CCNF gene, which encodes the cyclin F protein, in cohorts of patients with familial and sporadic ALS and frontotemporal dementia (FTD) ). Cyclin F is a part of a Skp1-Cul-F-box (SCF) E3 ubiquitin-protein ligase complex and is responsible for ubiquitylating proteins for degradation by the proteasome. In this study, we investigated the phosphorylation status of cyclin F and the effect of the serine to glycine substitution at site 621 (S621G) on E3 ligase activity. This specific mutation (S621G) was found in a multi-generational Australian family with ALS/FTD. We identified seven phosphorylation sites on cyclin F, of which five are newly reported including Ser621. These phosphorylation sites were mostly identified within the PEST  sequence located at the C-terminus of cyclin F. Additionally, we determined that casein kinase II (CK2) can phosphorylate Ser621 and thereby regulate the E3 ligase activity of the SCF(cyclin F) complex. Furthermore, the S621G mutation in cyclin F prevents phosphorylation by CK2 and confers elevated Lys48-ubiquitylation activity, a hallmark of ALS/FTD pathology. These findings highlight the importance of phosphorylation in regulating the activity of the SCF(cyclin F) E3 ligase complex that can affect downstream processes and may lead to defective motor neuron development, neuron degeneration and ultimately ALS and FTD.Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by progressive weakness, paralysis and muscle loss often resulting in patient death within 3–5 years of diagnosis. Recently, we identified disease-linked mutations in the  Around 20% of ALS patients show signs of frontotemporal dementia (FTD) and segregation of both ALS and FTD may be seen within individual families, particularly those with mutations in C9ORF72 [SOD1, TARDBP, FUS, UBQLN2, VCP, OPTN and C9ORF72 [Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common and convergent molecular and pathogenic features . ALS is  C9ORF72 ,4. Famil C9ORF72 , and dis C9ORF72 –13. The  C9ORF72 .CCNF gene that occur at similar frequency to mutations in TARDBP [CCNF mutations identified from ALS/FTD patients led to defective protein degradation and signature features of ALS pathogenesis in vitro including elevated ubiquitylation and increased levels of ubiquitylated ribonucleotide reductase M2 (RRM2) and TDP-43 [We have recently identified ALS–FTD linked mutations in the n TARDBP . A p.Serd TDP-43 .Cyclin F is a part of a Skp1-Cul-F-box (SCF) E3 ubiquitin-protein ligase complex which is an integral part of a cell's recycling system that ubiquitylates substrates for degradation by the proteasome. Ubiquitylation is carried out in three main steps: activation by E1s, ubiquitin conjugation by E2s and substrate ligation by E3s. This sequential cascade is the tethering of ubiquitin to lysine residues on proteins via different ubiquitin linkages (such as lysine 48 (Lys48) or lysine 63 (Lys63)), which in turn directs the tagged protein for various purposes including signalling and degradation.There are over 300 known E3 ligases , and it (cyclin F) E3 ligase activity. These findings provide insights into the molecular mechanisms of a newly discovered ALS/FTD gene CCNF, suggesting that the cyclin FS621G mutation affects Lys48-ubiquitylation activity, with potential consequences for protein degradation pathways that may drive disease development. The convergence of phosphorylation and disease-linked mutation of a single residue (S621G) indicates that this site is of particular importance for ALS/FTD.In this study, we investigated the phosphorylation status of cyclin F and the effect of the S621G mutation on its E3 ligase Lys48-specific ubiquitylation activity. We used a phosphoproteomics workflow to identify several novel phosphorylation sites within cyclin F, and identified a key role of CK2 phosphorylation on Ser621 in regulation of SCF2.2.1.Antibodies used in this study were: rabbit polyclonal anti-cyclin F , mouse monoclonal anti-mCherry , rabbit anti-mCherry , rabbit anti-Skp1 p19 (H-163) , mouse monoclonal anti-β-tubulin  and anti-phospho-CK2 substrate . Rabbit antibodies against phospho-cyclin F (immunogen: DQEpSEGEKEG) and pan-cyclin F (immunogen: DQESEGEKEG) were customized and purchased from Bethyl Laboratories. Recombinant casein kinase II was obtained from New England BioLabs. Plasmid DNA constructs were purchased from Genscript 2.2.6 cells in 100 mm plates and maintained in Dulbecco's modified Eagle's medium  supplemented with 10% heat-inactivated fetal bovine serum  and 1% antibiotics  in a 37°C heat-jacket humidified incubator with 5% CO2. Following 48 h of growth (approx. 80% confluence), HEK293 or Neuro-2a cells were transfected with DNA constructs as previously described [S621G cDNA fused to N-terminal mCherry was mixed with Lipofectamine 2000 (Life Technologies) and Opti-MEM (Life Technologies) for each plate and incubated at 37°C for 5 h. Cells were washed with warm PBS, re-fed with complete DMEM media and maintained for 24 h. Transfections were carried out in three biological replicates from different passages. For CK2 inhibition, cells were re-fed with complete DMEM media and treated for 18 h with CX4945 (4 µM) prior to harvesting. For CK2 RNAi knockdowns, 25 pmol of CK2α siRNA I  was co-transfected under the same conditions as with mCherry–cyclin F.Human embryonic kidney (HEK293) or Neuro-2a cell lines were plated at 1 × 10escribed . Briefly2.3.g (30 min at 4°C). Protein concentration was estimated using the Pierce BCA Reagent (Pierce Biotechnology). Typically, 1 µg of antibody per 500 µg of protein extract in NP40 buffer was used for immunoprecipitations. Protein A/G magnetic beads (Pierce) were used to capture the antibody : protein complex. Immunoprecipitation mixtures were washed with TBS containing 1% (v/v) NP40 (3×) to remove non-specifically bound proteins, and then resuspended in either (i) E3 ligase reaction buffer or (ii) 100 mM ammonium bicarbonate pH 8.0 for in-solution trypsin digestion and LC–MS/MS analyses. Triplicate immunoprecipitations were carried out from three biological transfected cell culture replicates.Cells were lysed and total protein was extracted with probe sonication  in NP40 (1% (v/v) Nonidet P-40 in TBS  or RIPA (1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS in TBS) buffer containing 2 mM EDTA, 10 mM N-ethylmaleimide, Complete protease inhibitor cocktail (Roche) and PhosStop inhibitor cocktail (Roche). Cellular debris was pelleted at 16 0002.4.N-morpholino) propane sulfonic acid (MOPS) running buffer  according to the manufacturer's instructions . One-dimensional SDS-PAGE separated proteins were transferred onto either nitrocellulose or PVDF membranes using the Bio-Rad Turbo Transfer apparatus . Blots were blocked with 3% (w/v) skim milk in TBS/T for 1 h and washed in TBS/T (3×). Primary antibodies were diluted in 3% (w/v) BSA in TBS/T and incubated with blots overnight at 4°C.For one-dimensional SDS-PAGE, denatured proteins (20 µg) were separated on 4–12% Bis–Tris pre-cast gels using a 3- [xcel. Graphs were made using GraphPad Prism 5 and Microsoft Excel.After incubation, membranes were washed in PBS/T (3×) for 10 min each before fluorescently labelled IRDye 800CW goat anti-rabbit IgG (1 : 10 000) was applied for 1 h at room temperature. Proteins were imaged using a Li-Cor Odyssey imaging system at the appropriate wavelength. Densitometry analysis was conducted using I Health)  and stat2.5.In vitro phosphorylation of naked DQESEGEKEG peptide  was carried out according to the manufacturer's instructions . Briefly, the naked peptide (0.2 mg) was resuspended in 180.6 µl of MilliQ water (1 nmol µl−1). Approximately 0.1 nmol of peptide was added to the reaction buffer  with recombinant CK2 (250 U) and 400 µM ATP. The in vitro phosphorylation assay was carried out in triplicate and incubated at 30°C for 2 h. Inhibition of CK2 phosphorylation was carried out in the presence of CX4945 (4 µM). The reaction mixture was stopped by addition of 2% (v/v) formic acid, desalted using a C18 Sep-Pak and eluted in 90% ACN, 0.1% formic acid and dried under vacuum centrifuge.2.6.18 Sep-Pak cartridge and eluted in 90% ACN, 0.1% formic acid and dried under vacuum centrifugation.Immunoprecipitated proteins were reduced and alkylated with 10 mM DTT and 55 mM iodoacetamide respectively, and digested with trypsin or Asp-N (1 : 50 enzyme : protein) overnight at 37°C. The digestion was inactivated by the addition of 2 µl of formic acid  formic acid). Tryptic or Asp-N digested peptides were cleaned and enriched on a pre-equilibrated C−1 glycolic acid, 80% (v/v) ACN, 5% (v/v) TFA and added to TiO2 beads  prewashed in the same solution [g to recover the beads. The supernatant was removed and re-added to a fresh batch of TiO2 beads and repeated. TiO2 beads from both incubations were washed 2× in 80 mg ml−1 glycolic acid, 80% (v/v) ACN, 5% (v/v) TFA, 2× in 80% (v/v) ACN, 1% (v/v) TFA, and 1× in 20% (v/v) ACN, 0.1% (v/v) TFA. The beads were briefly dried under vacuum centrifuge. Phosphopeptides were eluted sequentially with 1% NH4OH, 1% NH4OH/30% ACN and 1% NH4OH/50% ACN. The combined eluates were pooled and lyophilized under vacuum centrifugation.Lyophilized peptides were resuspended in 80 mg mlsolution . The mix2.7.18 material  at 5 µl min−1 for 6 min before being eluted onto an analytical column packed with Michrom Magic C18  using a nanoLC system (Thermo) with a nanoflow solvent delivery of 300 nl min−1. Each sample was separated on a 60 min gradient  with a flow rate of 300 nl min−1. The peptides were eluted and ionized into a Q-Exactive or LTQ Orbitrap Velos mass spectrometer (Thermo Fisher). The electrospray source was fitted with an emitter tip 10 µm  and maintained at 2.5 kV electrospray voltage. Precursor ions were selected for MS/MS fragmentation using a data-dependent ‘Top 10’ method operating in FTMS acquisition mode with HCD (Q-Exactive) or CID (Orbitrap) fragmentation. Precursor ions were selected for MS/MS fragmentation using a data-dependent ‘Top 10’ method operating in FT–FT acquisition mode with CID or HCD fragmentation. IT-MS and FT-MS on the Orbitrap Velos was carried out with a survey scan range between m/z 350–1800 Da with MS/MS threshold of 500 ions for CID, and MS/MS threshold of 5000 ions for HCD, with an isolation width of 2.0 Da and normalized collision energy of 35%.Peptide fractions were injected onto a 2 cm desalting trap column packed with YMC C6 ions in full MS; and MS/MS scans were carried out at 17 500 resolution with an AGC target of 2 × 105 ions. Maximum injection times were set to 120 and 60 ms, respectively. The ion selection threshold for triggering MS/MS fragmentation was set to 25 000 counts and an isolation width of 1.9 Da was used to perform HCD fragmentation with normalized collision energy of 27%.FT-MS analysis on the Q-Exactive was carried out with a 35 000 resolution and an AGC target of 1 × 10roteome Discoverer 1.4 software  incorporating the Mascot search algorithm . Peptide identifications were determined using a 10 ppm precursor ion tolerance and a 0.05 Da MS/MS fragment ion tolerance. Carbamidomethylation modification of cysteines was considered a static modification while oxidation of methionine, acetyl modification on N-terminal residues and phosphate on serine, threonine and tyrosine were set as variable modifications. MS/MS spectra were searched through Proteome Discoverer (Thermo Scientific) using the Mascot algorithm against the UniProt Homo sapiens database . The protein sequence for the mutated cyclin FS621G was added manually to a custom database which included the UniProt sequences. The data were processed through the Xtract and MS2 Processor nodes together with a direct search and the combined searches were sent to Percolator  for estimation of false discovery rates. Protein identifications were validated employing a q-value of 0.01  within the Proteome Discoverer software. Phosphosite occupancy was validated using the PhosphoRS 3.1 node within the PD 1.4 with a fragment mass tolerance of 0.5 Da [Spectra files (*.RAW) were processed using the Pf 0.5 Da . The dat2.8.2 and resuspended in 25 µl assay buffer . Immunoprecipitated mCherry, mCherry–cyclin FWT, mCherry–cyclin FS621G and pre-immune rabbit control IgG were then added to wells containing the Enzyme cocktail . Each ligase reaction was activated by the addition of ATP (0.4 mM) and incubated at RT for 30 min. Each well was washed with PBS/T (3×), incubated with detection solution 1 for 60 min at RT, followed by incubation with streptavidin–HRP for 60 min at RT. Luminescence was read on a FLUOstar OPTIMA reader (BMG Labtech).E3 ligase activity was determined using the E3LITE Customisable Ubiquitin Ligase ELISA Kit  as per the manufacturer's instructions. UBE2D3 was selected as the E2 enzyme and Lys48-ubiqitin was selected as the ubiquitin substrate. Briefly, empty mCherry vector, mCherry–cyclin F or pre-immune rabbit control IgG were immunoprecipitated from transfected HEK293 cells as described above. Protein A/G beads were washed (3×) in 100 mM Tris-HCl (pH 8.0), 10 mM MgCl3.(cyclin F) was phosphorylated and required for regulating its Lys48-specific E3 ligase activity. We determined that phosphorylation at Ser621 is carried out by CK2, and inhibition of CK2 with CX4925 blocks phosphorylation at this site and subsequently elevates SCF(cyclin F) E3 ligase activity. Further, a phosphomimetic created by mimicking constitutive phosphorylation of Ser621 through mutation to an aspartic acid (S621D) led to reduction in the ubiquitylation activity of cyclin F. Therefore, phosphorylation at Ser621 appears to act as a signal for cyclin F to suppress its E3 ligase activity, which can be regulated by controlling CK2 kinase activity or CK2 gene expression. Notably, we demonstrate that a recently identified S621G mutation in cyclin F which causes ALS/FTD [In this study, we identified that Ser621 on SCF ALS/FTD  also lea3.1.(cyclin F) complex is one of the many E3 ligases that are responsible for ubiquitylating proteins for UPS degradation. Using a commercial ELISA, we screened for four different E2 conjugating enzymes  that promiscuously select a large range of substrates [a). Neither UBE2L3 nor UBE2R1 showed Lys48-ubiquitylation activity, suggesting that these two E2s did not form the E1–E2–E3 triad with the immunoprecipitated SCF(cyclin F) complex. UBE2E3 showed E2 Lys48-conjugation activity approximately twofold above the background (no E2) . UBE2D3 showed high Lys48-specific ubiquitylation activity and was approximately eightfold higher than UBE2E3 and 16-fold higher than the background controls . Based on these data, UBE2D3 was selected as the E2 conjugating enzyme to be used in downstream E3 ligase activity assays. Immunoblots were carried out following E3 ligase activity assays to demonstrate equivalent levels of immunoprecipitated mCherry–cyclin F used for activity assays (a).The SCFbstrates  . Given that serine residues are well-described phosphosites that often regulate enzymatic activity, this suggests that the phosphorylation of the Ser621 site might regulate ubiquitylation activity of cyclin F . Accordingly, following complete dephosphorylation the E3 ligase activity of immunoprecipitated SCF(cyclin F) was significantly decreased in both mutant and wild-type cyclin F (b). This effect was greater in the wild-type  than the mutant cyclin FS621G . This led us to speculate that the activity of the SCF(cyclin F) complex was regulated by phosphorylation of multiple sites, including Ser621, which would explain the difference seen in the decreased activity of wild-type compared with mutant cyclin FS621G following dephosphorylation. Immunoblots were carried out following E3 ligase activity assays and demonstrated that the ubiquitylation activity measurements were performed on equivalent levels of immunoprecipitated mCherry–cyclin F (b).To determine the E3 ligase activity, wild-type and ALS/FTD mutant cyclin Fcyclin F b. This ecyclin F b.3.2.in vitro ubiquitylation assays (http://www.cbs.dtu.dk/services/NetPhos/). NetPhos predicted with high confidence 88 potential phosphorylation sites in cyclin F, which included Ser621. To determine phosphorylation sites on cyclin F, transfected mCherry–cyclin F was enriched by immunoprecipitation, digested with trypsin, phosphopeptides enriched by TiO2 beads, and analysed by LC–MS/MS. Using this digestion procedure, we identified two phosphorylation sites on cyclin F at Ser577 and Ser754 (2 and subjected them to LC–MS/MS. Using this strategy we observed specific diagnostic ions and loss of phosphate ions (PO4) to detect an additional four phosphosites on cyclin F: Thr588, Thr590, Ser621, Ser709 and Ser713 [The n assays  suggest d Ser754 . The regd Ser713  (table 1a) and wild-type mCherry–cyclin FWT (b) was confirmed by MS/MS fragmentation. To verify this phosphorylated sequence, we subjected a synthetic phosphorylated (c) and unphosphorylated (not shown) versions of the Asp-N digested peptide (DQESEGEKEG) to LC–MS/MS. We confirmed the MS/MS fragmentation spectra matched between both synthetic and immunoprecipitated peptides. We also immunoprecipitated mCherry–cyclin F and dephosphorylated the IP prior to LC–MS/MS analysis and observed MS/MS fragmentation patterns that were reminiscent of the unphosphorylated version of the peptide (d), further confirming that Ser621 is a phosphorylation site. The MS/MS spectra that confirm the phosphosites described in The phosphorylation status of immunoprecipitated mCherry–cyclin F containing the S621G mutation a and wilclin FWT b was conorylated c and unp peptide d, furtheWT were phosphorylated at Ser621 with negligible amounts detected in the mutant cyclin FS621G and empty vector transfected cells (e). We detected reduced phosphorylation by approximately 0.37-fold  of phospho-specific signal in cyclin FS621G mutant cell lysates compared with cyclin FWT lysates (e). We predicted that the non-phospho-specific antibody would detect less of the mutant cyclin FS621G than the wild-type cyclin F, and confirmed that the antibody reactivity observed was indeed cyclin F. These data confirm the mass spectrometric identification of phosphorylated cyclin F at Ser621. Taken together, using both trypsin and Asp-N digestion approaches seven phosphorylation sites were identified, of which five have not been reported previously  and two have been previously characterized  [Using an alternative method, we examined the phosphorylation of cyclin F by immunoblots using customized antibodies raised against the phosphopeptide sequence DQEpSEGEKEG and non-phosphorylated DQESEGEKEG (Bethyl Laboratories). Although there was very minor non-specific reactivity observed with the phospho-specific cyclin F antibody, we verified that cells expressing only mCherry–cyclin Fed cells e. We det lysates e. We pre3, S754) ,33  prior to proteolysis and is required for regulating cell cycle progression such that impairment of SCF function attenuates cyclin D1 ubiquitylation, promoting cyclin D1 overexpression and accelerated cell-cycle progression [(cyclin F) complex. Using the group-based prediction system (GPS) [Many SCF complexes function by ubiquitylating substrates in a phosphorylation-dependent manner  such thagression . Cyclin gression . Therefoem (GPS) , numeroua), we performed an in vitro phosphorylation assay with recombinant CK2 and ATP. The reaction was analysed by LC–MS/MS, which revealed phosphorylation on serine within the synthetic peptide that corresponds to Ser621 in cyclin F that eluted at 7.9 min (b). We repeated the in vitro phosphorylation assay in the presence of the CK2 inhibitor CX4945 and analysed the reaction by LC–MS/MS (c). Using extracted ion chromatograms (EICs) and matching retention times of 7.6 and 7.9 min to respectively identify the unphosphorylated and phosphorylated peptides and their specific masses, a mixture of 45% naked DQESEGEKEG peptide (m/z 554.2) and 55% phosphorylated DQEpSEGEKEG peptide (m/z 594.2) was observed. CX4945 treatment decreased recombinant CK2 phosphorylation activity by approximately 55% based on area-under-curve measurements (d). We suspect that the incomplete inhibition of CK2-mediated phosphorylation of Ser621 by CX4945 was likely caused by the presence of a 100-fold higher concentration of ATP than CX4945 (which was required for the in vitro ubiquitylation assay), as CX4945 acts by blocking the ATP-binding site in CK2 (and therefore CX4945 and ATP are competing for the same site in CK2) [Casein kinase II (CK2) was predicted to phosphorylate Ser621 with the surrounding sequences falling within the CK2 substrate motif (S–X–X–D/E). Using the synthetic naked peptide (DQESEGEKEG) which eluted at 7.6 min a, we per 7.9 min b. We repLC–MS/MS c. Using urements d. We sus in CK2) . Attempt(CyclinF) E3 ligase activity in the presence of CK2 inhibition by transfecting HEK293 cells with cyclin FWT and cyclin FS621G followed by treatment with CX4945 for 18 h. In cells treated with CX4945, immunopurified cyclin FWT had approximately 1.35-fold elevated Lys48-ubqiuitylation activity compared with the vehicle controls (a), while CX4945 treatment did not affect the activity of immunopurified mutant cyclin FS621G based on E3 ligase activity assays. We verified that equivalent levels of immunoprecipitated mCherry–cyclin F were used in the ubiquitylation assay, by probing for mCherry and cyclin F at the completion of the E3 ubiquitylation activity assays (a). We immunoblotted transfected cell lysates treated with CX4945 using the phospho-specific cyclin F (Ser621) antibody, and observed approximately 32% less Ser621 phosphorylation  in CX4945 treated cyclin FWT cells (b). To confirm that the CX4945 effectively impairs CK2 activity in these cell-based assays, we performed immunoblotting analyses to detect CK2 phosphorylated substrates containing the S–X–X–D/E motif. Inhibition of CK2 activity by CX4945 resulted in approximately 39% and 37% decreased phosphorylation of substrates in cells transfected with cyclin FWT and cyclin FS621G, respectively. The detection of residual CK2 substrates in cells treated with CX4945 is likely due to: (i) endogenous CK2 phosphorylation events that occurred prior to CX4945 treatment because CK2 is constitutively active regulating numerous cellular processes [d).Next, we examined SCFcontrols a, while y assays a. We immWT cells b. To conrocesses  and/or  in E3 ligase activity (c). We immunoblotted for phospho-specific cyclin F at Ser621 from transfected cell lysates, and observed approximately 0.61-fold less Ser621 phosphorylation  in CK2 RNAi knockdown cells expressing cyclin FWT compared with those with scramble siRNA control, further suggesting that CK2 is capable of phosphorylating this site (d).This finding was recapitulated by siRNA-mediated knockdown of CK2α expression in cells expressing mCherry–cyclin Factivity c. We immhis site d.S621G that may contribute to the abnormal hyperubiquitylation of proteins, which are a hallmark pathology of ALS and FTD. This suggests that phosphorylation of Ser621 serves to turn off cyclin F activity. To confirm this, we generated a phosphomimetic by mutating Ser621 to an aspartic acid (S621D). As expected, the phosphomimetic cyclin FS621D was approximately 0.8-fold less active than the wild-type cyclin F   . The con(cyclin F) complex E3 ligase activity. Notably, the ALS/FTD-causing S621G missense mutation confers elevated E3 ligase activity (We present a series of biochemical data that collectively suggest that CK2 phosphorylation of cyclin F at Ser621 is crucial for regulating SCFactivity . Our fin


To the Editor: Outbreaks of highly pathogenic avian influenza (HPAI) A(H5N1) virus among poultry were first reported in Africa in 2006, with initial reports from Nigeria (http://platform.gisaid.org) and GenBank databases , polymerase basic protein 2 (PB2), nucleoprotein, and neuraminidase (NA) genes . Sequencatabases , panel Aatabases , panel BOur phylogenetic analysis suggests that the Ghana HPAI H5N1 strains belong to clade 2.3.2.1c, as was reported for the 2015 Nigeria outbreak. Because of the high homology (>90%) between the Ghana and Nigeria strains, the HPAI H5N1 Ghana outbreak strain likely originated in Nigeria , panel ABecause the HPAI H5N1 virus of clade 2.3.2.1c has previously caused deaths in humans, the potential risk for transmission from infected poultry to humans is a major concern. Increased vigilance and rapid implementation of countermeasures are required to mitigate further virus adaptation and potential outbreaks among humans.Details of methods and of mutations in the highly pathogenic avian influenza A(H5N1) virus found in poultry in Ghana, 2015.


GLA (IVS4+919G>A) that causes aberrant alternative splicing resulting in Fabry disease has been reported, its molecular mechanism remains unclear. Here we reported that upon IVS4+919G>A transversion, H3K36me3 was enriched across the alternatively spliced region. PSIP1, an adapter of H3K36me3, together with Hsp70 and NONO were recruited and formed a complex with SF2/ASF and SRp20, which further promoted GLA splicing. Amiloride, a splicing regulator in cancer cells, could reverse aberrant histone modification patterns and disrupt the association of splicing complex with GLA. It could also reverse aberrant GLA splicing in a PP1-dependant manner. Our findings revealed the alternative splicing mechanism of GLA (IVS4+919G>A), and a potential treatment for this specific genetic type of Fabry disease by amiloride in the future.While a base substitution in intron 4 of  GLA gene at Xq22. The enzymatic defect leads to the accumulation of globotriaosylceramide (Gb3) and related glycosphingolipids throughout the body, causing multisystem disease [GLA gene, the intronic mutation at nucleotide 9331 (IVS4+919G>A) is reported to be a cardiac variant Fabry mutation [GLA transcript, generating a premature stop codon. The alternatively spliced transcript with 57 nt insertion is rarely expressed in most normal human tissues, but it is predominantly expressed in Fabry disease patients with the IVS4+919G>A mutation. Although the alternatively spliced transcript is reported to be responsible for the reduced enzyme activity causing Fabry disease, the mechanism of GLA splicing is unclear.Fabry disease (FD) is an X-linked lysosomal disorder caused by a deficiency of α galactosidase A (GLA), due to mutations in the  disease . Cardiacramide Gb and rela 9331 IVS+919G>A imutation . This incis sequence elements that define the exon-intron boundaries (the 5’ and 3’splice sites), and catalyzes the splicing reaction. Additional cis-acting elements, known as exonic and intronic splicing enhancers or silencers , also play a role in the regulation of splicing. Trans-acting factors, such as serine/argine-rich (SR) family proteins and heterogeneous nuclear ribonucleoproteins (hnRNPs), can bind to cis-acting elements and interact with the spliceosomal complex, thereby controlling the splicing outcomes [GLA (IVS4+919G>A) in Fabry disease from chromatin signatures to splicing machinery.Alternative splicing, a process that joins different 5’ and 3’ splice sites of an RNA transcript sequence, plays a major role in protein diversity. Splicing of pre-mRNA has been known to be regulated by the spliceosome and approximately 200 additional proteins . The sploutcomes , 9. In aoutcomes . Recent outcomes , 12. H3Koutcomes . H3K36meoutcomes . TherefoGLA were shown in The genetic organization and splicing pattern of GLA was quantified by real-time PCR using primer and probe sets targeting exon 4, intron 4 (cryptic exon), and exon 5. Schematic representation of position and sequence of primer/probe sets for real-time PCR are illustrated in GLA intron 4 might be involved in transcriptional activation rather than pre-mRNA splicing regulation. Further studies are needed to elucidate which modification on histone H3 is involved in the regulation of GLA alternative splicing.To investigate the correlation between histone modifications and alternative splicing, chromatin immunoprecipitation (ChIP) assays were performed using antibodies against a set of histone modifications in FD cells and normal cells. The relative enrichment of each histone modification on GLA splicing. To reveal whether proteins associated with the DNA template played a role in the regulation of GLA (IVS4+919G>A) splicing, streptavidin beads were used to pull down biotin-labelled DNA probes and its associated proteins. Three separate biotin-labelled DNA probes were synthesized and the sequences were shown in GLA splicing, we employed an shRNA approach to reduce the expression of HSP70 or NONO in FD cells. Knockdown efficiency was confirmed by western blot analysis  to enhance the expression of Hsp70 [GLA (IVS4+919G>A) splicing.Histone modifications can affect pre-mRNA splicing by directly recruiting an adaptor protein, which in turn recruits splicing factors to the nascent RNA. Thus, proteins associated with the DNA template might play a role in the regulation of analysis . Knockdoof Hsp70  enhancedGLA (IVS4+919G>A) alternative splicing, a biotin pull-down assay was performed. Three separate biotin-labelled RNA probe were synthesized and the sequence were shown in GLA (IVS4+919G>A) splicing  splicing in normal cells  in FD cells. RT-PCR and western-blot analysis demonstrated that amiloride induced the cryptic exon exclusion of GLA  splicing, and the phosphorylation levels of SF2/ASF and SRp20. The phosphorylation level of hnRNPs, however, was not affected , and mediated the alternative splicing in a PP1-dependant manner.Western blot analysis further revealed that amiloride down-regulated the expression and phosphorylation levels of SF2/ASF and SRp20 in a dose-dependent manner , but it activity . With thaffected . These rTo explore the effects of amiloride on histone modifications in the cryptic exon region, CHIP assays were performed. Although amiloride had no significant effects on H3K27me3 and H3K36me3, it reversed aberrantly elevated levels of H3K4me3, H3S10P and H3 acetylation, and aberrantly reduced levels H3K9me3 in FD cells . These dThe splicing mechanism of the IVS4+919G>A transversion in Fabry disease, which leads to a cryptic exon creation, is unclear. In this study, we clarified its mechanism from chromatin signatures to splicing machinery.GLA (IVS4+919G>A). We also demonstrated that H3K36me3, PSIP1, SF2/ASF and SRp20 associated with each other and played a role in regulating GLA (IVS4 + 919G>A) splicing. Therefore, upon IVS4+919G>A transversion, the enrichment of H3K36me3 may contribute to exon definition and recognition by PSIP1, which in turn recruits it splicing factors and results in the cryptic exon inclusion of GLA.Histone modifications are differentially distributed with respect to intron-exon boundaries, and this differential marking contributes to exon recognition and alternative splicing regulation , 33. ForGLA splicing might through slowing down RNA polymerase II elongation rate to provide enough time for the splicing factors to recognize the weak splicing site, resulting in the cryptic exon inclusion. Further studies are required to assess this hypothesis. NONO has been reported to bind to the polymerase II CTD and RNA transcript simultaneously [GLA. Moreover, we found NONO was associated with Hsp70, SF2/ASF, and SRp20, suggesting its splicing effect might through mediating interactions between transcriptions and splicing machineries.From DNA associated proteins analysis, we found Hsp70 and NONO could specifically recognize the alternatively spliced region containing the mutant sequence (IVS4+919A). Hsp70, well-known as cytosolic chaperone, has been demonstrated to play a role in repairing heat-disrupted splicing and in inhibiting nuclear translocation of the splicing kinases , 36. It aneously . It has GLA (IVS4+919G>A) enrichs the A/C predominance for the sequence, acting as an exonic splicing enhancer (ESE) and leading to the inclusion of the cryptic exon. Instead, Palhais et al. have described that GLA (IVS4+919G) harbors an hnRNP A1-binding exonic splicing silencer (ESS) that prevents the cryptic exon inclusion. From our results, we revealed that hnRNP A1 played an inhibitory role in GLA splicing in normal cells. Upon IVS4+919G>A transversion, an hnRNPA1-dependent splicing silencer motif was disrupted, resulting in an increased recognition of the alternative splice site by SR proteins, including SF2/ASF and SRp20, which further recruits the spliceosome to the cryptic exon, leading to the inclusion of 57-nucleotide of intron 4.Lai et al. have described that there are cryptic donor and accepter splice sites, which are not normally activated, in the cryptic exon of normal individuals. Ishii et al. have proposed that GLA, amiloride may play a role in Fabry disease treatment. Consistent with previous study, amiloride regulated the alternative splicing of GLA through a PP1-mediated splicing mechanism. In addition, amiloride induced NONO disassociated from the alternatively spliced region. PP1 has been shown to mediate the splicing effects of NONO through regulating its phosphorylation status. Hyperphosphorylated NONO participates in pre-mRNA alternative splicing, whereas dephosphorylated NONO participates in constitutive pre-mRNA splicing [GLA through the dephosphorylation of NONO.Amiloride is potentially a good agent for cancer therapy by modulating the alternative splicing of various cancer genes . By modusplicing . TherefoGLA splicing in normal cells. Upon IVS4+919G>A transversion, the expression of H3K36me3 is enriched on the cryptic exon area in Int4 of GLA. PSIP1, an adapter of H3K36me3, together with Hsp70 and NONO are recruited and forms a complex together with the splicing factor, SF2/ASF and SRp20. Besides, this transversion diminishes the splicing silencer motif recognized by hnRNP A1, resulting in an increased recognition of the alternative splice site by SR proteins. Hsp70, NONO, and SR proteins work together to facilitate the recognition of alternative splice site by splicing machinery, resulting in the inclusion of the cryptic exon (GLA (IVS4+919G>A) splicing through a PP1 dependent manner, and suggest its role in the treatment of the specific genetic type of Fabry disease. These significant findings reveal the alternative splicing mechanism of GLA (IVS4+919G>A), and a potential treatment for this specific genetic type of Fabry disease.In conclusion, we discover that hnRNP A1 plays an inhibitory role in the cryptic exon of tic exon . We also320, phospho-Akt at Ser473, phospho-Akt at Thr308 from Cell Signaling Technology; anti-hnRNP A1 from Sigma; anti-hnRNP A2B1 from Acris; anti-SRp20 and SF2/ASF from Thermo Scientific; anti-H3K4me3, anti-H3K9me3, anti-H3K27me3, anti-H3K36me3, anti-H3K9me3, anti-H3K9A, anti-H3K9A, anti-H3K23A, anti-H3K27A, anti-H3S10P, anti-histone H3, anti-actin, and anti-HSP70 from Abcam; anti-p54nrb/NONO from Affinity Bioreagents; anti-PSIP1 from Bethyl Laboratories.Antibodies were purchased from the following companies: anti-GLA from Santa Cruz; anti-PP1, Akt, phospho-PP1 at ThrThe study cohort included adult patients (aged 20 years and older) with Fabry disease diagnosed at Taipei Veterans General Hospital from 2015 to 2017. Epstein-Barr virus-transformed lymphoblast cell lines from FD patient with the IVS4+919G→A mutation and health person were obtained in accordance with an Institutional Review Board-approved protocol at the Taipei Veterans General Hospital (TVGHIRB-2015-04-010C) and informed written consent was obtained from each participant in accordance with the ethical guidelines of the Declaration of Helsinki.2. Amiloride was purchased from Sigma-Aldrich and was dissolved in DMSO  to make 500 mM stock solutions. 4β-Hydroxywithanolide E was provided by Professor Y.C., Wu, and was dissolved in DMSO to make 10 mM stock solutions. C646 and HAT inhibitor VII were purchased from Sigma-Aldrich and Millipore. Okadaic acid was obtained from Sigma-Aldrich. C646, HAT inhibitor VII, and okadaic acid were dissolved in DMSO to make 10 mM stock solutions. Serial dilutions were made in DMSO to obtain final dilutions for cellular assays.The Epstein-Barr virus-transformed lymphoblast cells were grown in RPMI-1640 medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum, 100 U/ml penicillin and 100 ug/ml streptomycin at 37°C in a humidified atmosphere containing 5% COThe GLA enzyme activity was measured according to the method described by Desnick et al . In brieGLA alternatively spliced mRNA isoforms, PCR was performed with forward primer 5’- GTCCTTGGCCCTGAATAG -3’ and reverse primer 5’- GTCCAGCAACATCAACAATT -3’. The PCR was performed with a denaturing step at 94°C for 5 minutes, then 35 cycles of 30 seconds at 94°C, 30 seconds at 58°C and 1 minute at 72°C, followed by a final 5 minutes at 72°C. The PCR products were separated on 2.5% agarose gel and the intensity of the PCR products were analyzed by LabWorks Image Acquisition and Analysis Software (UVP BioImaging Systems). DNA gel bands of these RT-PCR products were isolated for sequencing to verify the authenticity of spliced isoforms.We extracted mRNA from the cells using mRNA capture kit , and converted it into cDNA using RevertAid RT Reverse Transcription Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. For analysis of Cytoplasmic and nuclear fractions of cells were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to the manufacturer's protocol. Total cellular proteins were obtained using Pierce™ IP Lysis Buffer (Thermo Scientific). Protein samples were separated by SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Millipore). The membrane was blocked with 5% BSA and then exposed to the appropriate concentrations of primary antibodies at 4°C overnight. Following PBST washes, membranes were incubated in the appropriate horseradish peroxidase-conjugated secondary antibody for detection by chemiluminescence kit (Amersham Life Science). Intensity of the signals was measured using LabWorks software (UVP BioImaging Systems).Coimmunoprecipitation was performed with Pierce Crosslink Magnetic IP/Co-IP kits (Thermo Scientific) according to the manufacturer's protocol. Briefly, 5 μg antibody were covalently cross-linked to 25 μl Protein A/G Magnetic Beads. Equal amounts of cell lysates were immunoprecipitated with the antibody-crosslinked beads at room temperature for 2 hours. After washing, the precipitates were dissociated from the antibody-linked beads by a low-pH elution buffer and analyzed by Western blotting.GLA were conjugated with 50 μl of streptavidin resin (Pierce) at 4°C for 30 min. 250 μl of Biotin Blocking Solution was added to block available streptavidin site with free biotin. After three times washing with Tris Buffered Saline, a 300 μl reaction mixture containing 50 μl of nuclear extract and 50 μl of streptavidin beads conjugated with biotinylated RNA was incubated under specific conditions  at 4°C for 60 min. After extensive washing with NET-2 buffer  containing 0.25% (wt/vol) NP-40, resin-bound proteins were eluted by addition of 250 elution buffer, digested by enzyme and then subjects to Nanoscale capillary liquid chromatography tandem MS (LC-MS/MS) analysis.Ten micrograms of biotinylated DNA or RNA oligonucleotide corresponding to The pLKO shRNA vectors were obtained from the National RNAi Core Facility . Lentiviral packaging was performed according to the manufacturer's protocol. Briefly, lentiviral construct was transfected into HEK293T cells using pPACKH1 Lentivector Packing Kit and PureFection Transfection Reagent . Virus-containing medium was collected at 72 hours post-transfection. Titrated virus-containing media were used for cells infection.ChIP assays were performed with ChIP-IT® Express Chromatin Immunoprecipitation Kits (Active Motif) according to the manufacturer's protocol. In brief, cells were grown to 90% confluence in 150-mm dishes. After crosslinking, cells were lysed in ice-cold complete lysis buffer on ice for 30 min. Nuclei were pelleted at 2400 g for 10 minutes at 4°C, and were resuspended in complete shearing buffer. Chromatin was sheared into 100 bp to 1000 bp fragments by sonication. Ten micrograms of total chromatin was incubated overnight at 4°C with 1 μg antibody. After washing, the immune complex was eluted by adding 100 μl of elution buffer. Subsequently, 2 μl of 5 M NaCl is added to reverse the formaldehyde cross-linking at 65°C for 1.5 h. Following incubation with proteinase K, DNA was obtained and analyzes by real-time PCR. The PCR is performed in a final volume of 10 μl using a LightCycler instrument (Roche Diagnostics) according to the manufacturer’s recommendations. Real-time PCR was performed with the following prime/probe pairs:CCCTCTGTCCATTCATTCTTCForward Primer: GTCAAAGTCAGACAAGGTCCReverse Primer: TATTTGTTGACTTGTTACCATGTCTCCCCACTProbe: S1 FigWestern blot analysis showed the knockdown efficiency of HSP70 (A) and NONO (B) in FD cells. Histone H3 was used as an internal control.(TIF)Click here for additional data file.S2 FigReal-time PCR analysis of various target genes knockdown efficiency in FD cells (A) and normal cells (B), respectively. GAPDH gene was used as an internal gene. Data represent the means±S.D. of three independent experiments. *P<0.05, compared with shCon.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


The anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitides are autoimmune diseases associated with significant morbidity and mortality. They often affect the kidney causing rapidly progressive glomerulonephritis. While signalling by complement anaphylatoxin C5a though the C5a receptor is important in this disease, the role of the anaphylatoxin C3a signalling via the C3a receptor (C3aR) is not known. Using two different murine models of anti-myeloperoxidase (MPO) glomerulonephritis, one mediated by passive transfer of anti-MPO antibodies, the other by cell-mediated immunity, we found that the C3aR did not alter histological disease severity. However, it promoted macrophage recruitment to the inflamed glomerulus and inhibited the generation of MPO-ANCA whilst not influencing T cell autoimmunity. Thus, whilst the C3aR modulates some elements of disease pathogenesis, overall it is not critical in effector responses and glomerular injury caused by autoimmunity to MPO. The anti-neutrophil cytoplasmic antibody (ANCA) associated vasculitides (AAV) are diseases in which autoimmunity to the neutrophil granule proteins myeloperoxidase (MPO) or proteinase-3 (Pr3) can cause multi-organ injury, including rapidly progressive glomerulonephritis. The pathogenesis of AAV involves multiple steps. T and B cell tolerance to MPO or Pr3 is lost, resulting in the secretion of autoantibodies (ANCA). ANCA can bind to their cognate autoantigen on primed neutrophils, inducing them to activate and lodgThe complement system is an important component of innate immunity. Three pathways can activate complement: the classical, alternative and lectin. These pathways converge on the generation of a C3 convertase. C3a is a bioactive split product of C3 produced, along with C3b, by the action of the C3 convertases. C3a is rapidly inactivated by cleavage of the C-terminal arginine to form C3a-desArg. The cellular receptor for C3a, the C3aR, is a G-protein coupled receptor with 7 trans-membrane domains and high homology to the human C5aR1. Activation of the receptor leads to intracellular calcium mobilisation .Complement has emerged as an important mediator of disease in AAV. Murine studies revealed that complement, activated via the alternative pathway and signalling through C5aR is required for ANCA-induced neutrophil activation and glomerulonephritis –10. Suppin vitro [C3ar-/- mice[Although circulating levels of C3a are elevated in patients with active AAV , whetherin vitro . This isin vitro . Howeverin vitro , the genin vitro  and B cein vitro responsesin vitro  and mastin vitro  there arr-/- mice in two c-/- mice [2 at the end of experiments.All mice were on a C57BL/6 background. C3ar-/- mice  were kinTACAATATAGTCAGTTGGAAGTCAGCC), NeoA (TGGGCTCTATGGCTTCTGAGGCGGAAAG), and A201+ (GAGAATCAGGTGAGCCAAGGAGAAG). GoTaq Green Master Mix (Promega) was used for the PCR reaction. The PCR was run at 95°C for 1 minute, followed by 40 cycles of 95°C for 15sec, 57°C for 15sec, 72°C for 30sec, then a final elongation step at 72°C for 30sec and a holding step of 4°C. The primers C1 and NeoA yield a fragment of 537 bp denoting the C3ar-/- allele. Primers C1 and A201+ yield a fragment of 726 bp, denoting WT allele. All mice tested had the C3ar-/- genotype  (Sigma-Aldrich) was administered by a single intra-peritoneal injection. After 6 days, 24 hours before the end of experiments, mice were placed in metabolic cages for urine collection.To induce glomerulonephritis age matched 6–10 week old C57B/L6 mice and C3ar2 inhalation.Autoimmune anti-MPO glomerulonephritis mice was induced as previously described . BrieflyGlomerular abnormalities were assessed on periodic acid-Schiff (PAS) stained, 4μm, formalin fixed, paraffin embedded sections using coded slides. Abnormalities scored included mesangial proliferation, capillary wall thickening, glomerular necrosis  and crescent formation (defined as two or more cells visible in Bowman’s space). A minimum of 30 glomeruli per mouse were examined. To evaluate leukocyte infiltrate, kidneys were fixed in periodate lysine paraformaldehyde for four hours then washed with 20% sucrose. 5μm sections were stained with a three layer immunoperoxidase technique  using GK5 cells per well. Cells were incubated for 18 hours at 37°C with 10μg/ml heat inactivated recombinant murine MPO (rMPO). Total anti-MPO IgG was measured by ELISA on MPO coated plates, with IgG detected with sheep-anti mouse IgG-HRP (Sigma-Aldrich). Antibody subclasses were measured using subclass specific goat anti-mouse IgG-HRP . Serum BAFF was measured by ELISA . For assessment of B cell development, bone marrow was flushed from tibiae and femora of naïve mice and analysed by flow cytometry.Immunity was assessed on day 10 after immunisation with 20μg MPO in FCA and at day 21, at the end of the autoimmune anti-MPO glomerulonephritis model. The spleen and draining lymph nodes were harvested and a single cell suspension was obtained. IFN-γ and IL-17A ELISPOT was performed according to the manufacturer’s instructions  with 5x10The following antibodies were used for flow cytometry. Anti-CD4 (RM4.5) anti-CD25 (PC61), anti-CD44 (IM7), anti-CXCR5 (2G8), all BD. Anti-Foxp3 (FJK-16s), anti-PD-1 (J43) all Ebioscience. Anti-B220 (RA2-6B2), anti-CD23 (B3B4), anti-CD21 (7E9), anti-IgM (RMMM-1), anti-IgD (11-26c.2a), anti-CD138 (281–2) all Biolegend. Fixable viability stain 450 or propidium iodide (BD Biosciences) were used to exclude dead cells. Flow cytometry was performed on the Beckman Coulter Navios platforms and analysed using FlowJo software .Data are presented with each dot representing a mouse and a bar representing median value. Prism 6  software was used with analysis by Mann-Whitney U test. * p<0.05, **p< 0.01, ***p<0.001.C3ar-/- mice received anti-MPO IgG and LPS. Renal injury was studied after 7 days. Both groups of mice developed glomerulonephritis with a similar degree of histological glomerular injury . Having excluded this potential confounder, we induced autoimmune anti-MPO glomerulonephritis in WT and C3ar-/- mice. Both groups had similar severity of histological injury and albuminuria, with no differences in the numbers of neutrophils, macrophages or CD4+ T cells between WT and C3ar-/- mice , transitional (B220+IgM+IgDlow) or mature (B220+IgMintIgD+) was not different between WT and C3ar-/- mice. In mice immunised with MPO in FCA, there was no difference in the total number of splenic B220+ B cells, Follicular B cells (B220+CD21+CD23+), marginal zone B cells (B220+CD21+CD23low) or plasma cells (B220low/int CD138+) between groups  [+ cells that were CD44hi (activated T cells)  –36. We tT cells) , or CD253+ Tregs  were simn groups .+ activated or CD25+Foxp3+ Tregs were similar, both in the draining lymph nodes and spleen, as serum C3 in this strain has previously be described to be similar to that of WT mice [C3ar-/- mice had any numerical or developmental alteration in the B lymphocyte compartment. However, B cell number and development in the spleen and bone marrow appeared similar to WT mice.C3are strain . In humae strain ,51 whilse strain ,51 C3ar-tisation . In conttisation  or attentisation  humoral  WT mice ,40. Beca+Foxp3+ regulatory phenotype. This is in contrast to previous reports of elevated T regulatory cells in C3ar-/- mice[C3ar-/- mice used animal models that either require no adjuvant or use incomplete Freund’s adjuvant. It is possible that differences in the additional immune signals provided by the adjuvant may account for this discrepancy. Additionally, whilst C3aR expression in the mouse has previously been reported to be extensive in both myeloid and lymphoid lineages, this has been called into questions by recent findings using a C3aR reporter mouse in which expression of C3aR was evident on macrophages and some dendritic cell subsets but not bone marrow neutrophils, B or T cells [Given the previous descriptions of the important role that C3aR plays in influencing adaptive immunity we investigated the effect of absence of this receptor on the generation of T cell mediated immunity. We found no difference in the generation of Th1 or Th17 effector responses, or in the proportion of T cells that had a CD25r-/- mice,55. The  T cells ,57. In c T cells ,58–61. PIn summary, these data suggest a role for C3a in driving glomerular macrophage recruitment and suppressing humoral immunity in these pre-clinical models of AAV. However, the lack of attenuation of immune mediated glomerular injury in either model does not support the C3aR as a putative therapeutic target in this disease.S1 Fig-/- allele.Primers C1 and A201+ yield a fragment of 726 bp, denoting WT allele. Primers C1 and NeoA yield a fragment of 537 bp denoting the C3ar(TIFF)Click here for additional data file.S2 Fig-/- mice (n = 5-7/group). Anti-MPO IgG titres were higher in C3ar-/- mice compared to WT. Data shown as mean ± SEM.Anti-MPO IgG titres were measured by ELISA in WT and C3ar(EPS)Click here for additional data file.


Toxoplasma gondii vaccines, but are more susceptible to enzymatic degradation. As an effective delivery system, chitosan microspheres (CS) can overcome this obstacle and act as a natural adjuvant to promote T helper 1 (Th1) cellular immune responses. In this study, we use chitosan microparticles to deliver multiple antigenic epitopes from GRA10 (G10E), containing three dominant epitopes. When G10E was entrapped within chitosan microparticles (G10E-CS), adequate peptides for eliciting immune response were loaded in the microsphere core and this complex released G10E peptides stably. The efficiency of G10E-CS was detected both in vitro, via cell culture, and through in vivo mouse immunization. In vitro, G10E-CS activated Dendritic Cells (DC) and T lymphocytes by upregulating the secretion of costimulatory molecules (CD40 and CD86). In vivo, Th1 biased cellular and humoral immune responses were activated in mice vaccinated with G10E-CS, accompanied by significantly increased production of IFN-γ, IL-2, and IgG, and decreases in IL-4, IL-10, and IgG1. Immunization with G10E-CS conferred significant protection with prolonged survival in mice model of acute toxoplasmosis and statistically significant decreases in cyst burden in murine chronic toxoplasmosis. The results from this study indicate that chitosan microspheres used as an effective system to deliver a linked antigenic peptides is a promising strategy for the development of efficient vaccine against T. gondii.Multiple antigenic peptide (MAP) vaccines have advantages over traditional  Toxoplasma gondii is an important medical pathogen that infects approximately 30% of the global population. Generally, toxoplasmosis is asymptomatic in immune-competent hosts, however, it can result in severe symptoms in immunocompromised individuals due to cerebral cyst reactivation. Another potentially fatal presentation is vertical transmission in the fetus, which can result in encephalitis, neonatal malformations, or spontaneous abortion  vaccines containing different epitopes may prove a highly efficacious strategy in the development of T. gondii vaccines  . The release efficiency of these chitosan microparticles containing the MAP of GRA10 (G10E-CS) was detected in terms of peptide loading and releasing efficiency. As an adjuvant, the capacity of chitosan to activate dendritic cells and T lymphocytes was tested in vitro. Furthermore, the protective effect of G10E-CS vaccination on acute and chronic toxoplasmosis murine models was evaluated. This study aims to make a contribution to peptides vaccines against T. gondii by exploring chitosan microspheres, an effective delivery system, to enhance MAP vaccine immunogenicity.Herein, we present a novel attempt to combine highly immunogenic MAP from GRA10 with chitosan microspheres in the design of a vaccine against Female 6–8weeks-old BALB/c and C57BL/6 mice were purchased from Shandong University Laboratory Animal Centre  and housed in SPF conditions. All the mice were housed 5 per cage under pathogen-free conditions and were adequately supplied with sterilized water and food.Experiments were carried out in accordance with animal ethics approved by the Institutional Animal Care and Use Committee of Shandong University under Contract LL201602044. Humane endpoints were chosen to terminate the pain or distress of experimental animals via euthanasia.T. gondii were propagated in human foreskin fibroblast (HFF) cells in Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% fetal bovine serum (FBS)  as previously described  , GRA10221−244 (P2), and GRA10310−332 (P3), were selected with good hydrophilicity, high conformational flexibility, surface accessibility and strong antigenicity.The PROTEAN subroutine in the DNASTAR software package was used to predict the B cell epitopes of GRA10  as described previously  as described previously , GRA10161−169 (P5), and GRA10300−308 (P6) were chosen based on their predicted binding affinity to MHC I supertype molecules , GRA10170−184(P8), and GRA10180−194(P9) were chosen based on their predicted binding affinity to MHC II  was designed by linking the selected epitopes with the spacer sequence Gly-Ser  as previously described  chitosan microspheres and crosslinked by genipin as above described. The suspension was centrifuged  to collect the supernatant. Then, the non-bound G10E concentration in the supernatant was determined by Protein Quantitative Reagent Kit-BCA Method   as previously described  using a shaking air bath  for 15 d. After centrifuging , the microsphere precipitate at the bottom of the tube was collected to be analyzed by gel electrophoresis.6 cells/ml) were cultured for 7 days in 6-well plate  in RPMI 1640 medium  supplemented with 10% FBS , 50 μM penicillin and streptomycin, and 20 ng/ml GM-CSF and IL-4 . Half of the medium was replaced with fresh complete medium on day 3 and 5. At day 7, non-adherent cells were harvested and ready for use as mature DCs.Bone-marrow derived dendritic cells (DCs) were prepared as previously described  were seeded in 96-well plate  in the same medium as described above (including GM-CSF and IL-4) and cultured with CpG (4 μg/mL), CS, G10E or G10E-CS (10 μg/mL each) for 48 h. CpG, a TLR agonist that generally enhances CD40 and CD86 surface expression, served as a positive control. Cell suspension was subsequently stained with 0.3 μg/ml of anti-CD11c, 1.25 μg/ml of anti-CD86 and 2.5 μg/ml of anti-CD40 antibody   for 72 h  were plated at 2 × 10in vitro was evaluated by CCK-8 assay  conducted on the aforementioned dendritic cells (DCs) and T lymphocytes as previously described  of G10E-CS microspheres and incubated for 48 and 72 h at 37°C. Ten micro liters CCK-8 was added to the wells and the absorbance of the solution was measured at 450 nm in a Thermo Scientific Multiskan FC Microplate Photometer . Cell viability (%) was defined as the OD ratio in test group relative to controls. All experiments were carried out five times.The cytotoxicity of the G10E-CS microspheres n = 23 per group) and were immunized thrice via intramuscular injection at 2-week intervals with G10E (100 μg), G10E-CS microspheres containing 100 μg of G10E (667 μg) (LC = 15%) or empty CS microsphere (667 μg) . The challenged mice were monitored over a period of 21 days and observed daily for mortality.Two weeks after the last immunization, the protection of G10E-CS microspheres against acute and chronic At the same time, 10 mice per group were challenged orally with a sublethal dose of cysts (30 cysts) of the PRU strain (type II) to evaluate the effect of vaccination in mice with chronic toxoplasmosis. All surviving mice were euthanized at 45 days after challenge. Brains were collected and homogenized in 1 ml PBS. The number of cysts per brain was confirmed by counting eight 10 μl samples of each cerebral homogenate under an optical microscope.T. gondii-specific IgG, IgG1 and IgG2a antibodies in the serum samples as previously described . For each serum sample the assay was done in triplicate and average values were calculated.Serum samples were collected from the mice by retro-orbital bleeding at 0, 2, 4, and 6 weeks after immunization. Standard ELISA assay was used to determine the levels of 6 cells/ml in RPMI-1640 medium with 10% FBS and plated in 96-well plates in presence of the G10E peptides (10 μg/mL). Plates were incubated for 72 h at 37 °C. Lymphocyte proliferative activity was measured by a Cell Counting Kit-8  according to manufacturer's instructions. The absorbance was detected at 450 nm and the stimulation index (SI) was calculated.Two weeks after the last immunization, three mice per group were euthanized to obtain single splenocyte suspensions as described previously  of vaccinated mice were stimulated with G10E peptides (10 μg/mL) for 72 h. Then the suspensions were stained with anti-mouse CD3-APC, anti- mouse CD4-FITC and anti-mouse CD8-PE  for 30 min at 4 °C in the dark.The percentages of CD46 cells/ml) were also stimulated with G10E peptides for 72 h in the presence of Cell Stimulation Cocktail  containing Phorbol myristate acetate , Ionomycin (2 μg/ml), Brefeldin A (1 μg/ml), and Monensin (1 μg/ml) to inhibit the secretion of cytokine into the extracellular space. The cells were fixed using an Intracellular Fixation & Permeabilization Buffer Set Kit in accordance with the manufacturer's protocol  and then stained directly with anti-mouse CD4-FITC, anti-mouse CD8-PE, anti-mouse IL-2 (APC), anti-mouse IFN-γ (PerCP-Cyanine 5.5), anti-mouse IL-4 (APC), and anti-mouse IL-10 (PerCP-Cyanine5.5)  for 30 min at 4°C. All these cell population were analyzed by Cytoflex S Flow Cytometer  and the data were analyzed by CytExpert software.Splenocytes  and GraphPad Prism 7.0 . Differences between groups were analyzed using one-way ANOVA analysis. Student's T. gondii, in which GRA10192−215 (P1) was demonstrated to induce highest level of IgG  and GRA1080−94 (P7) stimulated the highest level of proliferation of lymphocytes in infected mice , GRA10161−169 (P5), and GRA1080−94 (P7) were chosen to construct the MAP G10E with linker GS , P7(CD8+ T cell epitope) or G10E were tested for B cell antibody level and T cell proliferation ability. The results showed that significant enhanced B cell and T cell immune responses were achieved in G10E vaccinated group  were prepared by emulsion cross-linking method. The purity of G10E was 98.4% detected by high performance liquid chromatography (HPLC) Figure , and itsThe zeta potential of the CS microspheres was positive 37.1 ± 5.2 mV while the G10E peptides was negative 31.7 ± 9.1 mV Table . After lThe loading capacity and encapsulation efficiency of the chitosan microparticles were analyzed. When the concentration of G10E reached 4 mg/ml, a high encapsulation efficiency (EE) of 71% was achieved, and the peptide loading was almost saturated (15.3%). Therefore, 4 mg/ml G10E peptides were selected as the optimal concentration Figure .We further evaluated the release of the entrapped G10E peptides from the chitosan microparticles. The crosslinked G10E-CS microspheres showed the more favorable and steady release profile Figure . Only 22in vitro, the expression of the co-stimulatory molecules CD40 and CD86 was detected by flow cytometry in stimulated DCs. Dramatic upregulation of CD40 and CD86 in DCs was observed after incubation with G10E-CS, G10E, CS, CpG (positive control) compared to PBS groups .In order to determine if chitosan can induce dendritic cells (DC) activation in vitro. As shown in Figure p < 0.01).We further confirmed that CD86 and CD40 could provide activate lymphocytes. Hence, we assessed T cell proliferation and cytokines levels in supernatant of C57BL/6 mice lymphocytes after coculture with the above stimulated DC cells p < 0.05). The level of IFN-γ induced by G10E-CS microspheres (p < 0.01) was particularly noteworthy  for different incubation times (48 or 72 h). For DCs and lymphocytes, the results demonstrated that all the G10E-CS microspheres showed cell viabilities higher than 90% of the negative control Figure . Hence, T. gondii specific IgG (p < 0.05)  Figure .p < 0.01) compared with G10E group. These results indicated that mice immunized with G10E-CS microspheres generated Th1 polarize immune response with more IgG2a than IgG1.Furthermore, the level of specific IgG1 and IgG2a sub-classes against G10E peptides was measured. As shown in Figure p < 0.01) and G10E (p < 0.05) was significantly higher than those of other groups. This result indicated that the T lymphocytes proliferation of mice vaccinated with G10E-CS was successfully stimulated.Antigen-specific proliferative responses of lymphocytes from immunized mice were detected by CCK-8 2 weeks after the third immunization and represented by the SI value as illustrated in Figure + or CD4+ T cells numbers after vaccination, the percentages of CD4+ and CD8+ T lymphocytes subsets in the spleen of immunized mice were evaluated by flow cytometry analysis. As shown in Figures + and CD4+ T cells were both increased in mice immunized with G10E-CS and G10E compared to other groups (p < 0.05). G10E-CS-immunized mice also had the highest ratio of CD8+/CD4+ (p < 0.05). G10E-CS microspheres, therefore, could elicit more activation of CD8+ and CD4+ T cells in immunized mice than other vaccine formulations.To determine CD8p < 0.01) and G10E (p < 0.05) immunized mice compared to that of CS and PBS . While only 3 days survival extension when mice immunized with only G10E without CS (10 days) compared to control group (p > 0.05).In order to evaluate if G10E-CS could confer effective protection against T. gondii was also evaluated by challenging the immunized mice with 30 cysts of the PRU strain orally. On day 45 post challenge, as shown in Figure p < 0.01). This result suggests that the G10-CS microsphere vaccine could significantly reduce the cyst form in mice chronically infected with T. gondii.The efficacy of this vaccine formulation in the context of chronic infection with + and CD4+ cell epitopes from T. gondii GRA10 (G10E). Chitosan microspheres were loaded with adequate G10E in an effort to protect the peptides from enzymatic degradation and release the G10E peptides stably. This complex was referred to as G10E-CS. In vitro, G10E-CS microspheres induced DC maturation and T cell activation with enhanced expression of costimulatory molecules (CD40 and CD86). In vivo, significantly prolonged survival and decreased brain cyst burdern were observed in mice vaccinated with G10E-CS, accompanied by strong humoral and cellular responses.In this study, we first screened and constructed a MAP encoding three dominant B, CD8+ T  is essential for parasite growth with potential immunogenic capability, and it had not been studied as a candidate vaccine antigen prior to the work presented herein. In this study, we used bioinformatics and immunological methods to select nine B cell or T cell mimotopes of GRA10. Among them, three strongest immunogenic B and T cell epitopes, namely B cell epitope (TQSPPESRKKRRRSGKKKRGKRSV), CD8+ T cell epitope (LGYCALLPL), and CD4+ T cell epitope(SGFSLSSGSGVSVVE), were selected to construct the MAP, G10E. Peptide-based vaccines have some weaknesses. For example, they are poor immunogens, in and of themselves, and very susceptible to enzymatic degradation. Therefore, they need the assistance of an effective delivery system    response, can directly suppress the growth of dividing tachyzoites and reactivation of the cysts of T. gondii  resulted in a more biased Th1 response as measured by antigenspecific IgG2a serum levels and splenocyte IFN-γ production than vaccination using the smaller bilosomes (250 nm)  compared to the other groups. These results, taken together, proved that our G10E-CS microsphere vaccine was able to protect against both acute and chronic T. gondii infection.The survival rate of vaccinated mice against T. gondii. Our results proved that chitosan microparticles loaded with MAPs could be a promising approach to vaccine development against T. gondii.In summary, in this study, G10E-CS microspheres have triggered a strong, mixed Th1/Th2 cellular and humoral immune response in immunized mice. At the same time, CS as a potent adjuvant, has also enhanced cellular immunity by eliciting Th1-type immune responses. Therefore, the humoral and cellular immune responses induced by G10E-CS microspheres were interrelated and synergistic in strengthening protection against A limitation of this study was that the loading capacity (LC%) of the chitosan microsphere was low (15.3%). This may be due to liquid paraffin on the surface of the microspheres, which was hard to clean completely, potentially affecting the peptides contact with the microspheres. Interestingly, this loading capacity was slightly higher than that observed with chitosan-based microspheres loaded with other protein (Li et al., HC and HY designed experiments. HY, JG, TW, and XS performed experiments and analyzed data. JG and HY wrote the manuscript. HC and YL revised the manuscript. CZ, HZ, and SH provided advice for experiment. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The authors would like to correct The authors have provided raw, uncropped blots for all panels included in In addition, Figures 2A, 2B, 2D, 3A, 3B, 5B and 5C underwent a cut and transfer before incubation that was not previously indicated in the figure legends. The corrected figure legends are included here for reference, and original raw gels post cut and transfer are also included as Supporting Information. Original raw blots are also included for Figures 2C and 5A.Figure 2. Testing for the activity of anti-Helix-3 antibodies: pAb RVC does not recognize PrPSc generated in prion infected brains. (A) Activity of pAb RVC and RTC, as compared to the established mAb 6H4, against normal brain homogenates from bovine, mouse, humans and hamster. (B) Human and mouse normal brain homogenates were immunoblotted with pAb RVC and RTC alone or in the presence of diverse Helix-3 PrP peptides . (C) Brain homogenates from mice, hamster and humans , were immunoblotted with mAb 6H4, with pAb RTC or RVC, or with rec Ab R1. (D) Mouse scrapie (RML) and human CJD brains (E200K) were digested with PK, processed for MMA reduction as described in the methods and immunoblotted with a PrP mAb 6H4, IPC2, or pAb RVC. For panels A, B and D, the same brain samples (Hu Mo or Bo) were run in consecutive sequences in big gels which were cut after transfer for incubation with different antibodies and peptides incubation, as described in the manuscript.Figure 3. pAb RVC does not recognize PrPSc generated in prion infected cells. (A) Brain  as well as normal and prion-infected cells, (N2a and GT1 infected either with the RML or the 22L prion strains) were extracted and immunoblotted with mAb IPC1 as compared to pAb RVC. (B) Effect of the MMA chemical reduction of proteinase K digested ScGT1 and Sc N2a cells on the PrP recognition by mAb IPC1, mAb IPC2 and pAb RVC. For all mouse part in panel A and for all samples in panel B, samples were run in consecutive sequences in big gels which were cut after transfer for incubation with different antibodies and peptides incubation, as described in the manuscript.Figure 5. HuPrP E200K is spontaneously oxidized. (A) Brain samples from scrapie infected mice and from humans suffering from familial E200K or sporadic CJD, were digested in the presence or absence of proteinase K and subsequently immunoblotted with mAb 6H4 or pAb RGM. The last 2 lanes of each gel comprise normal GT1 and proteinase K digested ScGT1 cells expressing a chimera Mo/Ha PrP form. (B) Human and mouse normal brain homogenates were immunoblotted with the RGM antibody alone or preincubated with several PrP peptides in the Helix-3 Met area. (C) Immunoblots of HuPrP(23–230) wt and E200K with 3F4 (recognizing the 109–112 region), DZS18 (recognizing oxidized Met residues in different proteins), IPC2 (recognizing non-oxidized M213) and RGM (recognizing non-oxidized M206). Blots were prepared in the absence of bmercaptoethanol. (D) Thermal stability of HuPrP(23–230) wt and E200K probed by the relative change in the ellipticity at 220 nm as a function of temperature. Insert: Far-UV CD spectrum of HuPrP(23–230) wt and E200K. For panels B and C, samples were run in consecutive sequences in big gels which were cut after transfer for incubation with different antibodies and peptides incubation, as described in the manuscript.S1 File(PPTX)Click here for additional data file.


SCH9, but not TOR1, allows newly-formed tetraploids to exhibit evolved phenotypes and knocking out SCH9 diminishes the evolved phenotypes. Furthermore, when cells were challenged with conditions causing ancestral cells to evolve aneuploidy, tetraploidy was maintained in the evolved lines. Our results reveal a determinant role for Sch9 during the early stage of polyploid evolution.Polyploidization has crucial impacts on the evolution of different eukaryotic lineages including fungi, plants and animals. Recent genome data suggest that, for many polyploidization events, all duplicated chromosomes are maintained and genome reorganizations occur much later during evolution. However, newly-formed polyploid genomes are intrinsically unstable and often quickly degenerate into aneuploidy or diploidy. The transition between these two states remains enigmatic. In this study, laboratory evolution experiments were conducted to investigate this phenomenon. We show that robust tetraploidy is achieved in evolved yeast cells by increasing the abundance of Sch9—a protein kinase activated by the TORC1 (Target of Rapamycin Complex 1) and other signaling pathways. Overexpressing  SCH9 enabled ancestral cells to display the evolved phenotypes and eliminating SCH9 diminished the evolved phenotypes. Finally, we show that evolved cells were able to maintain their genomes even under a condition that causes newly-formed tetraploids to evolve aneuploidy. Our results reveal that at the early stages after genome duplication, stable polyploidy can be achieved by fine-tuning a conserved key regulator coordinating multiple cellular processes.Polyploidy is frequently observed in eukaryotes, including in human liver cells and cancer. Evolutionary studies also suggest that polyploidy has contributed to species diversification and novel adaptation in fungi, plants and animals. However, artificially-constructed polyploids often display chromosome instability and quickly convert to aneuploids. This phenomenon conflicts with observations that many species derived from ancient genome duplications have maintained the extra number of chromosomes following polyploidization. What happened during the early stages of these polyploidy events that stabilized the duplicated genomes? We used laboratory evolution experiments to investigate this process. After being propagated in a rich medium at 23°C for 1000 generations, newly-constructed tetraploid yeast cells had evolved stable genomes. In addition, evolved cells acquired resistance to stresses specific to tetraploids and exhibited a more diploid-like transcriptome profile. Further analyses indicated that Sch9—the functional ortholog of mammalian S6 kinase involved in protein homeostasis, G1 progression, stress response and nutrient signaling—contributed to the evolved phenotypes. Evolved cells increased the protein abundance and stability of Sch9. Reconstitution experiments showed that overexpression of  Polyploid organisms that contain more than two sets of genomes are commonly found in natural eukaryotic populations (reviewed in ). VariatHowever, newly-formed polyploid genomes can perturb cellular homeostasis and are also intrinsically unstable , 11. ChrSaccharomyces cerevisiae is an excellent model organism for studying genome evolution. Yeast cells alternate between haploid and diploid states in their normal life-cycle t, in which P represents the relative intensity of Sch9 and T1/2 represents the half-life. The degradation constant [k = ln(1/2)/T1/2] was estimated as the slope of this linear regression line on the ln(Pt/P0)-t plot.The ancestral and evolved tetraploid SCH9  was designed using the online tool CRISPy (http://staff.biosustain.dtu.dk/laeb/crispy_yeast/) [The CRISPR-Cas system was modified from the previous methods , 55. The_yeast/) . The gRNgRNA-F1: ACCGTATTACCGCCTTTGAG,gRNA-R1: AAAACGTATCCTGATTCGACGATTTGATCATTTATCTTTCACTGCGGAG,gRNA-F2: CGTCGAATCAGGATACGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG,gRNA-R2: GAAGGGAAGAAAGCGAAAGG.SCH9 coding region and it also destroyed the PAM sequence to prevent repeatedly targeting by Cas9. The donor DNA was generated using the following two pairs of PCR primers,The donor DNA was used to introduce two premature stop codons (bp 40 to 45) into the donor DNA-F1: ACCCACTCTCACATAATCACC,donor DNA-R1: GTTGGTGTTGAGAGCTAAACTATCAATCCTGATTCGACGATTTTGATG,donor DNA-F2: CGTCGAATCAGGATTGATAGTTTAGCTCTCAACACCAACATCC,donor DNA-R2: CCTGACTAATAGCGGTGGTCG.The original plasmid carrying the high specificity Cas9 nuclease, eSpCas9(1.1), was obtained from Addgene (plasmid #71814) and the enzyme was further subcloned into the yeast pRS414 vector in our lab .sch9 null mutant, diploid and tetraploid cells were first transformed with the pRS414-eSpCas9(1.1) plasmid. The transformants were then transformed with the DNA mixture of the linearized gRNA plasmid (0.12 μg), gRNA insert fragment (0.3 μg) and donor DNA (0.6 μg). For each strain, single colonies were picked and checked by PCR using wild-type-specific and mutant-specific primers. Only the colonies carrying mutant sch9 were further examined using the Sanger sequencing. The correct clones were propagated in nonselective YPD medium to lose the plasmids and their ploidy was confirmed using flow cytometry. For each strain, we performed phenotypic assays for at least three sch9 null mutant clones and observed consistent phenotypes between clones.For constructing the http://www.ncbi.nlm.nih.gov/geo/) under the accession number GSE76595. The expression data are available at GEO under the accession number GSE 76596.The CGH array data are available at the NCBI Gene Expression Omnibus (GEO)  Ancestral and evolved diploid clones were serially diluted and spotted onto YPD plates. The plates were then incubated at 23°C, 36°C or 38°C until colonies were easily observed. No single colonies were found on the plate incubated at 38°C, even after one week. (B) Ancestral and evolved diploid cells were serially-diluted and spotted onto YPD plates containing different concentrations of rapamycin . The plates were then incubated at 23°C until colonies were easily observed.(EPS)Click here for additional data file.S2 FigExpression levels of all yeast genes of anc4N-4 and evo4N-4 were compared to that of ancestral diploids, and a higher correlation in the expression level was observed between ancestral diploids and evolved tetraploids.(EPS)Click here for additional data file.S3 FigCells from evolved tetraploid clones (A) and diploid clones (B) were measured for their cell size using flow cytometry. All the measurements were normalized to the cell size of ancestral clones.(EPS)Click here for additional data file.S4 FigSCH9 and TOR1 exhibited similar growth rates at 38°C. (B) Overexpression of kinase-dead (SCH9k.d.) or constitutively active (SCH92D3E) mutants does not affect the growth of diploid cells at high temperatures.(A) Unlike tetraploid cells, diploid cells with or without overexpression of (EPS)Click here for additional data file.S5 FigSSA4 or HSP42 (OE-SSA4 and OE-HSP42) were introduced into the anc4N-4 and evo4N-4 strains and tested for their effects on cell growth at 36°C.Multicopy plasmids carrying either (EPS)Click here for additional data file.S6 FigEight ancestral diploid clones (anc2N-1 to anc2N-8) and eight evolved diploid clones (evo2N-1 to evo2N-8) were propagated in regular YPD medium at 30°C for 200 generations and the evolved cultures were analyzed using flow cytometry. All the lines remained diploid.(EPS)Click here for additional data file.S7 FigEight newly-constructed isogenic tetraploid cells were propagated in YPD medium at 23°C with daily dilutions. Cell cultures at generation zero , 1000, 2000, 3000, 4000 and 5000 were analyzed using flow cytometry.(EPS)Click here for additional data file.S1 TableOnly genes with expression changes greater than two-fold (p < 0.005) are included. In the diploid data set, only one gene passed this criterion.(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 TableData for the rapamycin-treated cells were taken from the study by Hardwick et al. . Overlap(XLSX)Click here for additional data file.


The influenza epidemic in 2015–2016 in Russia is characterized by a sharp increase of influenza cases (beginning from the second week of 2016) with increased fatalities. Influenza was confirmed in 20 fatal cases registered among children (0–10 years), in 5 cases among pregnant women, and in 173 cases among elderly people (60 years and older). Two hundred and ninety nine people died from influenza were patients with some chronic problems. The overwhelming majority among the deceased (more than 98%) were not vaccinated against influenza. We isolated 109 influenza A(H1N1)pdm09 and one A(H3N2) virus strains from 501 autopsy material samples. The antigenic features of the strains were similar to the vaccine strains. A phylogenic analysis of hemagglutinin revealed that influenza A(H1N1)pdm09 virus strains belonged to 6B genetic group that had two main dominant subgroups during the 2015–2016 season. In Russia strains of the first group predominated. We registered an increased proportion of strains with D222G mutation in receptor-binding site. A herd immunity analysis carried out immediately prior to the epidemic showed that 34.4% blood sera samples collected in different regions of Russia were positive to A/California/07/09(H1N1)pdm09. We came to a conclusion that public awareness enhancement is necessary to reduce unreasonable refusals of vaccination. According to the World Health Organization (WHO) Regional Office for Europe, the influenza epidemic season started in week 51, 2015. The beginning of the season in Eastern Europe and Western Asia was marked by a spike of influenza cases compared to Western and South Europe. Five countries  reported a rising incidence of severe acute respiratory infections (SARI) cases [Predominance of A(H1N1)pdm09 subtype among circulating influenza viruses was observed in all Northern hemisphere countries except China where cocirculation of A(H1N1)pdm09, A(H3N2) and influenza B was registered. Some European countries  reported cases caused by influenza A(H3N2) viruses. According to FluNet, influenza B viruses cocirculated together with A(H1N1)pdm09 viruses in a number of countries.th week. Influenza A(H1N1)pdm09 virus was predominant during the whole period of epidemic. Laboratory monitoring confirmed more than 28,200 influenza cases including approximately 28,000 influenza A cases, of which 93% were caused by A(H1N1) virus [In Russia a morbidity spate started in the week 2 of 2016; and the influenza epidemic season finished by the 12We have been studying the condition of herd immunity and influenza epidemical situation in Russia since 2009–5. This Blood samples were obtained on condition of anonymity upon written informed donor consents. Sera were collected at the Sanitary-and-Epidemiological Centers of the Federal Service for Supervision of Consumer Rights and Human Welfare in 34 regions of Russia, 100–200 pieces per region. The places of collection located close to flyways and breeding grounds of wild waterfowls. Sera were collected from healthy donors. 5-ml blood samples were collected using disposable syringes or disposable plastic systems (vacutainers). The samples were transported to Vector State Research Center of Virology and Biotechnology (SRC VB Vector), Novosibirsk, in insulated shipping containers with cold packs. The collected samples were stored at –20°C before examination. The presence of antibodies to different types/ serotypes of influenza virus in the sera was tested following a standard technique, in hemagglutination inhibition (HI) test . TestingA/California/07/09(H1N1)pdm09, A/Switzerland/9715293/13(H3N2), B/Brisbane/60/2008 (Victoria lineage), B/Phuket/3073/2013 (Yamagata lineage) influenza viruses were kindly provided by the WHO Collaborating Center in Atlanta, the United States. The WHO Collaborating Center in Beijing, China, kindly furnished A/Anhui/01/2013(H7N9) virus. A/rook/Chany/32/2015(HPAI H5N1) virus (clade 2.3.2.1c.) was isolated by the authors in 2015 in Western Siberia [Samples were collected at the local Sanitary-and-Epidemiological Centers of the Federal Service for Supervision of Consumer Rights and Human Welfare after getting written informed consents from close relatives in accordance with the regulations of the Russian Federation. PCR-based diagnostics of raw material for influenza virus RNA was conducted in local laboratories, and then all the positive samples were sent to SRC VB Vector. Work with autopsy materials was approved by the Ethics Committee IRB 00001360 affiliated with SRC VB Vector   Files.After delivery to SRC VB Vector we tested all samples in PCR again and isolated strains in MDCK-London by infecting a monolayer of cells. For the purpose, the autopsy material was homogenized, a 10% suspension was prepared by adding the MEM culture medium (by Invitrogen), the suspension was filtered through a bacterial filter (0.22 μm pore diameter), and 200 μl were added to the culture flasks with a one-daymonolayer of MDCK cells, then we added medium MEM (Invitrogen), TPCK treatedtrypsin 2 μg/ml (Sigma), BCA0.2% and an antibiotic . The infected cells were incubated at 37°C. The viral replication was monitored visually for cytopathic effect in HI test with chicken, goose and guinea pig erythrocytes and with human erythrocytes of 0(I)Rh group as well as in real-time PCR. If necessary, each sample was passaged at least three times.Typing / subtyping of isolated influenza virus strains and studying their antigenic features were carried out in HI test by the technique recommended by WHO  using powww.megasoftware.net) with 1,000 bootstrap replicates.Sequencing was carried out at the Genomics Core Facility, the Institute of Chemical Biology and Fundamental Medicine, the Siberian Branch of the Russian Academy of Sciences, Novosibirsk. To determine a nucleotide sequence of viral genome, viral RNA was isolated using the SV Total RNA Isolation System  according to the manufacturer’s instruction. Reverse transcription reaction was carried out with Uni12 primers using AMV reverse transcriptase. The products of amplification were isolated using QIAquick gel extraction kit . DNA sequencing was performed at the Genomics Core Facility, Novosibirsk. An automatic sequenator 3130xl Genetic Analyzer  was employed to analyze the products. The nucleotide sequences were studied using Vector NTI Advance 10 program package  and entered into the GISAID database. The phylogenetic trees were constructed applying the neighbor-joining method with the Kimura 2-parameter model and 5.2 MEGA version  and A(H7N9) antigens even at 1:10 dilution.From December 2015 to April 2016 SRC VB Vector received 501 autopsy material samples from patients presumably died from influenza. Seventy-one per cent of all sera were collected in the European part of Russia, 22 per cent in the Urals and Western Siberia, and 7 per cent in Eastern Siberia and Russian Far East. Since the share of population in these regions is 74.2, 21.6 and 4.2 per cent respectively, it is obvious that the mortality rate caused by influenza was similar in all Russian regions. The majority of deaths fell within the 31–59 age group. This situation can be attributed to the fact that children under the age of 3 stay home. Preschoolers and school students are vaccinated at schools and day-care centers. University and college students, as well as the elderly have an opportunity to get a free vaccination. In autumn 2015 13.3 million children and adolescents and 25.9 million adults  were immunized under the free vaccination program. In total 45.3 million people (31.3% of the population) were vaccinated [PCR analysis showed that samples from 497 deceased were positive to influenza A(H1N1)pdm09 RNA; 4 samples were positive to influenza A(H3N2) RNA.We isolated 109 influenza A(H1N1)pdm09 and one influenza A(N3N2) virus strains from autopsy material from 501 died patients.All 109influenza A(H1N1)pdm09 virus strains were characterized in HI test with ferret sera anti-A/California/07/2009. Only the HI titer of A/Kaluga/13/2016 isolated from patient immunized with influenza vaccine on October 30, 2015 was 4-fold lower than the vaccine strain. Fifteen strains were additionally studied in HI test with serum of 38-year-old woman immunized on October 28, 2015 with Grippol trivalent vaccine (ser. 300815). The titer was below 20 before vaccination and reached 640 one month after immunization with A/California/07/2009 virus. According to the antigenic characteristics, all studied strains were similar to A/California/07/2009, which was recommended by WHO for developing the vaccine for the current epidemic season. The antigenic properties of A/Saratov/5/2016 (H3N2) isolated strain are similar to those of A/HongKong/4801/2014 (H3N2) strain with a 16-fold reduction of HI titer compared to A/Switzerland/9715293/13 strain . 107 invWe sequenced hemagglutinin (HA), neuraminidase (NA) and NS genes of 15 isolated A(H1N1)pdm09 viruses and 1 A(H3N2) virus  and builHA phylogenic analysis revealed that influenza A(H1N1)pdm09 virus strains belonged to the 6B genetic group that had two main dominant subgroups during 2015–2016. In Russia strains of the first subgroup dominated. Among fatal cases we registered an increase in proportion of strains with D222G mutation in the receptor-binding site.In all strains with a detected nucleotide sequence of NS gene we registered the following mutations: NS1 –N205S and NS2(NEP)–T48A which increase virulence  due to iFurthermore, we detected an NA gene mutation in one of the analyzed strains (A/Saint-Petersburg/41/2016) –S247N reducing sensitivity of this strain to NA inhibitor oseltamivir . NeverthWe found no changes in A/Saratov/5/2016 (H3N2) NA resulting in resistance to antineuraminidase drugs, and in NS that might enhance pathogenicity. HA of this strain belongs to the same 3c.2a genetic group as A/HongKong/4801/2014 (H3N2) vaccine strain .A(H1N1)pdm09 viruses have been circulating in the human community since 2009 and nine clades have been designated on HA phylogenetic tree. According to the European Centre for Disease Prevention and Control, “viruses in clade6, represented by A/St Petersburg/27/2011 and carrying amino acid substitutions of D97N, S185T and S203T in HA1 and E47K and S124N in HA2 compared with A/California/7/2009, have predominated worldwide with a number of subclades emerging. All viruses having characterized since September 2014 carry HA genes in 6B subclade, which is marked by additional amino acid substitutions of K163Q, A256T and K283E in HA1 and E172K in HA2 compared with A/California/7/2009, e.g. A/South Africa/3626/2013. A number of virus clusters have emerged within 6B clade and two of these have been designated as subclades: viruses in 6B.1 subclade are defined by HA1 amino acid substitutions S84N, S162N  and I216T, while those in 6B.2 subclade are defined by HA1amino acid substitutions V152T and V173I” [The European Centre for Disease Prevention and Control also states that “influenza A(H3N2) viruses circulated in 2015–2016 were poorly detected by reference-antisera to A/Switzerland/9715293/2013, i.e. vaccine strain recommended for usage in the Northern hemisphere in the 2015–2016 influenza season, although some viruses belonged to the same 3C.3a subclade as vaccine virus. Antisera to A/Hong Kong/4801/2014 virus recommended for developing influenza vaccines in the Southern hemisphere in 2016 and in the Northern hemisphere in 2016–2017 resulted in better detection of the analyzed viruses” .In 2015–2016 in Russia fatal influenza cases were primarily caused by A(H1N1)pdm09 virus, and in 99% of confirmed fatal influenza cases we detected RNA of that virus subtype. Interestingly, the antigenic features of the strains were similar to A/California/7/2009 being recommended by WHO for vaccines since 2009, i.e. in the last seven epidemic seasons. A number of researchers believe that the increased mortality rate from influenza A(H1N1)pdm09 is caused by D222G mutation in HA protein –14. HemaSome researchers state that this mutation is developed through selection from polymorphous influenza viruses. This selection is found in infected organisms, and at the stage of introduction of infection D222G mutation is neither present nor predominant . RegenerE391K mutation in HA gene (known as the 47th according to HA2 numeration) with capability to enhance virus pathogenicity has become typical for A(H1N1)pdm09 current strains . This muIn the 2015–2016 season, two new yet uncharacterized mutations D2E and E125D (GISAID) were detected in a segment of nonstructural protein (NS) of influenza virus. E125D mutation is located at the site of binding to cell proteins causing production of antiviral mRNA. Such mutation seems to block antiviral cell activity.Current strains include two more significant mutations in the segment of nonstructural protein in influenza viruses: T48A in NS2 and N205S in NS1 involved in enhancing antagonism to interferon alpha, which increases virulence . This paLow herd immunity to A(H1N1)pdm virus can be another important factor increasing the number of more severe and fatal influenza cases. After emergence and prevalence of influenza A(H1N1)pdm09 virus variant in 2009–2011 this subtype has not predominated in the subsequent epidemic seasons  except tConsequently, new properties of influenza A(H1N1)pdm09 virus and low herd immunity in the recent years have resulted in an increased number of severe and fatal cases in Russia in the 2015–2016 influenza epidemic. Thus, there were 25 fatal cases caused by influenza A(H1N1)pdm09  in the 2In Russia immunization against seasonal influenza is on the national vaccination schedule. The following population categories should be vaccinated: 6-month old babies, schoolchildren and university students; transport, healthcare and education workers, pregnant women, elderly people at the age of 60 and older, as well as patients with chronic problems. However, in many cases people refuse vaccination for no important reasons. Undoubtedly, awareness-raising activities among the population should be intensified to decrease the number of unreasonable refusals of vaccination. For instance, in the 2015–2016 season in Russia 20 children aged 0–10, 5 pregnant women, and 173 elderly people at the age of 60 and older died from influenza; and 299 deceased persons had chronic illness. The majority of those who died (> 98%) were unvaccinated against influenza.S1 File(PDF)Click here for additional data file.S2 File(DOCX)Click here for additional data file.