These cells express also Olig2 and Sox10, two oligodendrocyte lineage-specific markers, but not PDGFRα, a marker of oligodendrocyte progenitors. In agreement with oligodendroglial cells being a source of Shh in the adult mouse brain, we identify Shh transcripts by single molecule fluorescent in situ hybridization in a subset of cells expressing Olig2 and Sox10 mRNAs. These findings also reveal that Shh expression is more extensive than originally reported. The Shh-C9C5-associated signal labels the oligodendroglial cell body and decorates by intense puncta the processes. C9C5+ cells are distributed in a grid-like manner. They constitute small units that could deliver locally Shh to its receptor Patched expressed in GFAP+ and S100β+ astrocytes, and in HuC/D+ neurons as shown in PtcLacZ/+ reporter mice. Postnatally, C9C5 immunoreactivity overlaps the myelination peak that occurs between P10 and P20 and is down regulated during ageing. Thus, our data suggest that C9C5+CC1+ oligodendroglial cells are a source of Shh in the mouse postnatal brain.In the mature rodent brain, Sonic Hedgehog (Shh) signaling regulates stem and progenitor cell maintenance, neuronal and glial circuitry and brain repair. However, the sources and distribution of Shh mediating these effects are still poorly characterized. Here, we report in the adult mouse brain, a broad expression pattern of Shh recognized by the specific monoclonal C9C5 antibody in a subset (11–12%) of CC1 Sonic Hedgehog (Shh) is a secreted molecule implicated in neural patterning during embryogenesis. It acts at short- and long-range through its release as soluble multimers and its transport in extracellular particles or along filopodia . In the Activation of Shh signaling in astrocytes has been proposed to promote neuroprotection , to reduHere, we investigated the distribution pattern of Shh in the mouse brain using the monoclonal C9C5 antibody directed against human SHHN. We further characterized a broad distribution of C9C5 immunoreactive signal in brain tissues, identified a subset of mature oligodendrocytes as a source of Shh and showed that these cells constitute Shh-expressing domains in the brain.ERT2R26R-YFP and Ptc+/LacZ mouse lines have been described [Post-natal and adult C57BL/6J male mice were from Janvier Labs . Glast-Creescribed . Adult mHEK293 (ATCC) cells were cultured, transfected using X-tremeGENE9 (Sigma) with pRK5-mShh for the expression of mouse Sonic Hedgehog (mShh) (provided by Dr P. Beachy) or empty vector and harvested 48h later as described .Adult brain tissues from four mice were dissected, homogenized in RIPA Buffer (Thermo) and solubilized (1h30). Twenty μg of the cleared tissues supernatants and 1 μg of mock or mShh transfected HEK293 homogenates were subjected to SDS-PAGE and Western blotting . Proteinin situ hybridization was performed on frozen brain sections of three adult mice using the RNAscope® Multiplex Fluorescent Kit-v2 according to the manufacturer’s protocols . Specific probes were used to detect Shh (#314361), Olig2 (#447091-C2), Sox10 (#435931-C3) and GAD (#400951-C3) mouse mRNAs.Mice were deeply anesthetized with intraperitoneal injection of Xylazine Rompun®, 10 mg/kg) and Ketamine  and were perfused with 4% paraformaldehyde (PFA) in 0.2 M sodium phosphate, pH 7.4. After dissection, brains were post-fixed for 2 h in 4% PFA and cryoprotected in 10% sucrose in PBS until tissue sank (6–12 hrs) and then 30% sucrose in PBS until tissue sank. The tissues were embedded in Optimal Cutting Temperature (O.C.T.) compound Tissue-Tek (Sakura Finetek), frozen in liquid nitrogen cooled isopentane and kept at -80°C until use. Tissues were allowed to equilibrate to –20°C in a cryostat  for ~30 min and 14 μm coronal sections were prepared and mounted onto SuperFrost® ultra Plus slides (Thermo Scientific). For immunostaining, sections were incubated 1h in PBS, 0.25% Triton, 1% BSA and 1h with donkey anti-mouse Fab fragment . The primary antibodies were incubated overnight at 4°C: rabbit anti-SHHN , mouse anti-GFAP , goat anti-Olig2 , mouse anti-S100β , mouse anti-adenomatous polyposis coli (APC) , chicken anti-βgalactosidase , goat anti-Sox10 , rat anti-PDGFRα , sheep anti-Carbonic Anhydrase II (CAII) , rabbit anti-Iba1 , mouse anti-HuC/D . Sections were incubated with the appropriate secondary antibody  for 2h at room temperature. Images were acquired with a 40X objective (N.A. 0.75) using fluorescence (Leica DM2000) or confocal (Leica TCS-SP8) microscope. For the later, Z-stacks (9.6 μm), containing twelve Z-sections images were analyzed using LASAF (Bitplane AG) and reconstructed in ImageJ 1.39t  and Photoshop-CS3 (Adobe). C9C5 immunostaining was performed from the brain of twenty adult mice samples used in this study with similar results. The specificity of C9C5 antibody was assessed on adult mouse brain sections in three independent blocking experiments: C9C5 antibody was pre-incubated overnight at 4°C with 400 μg/ml of recombinant mouse ShhN fragment (residues 25–198) fused to glutathione-S-transferase (GST-mShhN) or of GST alone as control [ mg/kg an+ cells in the hypothalamic parenchyma was performed on sections obtained at the level of the median eminence from three animals at P4, P10 and 9 months stainings, or four animals at P20, 2 and 12 months, and was expressed as a number of cells per mm2. The number of C9C5+ counted cells per animal ranged between 0 and 4 at P4, 45 and 57 at P10, 41 and 70 at P20, 39 and 61 at 2 months, 4 and 8 at 9 months, and 0 and 3 at 12 months. Quantification of C9C5+CC1+Olig2+, C9C5+CC1+Sox10+, C9C5+CAII+ and C9C5+PDGFRα+ co-staining in the cerebral cortex and hypothalamic parenchyma was performed on sections obtained at the level of the median eminence from three adult mice except for Sox10 staining (n = 4). The number of counted cells per region and per animal ranged between 200 and 350 for CC1+ cells, 300 and 700 for Olig2+ cells, 300 and 600 for Sox10+ cells, 100 and 250 for CAII+ cells, 50 and 200 for PDGFRα+ cells. Quantification of βgalactosidase+S100β+ co-staining was performed in the hypothalamic parenchyma at the level of the median eminence from four adult mice. The number of S100β+ counted cells per animal ranged between 250 to 325 cells. Quantification of Shh+Olig2+ and Shh+Sox10+ co-stainings in the cerebral cortex and in the hypothalamic parenchyma was done on sections obtained at the level of the median eminence from three adult mice. The number of counted cells per region and per animal ranged between 120 to 300 Olig2+ cells and from 100 to 550 Sox10+ cells. Countings of co-localised stainings were done using ROI and multi-point ImageJ tools. Quantitative data are expressed as the mean ± standard error of the mean (SEM). Comparisons between two experimental groups were made using unpaired Student’s t-tests. A value of p < 0.05 was considered statistically significant. Graphs were drawn using GraphPad Prism 5.2 .Quantification of C9C5We sought to identify in the adult mouse brain, the protein expression and cell distribution of Sonic Hedgehog (Shh) using the monoclonal ‘C9C5’ antibody directed against the amino-terminal fragment of human SHH. From Western blot experiment analysis , S1 Fig,Immunofluorescent labeling with C9C5 revealed high immunoreactivity throughout most brain regions including the cerebral cortex, the hippocampus, in fiber tracts, in thalamic nuclei, in the tuberal region of the hypothalamus including the median eminence (ME)  and in t+ cells were also immunoreactive for the three oligodendroglial markers Olig2, Sox10 and CC1 , a marker of oligodendrocyte progenitor cells (OPCs) [+ cells in these various oligodendroglial cell populations in the hypothalamus and in the cerebral cortex are presented in + cells represented 11–12% of CC1+ cells and 3–6% of Sox10+ and Olig2+ cells. These data suggest that C9C5 labels a subpopulation of mature oligodendrocytes as shown in hypothalamic nuclei, the ME, the hippocampal and cerebral cortical layers and in the white matter of the corpus callosum   and for s (OPCs)  + GABAergic neurons  was strongly expressed in GFAP+  . In the tuberal region of the hypothalamus of Ptcin GFAP+  or S100βanimals)  astrocytanimals) . Using cstaining . These o+ cells in the brain during the postnatal period and in aged animals , Olig2+ and Sox10+ (3–6%), CAII- mature oligodendrocytes as quantified in the cerebral cortex and in the hypothalamus. These cells do not express the PDGFRα signal implicated in OPCs proliferation and in the inhibition of the expression of oligodendroglial maturation markers such as CC1 [Here, we report the widespread distribution pattern of the C9C5 immunoreactivity in the adult mouse brain and show that C9C5 antibody marks a subset of CC1h as CC1 , 20. Thuh as CC1 , 23, andh as CC1 , 25, andh as CC1 .+ and Sox10+ cells in the adult mouse brain using RNAscope which indicates that Shh is more widely expressed than originally reported [Interestingly, we also identify for the first time Shh mRNAs in Olig2reported , and sugreported , 28 reflreported .+ and Olig2+ oligodendrocyte progenitors occurs during remyelination [Studies using other Shh antibodies , 14, 29 +CC1+ cells are distributed in a grid-like manner with cells residing mostly in non-superposed domains. They constitute small Shh-expressing units that could distribute locally the protein in almost all brain regions and could represent a source for spreading Shh. Thus, our data suggest that besides neurons in the adult forebrain and cerebral cortex [+ and CC1+ cells in the postnatal mouse brain overlaps the myelination peak that occurs between P10 and P20 [Interestingly, the Shh signal labels the oligodendroglial cell body and decorates by intense puncta the processes extending at several cell diameters. Thus, Shh could be secreted to act locally on its receptor Ptc expressed by neighboring astrocytes and neurons. It is noteworthy that C9C5l cortex  and in tl cortex , oligode and P20  further +CC1+ cells belong and to characterize their properties in CNS inflammation and during remyelination [+Shh-associated signal should undoubtedly be taken into consideration when analyzing brain disorders associated to Shh signaling including brain tumors, neurodevelopmental disorders and neurodegenerative diseases [Recently, an unsuspected oligodendrocyte heterogeneity has been reported by RNA sequencing of oligodendrocyte lineage cells both in the juvenile and adult mouse brain , 33. Furlination . Thus, tdiseases .S1 FigTwenty μg of RIPA solubilized samples from cerebellum (Cb) and hypothalamus (Hpt) and 1μg of mock or mShh transfected HEK293 homogenates were subjected to Western blot and immunodetection with C9C5 antibody (1/1000). Chemiluminescence was acquired with Chemidoc apparatus. The exposure time is indicated below. The 22 kDa  and 47 kDa  bands are indicated. A single band corresponding to ShhN is detected in brain extracts after short and long time exposure, further supporting specificity of the C9C5 antibody.(TIF)Click here for additional data file.S2 Fig(A-B) Immunostaining with C9C5 and CC1 antibodies on a coronal section of the cerebellum. (B) Magnification of red box indicates that Purkinje cell bodies (white arrowheads), and their projections in the molecular layer are not labeled by the C9C5 antibody. Magnification of white box highlights a C9C5 positive cell expressing the oligodendroglial marker CC1 (white arrow) within the granular cell layer. Staining was replicated on three mice Mol, molecular cell layer; Gr, granular cell layer; Px, Purkinje cells. Scale bar: A, 100 μm. B, 50 μm.(TIF)Click here for additional data file.S3 Fig+ (white arrowhead), CAII+ (orange arrow), PDGFRα+ (white arrow) cells are presented in merge and single channels with the nuclear marker DAPI. Note that C9C5 positive cells are CAII and PDGFRα negative. Staining was replicated on three mice. Hpt, hypothalamus; DG, dentate gyrus of the hippocampus.(A-B) Immunostaining of coronal brain sections from an adult mouse of the hypothalamus at the level of the median eminence and of the dentate gyrus of the hippocampus with the C9C5 antibody (yellow) and the oligodendroglial markers (A) CC1 (green) and Sox10 (red) or (B) CAII (green) and PDGFRα (red). White boxes  highlight magnifications of the hypothalamic parenchyma and the granule cell layer of the dentate gyrus. (A) C9C5/CC1/Sox10 triple positive cells (white arrowhead) with stellate morphology are presented in merge and single channels with the nuclear marker DAPI. (B) C9C5(TIF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.


Avian influenza H7N9 viruses have been causing disease outbreaks in poultry and humans. We previously determined that propagation of H7N9 virus in virus-specific antiserum gives rise to mutant viruses carrying mutations A125T+A151T+L217Q in their hemagglutinin protein, enabling the virus to overcome vaccine-induced immunity. As predicted, these immune escape mutations were also observed in the field viruses that likely emerged in the immunized or naturally exposed birds. This study demonstrates that the immune escape mutants also (i) gained greater replication ability in cultured cells and in chicken embryos as well as (ii) increased acid and thermal stability but (iii) lost preferences for binding to human-type receptor while maintaining binding for the avian-like receptor. Therefore, they potentially pose reduced pandemic risk. However, the emergent virus variants containing the indicated mutations remain a significant risk to poultry due to antigenic drift and improved fitness for poultry. in vitro and in ovo. They had a lower pH of fusion and increased thermal stability. Of note, the serum escape mutant completely lost the ability to bind to human-like receptor analogues. Further analysis revealed that N-linked glycosylation, as a result of A125T or A151T substitutions in HA, resulted in reduced receptor-binding avidity toward both human and avian-like receptor analogues, and the A125T+A151T mutations completely abolished human-like receptor binding. The L217Q mutation enhanced the H7N9 acid and thermal stability while the A151T mutation dramatically decreased H7N9 HA thermal stability. To conclude, H7N9 AIVs that contain A125T+A151T+L217Q mutations in the HA protein may pose a reduced pandemic risk but remain a heightened threat for poultry.H7N9 avian influenza viruses (AIVs) continue to evolve and remain a huge threat to human health and the poultry industry. Previously, serially passaging the H7N9 A/Anhui/1/2013 virus in the presence of homologous ferret antiserum resulted in immune escape viruses containing amino acid substitutions alanine to threonine at residues 125 (A125T) and 151 (A151T) and leucine to glutamine at residue 217 (L217Q) in the hemagglutinin (HA) protein. These HA mutations have also been found in field isolates in 2019. To investigate the potential threat of serum escape mutant viruses to humans and poultry, the impact of these HA substitutions, either individually or in combination, on receptor binding, pH of fusion, thermal stability, and virus replication were investigated. Our results showed the serum escape mutant formed large plaques in Madin-Darby canine kidney (MDCK) cells and grew robustly IMPORTANCE Avian influenza H7N9 viruses have been causing disease outbreaks in poultry and humans. We previously determined that propagation of H7N9 virus in virus-specific antiserum gives rise to mutant viruses carrying mutations A125T+A151T+L217Q in their hemagglutinin protein, enabling the virus to overcome vaccine-induced immunity. As predicted, these immune escape mutations were also observed in the field viruses that likely emerged in the immunized or naturally exposed birds. This study demonstrates that the immune escape mutants also (i) gained greater replication ability in cultured cells and in chicken embryos as well as (ii) increased acid and thermal stability but (iii) lost preferences for binding to human-type receptor while maintaining binding for the avian-like receptor. Therefore, they potentially pose reduced pandemic risk. However, the emergent virus variants containing the indicated mutations remain a significant risk to poultry due to antigenic drift and improved fitness for poultry. A novel H7N9 low-pathogenicity avian influenza (LPAI) virus strain emerged in birds in China. This virus showed the ability to cause severe to fatal disease in humans through zoonotic transmission from infected birds, and the first human infection with H7N9 was reported in China in February 2013. Since then, there have been 1,568 confirmed human infections with an ∼40% case fatality rate  (referred to as Anhui/13)-like virus. Since its use, the number of poultry outbreaks along with human infections has dropped dramatically, with only three human infection cases reported in epidemic wave six during 2016 to 2017 and one human infection case reported in epidemic wave seven during 2017 to 2018 . The con in 2019 . It is,  in 2019 , 12. The in 2019 –16. Howein 2019 –.In this study, we systematically investigated the impacts of the emergent immune escape variants carrying substitutions A125T, A151T, and L217Q individually or in combination on the virus infectivity, receptor binding, pH of fusion, and HA thermal stability. The results define the potential risks from the contemporary field H7N9 AIV with those HA mutations to human and animal health.We previously showed that serially passaging the LPAI H7N9 avian influenza virus A/Anhui/1/2013 in the presence of homologous ferret antiserum selected the immune escape variants containing A125T+A151T+L217Q in the HA protein . The sub8 PFU/ml), while the Anhui/13 wild-type virus and its L217Q mutant replicated to the lowest titers (∼106 PFU/ml). To conclude, the serum escape mutants demonstrated enhanced replication both in vitro and in ovo, and the glycosylation at amino acid 123 or 149 contributed to virus replication fitness.To evaluate the effects of immune escape mutations in the HA on virus infectivity and replication fitness, we generated a series of reverse genetics-based mutant viruses carrying the indicated mutations A125T, A151T, and L217Q individually or in all possible combinations in the HA  by site-directed mutagenesis. Like the Anhui/13 wild-type virus, the L217Q mutant formed relatively small plaques. However, all of the other mutant viruses formed significantly larger plaques than the wild-type viruses  and B. TInfluenza viruses adapted to infect humans preferentially bind to human-like receptors , whereas avian influenza viruses adapted to infect birds preferentially bind to avian-like receptors  . The rec14–To evaluate the functional significance of the receptor-binding data by biolayer interferometry, we measured the elution of viruses from chicken red blood cells (RBCs) that possess both α-2,3- and α-2,6-linked sialic acid receptors . ConsistThe pH of fusion has been shown to play an important role in host adaptation and transmission. Consistent with the previous report, wild-type Anhui/13 triggered fusion at pH 5.6 or lower . The serIn addition to receptor binding and pH of fusion, HA stability also plays a vital role in AIV evolution . It has 28–Polymorphism analysis was performed on HA sequences retrieved from the GISAID  database from 1 January 2013 to 1 September 2019 in China. All mutations, apart from A125T+A151T and A125T+L217Q, were found in naturally occurring H7N9 isolates . The A15in vitro and in ovo, virus receptor-binding profile, pH fusion threshold, and HA thermal stability. The serum escape mutants containing A125T+A151T+L217Q substitutions replicated to significantly higher titers than the wild-type Anhui/13 virus both in vitro and in ovo at various time points. They completely lost human-like receptor binding while maintaining the avian-like receptor binding, fused at lower pH, and had higher HA thermal stability. Further analysis showed that A125T+A151T double mutations that resulted in N-linked glycosylation at amino acids 123 and 149 were responsible for the loss of human-like receptor binding. The L217Q substitution was responsible for the increased acid and thermal stability.In our previous study, serially passaging the H7N9 viruses (A/Anhui/1/2013) in the presence of homologous ferret antiserum resulted in the emergence of escape viruses containing mutations A125T+A151T+L217Q in the HA  at the Pirbright Institute. All of the influenza virus-related work was risk assessed and carried out adopting biosafety level 2 containment code of practice approved by The Pirbright Institute Biological Agents and Genetic Modification Safety Committee and the UK Health and Safety Executive.The recombinant H7N9 viruses containing HA and NA from LPAI H7N9 Anhui/13 virus or H7N9 HK125/17 virus and the internal gene segments from A/Puerto Rico/8/34 (H1N1) (PR8) virus were generated as previously described (2 atmosphere.The Madin-Darby canine kidney (MDCK), MDCK-SIAT1 (SIAT), human embryonic kidney 293T (HEK 293T), and Vero cells (ATCC) were maintained with Dulbecco’s modified Eagle’s medium (DMEM) (Gibco), supplemented with 10% fetal calf serum (FCS) (Gibco), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) at 37°C under a 5% COl-glutamine, 0.3% bovine serum albumin (BSA), 15 mM HEPES, 0.22% sodium bicarbonate, and 0.01% DEAE-dextran . The cells were further incubated at 37°C for 72 h before being fixed with 1% crystal violet (Sigma-Aldrich) in 20% methanol. The plaque images were taken by Panasonic camera FZ38, and 30 plaques were randomly selected for each virus to measure the plaque diameter.MDCK cells were infected with H7N9 AIV at a multiplicity of infection (MOI) of 0.001 and incubated at 37°C for 1 h. The cells were then washed once with phosphate-buffered saline (PBS) and replenished with DMEM containing 2 μg/ml tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin. The supernatants were taken at 24, 48, and 72 h postinfection and stored in −80°C before titration by plaque assay in MDCK cells. The plaque assay was carried out as previously described , and theTen-day-old embryonated chicken eggs were infected with 100 PFU of wild-type Anhui/13 virus or its variants. The eggs were incubated at 35°C for 48 h before allantoic fluid was harvested and titrated by hemagglutination (HA) assay.The HA titration assays were performed using 1% chicken red blood cells (RBC) as described in the WHO animal influenza training manual . BrieflyRecombinant H7N9 virus containing 32 HA units in a 50-μl volume was serially 2-fold diluted with PBS in 96-well V-bottom microtiter plates (Greiner). Each virus dilution was mixed with 50 μl of 1% chicken RBC. The plates were initially kept at 4°C for 1 h and then incubated at 37°C. The HA titer was recorded over 5.5 h.The recombinant H7N9 avian influenza viruses were diluted with allantoic fluid harvested from noninfected embryonated chicken eggs to 32 HA units/50 μl. The viruses were then left at 4°C as control or heated at 50°C, 50.7°C, 51.9°C, 53.8°C, 56.1°C, 58.0°C, 59.2°C, and 60°C for 30 min before HA assay.The pH of fusion for H7N9 AIV was determined by syncytium formation assays as previously described with slight modification . To deteViruses were purified as previously described . BrieflyTen microliters of the sucrose-gradient-purified viruses  was treP values of <0.05 were considered significant.Statistical analysis was performed using GraphPad Prism 8 (GraphPad Software). One-way analysis of variance (ANOVA) and two-way ANOVA were used to test differences between different groups. The two-way ANOVA (Tukey's multiple-comparison test) results for


C. elegans to humans.During mitosis, translation of most mRNAs is strongly repressed; none of the several explanatory hypotheses suggested can fully explain the molecular basis of this phenomenon. Here we report that cyclin-dependent CDK11/p58—a serine/threonine kinase abundantly expressed during M phase—represses overall translation by phosphorylating a subunit (eIF3F) of the translation factor eIF3 complex that is essential for translation initiation of most mRNAs. Ectopic expression of CDK11/p58 strongly repressed cap-dependent translation, and knockdown of CDK11/p58 nullified the translational repression during M phase. We identified the phosphorylation sites in eIF3F responsible for M phase-specific translational repression by CDK11/p58. Alanine substitutions of CDK11/p58 target sites in eIF3F nullified its effects on cell cycle-dependent translational regulation. The mechanism of translational regulation by the M phase-specific kinase, CDK11/p58, has deep evolutionary roots considering the conservation of CDK11 and its target sites on eIF3F from The online version of this article (10.1007/s00018-019-03436-3) contains supplementary material, which is available to authorized users. Translation is the last step of gene expression, and regulation of gene expression at the translational level plays important roles in various biological processes owing to its rapid and reversible nature and the necessity of controlling gene expression spatially. Translation is a complex process that requires many protein and RNA participants , 2. TraniMet) onto the 40S ribosomal subunit  and eIF3F mutants Fig. a were trTo investigate which amino acid residues are phosphorylated by CDK11/p58, we co-transfected the plasmids expressing eIF3F variants with those expressing CDK isoforms and then monitored cap- and IRES-dependent translation using the CrPV bicistronic reporter mRNA Fig. . As showAs a control, we performed co-expression experiments with CDK11/p46. As previously reported , co-exprWe investigated whether the CDK11/p58-dependent phosphorylation of eIF3F affects eIF3 complex formation using a co-immunoprecipitation method. We performed co-immunoprecipitation experiments after ectopic expressions of two components of the eIF3 complex . Alternatively, we performed co-immunoprecipitation experiments after ectopic expression of one of the eIF3F variants to monitor eIF3 complex formation with the endogenous components of eIF3 . The results indicate that both phosphorylated [reflected by phospho-mimetic mutants eIF3F(D3D4)] and unphosphorylated eIF3F [reflected by unphosphorylatable mutants eIF3F(A3A4)] are incorporated into the eIF3 complex to the same extent. It is noteworthy that eIF3B, which does not directly interact with eIF3F in the eIF3 complex , was alsTo further investigate the presence of phosphorylated eIF3F in the eIF3 complex, we performed sucrose gradient analyses with HeLa cells synchronized at the interphase or mitotic phase . As expected, eIF3B and eIF3F were mainly enriched in two fractions where eIF3 complex (fraction A) and the free forms of eIF3 components (fraction D) migrated during centrifugation . We analyzed the phosphorylated form of eIF3F in fractions A–D using Phos-tag SDS-PAGE. To our surprise, the phosphorylated eIF3F was not enriched in either fraction A or D. Instead, the phosphorylated eIF3F was enriched in fraction B having intermediate sedimentation velocity between the eIF3 complex and free eIF3F . This may indicate that the phosphorylated eIF3F composes an immature eIF3 complex which is lighter than the complete eIF3 complex (see below).We further investigated the effects of eIF3F phosphorylation by CDK11/p58 on M phase translation using newly established HeLa cell lines ectopically expressing either wild-type eIF3F [eIF3F(WT)] or unphosphorylatable eIF3F mutants [eIF3F(A1A2) and eIF3F(A3A4)] Figs. , 8. We sA striking difference in global translation was observed in M phase-arrested cells expressing eIF3F(A3A4), which cannot be phosphorylated by CDK11/p58 , nocodazole induces stress-related translational inhibition that obscures the magnitude of translation repression in M phase . TherefoWe monitored the global translation levels of control, eIF3F(WT)-expressing, and eIF3F(A3A4)-expressing cells at various time points after cell cycle resumption by western blotting using the SUnSET method Fig. a. The amConventional studies on cell cycle-dependent translational regulation , 14 and Even though several functions of CDK11/p58 during M phase have been suggested, none of them revealed that CDK11/p58 functions in translational regulation. Thus, we uncovered a novel function of CDK11/p58 in modulating general translation during M phase. A previous study showed that CDK11/p46 produced in apoptotic cells represses translation by phosphorylating eIF3F . HoweverNeurospora crassa eIF3 complex [S. cerevisiae), which suggests that eIF3F is not essential for translational activation by eIF3. Moreover, overproduction of eIF3F inhibits cap-dependent translation [In this study, we demonstrated that the mitotic phosphorylation of eIF3F by CDK11/p58 leads to translational repression Figs. , 8. We a complex . Therefonslation  Blocking eIF3F phosphorylation by knockdown of CDK11/p58 Fig. c or by o35S-methionine labeling of newly synthesized proteins using a cell cycle arrest-and-resumption approach with RO-3306.Recently, Tanenbaum and his colleagues showed that M phase-specific translational inhibition occurs using a different cell cycle blocker, RO-3306 (CDK1 inhibitor), and genome-wide ribosome profiling analysis . The autIn this study, we also performed a cell cycle arrest-and-resumption experiment using double thymidine block and puromycine labeling Fig. . We usedEven though mitotic translational repression is a well-known feature of mammalian cells, the physiological roles of translational modulation during cell cycle progression are not clearly understood. We speculate possible roles of translational repression during M phase. (1) Translation repression of some genes during mitosis is needed for cell cycle progression from M to next G1 phase. For example, the specific translational inhibition of Emi1 gene, an inhibitor of anaphase-promoting complex (APC) formation, is required for the activation of APC during M phase, which in turn is needed for cell cycle progression . (2) TraAn interesting relationship between cap-dependent and IRES-dependent translation during M phase was revealed via investigations into the role of a tumor suppressor gene, 14-3-3σ, in M phase-specific translational regulation . The autS. cerevisiae, but both CDK11 and eIF3F homologues exist from Schizosaccharomyces pombe (S. pombe) to humans. Furthermore, the region corresponding to CDK11/p46 exists in all CDK11 homologues from S. pombe to humans, and one of the CDK11/p46 phosphorylation sites in eIF3F (Thr119) exists in all eIF3F homologues . In contrast, neither CDK11/p58 nor its target phosphorylation site in eIF3F exists in S. pombe . This indicates that M phase-specific translational repression by an isoform of CDK11 may not occur in S. pombe. Both cyclin D family members and the region in CDK11 corresponding to CDK11/p58 are conserved from Caenorhabditis elegans (C. elegans) to humans, and one of the CDK11/p58 target sites in eIF3F (Ser258) is also conserved from C. elegans to humans . This may indicate that cell cycle-dependent translational regulation by a CDK11 isoform (CDK11/p58) functions from C. elegans to humans. Other cell cycle-dependent translational regulation mechanisms that have been suggested to function during M phase are not as well conserved evolutionarily as the CDK11 and eIF3F pair. For example, the CDK1 phosphorylation site in eIF4G1, which was suggested to function in M phase-specific translational repression [Translational regulation by CDK11 and eIF3F pair seems to have deep roots in evolutionary history. Neither CDK11 nor eIF3F homologue exists in pression , is conspression , exists Supplementary file1 (PDF 10232 kb)Supplementary file2 (PDF 102 kb)Supplementary file3 (XLSX 13 kb)Supplementary file4 (XLSX 12 kb)Supplementary file5 (XLSX 16 kb)Below is the link to the electronic supplementary material.


Influenza A(H1N1)pdm09 viruses have undergone rapid evolution, and in recent years the complementary and antagonistic effects of HA and NA have gathered more attentions; however, the effects of co-occurring mutations in HA and NA on the patients’ clinical characteristics are still poorly understood. In this study, we analyzed molecular epidemiology and evolution of A(H1N1) pdm09, explored co-occurring mutations of HA and NA, and investigated effect of co-occurring mutations on patients’ clinical features.A(H1N1)pdm09 was confirmed by reverse transcription-polymerase chain reaction. HA and NA genes were sequenced and phylogenetically analyzed. Clinical characteristics of the co-occurring mutations were analyzed statistically.2, P < 0.05).By analyzing the HA and NA gene sequences of 33 A(H1N1)pdm09 viruses during the 2015–2017 influenza season, we found that all the viruses shared high similarities to each other and the HA genes of these viruses exclusively belonged to subclade 6B.1A. Several unreported substitutions in HA and NA proteins were observed, furthermore, co-occurring mutations of HA-V169T, A278S, E508G, D518E and NA-V67I were detected in 30.3% (10/33) A(H1N1)pdm09 virus strains when comparing with vaccine strains A/California/07/2009 and A/Michigan/45/2015 (H1N1). Sore throat was significantly associated with co-occurring mutations in HA and NA of A(H1N1)pdm09 (χCo-occurring mutations in HA and NA were detected in A(H1N1)pdm09 isolated during 2015–2017 in Beijing. Symptomatically, sore throat was associated with co-occurring mutations in HA and NA of A(H1N1)pdm09. Therefore, studying the effect and mechanism of co-occurring mutations in HA and NA on patients’ clinical features is of note needed. No statistically significant difference was detected for other clinical characteristics. This result indicated that sore throat was associated with co-occurring mutations in HA and NA of A(H1N1)pdm09.Based on above substitution analysis of HA and NA genes, co-occurring mutations of HA-V169T, A278S, E508G, D518E and NA-V67I were detected in 30.3% (10/33) A(H1N1)pdm09 virus strains when comparing with vaccine strains A/California/07/2009 and A/Michigan/45/2015 (H1N1). To investigate the associations with clinical characteristics and the co-occurring mutations in HA and NA, we collected medical data from these ten patients in which the above co-occurring mutations were detected, including demographic characteristics, symptoms, laboratory test results, and whether complicated by pneumonia pdm09 strain has been one of the seasonal influenza strains and circulated seasonally in humans. Genetic evolution of seasonal influenza viruses is gradual. The close monitoring on the viruses are of note for the vaccine strain recommendations. Since 2009, persistent monitoring of the influenza viruses has been conducted in PKUPH in Beijing, China. We have previously studied the evolution of surface antigen site mutations using the viruses circulated in 2012–2014 , 20. DurN1pdm09 sComparing to the HA protein of A/California/07/2009 vaccine strain, A(H1N1)pdm09 viruses circulated in Beijing during 2012–2014 contained several substitutions in the key epitope  , 20, incCorrespondingly, substitutions in NA antigen epitope have been reported to occur in the A(H1N1)pdm09 viruses during 2012–2014, including NA- K84N, P93S, V106I L139V, I163T, V264I, E287K and S340F. while in this study, NA-V264I mutation occurred in all the A(H1N1)pdm09 viruses detected during 2015–2017. No mutations of NA-K84N, V106I, L139V, I163T, E287K, or S340F has been detected. Moreover, seven locus mutations were newly found, including A86T (1strain), F115L (4 strains), N449T (4 strains), S450V (4 strains), D451T (4 strains), T452L (4 strains), V453W (4 strains).Comparing with substitutions in the A(H1N1)pdm09 viruses in 2012–2014, several substitutions have not be detected in 2015–2017. It suggested that these variation sites may only be transient variation. On contrast, the increased prevalence of HA-K180Q and NA-V264I mutations from 2012–2014 to 2015–2017 in all strains suggested that several mutations may gradually become dominant over time under the pressure of human immune selection , 22.Many events such as recombination and infection of new hosts can disrupt the balance between HA and NA, but mutations in the HA and NA genes can make up for this imbalance –25. Co-oWhile our study highlighted the importance of investigating the effect and mechanism of co-occurring mutation of HA and NA; however, this study had several limitations. First, the study population were all from outpatients who were not severely ill, and the findings may not be generalized to hospitalized or severely ill individuals. Second, the medical data collection was limited, and many important clinical data such as further examination, period of treatment, and prognosis were not available. Third, the number of study patients may not be sufficient to draw the firm conclusions. Fourth, co-occurring mutations we observed had possibility of gradual accumulation of mutations, and we analyzed the co-occurring mutation of HA and NA through the clinical manifestations without mechanism. Therefore, further studies with a larger sample size and more clinical data will be needed to confirm and extend our findings. Evidences of increased fitness or epistasis between the HA and NA segments will also be needed in order to exclude gradual accumulation of mutations.In this study, we analyzed molecular evolution and amino acid variation characteristics of HA and NA of A(H1N1) pdm09 during the 2015–2017 influenza seasons in Beijing, founding these viruses shared common evolutionary lineages to the vaccine strain A/Michigan/45/2015 (H1N1). However, through our team’s monitoring on molecular evolution and amino acid variation of A(H1N1)pdm09 these years, some variation sites were only transient variation, while some from one to all strains, suggesting that persistent monitoring was needed. Co-occurring mutations of HA-V169T, A278S, E508G, D518E and NA-V67I were detected in 30.3% (10/33) A(H1N1)pdm09 virus strains when comparing with the two vaccine strains A/California/07/2009 and A/Michigan/45/2015 (H1N1). We also made progress on exploring the clinical characteristics of co-occurring mutations in HA and NA proteins of influenza A(H1N1)pdm09 virus. Above results highlight the fact that it is necessary to study the effect and mechanism of co-occurring mutations in the surface antigens HA and NA on patients’ clinical features, and one day those co-occurring mutations maybe used as biomarkers of clinical features to assist the clinical work.Additional file 1: Table 1. Nucleotide similarity of HA genes compared to vaccine strains.Additional file 2: Table 2. Amino acid similarity of HA genes compared to vaccine strains.Additional file 3: Table 3. Nucleotide similarity of NA genes compared to vaccine strains.Additional file 4: Table 4. Amino acid similarity of NA genes compared to vaccine strains.


Chromatin regulation ensures stable repression of stress-inducible genes under non-stress conditions and transcriptional activation and memory of stress-related genes after stress exposure. However, there is only limited knowledge on how chromatin genes are regulated at the transcriptional and post-transcriptional level upon stress exposure and relief from stress. We reveal that the repressive modification histone H3 lysine 27 trimethylation (H3K27me3) targets genes which are quickly activated upon cold exposure, however, H3K27me3 is not necessarily lost during a longer time in the cold. In addition, we have set-up a quantitative reverse transcription polymerase chain reaction-based platform for high-throughput transcriptional profiling of a large set of chromatin genes. We find that the expression of many of these genes is regulated by cold. In addition, we reveal an induction of several DNA and histone demethylase genes and certain histone variants after plants have been shifted back to ambient temperature (deacclimation), suggesting a role in the memory of cold acclimation. We also re-analyze large scale transcriptomic datasets for transcriptional regulation and alternative splicing (AS) of chromatin genes, uncovering an unexpected level of regulation of these genes, particularly at the splicing level. This includes several vernalization regulating genes whose AS may result in cold-regulated protein diversity. Overall, we provide a profiling platform for the analysis of chromatin regulatory genes and integrative analyses of their regulation, suggesting a dynamic regulation of key chromatin genes in response to low temperature stress. FLOWERING LOCUS C (FLC). The repression of FLC is achieved through the action of regulators of the Polycomb-group (Pc-G), which deposit and maintain the repressive tri-methylation of lysine 27 in histone H3 (H3K27me3) upon cold exposure , which transfer the acetyl group of acetyl Coenzyme A to the ϵ-amino group of lysine side chains . Histonethaliana . Methylathaliana . The secthaliana .In addition to histone modifications, the state of chromatin can also be affected by DNA methylation, which was found to be linked to gene repression . DNA metA. thaliana, 13 H2A variants (labeled HTA1-13) exist, and are clustered in four groups: H2A, H2A.X, H2A.W, and H2A.Z  is the only VRN gene known to be induced by cold, however, protein level analyses of Pc-G proteins involved in vernalization suggest post-transcriptional regulation of several genes including VRN2, CLF, FIE, and SWN  platform. We identify a potential role for Pc-G proteins in repressing stress-inducible genes under non-stress conditions and substantial transcriptional and post-transcriptional regulation of chromatin regulatory genes.Interestingly, genes involved in vernalization are largely not transcriptionally regulated under short-term (3 days) cold conditions. However, they may be alternatively spliced, resulting in potentially altered protein sequences. Based on data generated with the RT-qPCR platform, we have identified additional cold-inducible chromatin regulatory genes and genes specifically regulated during deacclimation, including DNA demethylases and histone variants.A. thaliana accession Col-0 seeds were sown in a single pot and grown on soil in a climate chamber with 20°C day-time temperature and 6°C night-time temperature in a 14-h light cycle with a light intensity of 180 µE m−2 s−1 and a humidity of 60% at day and 70% at night. After 1 week the plants were moved to a short-day climate chamber with the following conditions: 20°C/16°C day/night, 8 h day length, 180 µE m−2 s−1, humidity of 60%/75% day/night. The plants were kept under these short-day conditions for a week before transferring them to new pots (10 plants per 10-cm diameter pot). Afterwards, the plants were kept for another 7 days under short-day conditions before transfer to long-day conditions for another week. The conditions for long-day were 20°C day and 16°C night temperature with a day length of 16 h at a light intensity of 200 µE m−2 s−1. These 4-week-old plants were used in cold acclimation and deacclimation experiments. For cold acclimation, plants were moved for 3 days to a growth chamber with a constant temperature of 4°C and a day length of 16 h with a light intensity of 90 µE m−2 s−1 and a humidity of 70% to 80%  locus or the ACTIN7 (AT5G09810) locus, depending on the analyzed mark. Statistical significance was tested using one-way ANOVA followed by a Dunnett's multiple comparison test using GraphPad Prism7.Plants were grown for 21 days on ½ MS medium under SD conditions  and transferred to 4°C for 3 h or 3 days. Seedlings (0.5–1 g) were crosslinked in phosphate-buffered saline (PBS) containing 1% paraformaldehyde for 10 min under vacuum. The crosslink reaction was stopped by the addition of 0.125 M glycine. Samples were rinsed with ice-cold PBS solution and frozen in liquid nitrogen. Chromatin extraction was performed as previously described . The extACTIN2 (AT3G18780), PDF (AT1G13320), and TIP41 (AT4G34270) AT4G34270 and taken as the negative exponent to the base 2. Primer sequences can be found in For gene expression analysis, RNA was isolated from 100 mg of seedlings grown as described above, using the innuPREP Plant RNA Kit . RNA was quantified using a Nanodrop-1000 spectrophotometer (Thermo Scientific). Genomic DNA was removed using the DNase I kit (Thermo Scientific) and cDNA was synthetized using the RevertAid Reverse Transcriptase (Thermo Scientific). The quantitative PCR was performed as described above. The Ct values were normalized by subtracting the mean of the three housekeeping genes GAPDH (AT1G13440) (RNA was isolated from a pool of five samples consisting of 10 different plants using Trizol reagent (BioSolve BV). RNA was quantified using a NanoDrop-1000 spectrophotometer (Thermo Scientific) and DNA was removed from the samples using a RapidOut DNA Removal Kit (Thermo Scientific). The absence of genomic DNA was tested by qPCR with intron-specific primers (Intron MAF5 AT5G65080) . cDNA wa1G13440) . cDNA of1G13440) .Actin2 (AT3G18780), EXPRS (AT2g32170), GAPDH (AT1G13440), and PDF (AT1G13320) was measured for each sample on each plate  plants.RT-qPCR was performed for 135 genes of interest . Expressch plate . The Ct Heat maps were constructed in RStudio  using thPrimers were either designed in Primer3 or taken from the literature as indicated in A. thaliana Col-0 plants were stratified for 4 days at 4°C in the dark and then grown in hydroponic culture for 5 weeks. Growth conditions were 12 h light (150 μE m−2 s−1)/12 h dark with a constant temperature of 20°C. The 4°C cold treatment was started at dusk. Rosette material was harvested and pooled per sampling point.The dataset of differentially expressed cold-regulated genes was extracted from previously published data . Experimclavata3 mutant plants (Col-0 background). For this analysis the leaf samples were taken from plants grown for 9 weeks under short day conditions (8 h light/16 h dark).H3K27me3 targets were extracted from previously published data . In thisThe datasets were compared using the conditional formatting and filter function from Excel.th of July, 2011)   . This liy, 2011)  using thhttps://wyguo.shinyapps.io/atrtd2_profile_app/  with genes regulated quickly after cold exposure (3 h) or after long-term (3 d) cold  , WRKY48 (TOLERANT TO CHILLING AND FREEZING1 (TCF1), and the glutaredoxin GRXS4 (PINORESINOL REDUCTASE1 (PRR1) showed similar H3K27me3 (and H3) in ambient and cold temperatures  using chromatin immunoprecipitation . In addition, we extracted chromatin genes from the ChromDB  and analHTR2, HTR6, HTR11; HTA6, HTA12; H1.3) and all three paralogues of the DNA demethylase DEMETER, DML1/ROS1, DML2, and DML3  domain-containing protein 14 (JMJ14) (AT4G20400), Maternal affect embryo arrest 27/(JMJ15) (AT2G34880), JMJ17 (At1g63490), JMJ18 (AT1G30810), JMJ26 (At1g11950), VP1/ABI3-like 3 (VAL3) (AT4G21550), Probably E3-ubiquitin protein ligase (DRIPH) (AT3G23060), Chromomethylase 1 (CMT1) (AT1G80740), and male-gamete-specific histone H3 (MGH3) (AT1G19890) were not further considered, resulting in 135 investigated genes.To allow expression analyses of Pc-G, Trx-G, and associated genes, histone genes, and genes involved in DNA methylation under various conditions, we set up a RT-qPCR platform. The expression of the 144 selected genes was analyzed in samples from NA, ACC, and deacclimated (Deacc) plants after 2, 4, 6, 12, and 24 h of deacclimation. Due to extremely low expression levels throughout all samples, gene expression data for −ΔCt values are shown in Expression changes of these genes during cold acclimation and subsequent deacclimation are shown in five heat maps compiled according to the function of the respective proteins in epigenetic regulation. The corresponding normalized 2JMJ11/ELF6, JMJ19, JMJ22, JMJ27, JMJ28, and JMJ30 showed the highest log2 fold change compared to NA (The expression of 9 out of 19 genes encoding JUMONJI-type and lysine specific 1A-type (LSD1) histone demethylases was significantly upregulated after cold acclimation. Out of these nine genes, CLF, four WD40 repeat containing proteins MSI1-4 and VRN1. Altogether, 13 genes encoding Pc-G related proteins were significantly upregulated, while SWN, DRIP2/BMI1A, and VEL3 exhibited a downregulation at ACC. Most genes with a strong upregulation at ACC kept their higher expression levels over 12 h of deacclimation before they were significantly downregulated in comparison to ACC after 24 h . After 2, 6, and 12 h Deacc only two, four, and two of these genes, respectively, were still significantly induced with only SWI3A showing a stable significant induction over almost all time points. A significant transiently changed expression over two time points during deacclimation compared to NA was only evident for PDS5e at 6 h and 12 h. At 24 h Deacc expression changes caused by cold acclimation were mostly reversed and expression of all genes reached similar levels as under NA conditions, except for ULT1, which was significantly downregulated. ATX5 displayed a transient upregulation in comparison to ACC conditions till 12 h Deacc (The expression of 22 genes encoding Trithorax-group (Trx-G) related proteins and chromatin remodelers was also investigated  interactions, RNAi and DNA methylation was measured , or histone variants were investigated (HTA12 was upregulated at later time points of deacclimation (6 and 12 h Deacc) after exhibiting the largest decrease in expression after cold acclimation in comparison to NA. Consistently, this gene was significantly induced compared to ACC along the whole deacclimation time course  and ULT1 , there are likely different, gene specific mechanisms at work. Our analyses also revealed that H3K27me3 target genes (identified in warm conditions) are overrepresented among the late repressed genes in the cold  repression is not accompanied by increased H3K27me3. Also here, H3K27me3 may be increased at specific nucleosomes, which is observed for the H3K27me3 target gene FLOWERING LOCUS C (FLC) which carries H3K27me3 in the warmth but a higher coverage/spreading of H3K27me3 upon cold exposure/vernalization (FLC.Chromatin and chromatin modifications contribute to the regulation of stress-regulated genes at various layers: (1) the repression of stress-inducible genes in non-stress conditions (by repressive chromatin), (2) the activation or repression of genes immediately after stress exposure , (3) the sustained activation or repression in non-stress conditions after exposure to the stress, and (4) the transcriptional memory of a stress (in non-stress conditions), permitting primed gene regulation when exposed again to the stress . It is tse JMJ30 , the Trxand ULT1 , and divlization . The latIn general, our analysis indicated that cold acclimation had a strong influence on the expression of the selected epigenetics-related genes. This is in agreement with the finding that cold stress enhanced the accessibility of chromatin and bivalent histone modifications of active genes in potato . After 2JUMONJI-type and LSD1-type enzymes are histone demethylases. For several years histone methylation was thought to be irreversible until the discovery of human LSD1 . AlthougJUMONJI and LSD1 genes are linked to regulation of developmental transitions in A. thaliana. For example, histone demethylation of the FLC gene by JMJ30 and JMJ32 controls flowering at warm temperatures . PRC2 mediates the trimethylation of H3K27 (H3K27me3), which results in the repression of transcription through changes in chromatin organization , at ACC conditions might have led to an increase or redistribution of H3K27me3, resulting together with other changes in the increased freezing tolerance of A. thaliana. Supporting this hypothesis, induction of Pc-G genes during cold acclimation has also been reported previously. Brassica oleracea displayed induced expression of alfin-like transcription factors, which are interactors of PRC1, during 24 h at 4°C  which is only induced after prolonged cold (at least 10 d)  . We did and EMF2  revealedA. thaliana , encoding a protein catalyzing the limiting step in ABA synthesis, was observed, and a loss of ATX1 resulted in decreased NCED3 levels under drought stress , encoding an ATP-dependent chromatin remodeling complex, displayed opposite effects to ATX1 under drought stress, resulting in increased drought tolerance when the gene was non-functional, through repression of ABA INSENSITIVE5   and are involved in maintaining the cellular organization of the shoot apical meristem  . Thus, imeristem . SWI3A welopment . These rCMT2 displayed a continued increase in expression up to 12 h Deacc. Furthermore, DNA demethylases such as DEMETER have been previously linked to plant stress responses and DME also showed increased expression during deacclimation. In addition, deletion of three DNA demethylases  resulted in increased susceptibility to fungal pathogens and therefore a participation in the biotic stress response of plants was proposed  were still upregulated at 12 and/or 24 h Deacc, suggesting that deacclimation is accompanied by a resetting of DNA methylation.One group of DNA methylases adds a methyl group onto cytosine residues in higher eukaryotes and have been proposed to control gene expression in plants during development and regulate transposable elements and heterochromatin . Chromomproposed . InteresDCL1 was induced by cold acclimation whereas DCL3 was significantly upregulated only after 12 h Deacc. Expression analyses on DCL genes in rice showed differential responses comparing drought, cold, and salt stress  interacts with transcriptional adapter ADA2B, a transcriptional activator of HACs, and T-DNA insertions of GCN5 lower the induction of COR genes during cold acclimation , H2A.Z  and H2A.W  were included in the analysis, as well as the histone H1 variants H1.1, H1.2, H1.3, and HTR1 to HTR15 from the histone H3 . We gratefully acknowledge support to MP through the People Program (Marie‐Curie Actions) of the European Union's Seventh Framework Programme (FP7 People: Marie‐Curie Actions FP7‐MC‐IEF) under REA grant agreement 328,713. We acknowledge support by the German Research Foundation and the Open Access Publication Fund of the Freie Universität Berlin.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.


Of the eighteen hemagglutinin (HA) subtypes (H1–H18) that have been identified in bats and aquatic birds, many HA subtypes have been structurally characterized. However, several subtypes  still require characterization. To better understand all of these HA subtypes at the molecular level, HA structures from an A(H4N6) (A/swine/Missouri/A01727926/2015), an A(H8N4) (A/turkey/Ontario/6118/1968), an A(H11N9) (A/duck/Memphis/546/1974), an A(H14N5) A/mallard/Gurjev/263/1982, and an A(H15N9) ; A(H11N9); A(H14N5); A(H15N9); A(H4N6) While aHA and NA both play an important role during the virus life cycle. Influenza virus infection is initiated by HA binding to sialic acid receptors and mediates virus entry and fusion , while NPrevious studies identified a number of key receptor binding site (RBS) mutations of HA, responsible for switching avian/human receptor specificity in H1, H2 and H3 subtypes. In H1 subtypes, a Glu190Asp and Gly225Asp double mutation renders the HA capable of binding human α2-6 receptors . For H2 Phylogenetic analysis reveals that all HA subtypes can be separated into two groups, and each group further divided into subgroups  Gamblin.Figure 1Indeed, almost four decades have elapsed since the first crystal structure of influenza virus HA was determined and it facilitated an understanding of the structural identification of the major antigenic sites and the effects of natural variation . Among a22.1N-acetylglucosamines (GlcNAc) (1JSD). Previous studies of HA structures have shown some group-specific features at the interhelix loop and the rigid-body orientation of the globular domain . Both H8 and H11 are group-1 HAs , but the(GlcNAc) A. With Cr domain . Here wer domain A. Differr domain . Howeverr domain B. The inr domain ; therefo4KPQ) and H16 HA (PDB ID: 4F23), the RMSD values are 1.44Å and 1.55Å respectively. Among the five predicted glycosylation sites , only Asn23 on HA1 has interpreted density for glycan  viruses have been isolated from different species of waterfowls that are located globally. The avH11 HA structure was determined at 2.8Å : it showr glycan B.2.23EYJ) and H15N9 (PDB ID: 5TG8) HAs have been published before (3EYJ) did not report any interpretable density for carbohydrates. The avH15 HA structure was determined at 2.5Å (N-acetylglucosamines (GlcNAc)  A/mallard/Gurjev/263/1982 HA, and an A(H15N9) A/wedge-tailed shearwater/Western Australia/2576/1979 HA to assess their structural and binding properties. H14 and H15 are two AIV subtypes with only 45 and 22 sequences deposited in the GISAID database. Crystal structures of an H14N6 virus  D, consis(GlcNAc) . Superim(GlcNAc) B. The st(GlcNAc) . Moreove(GlcNAc) D. In add(GlcNAc) .2.3Three HA receptor binding sites (RBS) are present in the HA trimer and are located at the top of each monomer. Including the 5 HAs discussed here, three major structural elements form the RBS binding pocket in all current HAs: a 190-helix (residues 188 to 194 based on H3 numbering), a 220-loop (residues 221 to 228), and a 130-loop (residues 134 to 138). In addition, highly conserved residues  are also present in the base of the pocket of all 5 HAs and have been shown to affect receptor binding . When us2.4To improve our understanding of the interactions between these avian viruses and host receptors, glycan-binding studies of avH8, avH11, avH14 and avH15 recHAs were carried out using version 1 glycan microarrays. Results revealed a strict α2-3 linked sialic acid binding for all four avian subtype HAs A–D. Only2.5More than 2200 H4 HA sequences are available in the GISAID database and these sequenced H4 viruses have been found associated with all 9 NAs. There is also one documented example of a highly pathogenic A(H4N2) virus isolated from a quail in the U.S.  . While dN-acetylglucosamine (GlcNAc) attached. A larger glycan was interpretable at Asn162 because this glycan makes contact with the main chain nitrogen of an adjacent chain (Trp219) within the same trimer and residues Gln122 and Asn168 in different trimers within the crystal. The overall structure of swH4 HA resembles other available HA structures (5XL1) and swine H4N6  are 0.36 Å and 1.03Å, respectively.In this report, the structure of swH4 HA has been determined at 2.2 Å . There aructures A. With CGlycan microarray analysis of swH4 with version 1 microarrays, revealed a restricted binding to α2-6 glycans, in particular the biantennary α2-6 di-LacNAc sialoside  B. To fur3Hemagglutinin is the most important surface glycoprotein of influenza that enables the virus to bind to host cell receptors and gain entry into the host cell. The first three-dimensional structure of HA was published in 1981 by Wilson, Wiley and Skehel . Since tIn this study, we report the first avH8 and avH11 HAs to be structurally characterized. The overall structure of these two subtypes HAs show high similarity with all other available HAs, especially the group-1 HAs. Rotation of the head domain does not seem to relate to any of the HA functions, such as receptor binding, and analysis of these rotations revealed no distinct subgroup clustering . SimilarGlycan microarray technology is a very useful tool to study HA binding specificities. The results of avian recHAs used in this study show that all avHAs have strict avian-like receptor binding preferences. Swine are suggested to be a genetic mixing vessel due to the frequent co-infection and recombination of influenza viruses from various sources and lineages . InfluenThe requirements for virus host switching are complicated and changing receptor binding specificity is not the only major barrier for adaptation of avian influenza viruses to humans. With swine as a possible intermediate host, the swH4 virus HA has the Leu223 and Ser225 constellation and thus the H4 virus has the potential to adapt further or reassort with human viruses in pigs.4In this study, we presented structural and glycan binding data of four avHAs and one swHA. While all the HAs have a similar overall structure, they also maintain group-specific structural features of different subtype HAs, such as an extended 150-loop for the H15 and similar interhelix loop variations. Although the avHAs revealed strict receptor binding preferences, the swine H4 however showed markedly reduced binding to avian-type receptors and weak binding to human receptors. While this virus may not yet be considered a virus with pandemic potential, the H4 subtype should be included in the list of viruses to continue watching. With the growing number of human and zoonotic infections by AIVs, it raises the public health concern that one or more of these viruses will eventually evolve into a variant capable of causing another global pandemic. Continued surveillance of domestic and wild bird populations as well as domestic animals is critical. Moreover, the transmission of AIVs to swine should be a warning sign, and these types of incidents should be monitored and investigated in detail.55.1Sf9 insect cells.cDNAs encoding the mature ectodomains of each HA . For crystallization trials, HAs were concentrated to 13-17 mg/mL during buffer exchanging into 10 mM Tris-HCl, 50 mM NaCl, pH 8.0.5.3Either a Topaz™ Free Interface Diffusion (FID) Crystallizer system  or a Formulatrix NT8  were used to set up the initial crystallization screens. Conditions in which crystals were observed were optimized using a modified method for microbatch under oil  at 20 °C5.4HA structures were solved by molecular replacement with Phaser . Initial5.5RecHA glycan microarray analyses have been described previously . BrieflyA second array (version 2) containing only a limited set of glycans was also used for analyzing the H4 recHA. These glycans, a mix of linear and biantennary α2-3 and α2-6 linked sialosides of different lengths (from 1 to 4 LacNAc repeats), were imprinted on the array at different concentrations  to assess both specificity and avidity. This alternate glycan array slide was processed using the same procedure as described above. 5.6www.pdb.org) under the accession codes listed in All HA models with their atomic coordinates and structure factors are available from the RCSB PDB database (Hua Yang, Paul J. Carney, Jessie C. Chang: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.James Stevens: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper.10.13039/100000030Centers for Disease Control and Prevention.This work was supported by the The authors declare no conflict of interest.No additional information is available for this paper.


During the current second wave of COVID-19, the radiologists are expected to face great challenges in differentiation between COVID-19 and other virulent influenza viruses, mainly H1N1. Accordingly, this study was performed in order to find any differentiating CT criteria that would help during the expected clinical overlap during the current Influenza season.. Regarding HRCT findings of airway involvement , all showed significant p value (ranging from 0.008 to 0.04). On the other hand, HRCT findings of parenchymal or alveolar involvement (mainly ground glass opacities) showed no significant relation.This study was retrospectively conducted during the period from June till November 2020, on acute symptomatic 130 patients with no history of previous pulmonary diseases; 65 patients had positive PCR for COVID-19 including 50 mild patients and 15 critical or severe patients; meanwhile, the other 65 patients had positive PCR for H1N1 including 50 mild patients and 15 critical or severe patients. They included 74 males and 56 females (56.9%:43.1%). Their age ranged 14–90 years (mean age 38.9 ± 20.3 SD). HRCT findings were analyzed by four expert consultant radiologists in consensus. All patients with COVID-19 showed parenchymal or alveolar HRCT findings; only one of them had associated airway involvement. Among the 65 patients with H1N1; 56 patients (86.2%) had parenchymal or alveolar HRCT findings while six patients (9.2%) presented only by HRCT signs of airway involvement and three patients (4.6%) had mixed parenchymal and airway involvementHRCT can help in differentiation between non-severe COVID-19 and H1N1 based on signs of airway involvement. Human infection by coronavirus family was believed to be attributed to six major types, two types of which caused severe respiratory symptoms. The first is severe acute respiratory syndrome (SARS), seen in south China in 2003, and the other is Middle East respiratory syndrome (MERS), seen in Saudi Arabia in 2012 [Clinical picture of COVID-19 and H1N1 may overlap as both cause variable degrees of respiratory symptoms ranging from mild (flu like) up to severe  , 4. AlsoDuring the current second wave of COVID-19, the radiologists are expected to face great challenges in differentiation between COVID-19 and other virulent influenza viruses, mainly H1N1. Accordingly, this study was performed in order to find any differentiating CT criteria that would help during the expected clinical overlap during the current Influenza season.2 saturation at room air > 93%) and 15 critical or severe patients . The other 65 patients had positive PCR results for H1N1 patients (registered during the period from October 2018 till November 2020), including 50 mild or moderate patients and 15 critical or severe patients as well. They included 74 males and 56 females (56.9%:43.1%). Their age ranged 14–90 years (mean age 38.9 years ± 20.3 SD).This study was retrospectively conducted, during the period from June till November 2020, on acute symptomatic 130 patients. Among them, 65 patients had positive PCR results for COVID-19 (registered during the period from February till May 2020), including 50 mild or moderate patients  acute illness with respiratory symptoms either mild or severe based on the degree of hypoxia and the need for external respiratory support, (2) HRCT for positive COVID-19 patients, and (3) HRCT for positive H1N1 patients. All patients should have a confirmed PCR diagnosis of the disease entity in order to be enrolled in the study.Exclusion criteria were as follows: (1) degraded quality of CT scans due to patient unavoidable tachypnea with respiratory motion artifacts; (2) past history of chronic airway disease such asthma, COPD, or bronchiectasis due to possible overlap with the CT findings of infection and expected bias; and (3) known patients with secondary bacterial infection, proved laboratory by culture/sensitivity tests.CT examinations were conducted using multiple MDCT machines including: GE LightSpeed Plus 4 slice CT scanner (USA), Philips Brilliant-16 (USA), Siemens SOMATOM Emotion 16 and Siemens SOMATOM Sensation 64 (Germany), Canon Medical Systems; Toshiba Aquilion 64 and Toshiba Aquilion CXL/CX 128 (USA).CT scanning parameters were as follows: slice thickness, 1–2.5 mm; FOV = 350mm × 350 mm; tube rotation, 0.6–0.9 s; detector collimation, 1 mm; helical mode (volumetric HRCT); kVp and mA per slice, 120–130 kVp and 200–400 mA, according to the type of MSCT machine used; the weight of the patient; and the clinical indication.Intra-venous contrast administration was done to 11 patients only as clinically requested. It was non-ionic iodinated contrast (350mg/ml concentration and 70–90 ml volume at 4–5 ml/s injection rate). No major reactions were reported. Only minor reactions were reported such as sense of hotness and nausea, which needed noting but assurance.A)Site of the pathology: unilateral or bilateral—focal, multi-focal, or diffuse.B)HRCT findings of pulmonary parenchymal or alveolar involvement including ground glass opacities (GGOs), consolidative changes, interlobular septal thickening, and “crazy paving pattern”.C)HRCT findings of airway involvement including bronchial wall thickening, mucous plugging, and traction bronchiectasis/bronchiolectasis. This is in addition to secondary air trapping with or without “head cheese pattern.”D)Relevant CT findings including pleural, pericardial, and nodal lesions.CT images were assessed in consensus by four consultant radiologists . They were blinded to the final clinical diagnosis. Image analysis in axial, sagittal, and coronal planes was done using both maximum intensity projection (MIP) and minimum intensity projection (Min-IP) reconstructions. The following CT features were compared between each pathological process:p value < 0.05 was considered statistically significant. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of HRCT were estimated for differentiation of H1N1 from COVID19.The prevalence rate of HRCT findings was estimated as the percentage of patients showing each criteria or abnormality. Data were compared using a chi-square test and Sixty-four patients with COVID-19 showed pure lung parenchymal or alveolar pattern of the disease. Only one patient (1.5%) presented by mixed parenchymal pattern and airway involvement (manifested by tree in bud nodules). Fifty-six patients with H1N1 (86.2%) showed parenchymal or alveolar pattern of the disease while six patients (9.2%) presented mainly with airway disease manifestations and three patients (4.6%) showed mixed parenchymal and airway involvement . Traction bronchiectasis was seen in five patients with H1N1 (7.7%). Mucous plugging was seen in four patients with H1N1 (6.2%). Air trapping was seen in seven patients with H1N1 (10.8%). “Head cheese pattern” with mixed air trapping and ground glass attenuation was seen in three patients with H1N1 (4.6%)  signs related to topographic picture of the diseases, (B) signs of lung parenchymal or alveolar involvement, (C) signs of airway involvement, and (D) signs related to pleuro-pericardial or nodal involvement.(A)Regarding the topographic HRCT findings(B)Regarding the HRCT findings of parenchymal or alveolar involvementPrevalence rate of mixed peripheral and proximal lobular lung involvement was higher in H1N1  compared to COVID-19 . On the other hand, prevalence rate of sub-pleural predilection was higher in COVID-19  compared to H1N1  (Table (C)Regarding the HRCT findings of airway involvementGGOs were found in all COVID-19 patients and 90.8% of H1N1 patients. In mild cases, prevalence of pure ground glass nodules or patchy opacities (GGOs) was slightly higher in COVID-19  compared to H1N1  Fig. . Prevale(D)Regarding the other relevant HRCT findingsTree in bud nodules (sign of airway involvement) were seen in nine patients with H1N1 (13.8%) compared to only one patient with COVID-19 (1.5%). All of them had mild clinical presentation Table .(D)RegaPleural effusion and pericardial effusion, despite being uncommon CT finding and minimal in amount, were found more in H1N1 than COVID-19. Significant lymph node enlargement (short axis diameter exceed 1 cm) was noted in seven critical COVID-19 patients and five critical H1N1 patients Table .p value (ranging from 0.008 to 0.04). On the other hand, other HRCT findings related to topographic features of the disease and other HRCT findings which were corresponding to parenchymal lung disease and accompanied both mild and severe clinical presentations showed non-significant p value > 0.05  showed significant 05 Table .Among overall 65 patients proved with COVID-19, 20 patients were falsely diagnosed as H1N1. While among overall 65 patients proved with H1N1, 31 patients were falsely diagnosed as COVID-19. Accordingly and in overall, HRCT showed 50% sensitivity, 69.2% specificity, 61.5% positive predictive value (PPV), and 58.4% negative predictive value (NPV) for differentiation of H1N1 from COVID-19.Among those nine patients proved with H1N1 having airway involvement , only one patient was falsely diagnosed as COVID-19. Meanwhile, the only one patient that had COVID-19 with mixed parenchymal and airway involvement was falsely diagnosed as H1N1. Accordingly, presence of HRCT findings of airway involvement in mild cases raise both HRCT sensitivity and PPV for differentiation of H1N1 from COVID-19 up to 88.9%.A schematic flow-chart is summarizing the results of the study among 100 mild patients and 30 severe patients Fig. .Fig. 6APCR tests for COVID-19 patients are not always positive and HRCT findings are not always pathognomonic , 8. Hencp value, the presence of airway involvement can highly suggest H1N1 viral pneumonia over COVID-19 after exclusion of past history of large or small airway disease such as asthma, COPD, or bronchiectasis.Large and small airway involvement was strikingly missing in 98.5 % of COVID-19 patients who strictly presented by other HRCT findings related to pulmonary parenchymal or alveolar involvement. This is slightly different from Chen et al.’s  study whp value analysis, site of the pathology is non-differentiating parameter.This study matches Marchiori et al.  and Liu p value analysis, HRCT findings of parenchymal or alveolar disease in mild patients are non-differentiating parameters. Among severe patients (mainly patients with DAD and ARDS), “crazy paving pattern” was also depicted in both COVID-19 and H1N1 patients, with slight higher percentage in H1N1. This is concordant with Pritt et al. [p value analysis, HRCT findings related to parenchymal or alveolar involvement in severe patients are non-differentiating parameters.Regarding the lung parenchymal or alveolar pattern of the disease and keeping with Dawoud et al. , Liu et t et al.  who repop value analysis, HRCT findings of airway disease in mild patients are good differentiating parameters.This study again go side by side to Marchiori et al.  regardinPleural effusion was noted in 10% of H1N1 patients, agreeing with Koo et al.  while onPericardial effusion was seen in a small percentage of COVID-19 patients presenting with myocarditis, similar to findings of Inciardi et al. . It was This study disagreed with Bernheim et al.  that cavThis study disagreed with Hu et al.  that lymThis study was limited by small number of patients proved with H1N1 at single center; hence, further large group future studies are encouraged.HRCT can help in differentiation between non-severe COVID-19 and H1N1 based on signs of airway involvement. Pulmonary parenchymal as well as pleural and pericardial involvement are not reliable differentiating CT parameters. Presence of cavitation or significant lymph node enlargement also are not reliable differentiating parameters. At severe stages, both COVID19 and H1N1 are considered as great mimics and non-differentiable on CT basis.


Acipenser dabryanus) was first determined in this study. The mitochondrial DNA is circular molecule  with a typical vertebrate mitochondrial gene arrangement, including 13 protein-coding genes, 2 ribosomal RNA (rRNA) genes, 22 transfer RNA (tRNA) genes, and 1 non-coding control region (D-loop). All genes were encoded on the heavy strain except for ND6 and eight tRNA genes. The overall base composition of the heavy strain was A (30.2%), T (23.7%), C (29.7%), G (16.4%), and with an AT bias of 53.9%. Phylogenetic analysis based on the mitochondrial genomes of 10 related species using MEGA 5.1 indicated that A. dabryanus and A. sinensis are the most closely related species.The complete mitochondrial genome of Dabry’s sturgeon ( This gene arrangement of Dabry’s sturgeon is similar to the typical vertebrate mitogenomes . The overall base composition of the heavy strain was A (30.2%), T (23.7%), C (29.7%), G (16.4%), with a slight bias to AT (53.9%). Twelve of the protein-coding genes share the start codon ATG, except for COI, which begins with GTG. Ten protein-coding genes use TAA or TAG as stop codon, although three of the genes  require T–– or TA– as incomplete stop codon.The sampled specimen was collected from Xiangjiaba breeding and releasing station  in January 2016, China. This specimen was stored in Institute of Chinese Sturgeon with the accession number: YC20160118F02. Total genomic DNA was extracted from caudal fins by a traditional phenol-chloroform method (Sambrook & Russell A. dabryanus and A. sinensis are the most closely related species (The mtDNA sequences of 10 species of fishes were downloaded from GenBank, Amia calva as an out-group. Phylogenetic analysis based on the mitochondrial genomes of 10 related species by using MEGA 5.1 (Kumar et al.  species .


Breast cancer (BC) is known to be the most prevalent cancer among women. One-carbon metabolism disturbance might play an important role in the etiology of BC. The present study aimed to investigate the thymidylate synthase (TYMS), 5-methyltetrahydrofolate-homocysteine methyltransferase (MTR), and methionine synthase reductase (MTRR) variants as good candidates for studying the role of genetic variants of folate metabolizing enzymes in the risk of BC.TYMS 2R/3R (rs34743033), MTR c.2756A>G (rs1805087), and MTRR c.66A>G (rs1801394) variants were detected by polymerase chain reaction (PCR), PCR-restriction fragment length polymorphism (RFLP), and a designed amplification-refractory mutation system (ARMS) method, respectively.The present case-control study includes 100 BC patients and 141 healthy females. The TYMS enhanced the risk of BC by 2.84-fold (P<0.001). In the presence of TYMS 3R/3R, compared to TYMS 2R/3R, there was a trend toward enhancing the risk of metastasis by 4.15-fold . The frequencies of MTR c.2756A>G and MTRR c.66A>G variants were not significantly different among patients and controls.The 3R allele of TYMS 3R is a risk allele for susceptibility to BC and this allele may increase the risk of metastasis in BC patients. .We observed that the  Breast cancer (BC) is the most prevalent cancer among women accounting for 16% of all cancers in females , in whicGenetic alterations in a multistep process result in normal epithelial cells transformation of mammary into highly malignant cells . PopulatSince one-carbon metabolism (OCM) has a major role in the DNA synthesis and methylation and interaction between genetic and epigenetic processes, OCM disturbance might have an important role in the etiology of BC . The patThe 5,10-methylene tetra hydrofolate (THF) is the common substrate for two key enzymes of TYMS and methylenetetrahydrofolate reductase (MTHFR). These enzymes catalyze the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) and flavin adenine dinucleotide (FAD)-dependent reduction of 5,10-methylene THF to 5-methyl THF, respectively . The dTMTYMS 3R, compared with 2R, there is 2.6-fold more TYMS expression and enhancing the TYMS enzymatic activity.The TYMS gene locates at 18 p11.32. The important target for the chemotherapy drugs such as 5-fluorouracil (5-FU) is TYMS . In the MTR c.2756A>G (rs1805087) reduces the enzyme activity due to the conversion of aspartate to glycine in the protein, and induces the increase of homocysteine and DNA hypomethylation  results in replacement of isoleucine with methionine at amino acid 22 in the protein , MTR c.2756A>G (rs1805087), and MTRR c.66A>G (rs1801394) gene variants in susceptibility to BC among a population with Kurdish ethnic background from Western Iran.The aim of preset study was to investigate the role of SampleWe studied 100 BC patients  aged 49.5±10.2 years (29–79 years) and 141 healthy females aged 38.7±9.4 years (range 28–70 years). Diagnosis of BC was based on standard clinical, radiological, and histological parameters. All patients had been admitted to a university hospital affiliated to the Kermanshah University of Medical Sciences (KUMS), Iran. The ethnic background of both patients and controls was Kurdish. The Ethics Committee of KUMS approved the study. All patients and healthy controls agreed to participate in the study and a written informed consent was obtained from all participants prior to conducting the study. The study was in accordance with the principles of the Helsinki II declaration. GenotypingFrom each participant, 5 ml of whole blood with anticoagulant of Ethylenediaminetetraacetic acid (EDTA) was taken. Using treatment of whole blood with proteinase K and subsequent phenol–chloroform extraction and ethanol precipitation, genomic DNA was isolated from leukocytes . The concentration and purity of DNA were determined by a Nanodrop spectrophotometer . Thymidylate SynthaseTYMS was amplified by polymerase chain reactions (PCR) using the forward primer 5´GTG GCT CCTGCG TTT CCC CC 3´and the reverse primer 5´CCA AGCTTG GCT CCG AGC CGG CCA CAG GCA TGG CGCGG 3´to detect the tandem repeat sequences in the 5´-terminal of the TYMS regulatory region (TYMS 2R/3R (rs34743033). The parameters of PCR thermal cycling were: 1 cycle at 94oC for 5 min, 40 cycles by 94oC for 60s, 63oC for 60s, and 72oC for 60s followed by final extension at 72oC for 10 min. In the presence of homozygotes of double repeat (TYMS 2R2R) a 220-bp fragment is produced, while heterozygotes (TYMS 2R3R) produce two fragments of 220- and 250-bp. In the presence of homozygous TYMS triple repeat (TYMS 3R3R) a fragment with 250-bp is produced (The genomic region of produced  12)..TYMS wasMTR c.2756A>GMTR c.2756A>G polymorphism was identified using the forward primer 5´TGT TCCAGA CAG TTA GAT GAA AAT C 3´and 5´GAT CCA AAG CCT TTT ACA CTC CTC 3´as reverse primer. The parameters of PCR thermal cycling were: 1 cycle at 94oC for 5 min, 40 cycles by 94oC for 60s, 53oC for 60s, and 72oC for 60s followed by final extension at 72oC for 10 min. The 211-bp PCR product (10 to 15 µl) was digested using five units of the Hae III restriction enzyme at 37ºC overnight. The presence of MTR c.2756A>G polymorphism creates a recognition sequence for Hae III, which digests the PCR product with 211-bp into 131- and 80-bp fragments (The ragments .MTRR c.66A>GMTRR c.66A>G was detected by a designed amplification-refractory mutation system (ARMS) method. We designed primers with Oligo7 software that consisted of a primer for wild allele as 5'- TGTACCACAGCTTGCTCACTT-3', a primer for mutant allele with the sequence of 5'- TGTACCACAGCTTGCTCACTC-3', and a common primer of 5'- TGAAGTGATGAGGAGGTTTC-3'. The parameters of thermal cycler for gene amplification were: 1 cycle at 94°C for 5 min; 35 cycles: denaturation (94°C for 35 s), annealing (58°C for 35 s), extension (72°C for 45 s), and final extension (72°C for 5 min). The PCR product of wild and/or mutant allele was a fragment with 90-bp. A fragment with 506-bp from prothrombin gene was amplified using the primers 5' GCA CAG ACG GCT GTT CTC TT 3' and 5' ATA GCA CTG GGA GCA TTG AAG C 3', and was used as internal standard . Odds ratios (OR), that estimates the relative risk of disease, and 95% confidence interval (CI) were detected using the SPSS software logistic regression. Quantitative data were compared using Student’s t test. The level of P-value<0.05 was used for the statistical significance. The SPSS version 16 was used for statistical analyses .Using allele frequency calculation method, the frequency of the alleles was calculated. Difference in the alleles and genotypes frequency between both groups and its significance were tested using the χAmong participants, 39 individuals (39%) had a family history of cancer among first degree relatives. Based on the available files from 66 patients, there were 51 patients (77.3%) with lymph node metastasis. Also, there were 13 patients (19.7%) with tumor stage I, 42 patients (63.6%) with tumor stage II, and 11 patients (16.7%) with tumor stage III. Further, based on available data from files of 82 patients, the histological type of tumor was as 73 (89%) with invasive ductal carcinoma, 8 (9.8%) with invasive lobular carcinoma, and 1 (1.2%) with in situ type.TYMS 2R/3R in patients with BC and controls are indicated in TYMS 28-bp repeat 2R2R, as a reference genotype, was identified in 9 BC patients (9%), 2R3R was observed in 38 patients (38%), and 3R3R was identified in 53 patients (53%). The frequencies of 2R2R, 2R3R, and 3R3R genotypes in 141 healthy individuals were 20.6, 63.8, and 15.6%, respectively. The frequency of 2R allele among patients and healthy individuals were 28 and 52.5%, respectively (P<0.001). Higher frequency of 3R allele in patients enhanced the BC risk   .TYMS 3R3R  was detected compared to 2R3R . The presence of TYMS 3R/3R, compared to 2R/3R, tended to elevate the risk of metastasis by 4.15-fold In BC patients with metastasis, a significantly higher frequency of MTR 2756A>G genotypes and alleles in patients and controls are depicted in MTR AA was found in 59 patients (60.2%), the AG genotype in 35 patients (35.7%) and the homozygous genotype of GG were identified in 4 patients (4.1%). In healthy controls, 75 subjects (53.2%) had the AA genotype, 52 subjects (36.9%) had the AG genotype, and 14 subjects (9.9%) had the GG genotype. The frequency of G allele was 21.9% in patients and 28.4% in controls (P=0.1) (Distribution of  (P=0.1) . MTRR 66A>G in patients and healthy individuals are depicted in MTRR GG genotype was 16.1% in patients compared to 13.5% in controls. The frequency of MTRR G allele was not significantly different among BC patients and controls   .TYMS 3R), compared with two copies of tandem repeats (2R), there is higher TYMS expression enhancing the TYMS enzymatic activity . In silico analysis revealed that the MTR 2756A>G could induce benign damage to the MTR protein while the MTRR 66A>G indicated a deleterious effect on MTRR protein. Regarding the action of MTR and MTRR together in a 1:1 stoichiometric ratio to produce the holoenzyme complex, it seems the variant alleles of MTR and MTRR act in synergy in modulation of BC risk (In our study, we observed the lack of association between  females , 31-33. enotypes . In a me (et al.  considersk of BC . Also, i (et al.  which co BC risk .TYMS 3R allele may be a risk factor for BC development in our population. Also, in the presence of TYMS 3R/3R, a tendency and increased risk for BC metastasis may exist. However, MTR 2756A>G and MTRR 66A>G variants are not significantly associated with the risk of BC development. In addition, there is no significant association between these genetic variants with the histopathological characteristics of the patients.The results of the present study indicates that the


Vaccination with LAIV of H5N2, H2N2, H7N3, and H7N9 subtypes led to 7%–29.6% NI antibody seroconversions in the absence of HI antibody conversions. There was relatively low coordination of hemagglutinin (HA) and NA antibody responses (r = 0.24–0.59). Conclusions: The previously noted autonomy for HI and NI immune responses was confirmed when assessing the immunogenicity of LAIVs. Combining the traditional HI test with the detection of NI antibodies can provide a more complete assessment of LAIV immunogenicity.Background: Currently, the immunogenicity of influenza vaccines is assessed by detecting an increase of hemagglutination inhibition (HI) antibodies. As neuraminidase (NA)-based immunity may be significant in protecting against influenza infection, detection of neuraminidase inhibiting (NI) antibodies may improve the assessment of the immunogenicity of influenza vaccines. Methods: We investigated the immune response to NA in people after immunization with live influenza vaccines (LAIVs). A number of A/H7NX or A/H6NX viruses were used to detect NI antibodies, using an enzyme-linked lectin assay (ELLA). Results: Seasonal LAIV immunization stimulated an increase in NI antibodies not only to homologous A/H1N1 influenza, but also to A/H1N1pdm09 and A/H5N1 influenza. After A/17/California/09/38 (H1N1) pdm09 LAIV vaccination, there was no statistical relationship between post-vaccinated antibody seroconversion and two surface glycoproteins in serum samples obtained from the same individuals ( Influenzavirus, the family Orthomyxoviridae) is of interest to science and for clinical practice. It can be used when examining the levels of collective immunity to new influenza viruses, and for assessing the immunogenicity of influenza vaccines [The determination of antibodies that inhibit neuraminidase (NA) in the influenza virus (the genus vaccines . Currentvaccines . Howevervaccines . For thevaccines  is used.http://www.biodiem.com/wp-content/uploads/2020/02/20-02-28-LAIV-influenza-vaccine-approved-in-China.pdf). The efficacy of intranasal LAIVs is up to 80% in children under six years of age, and 40% in adults [LAIVs have been used in Russia since the 1960s and were licensed for use in North America and Europe in 2003 . Recentln adults ,6.As seasonal influenza vaccines cannot protect against infection with avian influenza viruses, the World Health Organization (WHO) carries out monitoring and analysis of potentially pandemic zoonotic influenza viruses in the development of appropriate vaccines .In the current study, we used the enzyme-linked antibody assay (ELLA) to study the formation of antibodies to NA of pandemic and potentially pandemic influenza viruses after LAIV immunization.This study involved a retrospective analysis of serum samples from clinical trials of pandemic and potentially pandemic LAIVs based on A/Leningrad/134/17/57 (H2N2) MDS .The genome composition of almost all vaccine strains was 6:2, except A/17/duck/Potsdam/86/92 (H5N2), which had a 7:1 genome composition; the NA gene of A/17/duck/Potsdam/86/92 (H5N2) was inherited from A/Leningrad/134/17/57 (H2N2) MDS.We also investigated sera obtained from volunteers who were 18–20 years old in the fall of 2005, before and 21 days after vaccination with seasonal trivalent live influenza vaccine (LAIV) 2005–2006. The trivalent vaccine was manufactured by “Microgen”  and contained A/17/New Caledonia/99/145(H1N1), A/17/California/04/06(H3N2), and B/60/Jilin/01/1 (B/Yamagata-like) [Additionally, we tested the archive serum samples collected in the fall of 2010 and 2011 from routine tests, as well as the sera from patients of the Institute for Experimental Medicine Medical Research Center collected in the 2016, and the paired sera from influenza convalescents provided by the Institute of Influenza. The patients were fully informed of the research procedures and any risks associated with participation, and consented to participate in these scientific projects. None of the authors collected any of the samples used in the study, or had access to the confidential information of patients; data were anonymized before being made available to the authors. The sera were stored frozen at −20 °C until testing. Following obtaining the approval from the Local Ethics Committee in the Institute of Experimental Medicine, No. 3/17 of 30 November 2017, these sera were provided for serological tests.To evaluate serum NI antibodies using the enzyme-linked lectin test (ELLA), we developed a number of A/H7NX or A/H6NX viruses .To detect NI antibodies after vaccination with LAIVs based on the A/17/mallard/Netherlands/00/95 (H7N3) vaccine strain, we used the A/tern/South Africa/61 (H5N3) influenza virus.All viruses were cultured in the allantoic cavity of 10-day-old developing chicken embryos (CEs), which were provided by the Sinyavino poultry farm , at 33 °C for 48 h. For ELLA, the viruses were purified and concentrated on a stepwise 30%/60% sucrose gradient, then aliquoted, and stored at −70 °C.We evaluated NI antibodies using ELLA as previously described . BrieflyThe HI test with blood sera was carried out using a 0.75% suspension of human red blood cells (Group “0”) in “U”-bottom 96-well polymer plates, using previously published procedures . The invDetermination of neutralizing antibodies was carried out using the microneutralization (MN) test in Madin–Darby canine kidney (MDCK) cells with no-RDE-treated sera, as previously described .p-value < 0.05 was considered to be statistically significant.Data were analyzed using Statistica software, version 6.0 . Antibody levels were presented as geometric mean titers (GMTs). For statistical analysis, antibody titers were expressed as log2 inverse final dilutions. Comparisons of two independent groups were carried out using the non-parametric Kolmogorov–Smirnov two-sample test. To compare several independent groups, we used the Kruskal–Wallis analysis of variance (ANOVA), followed by multiple pairwise comparisons based on the Kruskal–Wallis sums of ranks. Comparisons of two dependent variables were performed using the Wilcoxon matched pairs test. Fisher’s exact two-tailed test was carried out in the case of nominal variables. A nonparametric measure of the statistical relationship between the two variables was performed using the Spearman rank correlation coefficient (r). A p > 0.05). Pre-vaccination HI antibody titers to new vaccine strains did not exceed the lower detection limits of the HI method, which was determined by the initial serum dilution (1:5–1:10). Revaccination was carried out 21 or 28 days after the first vaccination; the same period after revaccination, blood serum was collected [According to the HI test and ELLA, the initial immunological status of the vaccine and placebo groups of volunteers did not differ  and the drift variant of the pandemic virus A(H1N1)pdm09—the A/South Africa/3626/2013 (H1N1)pdm09 influenza virus .The lowest preexisting NI antibody levels were revealed against A/Anhui/1/13(H7N9) and A/South Africa/3626/13(H1N1)pdm09 influenza viruses .Despite the fact that the A/mallard/NL/12/00(H7N3) virus was never in the circulation, pre-existing antibodies to the NA of the N3 subtype were detected.p < 0.05). These data confirm previously obtained information on the cross-reaction of N1-directed antibodies acquired as a result of infection and vaccination with inactivated vaccines [https://www.who.int/influenza/vaccines/virus/recommendations/summary_a_h1n1pdm09_cvv_nh1617.pdf). From 2009, A/California/7/09(H1N1)pdm09-like viruses circulated, which, from 2010 to 2016, were consistently included in vaccines. Not surprisingly, the level of collective immunity to A/California/7/09(H1N1)pdm09 was, by 2016, significantly higher than that to A/South Africa/3626/13(H1N1)pdm09. It was shown that, in the 2015–2016 epidemic season, the levels of NI antibodies ≥1:40 to A/California/7/09(H1N1)pdm09 amounted to about 30% [Interestingly, before the start of A/H1N1pdm09 circulation, only 7.1% of examined persons had A/California/7/09(H1N1)pdm09-specific NI antibodies, which obviously were cross-reacting antibodies caused by contacts with previously circulating A/H1N1 strains. However, after the pandemic virus entered circulation, the titers of NI antibodies increased not only against A/H1N1pdm09, but also for the NA of the A/Vietnam/1203/04(H5N1) avian influenza virus. In 2011, 46% of the examined volunteers had cross-reacting NI antibodies to A/Vietnam/1203/04(H5N1), compared with 16% in 2005 . This confirms the previously obtained data on the age-related differences in the levels of pre-existing antibodies to NA, which may be associated with first contacts with influenza viruses [A higher proportion of persons with pre-existing NI antibodies against A/H2N2 1966 (year of isolation) was found among elder volunteers compared with younger people ( viruses .There has been relatively low coordination of HA and NA antibody responses (r = 0.24–0.59) in clinical studies of LAIVs. These data confirm the previously obtained data on NI antibodies as independent indicators of influenza immunity, other than hemagglutination inhibiting antibodies . In the n = 56; p < 0.001) and avian A/H5N1 influenza virus .In the next part of the study, we analyzed the cross-reactive properties of antibodies after immunization with seasonal trivalent LAIV. The phylogenetic analysis of NA amino acid sequences of avian A/H5N1 and epidemic and pandemic A/H1N1pdm09 influenza strains demonstrated the higher homology between amino acid sequences of H5N1/2004 and H1N1/pdm09 NA than between NAs of avian A/H5N1 and epidemic A/H1N1 viruses, which had circulated before 2008 (87.9% vs. 82.6%) b. Figurep = 0.01). Post-vaccination titers of HI antibodies after administration of A/17/California/09/38(H1N1)pdm09 LAIV were only slightly higher than pre-vaccination titers pdm09 LAIV with fourfold or more HI antibody rises. It was shown that on day 6–8 after onset of infection, the levels of NI antibodies were significantly higher than at the beginning of the disease (n titers .p = 0.24), although after infection, this ratio was statistically significant (p < 0.0001).Fisher’s exact test revealed no statistical relationship between post-vaccinated antibody seroconversion and two surface glycoproteins of the H1N1/2009pdm09 virus in serum samples obtained from the same individuals after vaccination pdm09 influenza virus after natural infection were higher compared with LAIV vaccination.We demonstrated an increase in the titers of NI antibodies to A/H5N1 virus, not only in patients with an increased level of NI antibodies to the H1N1/2009pdm09 influenza virus, but also when vaccinated with a seasonal LAIV. These data confirm the cross-reactive properties of the NA of the N1 subtype, not only after infection, but also following vaccination with LAIV.A rather low level of coincidence of the antibody levels to HA and NA in the same serum samples after immunization with LAIV confirmed the previously noted autonomy for HI and NI immune responses when assessing the immunogenicity of LAIV. NI antibodies produced in response to LAIVs vaccination may play an independent protective role .Higher proportions of pre-existing NI antibodies against distant A/H2N2 influenza viruses among elderly volunteers compared with younger people suggests that determining NI antibodies can help identify the most susceptible populations for priority vaccination.The absence of antibodies to the A/H7N9 influenza virus among people aged 18–49 indicates their susceptibility to infection, and the need for vaccination in the event of a pandemic threat. Despite the fact that, to other viruses, when there were no HI antibodies, a level of pre-existing antibodies to NA was detected, even to the N3 subtype, which had not previously been registered as in circulation, no preexisting NI antibodies against N9 were detected. This may be because of such features of N9 as an additional sialic acid binding site . When vaA number of LAIVs developed against pandemic and potentially pandemic influenza viruses have effectively elicited an immune response in the seronegative population. After double immunization, an increase in inhibition of the hemagglutinating and neuraminidase activity of influenza viruses, as assessed by post-vaccination sera, was revealed. Combining the traditional HI test with the detection of NI antibodies can provide a more complete assessment of the LAIV immunogenicity. NI antibodies to N1 have a certain degree of cross-reactivity against antigenically distant strains. Low preexisting NI antibodies against newly emerged influenza viruses may indicate a high-risk population for priority vaccination.