl operon is controlled mainly by the two-component system CovS/CovR (csrS and csrR genes)  . The cylermB gene  analysis  allowed SSWT00000000 (BioSample no. SAMN11415932), SSWU00000000 (BioSample no. SAMN11415933), SSWV00000000 (BioSample no. SAMN11415934), and SSWW00000000 (BioSample no. SAMN11415935), respectively.The draft genome sequences of strains W8A2, W8A6, W10E2, and W10F3 have been deposited in DDBJ/ENA/GenBank under the accession no.