The strictly regulated unidirectional differentiation program in some somatic stem/progenitor cells has been found to be modified in the ectopic site (tissue) undergoing regeneration. In these cases, the lineage barrier is crossed by either heterotypic cell fusion or direct differentiation. Though studies have shown the role of coordinated genetic and epigenetic mechanisms in cellular development and differentiation, how the lineage fate of adult bone marrow progenitor cells (BMPCs) is reprogrammed during liver regeneration and whether this lineage switch is stably maintained are not clearly understood. In the present study, we wanted to decipher genetic and epigenetic mechanisms that involve in lineage reprogramming of BMPCs into hepatocyte-like cells. Here we report dynamic transcriptional change during cellular reprogramming of BMPCs to hepatocytes and dissect the epigenetic switch mechanism of BM cell-mediated liver regeneration after acute injury. Genome-wide gene expression analysis in BM-derived hepatocytes, isolated after 1 month and 5 months of transplantation, showed induction of hepatic transcriptional program and diminishing of donor signatures over the time. The transcriptional reprogramming of BM-derived cells was found to be the result of enrichment of activating marks (H3K4me3 and H3K9Ac) and loss of repressive marks (H3K27me3 and H3K9me3) at the promoters of hepatic transcription factors (HTFs). Further analyses showed that BMPCs possess bivalent histone marks (H3K4me3 and H3K27me3) at the promoters of crucial HTFs. H3K27 methylation dynamics at the HTFs was antagonistically regulated by EZH2 and JMJD3. Preliminary evidence suggests a role of JMJD3 in removal of H3K27me3 mark from promoters of HTFs, thus activating epigenetically poised hepatic genes in BMPCs prior to partial nuclear reprogramming. The importance of JMJD3 in reprogramming of BMPCs to hepatic phenotype was confirmed by inhibiting catalytic function of the enzyme using small molecule GSK-J4. Our results propose a potential role of JMJD3 in lineage conversion of BM cells into hepatic lineage. Cre-LoxP system in lethally irradiated FAH-/- transgenic mouse, with high selection pressure for the replacement of hepatocytes, it was confirmed that BM-derived hepatocytes were the product of fusion heterokaryons, generated by polyploidization followed by ploidy reduction  by administering acetaminophen (500 mg/kg body weight) through intra-peritoneal injection. Bone marrow cells were isolated from healthy femurs and tibiae of 6–8 weeks old female eGFP-expressing Bl6/J [C57Bl6/J-Tg(UBCGFP) 30Scha/J] mice from which Lin- cells were separated by MACS following a negative selection method using mouse hematopoietic progenitor cell enrichment kit  which consist of antibody cocktail containing CD5, CD11b, CD19, CD45R, 7–4, Ly-6G/C (Gr-1) and Ter119 antibodies. Before harvesting bone marrow cells, euthanasia of mice was carried out by inhaling excess CO2. Within 30 h of liver injury, 0.25 × 106 Lin- eGFP cells were transplanted into HA mice through tail vein. CD45+ and CD45- cells were isolated from the Lin- eGFP cells using FACS Aria III  and 0.125 × 106 cells were transplanted in each mouse. Animals were procured from The Jackson Laboratories; all experiments were conducted as per procedures approved by the Institutional Animal Ethics Committee, National Institute of Immunology, New Delhi.Acute liver injury was induced in six to eight weeks old hemophilia A (HA) male mice [(Ct(1% input)–log x 100%.Cells were pelleted and resuspended in 500 μl of PBS containing 20 mM sodium butyrate. Cells were fixed at room temperature with 1% formaldehyde for 8 minutes, and then quenched with 1.25 M glycine. Cells were sonicated using Misonix sonicator under the following conditions to obtain chromatin fragments between 200–700 bp size: Hepatocytes– 65 Amplitude ; Lin primers  and the - BM cells were cultured in the presence of Williams E medium (Gibco-BRL) containing 10% fetal bovine serum, 1× insulin-transferrin-selenium supplement , 20 ng/ml hepatocyte growth factor, 30 ng/ml oncostatin M, 10 ng/ml epidermal growth factor , 10-8M dexamethasone, 10-4M ascorbic acid-2-phosphate (Sigma- Aldrich). Depending on the nature of the experiment, 100 nM GSK-J4 (Sigma-Aldrich) was added in the medium. Prior to culture, plates were coated with laminin-hyaluronic acid-collagen I.LinStaining was performed using PAS reagent (Sigma Aldrich). Culture medium was removed and adhered cells were washed with PBS and air dried. Cells were then fixed with formalin-ethanol fixative for 1 minute at room temperature and rinsed with tap water. Periodic acid was added to cells and left for 5 minutes at room temperature. Cells were washed with distilled water, Schiffs reagent added and left for another 15 minutes at room temperature. Cells were washed and counterstained with hematoxylin for 90 seconds, washed with tap water, air dried and examined microscopically.Indocyanin green dye (Sigma Aldrich) was added to the cells in culture at a concentration of 1 mg/ml and incubated at 37°C for 1 h. Medium with ICG was discarded and cells were washed with PBS and observed under microscope for cellular uptake of ICG. After that cells were further cultured in fresh medium 6 h at 37°C. The PBS-washed cells were observed again under microscope for release of the dye.Results of multiple experiments were reported as mean ± SEM. Student’s t-test was carried out to calculate the significance between the means of both groups and p < 0.05 was considered as significant. All analyses were carried out using GraphPad Prism software, Version 5.02.- BMCs from female to hemophilia A (HA) male mice, the immuno-histochemical analysis of liver sections revealed the presence of donor-derived cells expressing hepatic markers, like albumin and HNF4α  of eGFP-expressing Linpression .in situ hybridization (FISH) to identify XY chromosomes in sorted donor-derived cells (top right), whereas few nuclei bearing multiple X chromosomes along with Y chromosome were also observed (bottom). Cytogenetic analysis using 500 nuclei per mice revealed 10% of total donor-derived cells got fused with recipient cells that contained multiple X chromosomes along with Y chromosome. Whereas, remaining 90% of these were devoid of Y chromosome, suggesting that most of these donor-derived hepatic cells possessed female chromosome  and major hepatic transcription factors  in Lin- BM cells and BM-derived hepatic cells with respect to primary hepatocytes. The expressions of these genes were observed in BM-derived hepatocytes albeit to varying degrees  and acute inflammatory response  , cell cycle and cell division , transcriptional regulation  and chromatin organization  as shown in Cbx4, RNASEL, etc.) and chromosome reorganization   . The comd Hmgcs1 . The maj7, etc.) .- cells  in BM-derived hepatic cells.The real time PCR results validated microarray data and ensured similarity in major functional gene expressions among BM-derived and primary hepatocytes . It is iHNF4α, CEBPα, HNF1α, HNF3α, CEBPβ, HNF6, HNF3β and GATA4 in BM-derived hepatocytes after 5 months of Tx. As Lin- BM cells used for transplantation have both Lin-CD45+ (hematopoietic) and Lin-CD45-  fractions, we isolated these two fractions  like ractions  and tranractions . Since bnd GATA4 . H3K9Ac BM cells .-CD45+ and Lin-CD45- cells. In primary hepatocytes only a minimal enrichment of H3K27me3 mark was observed at these promoters, whereas in BM-derived hepatocytes H3K27me3 mark was still present at the promoters of CEBPα, HNF3α and CEBPβ. Other promoters like HNF4α, HNF1α, HNF6, HNF3β and GATA4 showed only minimal levels of H3K27me3 enrichment  at the promoters of all major hepatic transcription factors in Linrichment . H3K9me3atocytes .CD45 and down-regulation of GATA2 genes in BM-derived hepatocytes, we determined the change of histone mark occupancy at the promoter levels. No significant change in the enrichment levels of H3K4me3 and H3K9Ac marks were noticed, whereas H3K27me3 (8 and 53% of input) and H3K9me3 (15% and 25% of input) levels were significantly enriched at promoters of CD45 and GATA2 in BM-derived hepatocytes compared to Lin-CD45+ cells , an H3K27me3 demethylase, and 1000-fold reduction in the expression of EZH2, an H3K27 methyltransferase was found in BM-derived hepatocytes compared to Lin- BM cells  heterotypic fusion that lead to nuclear reprogramming towards hepatic lineage and (b) liver regeneration cues which trigger nuclear reprogramming in favor of hepatic differentiation. Liver regeneration by BM-derived cells has been documented for many years –12, 15; - BMCs for longer duration than earlier [Here we have studied the reprogramming of Lin earlier , so that earlier , 24–27, -CD45+) signature genes like GATA2 and CD45, which was also evidenced in induced hepatocytes obtained from fibroblasts [The comparative analyses of human hepatocyte-like cells, derived from ESCs or iPSCs, have revealed that despite significant similarities in gene expressions, there were considerable differences in the expressions of cytochrome P450 enzymes as compare to primary hepatocytes . In anotroblasts  and in hroblasts .- fraction of BM cells. These results were in support of the expression of HTFs in BM-derived hepatocytes. The findings also substantiate the role of chromatin modifications in driving differentiation or nuclear programming as clearly seen in several other systems [-CD45+ BM cells at most of the HTF promoters, which were also associated with H3K27me3 mark. It is known that bivalent mark is the presence of activating H3K4me3 and repressive H3K27me3 mark at the promoter that keeps the gene poised for differentiation under inductive conditions [-CD45+ BM cells. Favorable conditions that facilitate the loss of H3K27me3 mark from these sites can lead to the activation of their transcription and promote reprogramming of BM progenitors to hepatocytes. Increase of silencing marks (H3K27me3 and H3K9me3) at hematopoietic gene promoters in BM-derived hepatocytes implied that epigenetic changes during nuclear reprogramming also induced repressive alterations at donor signature gene promoters.Chromatin modifications have been shown to constitute an additional layer of transcriptional regulation. In this study, analysis of enrichment of various histone marks at the promoter regions of developmentally important hepatic transcription factors showed that permissive histone marks (H3K4me3 and H3K9Ac) were enriched at these loci whereas repressive marks (H3K27me3 and H3K9me3) were only minimally present at these sites in primary hepatocytes and BM-derived hepatocytes when compared to the Lin systems –32. Anotnditions . The pre-CD45+ and Lin-CD45- BM cells. EZH2 facilitated establishing of H3K27me3 mark at these sites through its H3K27 methyltransferase activity in Lin- BM cells. Down-regulation of EZH2 in hepatic progenitor cells has been reported to induce differentiation and maturation into functionally active hepatocytes [GATA2 and CD45 promoters in Lin-CD45+ BM cells, however during reprogramming recruitment of EZH2 led to establishment of H3K27me3 mark at these sites to down-regulate/silence their expression.Among all the chromatin remodeling enzymes analyzed, an interesting inverse co-relation was observed between JMJD3 and EZH2 that regulate methylation status of H3K27. The up-regulation of JMJD3 and down-regulation of EZH2 suggest an important role for JMJD3 in removal of H3K27me3 marks from promoters of cells that undergo reprogramming. This hypothesis was supported by ChIP-qPCR results, which showed increase in binding of JMJD3 to the HTF promoters in both primary and BM-derived hepatocytes whereas these loci were occupied by EZH2 in the Linatocytes . On the atocytes  and alsoatocytes  and lungatocytes , which a- BM cells in vivo. A few reports have suggested that cell fusion can lead to changes in DNA methylation patterns, which instruct nuclear reprogramming in heterokaryons leading to lineage conversion [In vitro hepatic differentiation experiments of Lin- cells clearly demonstrated the propensity of a smaller fraction of BMCs to differentiate into functionally active hepatocyte-like cells. ChIP experiments in the presence of JMJD3 inhibitor has confirmed that the mechanism by which JMJD3 promoted hepatic differentiation was through active demethylation of H3K27me3 from HTF promoters rather than functioning as a transcription factor, though GSKJ4 may have certain non-target inhibitory effects that was not analyzed in this study. Further elaborate studies are needed by generating conditional JMJD3 knockout mouse model to dissect the stage-specific mechanisms involved in fetal liver development and BM-specific deletion of JMJD3 prior to transplantation will validate the in vivo functional role during liver regeneration. Another limitation of this study was that we have not separately characterized the hepatocytes obtained from Lin-CD45+ and Lin-CD45- transplanted groups, which needs to be pursued in the future. Furthermore, elaborate and systematic characterization is needed to determine the differences in the lineage conversion mechanism and factors involved when these two cell fractions are separately transplanted.Induction of epigenetic reprogramming may occur both in fusion-dependent and fusion-independent pathways follow for the formation of hepatocytes from Linnversion , 39. Sin- BMCs to hepatocytes in regenerating liver.The comprehensive gene expression analysis revealed the dynamic transcriptional changes in BM-derived hepatocytes and their similarity with the primary hepatocytes after reprogramming. Targeted epigenetic analysis of some important histone modifications at gene promoters of major HTFs provided valuable insights in the changes of their chromatin landscape and the role of chromatin modifying enzymes in induction of cellular reprogramming. For the first time, this study implicates the potential role of JMJD3 in epigenetic reprogramming during lineage conversion of LinS1 Fig- BM cells. After 5 months of transplantation the liver sections were stained with eGFP and vimentin specific antibodies.Mice were transplanted with Lin(TIF)Click here for additional data file.S2 Fig(A) Single cell suspension of hepatocytes was made by two-step collegenase perfusion and the donor BM-derived hepatocytes were isolated by flow cytometry on the basis of eGFP marker. (B) The sorted cells were stained for GFP and albumin markers  to further analyze the purity of post sort samples.(TIF)Click here for additional data file.S3 Fig- BMCs . (B) Venn diagram illustrating the overlap in differential gene expression profile between BM–derived hepatocytes after 1 month and 5 months of transplantation relative to Lin- BMCs.(A) Hierarchical clustering of differentially regulated genes in BM derived hepatocytes– 1 month  and 5 month , primary hepatocytes  with respect to control–Lin(TIF)Click here for additional data file.S4 Fig- BM cells. (C) Gene ontology analysis of the commonly up-regulated genes in BM derived hepatocytes after 1 and 5 months of transplantation with respect to Lin- BM cells. Number of experiment (n) = 2.Gene ontology analysis of the total up-regulated genes in BM derived hepatocytes after (A) 1 month and (B) 5 months of transplantation with respect to Lin(TIF)Click here for additional data file.S5 Fig- BM cells. (C) Gene ontology analysis of the commonly down-regulated genes in BM derived hepatocytes after 1 and 5 months of transplantation with respect to Lin- BM cells. Number of experiment (n) = 2.Gene ontology analysis of the total down-regulated genes in BM derived hepatocytes after (A) 1 month and (B) 5 months of transplantation with respect to Lin(TIF)Click here for additional data file.S6 Fig- BM cells after 1 and 5 months of transplantation. Number of experiment (n) = 3.(A) Functional annotation of these genes obtained by DAVID Bioinformatics Resources 6.7. Number of experiment (n) = 2. (B) Heat map of hematopoietic genes, the expression of which is retained in donor derived hepatocytes even after 5 months of transplantation. Number of experiment (n) = 2. (C & D) Fold change in expression of few specific hepatic genes in BM-derived hepatocytes relative to Lin(TIF)Click here for additional data file.S7 Fig(A) Flow cytometric analysis for CD45+ and CD45- fractions of cells present in Lin- BMCs. (B) Engraftment of Lin-CD45+ and Lin-CD45- fractions of Lin- cells in damaged liver of mice after 1 month of transplantation and albumin expression by the engrafted cells. Number of mice per group = 3.(TIF)Click here for additional data file.S8 Fig-CD45+ and Lin-CD45- BM cells served as negative controls and primary hepatocytes as positive control. ChIP-qPCR analyses of (A) H3K4me3 (B) H3K9Ac (C) H3K27me3 and (D) H3K9me3 at the promoters of hematopoietic genes in BM-derived hepatocytes. Enrichment of the marks in the immuno-precipitated samples over input samples has been calculated. Number of experiment (n) = 3. BM-derived hepatocytes are isolated after 5 months of transplantation for ChIP-qPCR analysis. Lin(TIF)Click here for additional data file.S9 FigJMJD3 in livers harvested day 1 and day 4 post induction of injury by acetaminophen relative to that of primary hepatocytes. (B) Immuno-histochemical analysis of liver cryo-sections to study expression of JMJD3 in hepatocytes after liver injury . p value < 0.05 was considered as significant change. Number of animals used for analysis in each group = 5. Data are represented as mean ± SEM.(A) Expression of (TIF)Click here for additional data file.S10 Fig(A) Expression of JMJD3 in fetal livers of 13 and 18dpc mouse embryos relative to expression in normal adult liver. (B) Expression of EZH2 in fetal livers of 13 and 18dpc mouse embryos relative to expression in normal adult liver. (C) Immuno-histochemical analysis of 18dpc fetal liver cryo-sections to study expression of JMJD3 during liver development . Number of mice = 3.(TIF)Click here for additional data file.S11 Figin vitro differentiated cells was performed using Image J 1.48 version. Number of experiments = 3. (D) Functional characterization of hepatocytes derived from Lin- BMCs. Periodic acid schiff staining was performed in control Lin- BMCs, test samples in which Lin- BMCs were cultured under hepatic differentiation conditions for 30 days and primary hepatocytes (upper panel) . Indocyanin green uptake was assessed in these cells after 30 days of culture in control and test with primary hepatocytes as positive control (lower panel) . Number of experiments = 3. Data are represented as mean ± SEM.(A) Lin- BMCs were isolated and cultured on plates coated with hyaluronic acid, laminin and collagen I under hepatic differentiation conditions. After 14 days of culture cells were harvested and RNA was isolated. Expression of hepatic markers like HNF4α, HNF3α, albumin, E cadherin and CK18 was determined. Expression levels were calculated relative to Lin- BMCs. *denotes that no Ct was observed in these samples. Number of experiments = 3. (B) Expression of hepatic markers like HNF4α, albumin and E cadherin after 14 days of culture was determined by immuno-cytochemistry . (C) Quantification of (TIF)Click here for additional data file.S12 FigExpression of hepatic markers (A) albumin and (B) CK18 and induction of JMJD3 as analyzed by immuno-cytochemistry in cyto-spun cells after 14 days of culture  in presence and absence of GSKJ4. (C) Change in nuclear levels of H3K27me3 after 14 days of culture of Lin- BMCs under hepatic differentiation conditions with and without GSKJ4 was determined by immuno-cytochemical analysis . Cyto-spun cells were stained with rabbit anti-H3K27me3/anti-rabbit Alexa Fluor 594. Number of experiments = 3.(TIF)Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S3 Table(DOC)Click here for additional data file.S4 Table(DOC)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


During the 2016–17 influenza season  in the United States, influenza activityCDC receives influenza test results from public health and clinical laboratories located in all 50 states, Puerto Rico, and the District of Columbia through U.S. World Health Organization (WHO) Collaborating Laboratories and the National Respiratory and Enteric Virus Surveillance System. During October 2, 2016– May 20, 2017, clinical laboratories tested 865,168 specimens for influenza virus: 121,223 (14.0%) specimens tested positive for influenza virus , includiPublic health laboratories tested a total of 84,303 specimens during October 2, 2016–May 20, 2017, and 40,728 were positive for influenza, including 31,736 (77.9%) influenza A and 8,992 (22.1%) influenza B viruses . Among tAge data were available for 36,426 of the influenza-positive patients tested by public health laboratories. Overall, 2,912 (8.0%) persons were aged 0–4 years, 11,066 (30.4%) were aged 5–24 years, 10,872 (29.8%) were aged 25–64 years, and 11,576 (31.8%) were aged ≥65 years. Influenza A(H3N2) viruses were predominant among all age groups, accounting for 70% of viruses identified among persons aged 0–4 years and 80% of viruses reported among persons aged ≥65 years. The largest proportion of reported influenza B viruses occurred in persons aged 5–24 years; influenza B viruses accounted for 28% of the viruses reported for that age group.Three human infections with novel influenza A viruses were reported to CDC during the 2016–17 influenza season. The first was an infection with an influenza A(H1N2) variant (H1N2v) virusThe second case, a human infection with a North American lineage avian influenza A(H7N2) virus, was reported to CDC during the week ending December 24, 2016 (week 51). The patient reported close, prolonged unprotected exposure to the respiratory secretions of infected, sick cats at a New York City animal shelter. This is the first avian influenza A(H7N2) virus infection in humans identified in the United States since 2003 and the first known human infection with an influenza A virus acquired through exposure to an ill cat. The patient was mildly ill, not hospitalized, and recovered completely.The third case, an infection with an influenza A(H3N2) variant (H3N2v) virus, was detected through the Department of Defense Global Laboratory–based Influenza Surveillance Program and reported by Texas during the week ending April 29, 2017 (week 17). The patient reported contact with swine at an agricultural event the week preceding illness onset, was not hospitalized, and fully recovered.WHO collaborating laboratories in the United States are requested to submit a subset of influenza-positive respiratory specimens to CDC for further virus characterization. CDC characterizes influenza viruses through one or more laboratory tests, including the following: genomic sequencing, antigenic characterization by hemagglutination inhibition (HI), or neutralization assays. Historically, until vaccine effectiveness estimates are available,For the 2016–17 season, CDC genetically characterized 2,476 influenza viruses (311 influenza A(H1N1)pdm09, 1,280 influenza A(H3N2), and 885 influenza B viruses) collected by U.S. laboratories since October 1, 2016. The hemagglutinin (HA) gene of 309 (99%) of the 311 influenza A(H1N1)pdm09 viruses analyzed belonged to the predominant 6B.1 genetic subgroup, and the remaining two (1%) belonged to genetic group 6B. The HA gene of 1,187 (93%) influenza A(H3N2) viruses analyzed belonged to the 3C.2a genetic group, and the remaining 93 (7%) belonged to the 3C.3a genetic group. Of note, the 3C.2a genetic group includes an emerging subgroup known as 3C.2a1. The HA genes of 495 influenza B/Yamagata-lineage viruses analyzed all belonged to genetic group Y3. The HA genes of 390 influenza B/Victoria-lineage viruses all belonged to genetic group V1A. However, 78 (20%) of the 390 B/Victoria-lineage viruses have several amino acid changes, including two amino acid deletions at positions 162 and 163 in the HA gene, which alter the antigenic properties of these viruses. Viruses with these changes are currently being referred to as the “B/Victoria deletion variant subgroup.”CDC has antigenically characterized 1,824 influenza viruses collected by U.S. laboratories since October 1, 2016 (296 influenza A(H1N1)pdm09, 772 influenza A(H3N2), and 756 influenza B viruses). Among the 296 A(H1N1)pdm09 viruses, 294 (99.3%) were antigenically characterized as A/California/7/2009-like, the influenza A(H1N1)pdm09 component of the 2016–17 Northern Hemisphere vaccine. Among the influenza A(H3N2) viruses, 730 (94.9%) were antigenically characterized as A/Hong Kong/4801/2014-like, a genetic group 3C.2a virus recommended as the A(H3N2) component of the 2016–17 Northern Hemisphere vaccine. Among 42 viruses that were antigenically different from A/Hong Kong/4801/2014-like viruses , 36 (85.7%) belonged to genetic group 3C.3a, represented by the A/Switzerland/9715293/2013 reference virus, which was included as the A(H3N2) component of the 2015–16 Northern Hemisphere vaccine.Among influenza B viruses, 327 B/Victoria-lineage viruses were antigenically characterized using postinfection ferret antisera and among these, 283 (86.5%) were antigenically characterized as B/Brisbane/60/2008-like, a recommended influenza B component of the 2016–17 Northern hemisphere trivalent and quadrivalent influenza vaccines. Among the 44 B/Victoria linage viruses that had reduced titers against B/Brisbane/60/2008-like viruses, 39 (88.6%) belong to the B/Victoria deletion variant subgroup. All 429 (100%) B/Yamagata-lineage viruses tested were antigenically characterized as B/Phuket/3073/2013-like, the recommended influenza B component of the 2016–17 Northern Hemisphere quadrivalent influenza vaccines.CDC tested 2,569 influenza virus specimens (304 influenza A(H1N1)pdm09, 1,303 influenza A(H3N2), and 962 influenza B viruses) collected in the United States during October 1, 2016–May 20, 2017 for resistance to the influenza neuraminidase inhibitor antiviral medications oseltamivir, zanamivir, and peramivir, which are currently recommended for use against seasonal influenza. All 2,569 influenza viruses tested were found to be susceptible to all three of these antiviral medications. An additional 34 influenza A(H1N1)pdm09 viruses were tested for resistance to oseltamivir and peramivir and an additional 1,083 influenza A(H3N2) viruses were tested for resistance to oseltamivir and zanamivir; all were found to be susceptible to these antiviral medications.Data collected through the U.S. Influenza Vaccine Effectiveness Network during November 28, 2016–April 14, 2017, indicate that influenza vaccination this season reduced the overall risk for influenza-associated medical visits by 42% (95% CI = 35%–48%). Vaccine effectiveness against the predominant influenza A(H3N2) viruses was 34% (95% CI = 24%–42%) and vaccine effectiveness against influenza B viruses was 56% (95% CI = 47%–64%).The Food and Drug Administration’s Vaccines and Related Biologic Products Advisory Committee (VRBPAC) has recommended that the 2017–18 influenza trivalent vaccine to be used in the United States contain an A/Michigan/45/2015 (H1N1)pdm09-like virus, an A/Hong Kong/4801/2014 (H3N2)-like virus, and a B/Brisbane/60/2008-like (B/Victoria lineage) virus. It is recommended that quadrivalent vaccines, which have two influenza B viruses, contain the viruses recommended for the trivalent vaccines, as well as a B/Phuket/3073/2013-like (B/Yamagata lineage) virus , when 47 jurisdictions reported widespread influenza activity. From 2011 to 2016, the peak number of jurisdictions reporting widespread influenza activity ranged from 20 during the 2011–12 season to 48 during the 2012–13 season.State and territorial epidemiologists report the geographic distribution of influenza in their jurisdictionsCDC monitors hospitalizations associated with laboratory-confirmed influenza infections in adults and children through the Influenza Hospitalization Surveillance Network (FluSurv-NET),The cumulative hospitalization rate during October 1, 2016–April 30, 2017, was 44.1 per 100,000 population among children aged 0–4 years, 16.7 among children and adolescents aged 5–17 years, 19.8 among adults aged 18–49 years, 65.1 among adults aged 50–64 years, and 290.5 among adults aged ≥65 years. Among all hospitalizations, 14,185 (78.0%) were associated with influenza A virus infections, 3,873 (21.2%) with influenza B virus infections, 62 (0.3%) with influenza A virus and influenza B virus co-infections, and 64 (0.4%) with an influenza virus for which the type was not determined. Among the 5,383 patients for which influenza A subtype information was available, 5,276 (98.0%) were infected with influenza A(H3N2) viruses and 107 (2.0%) were infected with influenza A(H1N1)pdm09 viruses.Complete medical chart abstraction data were available for 7,315 (40.2%) hospitalized adults and children with laboratory-confirmed influenza as of June 9, 2017. Among 6,838 hospitalized adults with complete medical chart abstraction, 6,434 (94.1%) had at least one reported underlying medical condition that placed them at high risk**** for influenza-associated complications. The most commonly reported underlying medical conditions among adults were cardiovascular disease (51.8%), metabolic disorders (44.4%), obesity (34.8%), and chronic lung disease (30.1%). Among 477 hospitalized children with complete medical chart abstraction, 269 (56.4%) had at least one underlying medical condition; the most commonly reported of these were asthma (26.4%) and neurologic disorder (23.2%). Among 447 women of childbearing age (15–44 years) hospitalized with laboratory-confirmed influenza infections, 119 (26.6%) were pregnant.CDC tracks pneumonia and influenza (P&I)–attributed deaths through the National Center for Health Statistics (NCHS) Mortality Reporting System. The percentages of deaths attributed to P&I are released 2 weeks after the week of death to allow for collection of sufficient data to produce a stable P&I mortality percentage. Weekly mortality surveillance data include a combination of machine-coded and manually coded causes of death collected from death certificates. During the 2016–17 season, there was a backlog of data requiring manual coding within the NCHS mortality surveillance data. Work is underway to reduce and monitor the number of records awaiting manual coding. The percentages of deaths attributable to P&I are higher among manually coded records than the more rapidly available machine coded records and might result in initially reported P&I percentages that are lower than percentages calculated from final data.†††† for 12 consecutive weeks from the week ending December 31, 2016 through the week ending March 18, 2017 (weeks 52–11). Mortality attributed to P&I peaked twice, once at 8.2% of all deaths during the week ending January 21, 2017 (week 3) and once at 8.1% during the week ending February 25, 2017 (week 8). During the 2011–12 through 2015–16 seasons, the peak weekly percentages of deaths attributable to P&I ranged from 8.7% during the 2011–12 season to 11.1% during the 2012–13 season.During the 2016–17 season, based on data from NCHS, the proportion of deaths attributed to P&I was at or above the epidemic thresholdCDC monitors pediatric influenza deaths through the Influenza-Associated Pediatric Mortality Surveillance System. As of June 9, 2017, a total of 98 laboratory-confirmed influenza-associated pediatric deaths occurring during the 2016–17 season had been reported to CDC from Chicago, New York City, and 28 states. Of these 98 deaths, 46 were associated with an influenza A(H3N2) virus infection, three with an influenza A(H1N1)pdm09 virus infection, 14 with an influenza A virus for which no subtyping was performed, 34 with an influenza B virus infection, and one with an influenza virus for which the type was not determined. Since influenza-associated pediatric mortality became a nationally notifiable condition in 2004, the total number of influenza-associated pediatric deaths per season has ranged from 37 to 171, excluding the 2009 pandemic, during which 358 pediatric deaths were reported to CDC during April 15, 2009–October 2, 2010.The 2016–17 influenza season was notable for the predominant circulation of influenza A(H3N2) viruses. Nationally, influenza activity peaked in mid-February, with influenza A(H3N2) viruses predominant early in the season through the week ending March 25, 2017 (week 12). Influenza B viruses became the predominant virus starting during week 13  and remained the predominant virus through the end of May. The timing of peak influenza activity varied across the United States. Influenza activity peaked at least 1 month earlier (week 52 to week 2) in the western United States  than in the rest of the country. During the 2016–17 season, severity indicators  were within the range that has been observed during previous seasons when influenza A(H3N2) viruses predominated. Previous influenza A(H3N2)–predominant seasons have been associated with increased hospitalizations and deaths compared with seasons that were not influenza A(H3N2)–predominant, especially among children aged <5 years and adults aged ≥65 years  against illness caused by influenza A(H3N2) viruses and 56% (95% CI = 47%–64%) against illness caused by influenza B viruses were similar to previous seasons when recommended vaccine viruses have been well matched to  circulating viruses, including the lower effectiveness observed against well-matched A(H3N2) viruses. Evidence of reduced protection against A(H3N2) viruses, even when vaccine viruses and circulating viruses are well matched, has been observed since the 2011–12 season. In general, vaccination with inactivated influenza vaccine has offered better protection against influenza A(H1N1) and influenza B viruses . Additional information regarding influenza viruses, influenza surveillance, influenza vaccine, influenza antiviral medications, and novel influenza A infections in humans is available online (https://www.cdc.gov/flu).Influenza surveillance reports for the United States are posted online weekly  viruses were identified most frequently, but influenza A(H1N1)pdm09 and influenza B viruses were also reported. Data collected from November 28, 2016 to April 14, 2017, indicate that influenza vaccination this season reduced the overall risk for influenza-associated medical visits by 42% (95% CI = 35%–48%). The composition of the 2017–18 influenza vaccine has been updated to better match circulating influenza viruses.Annual influenza vaccination is recommended for all persons aged ≥6 months and remains the most effective way to prevent influenza illness. Antiviral medications are an important adjunct to vaccination in the treatment and prevention of influenza. Early treatment with neuraminidase inhibitor antiviral medications is recommended for patients with severe, complicated, or progressive influenza illness and those at higher risk for influenza complications, including adults aged ≥65 years.


However, introduction of a homozygous mutation at the drug's target site, the cysteine 528 residue inside the XPO1 cargo-binding pocket, by genetic engineering, confers resistance to selinexor. Here we investigated whether this resistance to selinexor is recessive or dominant. For this purpose we have engineered multiple leukemia cell lines containing heterozygous or homozygous C528S substitutions using CRISPR/Cas9-mediated genome editing. Our findings show that heterozygous mutation confers similar resistance against selinexor as homozygous substitution, demonstrating that SINE resistance can be obtained by a single and dominant mutation of the cysteine528 residue in XPO1.Exportin-1 (CRM1/XPO1) is a crucial nuclear export protein that transports a wide variety of proteins from the nucleus to the cytoplasm. These cargo proteins include tumor suppressors and growth-regulatory factors and as such XPO1 is considered a potential anti-cancer target. From this perspective, inhibition of the XPO1-mediated nuclear export by selective inhibitor of nuclear export (SINE) compounds has shown broad-spectrum anti-cancer activity. Furthermore, the clinical candidate SINE, selinexor, is currently in multiple phase I/II/IIb trials for treatment of cancer. Resistance against selinexor has not yet been observed in the clinic, but  N-azolylacrylate derivative small-molecule inhibitors of the XPO1-mediated nuclear protein export [528 residue through a Michael type addition, thereby preventing the interaction of cargo with XPO1. Currently, the leading candidate of the SINE, selinexor (KPT-330), is under investigation in multiple stage I/II/IIb clinical trials. Natural or acquired resistance to selinexor has not been observed in the clinic, but in an attempt to select for resistance against these compounds in vitro, Crochiere and colleagues treated HT-1080 cells with gradually increasing concentrations of the drug [528 residue located in this cleft, is highly conserved amongst higher eukaryotes. However, recently, in an effort to validate the SINE-XPO1 drug-target interaction, we were able to successfully engineer the genome of Jurkat leukemic T-cells to substitute cysteine528, the anchor point of selinexor inside the cargo-binding pocket of XPO1, with a serine. These homozygous genome-edited XPO1C528S Jurkat cells were viable and showed a >250 fold resistance to SINE compounds, demonstrating that the anti-cancer activity of SINE compounds effectively results from XPO1 inhibition [in cellulo.Human exportin 1 (XPO1), also known as chromosome region maintenance 1 protein (CRM1), is a ubiquitously expressed member of the importin-β family of nucleocytoplasmic transport proteins . XPO1 mediates the export of many different cargo proteins from the nucleus to the cytoplasm, including tumor suppressive or growth regulatory related proteins such as nucleophosmin, p53, p21, pRb, BRCA1, APC and FOXO family proteins, but also viral proteins, such as HIV-1 Rev, are transported by XPO1  and XPO1n export -10. Theythe drug . Resistahibition . In thisXPO1 targeting single guide RNA together with a 135 bases single-stranded oligodeoxynucleotide repair donor template containing the TGT to TCA mutation in addition to 3 silent mutations  (classified as hemizygous)  Figure , Table 1C528S protein, wild-type XPO1 was extracted out of the cells with a biotinylated SINE compound, KPT-9058  is a crucial protein involved in the nuclear export of many cellular proteins , includif cancer , 9, 14. f cancer , 8. Espef cancer .528 residue located in the hydrophobic cargo-binding pocket of XPO1 [528 to ser substitution conferred cells with >100-fold resistance to selinexor. Using the biotinylated SINE compound KPT-9058 we showed that in heterozygous mutant cells, wild-type and mutant XPO1 are expressed in a 50/50 distribution. The XPO1C528S protein expressed from the mutant allele in these heterozygous mutants is sufficient to elicit complete resistance to selinexor, demonstrating the dominant nature of this resistance mutation. Furthermore, this dominant resistance also demonstrates that 50% of the total XPO1 protein pool is sufficient for cellular homeostasis of the cell. However, the cysteine substitution appears to affect the cell proliferation slightly, which might be explained by an altered affinity of mutant XPO1C528S to a subset of cargo-proteins, which in turn might alter the nuclear-cytoplasmic distribution of these XPO1 cargo proteins.The drug has been shown to specifically and selectively interact with the cysteine of XPO1 . Althougin vivo. Several studies focused on patient samples have suggested that recurrent mutation of residue E571 located in the NES binding groove of XPO1 might have prognostic or therapeutic value for different types of cancer [528 residue is highly conserved and mutation has not been observed in in vivo models. Additionally, selinexor is currently under investigation in many different clinical trials focused on its anticancer activity, but to our knowledge none of these trials have examined the appearance of cysteine528 mutations in the different treatment regimens. Nevertheless, here we have shown that heterozygous substitution of this single residue is sufficient to confer cells with resistance against selinexor. Interestingly, this mutation also resulted in decreased cellular growth rates, suggesting this mutation might reduce cancer cell fitness. However, further in vivo studies are needed to fully elucidate the possible effect of cysteine substitutions on cellular homeostasis, cancer cell fitness or tumor growth rate and selinexors anticancer activity.Little is known on possible resistance mechanisms to selinexor f cancer -20. Howef cancer . Furtherf cancer , but the528 can arise in the clinic. Overall our results demonstrate that heterozygous genomic mutation of cysteine528 in XPO1 is sufficient for the development of a resistance mechanism to selinexor and further highlight the applicability of CRISPR/Cas9 genome editing for recessive and dominant drug-target interaction and validation studies.To summarize, this study provides important predictive information of a possible resistance mechanism that could arise upon selinexor treatment. However, it remains to be seen whether a heterozygous or homozygous mutation of the XPO1 cysteine2 flasks at 37°C and 4,5% CO2. Cells were grown in complete RPMI 1640 (Jurkat) or IMDM medium  containing 2 mM L-glutamine and supplemented with 10% fetal bovine serum and 20 μg/mL gentamicin. Cells were kept between 3 to 15 x 105 cells per mL.HL-60, Jurkat and K-562 cells were obtained from ATCC. All cells were cultured in 75cmxg and resuspended in Resuspension Buffer R  according to the instructions. Purified and highly concentrated DNA plasmids expressing Cas9-NLS (0.5 μg), single guide RNA targeting XPO1 (0.5 μg) and the donor cys528ser oligonucleotide (1 μg) were added to the resuspended cells. Next the mixture, containing 200.000 cells, was electroporated with the following settings: 10 μL, 1350V, 10 ms and 3 pulses. Following electroporation, cells were immediately plated in 1 mL of antibiotic free RPMI 1640 or IMDM Medium  containing 10% fetal bovine serum in a 24-well plate and cultured at 37°C in a CO2 incubator. After two days the medium was refreshed and KPT-185 was added to a final concentration of 100 nM. Cells were then maintained and grown in the presence of the compound over a period of 1-3 weeks following standard cell culture guidelines. After this period, surviving cells were harvested and cultured further. When cultures were sufficiently grown, cells were plated at a density of 0.5 cells/well in 96-well plates in 20% FBS containing medium to obtain single cell derived colonies. Colonies were grown for 2-6 weeks and were regularly screened. Ultimately, colonies were harvested and the genomic DNA was extracted for Sanger sequencing.HL-60, Jurkat, and K-562 cells were transfected using electroporation with a Neon Transfection system . Briefly, cells were collected by centrifugation at 400XPO1 near the cys528 coding region contains the following targeting sequence: 5′ GGATTATGTGAACAGAAAAG 3′-AGG (bold indicates the NGG protospacer adjacent motif). The oligonucleotide used for homologous recombination consisted of a 135 bases single stranded deoxynucleotide molecule containing two outer arms (50-80bp) homologous to the XPO1 genomic region flanking the codon for cysteine528 of XPO1 and was synthesized by Integrated DNA Technologies. The oligonucleotide contained three silent mutations near the PAM site and two point mutations at the cys528 coding triplet to provide the template for the Cys528Ser mutation and consisted of the following sequence: 5′-GCTAAATAAGTATTATGTTGTTACAATAAATAATACAAATTTGTCTTATTTACAGGATCTATTAGGATTATCAGAACAGAAgcGcGGCAAAGATAATAAAGCTATTATTGCATCAAATATCATGTACATAGTAGG-‘3 .The optimized sgRNA construct targeting XPO1, was amplified by PCR with the following primers: fwd: 5′-TCTGCCTCTCCGTTGCTTTC, rv: 5′-CCAATCATGTACCCCACAGCT. After PCR, the products were sequenced by Sanger Sequencing (Macrogen) using a forward and reverse primer .When single cell derived colonies in 96-well plates were sufficiently grown, cells were washed and then lysed in Bradley lysis buffer at 56°C (10 mM Tris-HCl (pH 7.5), 10 mM EDTA, 0.5% SDS, 10 mM NaCl and 1 μg/mL proteinase K). The genomic DNA was extracted from the lysate using ethanol-salt precipitation coupled to centrifugation. The target site, the DNA sequence around 6 Jurkat cells were plated in 1.5 mL of RPMI supplementend with 2 mM L-glutamine, 10% FBS and 20 μg/mL gentamicin inside 6-well plates. After a few hours inside the incubator, 1.5 mL of RPMI containing 2 μM of KPT-9058 was added to the cells . Cells were incubated for 2 hours and then collected by centrifugation (400xg) and washed in ice-cold PBS. Cell pellets obtained after centrifugation (400xg) were lysed on ice in RIPA buffer supplemented with 1x HALT protease inhibitors (Thermo Scientific) and then cleared from debris by centrifugation at 20.000xg for 10 min at 4°C. Protein concentrations were measured using a colorimetric BSA protein assay (Pierce). A fraction of the cell lysis mixture was taken for quantification of β-tubulin and XPO1 total protein. The rest of the extracts were allowed to bind to Dynabeads MyOne Streptavidin T1 (Life Technologies) by rotating overnight at 4°C in order to capture KPT-9058. Following overnight incubation, the beads were washed 5 times in modified RIPA buffer , 0.1% sodium deoxycholate, 1 mM EDTA) and subsequently boiled for 10 minutes in 0.5% SDS containing 1x sample buffer (Protein Simple) to elute captured proteins. Proteins were finally separated by size (12-230 kDa) and visualized on a Wes system (Protein Simple) with an anti-rabbit HRP conjugated antibody detecting the primary XPO1  and β-Tubulin  rabbit antibodies. Protein signals were visualized and quantified with the Compass software, v2.7.1 (Protein Simple).For pull down of XPO1 out of cells using KPT-9058, 5x10Growth rates of mutant and wild-type cell lines were determined with the CyQuant Direct Cell Proliferation kit (ThermoFisher Scientific). Initially a standard curve was obtained to determine the relation of the fluorescent signal to the amount of cells present in the sample. For the experiment, 20.000 Jurkat cells or 10.000 K-562 and HL-60 cells were seeded in different 96-well plates on day 0 in complete RPMI or IMDM medium respectively. The cells were allowed to grow over a period of 3-4 days and the amount of cells was determined every 24 hours by measuring fluorescence of the CyQuant Direct Cell Proliferation reagent with a SaFire II microplate reader according to the manufacturer's instructions. The obtained data points were compared to the previously obtained standard curves and cell numbers were normalized to day 0. Growth curves were then obtained by fitting the data with an exponential growth curve in GraphPad Prism and statistical significance was determined. The experiment was performed in triplicate and repeated at least once. For the determination of average cell sizes, all cells were analyzed during 3 different passages with the LUNA automated cell counter (Logos Biosystems). Statistical significance between cell sizes was determined with ANOVA.2. Cell viability was assessed with the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay reagent (Promega) according to the manufacturer's instructions. Absorbance of the samples was measured at 490 nm using a SaFire II microplate reader (Tecan). All assays were performed in duplicate and each experiment was repeated at least three times. The data was analyzed using a log-based 4 parameter model in GraphPad Prism and the bottom value for the EC50 calculation was set by hand for the mutant K-562 cells.Cell viability assays were performed by plating 10.000-20.000 cells in 96-well plates containing DMSO or a dilution of KPT-330. Cells were incubated for 72 hours at 37°C and 4.5% COJurkat cells were plated in a 96-well imaging plate  and treated with a dilution series of selinexor or carrier (DMSO) for 3 hours. After treatment, cells were washed with PBS and fixated for 10 minutes in 4% PFA. The cells were then washed and remaining PFA was inactivated with PBS with 0.1M glycine. Cells were permeabilized with PBS containing 0.1% Triton X-100 for 10 minutes. Subsequently, the cells were washed 3 times with PBS and then blocked at 37°C for 2 hours in 10% Normal Goat Serum. The primary anti-human RanBP1  antibody was then added and incubated for 1 hour. After primary staining, the cells were extensively washed and the secondary antibody, Alexa Fluor 488 , was incubated for 1 hour in the dark. Finally, cells were washed and the nuclei were counterstained with DAPI. The plate was imaged using the green and blue channels of an ArrayScan XTI High Content Reader (ThermoFisher Scientific). Nuclear and cytoplasmic compartments were segmented and their average pixel intensities were quantitated with the HCS Studio software. The obtained nuclear/cytoplasmic ratios were analyzed with GraphPad Prism. The experiment was repeated two times.


TCF7L2 rs7901695, FTO rs8050136, HHEX rs5015480, SLC30A8 rs13266634 and IGF2BP2 rs4402960) were observed in this study population. Furthermore, 14 of the tested SNPs had odds ratios (ORs) in the same direction as the previously published studies, suggesting that these variants can potentially be used in the Greek-Cypriot population for predictive testing of T2D. In conclusion, our findings expand the genetic assessment of T2D susceptibility loci and reconfirm five of the worldwide established loci in a distinct, relatively small, newly investigated population.Type 2 diabetes (T2D) has been the subject of numerous genetic studies in recent years which revealed associations of the disease with a large number of susceptibility loci. We hereby initiate the evaluation of T2D susceptibility loci in the Greek-Cypriot population by performing a replication case-control study. One thousand and eighteen individuals  were genotyped at 21 T2D susceptibility loci, using the allelic discrimination method. Statistically significant associations of T2D with five of the tested single nucleotide polymorphisms (SNPs) ( Type 2 diabetes (T2D) is a chronic complex heterogeneous disease of glucose metabolism caused by multiple genetic, epigenetic and environmental factors . It is cPPARG, KCNJ11 and TCF7L2 were replicated in most populations, thus being established as T2D associated genes , FTO rs8050136 [OR (95% CI) 1.34 (1.11–1.61) p-value = 0.002], HHEX rs5015480 [OR (95% CI) 1.36 (1.13–1.62) p-value = 0.001] and SLC30A8 rs13266634 [OR (95% CI) 1.31 (1.08–1.6) p-value = 0.007]. Further analysis with adjustment for age, gender and BMI showed statistically significant associations with five variants; in addition to the previous four, association with locus IGF2BP2 rs4402960 [OR (95% CI) 1.24 (1.01–1.53) p-value = 0.04] was also observed (TCF7L2 [OR (95%CI) 1.35 (1.1–1.64) p-value = 0.003], slightly weakened the association with HHEX [OR (95%CI) 1.38 (1.13–1.69) p-value = 0.002] and FTO [OR (95% CI) 1.33 (1.08–1.63) p-value = 0.006] and significantly weakened the association with SLC30A8 [OR (95% CI) 1.31 (1.05–1.63) p-value = 0.02]. The 19 SNPs were tested for association with T2D and the results are presented in observed . FurtherTHADA rs 7578597 and TCFL2 rs7901695) were associated with earlier age of diagnosis at nominal significance levels (p < 0.05), these did not survive Bonferroni correction [p < 0.0026 (0.05/19)]. For each extra risk allele of SNP rs7578597, the mean age of diagnosis was reduced by 3.81 years (p = 0.006) and for each extra risk allele of rs7901695, the mean age of diagnosis was reduced by 1.91 years (p = 0.005).Association analyses were also performed between the 19 SNPs and age of T2D diagnosis using linear regression and adjusting for gender and BMI . AlthougType 2 Diabetes is a chronic complex disease of glucose metabolism with a strong genetic contribution and a worldwide prevalence that reaches epidemic proportions . Many GWWe performed the first genetic study of T2D in the Greek-Cypriot population, a population of Caucasian origin. Compared to other Caucasian populations the Cypriot population is genetically distinct with significant differences from the Northern European populations ,40, and TCF7L2, rs8050136 in FTO, rs5015480 near HHEX, rs13266634 in SLC30A8 and rs4402960 in IGF2BP2) showed statistically significant associations with T2D in this population after adjustment for age, gender and BMI. Previous studies on SNP rs8050136 (FTO) report that the association with T2D is abolished after adjustment for BMI, thus indicating that the association is probably mediated through a primary effect on BMI, a finding that necessitates the inclusion of BMI as a covariate in similar analyses [FTO) with T2D. This result indicates that the association is likely not mediated through BMI in the Greek-Cypriot population, also in agreement with other published studies [This study focused on an initial evaluation of 21 known T2D common susceptibility loci, most of which were identified in Caucasian populations. Five of the tested loci , which was initially identified in the Japanese population [Nine of the non-statistically significant variants are affecting the disease in the same direction as previously reported in other populations, suggesting that these associations might not have been identified in this population due to reduced power. However, lack of association may also be attributed to true genetic diversity. At least for the significant loci and for the majority of the loci affecting the disease in the same direction, the obtained risk allele frequencies (RAF) and effect sizes in the Greek-Cypriot population, are similar, with overlapping confidence intervals with the published studies. The highest difference in the obtained RAF compared to the published study data was observed for SNP rs2237892  and rs7901695 (TCF7L2) were detected at nominal significance, however both associations did not survive Bonferroni correction.The association of age at T2D diagnosis with the 19 SNPs was further tested. Previous studies in other populations revealed associations of the age at T2D first diagnosis with specific loci, including variants in the EM genes ,44,46,47TCF7L2 and rs4402960 in IGF2BP2) are common with replicated associations in the Greek-Cypriot population. Additional SNPs (rs13266634 in SLC30A8 and rs8050136 in FTO) replicated with significant association in this study, were not statistically associated with T2D in the Lebanese population [HHEX) in the Cypriot population. Furthermore, a lack of association in the Lebanese population is reported for five of this study’s non-significant SNPs  [CDKAL1 and rs34872471 in TCF7L2) were reported with genome-wide significant association [To our knowledge, similar replication studies in neighbouring populations have only been performed in the Lebanese population ,48,49 anpulation . The Leb0010131) , for thrC1q SNP rs2920001 [IL-6 SNP rs1800795 were identified in the Greek population [TCF7L2 gene variant (rs7903146) was obtained through the study. Overall, published studies do not allow for a comparison between the Greek and the Greek-Cypriot populations due to the absence of any common study SNPs or diabetes study type.A few small studies relevant to T2D susceptibility or to disease complications have been reported in the Greek population focused on loci that were not identified through GWAS ,52. Assos2920001  and diabpulation . An assoCAPN10 variants [IRS1 variant [ABCC8 variants [ADIPOQ variants [KCNJ11 variant rs5219 [MTHFR [IL-4, IL-13 [GSTP1 [A replication study has not been performed in any of the remaining neighbouring populations. The majority of studies in the Turkish population were focused on variants in single genes, which identified the association of T2D with variants ,55, an I variant , some ABvariants  and somevariants . In one t rs5219 , in agre9 [MTHFR , IL-4, I4, IL-13  and GSTP3 [GSTP1  variantsTCF7L2, HHEX, SLC30A8 and IGF2BP2), have been associated with impairments in insulin secretion or sensitivity [FTO and IGF2BP2), have been associated with regulation of adipogenesis [TCF7L2, FTO and HHEX, were altered in human islets from patients with T2D compared to controls without diabetes. New target genes with altered DNA methylation and expression in human T2D islets contributing to impaired insulin and glucagon secretion, have also been identified [The majority of T2D susceptibility loci including four of the current study replicated loci  are replicated in the Greek-Cypriot population. Fourteen of the nineteen loci tested had ORs in the same direction as previously published, suggesting that these variants can potentially be used in the Greek-Cypriot population for predictive testing of T2D. This study initiates the genetic assessment of T2D in a distinct newly investigated population. Statistically significant associations of T2D with five established loci (


These 146S-specific VHHs were specific for particular O serotype strains and did not bind strains from other FMDV serotypes. Here, we describe the isolation of 146S-specific VHHs against FMDV SAT2 and Asia 1 strains by phage display selection from llama immune libraries. VHHs that bind both 12S and 146S particles were readily isolated but VHHs that bind specifically to 146S particles could only be isolated by phage display selection using prior depletion for 12S particles. We obtained one 146S-specific VHH—M332F—that binds to strain Asia 1 Shamir and several VHHs that preferentially bind 146S particles of SAT2 strain SAU/2/00, from which we selected VHH M379F for further characterization. Both M332F and M379F did not bind FMDV strains from other serotypes. In a sandwich enzyme-linked immunosorbent assay (ELISA) employing unlabeled and biotinylated versions of the same VHH M332F showed high specificity for 146S particles but M379F showed lower 146S-specificity with some cross-reaction with 12S particles. These ELISAs could detect 146S particle concentrations as low as 2.3–4.6 µg/l. They can be used for FMD vaccine quality control and research and development, for example, to identify virion stabilizing excipients.Intact (146S) foot-and-mouth disease virus (FMDVs) can dissociate into specific (12S) viral capsid degradation products. FMD vaccines normally consist of inactivated virions. Vaccine quality is dependent on 146S virus particles rather than 12S particles. We earlier isolated two llama single-domain antibody fragments (VHHs) that specifically recognize 146S particles of FMDV strain O Foot-and-mouth disease (FMD) is an animal disease that is caused by a picornavirus, FMD virus (FMDV), which encompasses seven serotypes: A, O, C, Asia1, SAT1, SAT2, and SAT3. Infection with any one serotype does not produce significant humoral immunity against other serotypes. In FMD endemic areas vaccination is used as a preventive method –7.1 Manisa  centrifugation . Novel m1 Manisa , 18. In  samples . However1 Manisa that were earlier isolated from llama immune libraries . Buffer was exchanged to PBS and free biotin removed by at least three consecutive 10-fold dilutions and concentration using Amicon Ultra 3-kDa molecular weight cutoff concentration devices . The VHH2 M3ggsVI4Q6E specific for 12S particles has been previously described /67, A Turkey/98, and A24 Cruzeiro/Brazil/55 were used for antigen production. FMDV antigen originated from the virus production facilities in Lelystad. FMDV was cultured using BHK-21 cells grown in suspension in industrial size bioreactors. FMDV present in the clarified culture was inactivated with 0.01 M binary ethylenimine and concentrated using two consecutive polyethylene glycol-6000 precipitations, resulting in crude antigen. Infectious FMDV was prepared at laboratory scale by growing them on monolayers of BHK-21 cells and harvesting the culture supernatant without further treatment. Work with infectious FMDV was done in a high containment unit at Wageningen Bioveterinary Research using appropriate measures for virus handling and waste disposal to prevent FMDV introduction into the environment. Wageningen Bioveterinary Research is authorized to work with live FMDV according to EU directive 2003/85/EC.The FMD vaccine strains Asia 1 Shamir/Israel/89, SAT2 SAU/2/00, Og. The gradients were fractionated into 20 0.61 ml aliquots and the absorbance at 254 nm was determined to identify the 146S peak. The 146S concentration in milligrams per liter was then calculated by multiplying the absorbance at 254 nm with 126.7. 12S particles were prepared from 146S preparations by acidification (12SA) or heat treatment for 1 h at 56°C (12SH) as previously described  when employing depletion. Bound phages were finally eluted by incubation with 1 mg/ml trypsin in PBS for 30 min at 37°C and immediately transduced to Escherichia coli TG1 [(F′ traD36 proAB lacIqZ ΔM15) supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5(rK−mK−)] cells. In each selection round, we performed a phage ELISA simultaneous with the phage display selection for evaluation of the phage display selection. For this purpose, a duplex plate containing similar concentrations and types of antigen and phage was incubated with a peroxidase-conjugated mAb against M13 phage instead of incubation with trypsin. The amount of bound antigen-specific phage was then measured by phage ELISA.Phage display selections were performed by two consecutive rounds of biopanning  in polysE. coli TG1 cells, individual colonies were picked and the VHH genes were induced with 1 mM isopropyl β-d-thiogalactopyranoside. Recombinant VHHs, extracted from the periplasm, were tested for binding to FMDV antigens at 10-fold dilution as described below in Section “After the second round of panning phages was transduced to Sequence analysis of the VHH encoding region was performed, as previously described . The ded2PO4; 8.1 mM Na2HPO4; pH 7.4) or PBSTM for 1 h at RT. The 146S concentration of the FMDV antigens was determined by measuring the absorbance at 254 nm as described above. The concentration of the 12S antigens was derived from the 146S concentration of the sample from which it was prepared assuming complete conversion of 146S into 12S particles. Plates were next incubated with either phage-displayed VHH, E. coli-produced VHH, or yeast-produced biotinylated VHH, and subsequently with a suitable specific peroxidase conjugate, using the same buffer as used for incubation with FMDV antigen (ELISA-buffer or PBSTM). The peroxidase conjugate was subsequently detected by staining with 3,3′,5,5′-tetramethylbenzidine. After stopping the reaction by addition of 0.5 M H2SO4 (50 µl per well) the absorbance at 450 nm was measured using a Multiskan Ascent spectrophotometer .Three different ELISA procedures were used. We first describe the basic ELISA procedure that was commonly used in these three procedures. ELISAs were performed by coating high-binding polystyrene 96-well plates (Greiner) with 0.1–1 mg/l of unlabeled VHH, VHH2, or mAb in 50 mM carbonate/bicarbonate buffer, pH 9.6 (coating buffer), overnight at 4°C. Coating and subsequent incubations were performed using 100 µl per well. After washing, the coated plates were incubated with FMDV antigens in ELISA-buffer  and FMDV antigen (0.5 mg/l) as described in Table  mg/l andE. coli-produced soluble VHH plates were preferably coated with 1 mg/l VHH and then subsequently incubated with 1 mg/l FMDV antigen in ELISA buffer. Bound FMDV antigens were detected by incubation with 10-fold diluted E. coli-produced soluble VHH, which contains a myc-tag, and a peroxidase-conjugated mAb against the c-myc tag .For ELISA using A preparations of FMDV antigen were used as standards in the ELISA employing M3ggsVI-4Q6E. Standards were serial twofold dilutions of 1 mg/l start concentration. Plates were next incubated with 0.25 mg/l of biotinylated VHH(2). Bound biotinylated VHH(2) was detected with 1 mg/l PO-conjugated streptavidin . Absorbance data were evaluated using an Excel® spreadsheet template . A four-parameter logistic curve was fitted to absorbance and FMDV antigen concentrations of standards by non-linear least squares using the Excel® solver tool. The FMDV antigen concentration in unknown samples was then determined by interpolation. For determination of the quality of binding of VHHs to particular FMDV antigens these antigens were titrated in twofold dilution series. The effective antigen concentration required to reach a particular absorbance value was then interpolated after four-parameter logistic curve fitting of absorbance and antigen concentrations.For DAS ELISA to measure the FMDV antigen concentration plates were coated with 0.5 mg/l of unlabeled VHH or VHH2. These plates were then incubated with serial 2-fold dilution series of FMDV antigen preparations in ELISA buffer. Normally, standards of untreated FMDV antigen were included in the ELISAs for quantification of 146S particles. However, 12SThe limit of detection (LOD) of ELISAs was measured by titrating FMDV antigens in triplicate and interpolating the VHH concentration required to reach the average and three times the SD of nine blank measurements without antigen.1 Manisa, A Turkey, A22 Iraq, and Asia 1 Shamir. We isolated many FMDV O1 Manisa binding VHHs from this llama by phage display employing biotinylated FMDV antigen captured with streptavidin-coated magnetic beads . After sequence analysis (Table A particles (see below), it was replaced by a novel isolated VHH that was highly produced in yeast, M311F, for capture of SAT2 SAU/2/00 146S particles. Furthermore, novel selections were performed using depletion with immobilized 12SA particles instead of competition with soluble 12SA particles. Using these novel selection procedures we retrieved seven unique SAT2 SAU/2/00 binding VHHs comprising three novel CDR3 groups (Table To improve the selection process of 146S-specific VHHs we purified 146S particles by SDGs. The purified 146S particles were then used for both novel llama immunization and phage display selection of VHHs. One llama (3049) was immunized with FMDV Asia 1 Shamir 146S and another llama (3050) with SAT2 SAU/2/00 146S. As observed with many other proteins  FMDV losN-glycosylation sites to prevent compromised antigen binding due to fortuitous N-glycosylation. The purified VHHs were biotinylated for use in DAS ELISA in combination with the same unlabeled VHH for coating. To assess the specificity of the biotinylated VHHs for the different FMDV particles we performed DAS-ELISAs with a titration series of untreated antigen, 146S particles and 12SH. Similar to earlier methods  show higher 146S specificity with ratios of 10–28  present in SDG fractions. As controls, we also analyzed each fraction using VHHs that detect both 12S and 146S particles of Asia 1 Shamir, SAT2 SAU/2/00, or O1 Manisa purified 146S particles were again separated on SDG a peak was seen in fractions 15–18 that corresponds to 146S particles (Figures A particles. When crude antigen was separated on SDG some antigenic material was detected in fractions 6–14 by ELISA (Figures A and 146S particles, although 75S particles would be expected to form a sharper peak. The amount of putative 75S particles as compared to 146S particles is especially high for strain Asia 1 Shamir (Figure 1 Manisa (Figure 1 Manisa the material in fractions 1–15 was plotted on a scale of 0–0.5 mg/l on the right axis (Figure 1 Manisa (Figure A particle reactivity in the M311F ELISA and the use of untreated antigen that predominantly consisted of 146S particles as a standard. Acidified O1 Manisa antigen consisted only of 12S particles, indicating full dissociation of 146S and 75S particles into 12S (Figure When FMDV Asia 1 Shamir, SAT2 SAU/2/00, or OS Figure .1 Manisa 146S particles (Figures A particles are detected with reduced efficiency as compared to the M311F ELISA (Figures A particles, although with reduced efficiency compared to 146S particles, is consistent with the relatively low 146S specificity observed for M379F.The M170F DAS ELISA detects only O Figures C,F,I,L.  Figures A,D,G,J.  Figures B,E,H. Th1 Manisa since strain SAT2 SAU/2/00 is not detected by M3 ELISA (Figures 1 Manisa infectious virus contained a considerable putative 75S peak (Figure To elucidate the nature of the putative 75S particles we fractionated infectious FMDV on SDG and determined the presence of infectious virus in each fraction by plaque assay. In addition, we determined the concentration of total FMDV antigen present in each fraction by M3 ELISA after acidification. The M3 ELISA facilitates reliable quantification of 12S, 75S, and 146S particles against a 12S standard. This analysis was only done for strains Asia 1 Shamir and OTaenia solium antigen without cross-reacting with other Taenia species was earlier described (We aimed to isolate VHHs that specifically bind to FMDV 146S particles of strains SAT2 SAU/2/00 and Asia 1 Shamir. For this purpose, llamas 3049 and 3050 were immunized with SDG purified 146S particles of these strains. We preferentially used a 12S depletion step in phage display selections to enrich for 146S-specific VHHs. In this manner, we isolated three novel SAT2 SAU/2/00 binding VHHs showing reasonable 146S specificity and one novel Asia 1 Shamir binding VHH showing high 146S specificity. However, when using a depletion step there is some risk of detachment of captured 12S particles and carryover of this material into the wells containing 146S particles for panning, resulting in binding to capturing VHH and unwanted enrichment for 12S binding phage clones. Since 12S binding VHHs are generally not serotype specific this can be prevented by using 12S particles from another strain for depletion in combination with a serotype-specific VHH for capturing 146S particles. We currently favor this approach for isolation of 146S-specific VHHs. Selection of VHHs specifically recognizing escribed . Furtherescribed . Togethe1 Manisa binding VHH M170F (We developed two DAS ELISAs that utilize the same yeast-produced VHH for coating, as well as detection when in a biotinylated form, for quantification of FMDV 146S particles. The ELISA with M379F VHH for quantification of SAT2 SAU/2/00 FMDV required 28-fold higher 12S concentrations than 146S concentrations to reach similar absorbance values. Thus, it shows reasonable specificity for 146S particles. However, it cannot be used for quantification of 146S particles in the presence of high concentrations of 12S particles due to cross-reaction with 12S particles. Since the 12S content of vaccines is generally less than 20% of total FMDV antigen , such spHH M170F  and two HH M170F , 16. ThiHH M170F . The 1461 Manisa reveals that some immunoreactive material fractionates between the 12S and 146S peaks (Figure 1 Manisa FMDV confirms that this material is not infectious (Figure Double antibody sandwich enzyme-linked immunosorbent assay analysis of SDG fractionated inactivated FMDV antigen of strains SAT2 SAU/2/00, Asia 1 Shamir, and Os Figure . These ps Figure  is inacc1 Manisa FMDV using the M170F ELISA (The ELISAs developed here for quantification of 146S particles of strains Asia 1 Shamir and SAT2 SAU/2/00 can be used for quality control of FMD vaccines during and after manufacturing. There is a need for such quality control as many locally produced FMD vaccines show poor quality , 46. Sev0F ELISA . As comp0F ELISA  our ELISLlama immunizations were performed after ethical review by Wageningen Bioveterinary Research and in accordance with Dutch national guidelines on animal use.MH and EP performed experiments. MH, JS, PE, BC, and AD conceived and designed experiments and analyzed data. MH, JS, and AD wrote the manuscript. All the authors read and critically reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer, MG, and handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.


S100A7 is expressed in many squamous cell carcinomas (SCCs). Our previous study revealed that S100A7 was dramatically induced in several SCC cells and activation of the Hippo pathway significantly promoted S100A7 in epidermoid carcinoma cells. However, whether the Hippo pathway regulates S100A7 expression in SCCs remains largely unknown. Here, we uncover that S100A7 induction by the Hippo-YAP pathway displays different characteristic in cervical and glossopharyngeal SCC. In well differentiated HCC94 cervical cells and FaDu pharyngeal cells, S100A7 is easily induced by both suspension and dense culture, which is accompanied by an increase in YAP phosphorylation and a decrease in nuclear YAP. Strikingly, these correlations of S100A7 and YAP reverse after recovery of cell attachment or relief from dense culture. Further examination finds that S100A7 induction is significantly repressed by nuclear YAP, which is validated by activation or inhibition of the Hippo pathway via loss- and/or gain-of- LATS1 and MST1 function. Subsequently, we prove that TEAD1 is required for YAP transcriptional repression of S100A7. However, S100A7 is hardly induced in poorly differentiated SiHa cervical cells and NCI-H226 pulmonary cells even in suspension or activation of the Hippo pathway. More importantly, cervical and lingual SCC tissues array analyses show that S100A7 expression displays the positive correlation with pYAP-S127 and the negative correlation with nuclear YAP in the majority of well differentiated but not in poorly differentiated tissues. Collectively, our findings demonstrate that the different induction of S100A7 toward activation of the Hippo pathway mainly depends on the degree of cell differentiation in cervical and glossopharyngeal SCC. Squamous cell carcinomas (SCCs) are the most common cancer and can be very aggressive and metastatic. S100A7 (psoriasin) belongs to the S100 multigenic family of calcium-modulated proteins of the EF-hand type and is originally identified in psoriatic keratinocytes. SubsequeThe Hippo pathway is a newly established tumor suppressor pathway that plays a central role in tissue homoeostasis. At the cHere we report that S100A7 is inducible in well differentiated HCC94 and FaDu SCC cells but not in poorly differentiated H226 and SiHa cells. We further demonstrate that S100A7 induction in HCC94 and FaDu SCC cells is repressed by YAP/TEAD1 via activation of the Hippo pathway. The negative correlation of S100A7 expression and nuclear YAP is detected in well differentiated cervical and glosspharyngeal SCC cells and tissues. Thus, our findings provide a new insight for understanding the characteristic of S100A7 induction by the Hippo-YAP pathway in cervical and glossopharyngeal SCC.A full description of materials and methods, including Plasmids and Reagents, Western blot, Immunofluorescence staining, Immunohistochemistry, MTT assay and Statistical analysis was described in 2) and at high cell density .Human squamous carcinoma cell lines HCC94, FaDu, SiHa and NCI-H226 were purchased from the Chinese Academy of Sciences Committee Type Culture Collection Cell Bank and were authenticated by short tandem repeat analysis at HK Gene Science Technology Co. . All cells were cultured according to the corresponding culture methods of the ATCC and Chinese Academy of Sciences Committee Type Culture Collection Cell Bank. Cell suspension cultures were obtained as described in our previous studies. CulturesTo silence the expression of YAP, LATS1, MST1, TEAD1, TEAD2, TEAD3 and TEAD4, all siRNAs as well as the non-targeting control siRNA were purchased from Gene Pharma  and transfected using the Transfection Reagent  according to the manufacturer's protocol. For each gene, two individual siRNAs were used .Total RNA was extracted from cells for the generation of single-stranded cDNA. Quantitative RT-PCR (qPCR) was performed using an ABI 7300 Real-time PCR System  with the Power SYBR® Green PCR Master Mix (Life Technologies Ltd) in a final volume of 20 μL. GAPDH was used as an endogenous control for each sample. The primers used for each of the genes are listed .Lingual SCC (No. OR601b) and uterine cervix SCC (No. CR803) tissue microarrays were purchased from Xi’ an Alenabio Company . All patients with cancer had received a pathological diagnosis and none had received prior therapy. All cancer tissues were obtained from surgically treated patients who gave their written informed consent to participate in this study and the Ethics Committee of the General Hospital of PLA Rocket Force (No. KY2015031) approved this protocol.CTGF (Connective tissue growth factor) and CYR61 (Cysteine-rich angiogenic inducer 61), two direct endogenous markers of YAP, were analyzed by quantitative RT-PCR (qPCR) as readout of YAP activity. Consistent with the increased YAP phosphorylation, CTGF and/or CYR61 transcription are significantly suppressed in suspended and dense HCC94 and FaDu cells Click here for additional data file.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S1 Fig2; Dense: 75 000–100 000 cells/cm2. After two days, cells were visualized by AxioObserverD1. Scale bar, 200μm.(a) HCC94, FaDu and SiHa cells were seeded to obtain sparse and dense cells. Sparse: 7 500 cells/cm(TIF)Click here for additional data file.S2 Fig(a-d) The silencing efficiency of LATS1 and MST1 siRNAs is detected by western blotting (Upper panel: a and b) and qPCR (Lower panel:c and d).(TIF)Click here for additional data file.S3 FigCTGF, CYR61 and S100A7 are analyzed by qRT-PCR. siLATS1+S48h (or D48h) indicates that cells are cultured in suspension (or dense) for 48 h after silencing of LATS1. siMST1+S48h (or D48h) indicates that cells are cultured in suspension (or dense) for 48 h after losing of MST1. H: HCC94 cells; F: FaDu cells. Error bar, SD of three different experiments. *P<0.05, **P<0.01; t-test(a-d) The mRNA levels of (TIF)Click here for additional data file.S4 FigCTGF, CYR61 and S100A7 in LatB (a) or Cyto D (b) treated HCC94 and FaDu cells is detected by qRT-PCR. Error bar, SD of three different experiments. *P<0.05, **P<0.01; t-test. (c) C3 (1 μg/ml) was added to HCC94  and FaDu  cells with serum-free growth medium for 4 h prior to harvesting for qRT-PCR. H: HCC94 cells; F: FaDu cells. Error bar, SD of three different experiments. *P<0.05, **P<0.01; t-test. The expression of (TIF)Click here for additional data file.S5 Fig(a-c) RT-PCR analyses for S100A7 expression after TEAD2 (a), TEAD3 (b) and TEAD4 (c) silencing in normal attached HCC94 cells. β-actin was used as a loading control.(TIF)Click here for additional data file.S6 Fig-/YAP+ tissues. Sample 23–25 are S100A7+/pYAP-S127-/YAP- tissues.(a) Sample 1–17 are S100A7+/pYAP-S127+/YAP+ tissues.Sample 18–22 are S100A7+/pYAP-S127(TIF)Click here for additional data file.