To determine whether deletion of Rb specifically in keratin (K) 18 positive TEC was sufficient for thymocyte hyperplasia, we conditionally inactivated Rb and its family members p107 and p130 in K18+ TEC in genetically engineered mice . We found that thymocyte hyperproliferation was induced in mice with Rb inactivation in K18+ TEC, while normal T cell development was maintained; suggesting that inactivation of Rb specifically in K18+ TEC was sufficient and responsible for the phenotype. Transplantation of wild type bone marrow cells into mice with Rb inactivation in K18+ TEC resulted in donor T lymphocyte hyperplasia confirming the non-cell autonomous requirement for Rb proteins in K18+ TEC in regulating T cell proliferation. Our data suggests that thymic epithelial cells play an important role in regulating lymphoid proliferation and thymus size.Thymic epithelial cells (TEC), as part of thymic stroma, provide essential growth factors/cytokines and self-antigens to support T cell development and selection. Deletion of Rb family proteins in adult thymic stroma leads to T cell hyperplasia  Thymic stroma is very heterogeneous, consisting of cortical thymic epithelial cells (cTEC), medullary thymic epithelial cells (mTEC), fibroblasts, macrophage, dendritic and endothelial cells , 2. Epitof K8/18 –11, and of K8/18 . While iof K8/18 .121) in specific K18 subtype  [TgK18GT121 mice with mice expressing Cre-recombinase inactivated all three Rb family members in K18-expressing TEC. We found that inactivation of Rb-TS in K18+ TEC is sufficient to promote T cell proliferation in a non-cell autonomous manner without disrupting T cell development.Rb and its family members (p107 and p130) are central regulators of the cell cycle. It has been demonstrated previously that inactivation of Rb tumor suppression (Rb-TS) (Rb and its family members p107 and p130) in multiple epithelial tissues and brain astrocytes initiates tumorigenesis in genetically engineered mice (GEM) by increasing proliferation and apoptosis mainly through a cell-autonomous mechanism –17. The 18 mice) . BreedinK18 mice (TgK18GT121) [β-actin Cre [R26CreER [PbCre4 [K18;R26CreER mice without tamoxifen treatment, which was sufficient to induce the transgene expression. Thus, K18;R26CreER mice were not treated with tamoxifen. R26YFP mice [β-actin Cre transgenic mice to harvest T cells for FACS analysis at 2 months of age. All bone marrow transplantation recipients were pretreated with acid water and antibiotics seven days before transplantation. Animals that did not receive bone marrow cells were moribund 12–15 days after irradiation due to failure of hematopoietic reconstitution.18GT121)  were croctin Cre , R26CreE [PbCre4  mice .This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committees (ACUC) at the University of North Carolina-Chapel Hill and at the National Cancer Institute (NCI)-Frederick (Permit Number: 11–030). All animals in this study were monitored daily and provided wet food when mice showed early evidence of sickness. Unexpected deaths were not observed. All mice were euthanized by CO2 asphyxiation per the “121) incubation overnight. Mixed Alexa fluor 488, 594, and 633  served as secondary antibodies. Nuclei were stained with DAPI. Images were captured using Zeiss light, immunofluorescence, or confocal microscopes.Thymuses were dissected and fixed overnight in 10% neutral buffered formalin, transferred to 70% ethanol, and routinely processed and embedded in paraffin. Four μm sections were stained with haematoxylin and eosin (H.E.) for histopathological examination. Immunohistochemistry (IHC) and immunofluorescence (IF) analyses were performed as previously described . AntibodT lymphocytes were mechanically dissociated from thymus using frosted glass slides in DMEM with 5% FBS. Red blood cells were lysed using ACK buffer, and passed through 40 μm mesh filter. T cells were incubated with Fc Block (BD Biosciences) in 3% fetal bovine serum (FBS) (v/v) in PBS for 20 minutes on ice. 1–2 million cells were incubated with or without fluorescent-conjugated antibodies that recognize CD4, CD8, CD45, B220, or isotype control antibodies (BD Biosciences) in the dark for 30 minutes at 4°C. Cells were washed 3x and resuspended in 1% FBS (v/v) in PBS. For FACS sorting, CD45 stained cells were sorted using BD FACSAria II SORP cell sorter (BD Biosciences). CD45+ T cells and CD45- thymic stromal cells were subjected to RNA extraction using Ambion RiboPure RNA purification Kit (Thermo Fisher Scientific) and RT-PCR (Supplementary Methods). For CD4 and/or CD8-stained cells, they were fixed with paraformaldehyde at a final concentration of 1% (v/v). Cells were then run on Dako CyAn ADP flow cytometer or BD FACSCanto II Analyzer, and analyzed using FlowJo software . At least 30,000 viable events were collected for analysis.C57BL/6 (WT) recipients were transplanted with 2x106 bone marrow cells from either Ly5.1+ K18;PbCre4 (K18) or WT mice by tail vein injection. The majority of the bone marrow cells used in our transplantation studies include hematopoietic stem and progenitor cells and mature hematopoietic cells . In addition, this population contains very few stromal cells of mesenchymal and endothelial lineages. For reciprocal transplantations, Ly5.2+ WT bone marrow cells were transplanted into irradiated Ly5.1+ K18;PbCre4 (K18) or WT recipients.Irradiated (10 Gy) 3 month old Ly5.2+ Student t test was performed to evaluate the statistical significance. P < 0.05 was considered statistically significant.RT-PCR and CBC analysis are described in the supplementary information .TgK18GT121; K18 mice), in which loxP-flanked eGFP stop cassette upstream of truncated SV40 T antigen  was driven by K18 regulation in a bacterial artificial chromosome (TgK18GT121 mice (TgK18GT121 mice to mice ubiquitously expressing Cre-recombinase (β-actin Cre and R26CreER)  . Transge medulla . Double/ medulla . Interes5+ cells , suggestK18;β-actin Cre and K18;R26CreER mice resulted in median survival of 94 and 41 days, respectively   , indicatK18 mice showed thymic hyperplasia  with low incidence of T-cell lymphoblastic lymphoma (2%)  . Overalloma (2%) , and theoma (2%) . Ki67 stoma (2%) , which wferating . This waCre mice .K18;Cre mice compared to wildtype by FACS analysis . Consistently, CD45+ thymocytes did not express eGFP mRNA by RT-PCR, while thymic stroma did (121 mRNA in bone marrow (BM) or spleen in Cre-induced K18 mice since all thymocytes were derived from BM  bone marrow cells into lethally irradiated Ly5.2+ WT recipients. There was no difference in weight or cellularity in recipient thymuses at 4–5 month post transplantation  of TEC. The CD4/CD8 profile was not altered in K18 recipients  [K18 recipients compared to WT recipients  . Interescipients , which m121 mouse model [121 mice for thymic hyperplasia-specific cause of death was longer than K18-driven T121 mice (7 month vs. 3 month), which was likely due to fewer K19+ T121-expressing cells. Furthermore, overexpression of cell cycle regulators (e.g. cyclin D1 or D2) in K5+ mTEC also led to the similar phenotype including cTEC, mTEC and T cell proliferation [Garfin et al. showed that although RB and its family members were deleted in both lymphoid lineage and thymic stroma using Mx1-Cre mice, the enlarged thymus phenotype was due to the proliferation of thymic stroma . Howeverse model . The medferation , 31–33. in vitro [The precise mechanism governing epithelial control on lymphoid proliferation in this model is unknown. Studies have shown a critical role of transcription factors on the regulation of TEC development  –39. As ain vitro , and actin vitro . In addiin vitro –45. Thisin vitro . The facin vitro [Our data suggest that Rb functions to regulate the proliferation of thymic epithelial cells, and inactivation of Rb and its family proteins in K18+ TEC extrinsically promotes the hyperproliferation of thymocytes, without affecting T cell development. Future studies will be aimed to further examine whether the modified K18+ TEC are functioning to produce molecules of known functional importance  and to promote correct positive and negative selection or tolerance, and whether T cells produced in contact with these modified epithelial cells are functionally normal. It is also our great interest to assess whether genetically modified K18+ thymic epithelial cells can be used to replace OP9-DL1 or OP9-DL4 co-culture system for supporting T cell development in vitro –52.S1 FigWT thymic cortex and medulla. (B) T121 IHC staining in thymic cortex and medulla in K18;β-actin Cre, K18;R26CreER, and K18;PbCre4 mice. (C) T121 IHC staining in thymuses of 1 and 2 month old K18;PbCre4 mice. Left panel: without primary antibody control. mo: month.(A) IHC staining of K5 and K19 antigens in (TIF)Click here for additional data file.S2 FigPbCre4 males by RT-PCR. Total RNA was extracted from thymus, bladder, and prostate . HPRT was used as loading control. (B) Representative flow cytometry plots of CD4 and CD8 expression in thymocytes from uninduced-K18 and induced-K18 mice. Only 2% of induced-K18 thymuses showed altered CD4 and CD8 profile. (C) Representative immunofluorescence images of T121 (green), K18 (yellow), K5 (red), and DAPI (blue) in cortex (C) and medulla (M) delineated by white dotted lines in induced-K18;Cre thymus. Right: higher magnification of left image. (D) Representative H.E. image of lymphoma developed in 2% of induced-K18 mice. (E) Representative images of T121 (green) and K18 (red) immunostaining in cultured thymic stroma cells derived from K18;PbCre4 mice demonstrating that T121 was expressed in K18 positive cells. Nucleus was counter-stained with DAPI as blue.(A) Cre mRNA levels in (TIF)Click here for additional data file.S3 FigRepresentative immunofluorescence images of Ki67 (green), K18 (red), and DAPI (blue) in thymic cortex and medulla. *Cells are positive for both Ki67 and K18.(TIF)Click here for additional data file.S4 FigK18;PbCre4 and C57BL/6 wildtype (WT) donors were transplanted into 3 month old Ly5.2+ lethally irradiated WT recipients. Total thymic cellularity in reconstituted recipients with either WT or K18;PbCre4 bone marrow cells as donor compared to intact WT control. (B) Bone marrow cells from Ly5.2+ C57BL/6 WT donors were transplanted into 3 month old Ly5.1+ lethally irradiated K18;PbCre4 (K18) and WT recipients (Rec). Flow cytometry analysis showed percentage of B220+ cells in spleen (top) and bone marrow  of WT Rec and K18 Rec. WT Rec were 4–5 month post transplantation, and K18 Rec were 4 month post transplantation. mo: months post transplantation. P < 0.05 is considered statistically significant.(A) Bone marrow cells from Ly5.1+ (TIF)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Defects in chromatin proteins frequently manifest in diseases. A striking case of a chromatin-centric disease is NUT-midline carcinoma (NMC), which is characterized by expression of NUT as a fusion partner most frequently with BRD4. ChIP-sequencing studies from NMC patients revealed that BRD4-NUT (B4N) covers large genomic regions and elevates transcription within these domains. To investigate how B4N modulates chromatin, we performed affinity purification of B4N when ectopically expressed in 293-TREx cells and quantified the associated histone posttranslational modifications (PTM) using proteomics. We observed significant enrichment of acetylation particularly on H3 K18 and of combinatorial patterns such as H3 K27 acetylation paired with K36 methylation. We postulate that B4N complexes override the preexisting histone code with new PTM patterns that reflect aberrant transcription and that epigenetically modulate the nucleosome environment toward the NMC state. Histones assemble genomic DNA into chromatin and display many posttranslational modifications (PTMs) such as lysine acetylation. Nearly all PTMs are enzymatically regulated, for instance by lysine acetyltransferases (KATs) such as EP300. In addition PTMs can also be recognized by other non-histone proteins containing specific domains, for instance by bromodomain proteins such as BRD4. Many of these factors themselves regulate gene expression . ConsequNUT gene , which is normally restricted to the germline . Whi. Whi8]. short, and BioTAP-B4N , where the single acetyl group can localize to either K18 or K23 and where these two isoforms can co-elute, we additionally relied on MS/MS quantification. We examined the average MS/MS across the chromatographic peak and determined the relative fragment ion ratios characteristic of either isoform.To determine the enriched histone PTMs, we performed label-free relative quantification of the various modified peptides. We integrated the chromatographic peaks of each peptide and normalized the area against all modified forms of the same peptide backbone. This method of quantification differs from spectral counting, which measures the total number of MS/MS collected. If our baits did not enrich for specific histone PTM patterns, we would expect to recover essentially background levels of PTMs that are of a similar relative distribution as the genomic input. For certain peptides such as the mono-acetylated H3 18–26 peptide (K16R and AKAKS16R) at a consistent frequency. We observed that the fold difference in the T16/S16 peptide levels did not differ by more than 20% of the sample mean value  and tri-methylation (me3) upon induction, most PTMs remain unchanged . Thus, Pshort PTM patterns . Although B4N did not enrich for mono-acetylation relative to the other baits, B4N did enriched for di-acetylation more than 8-fold over BRD4short  that spans K5, K8, K12, and K16 from the pulldowns. BRD4short significantly enriched for H4 tri-acetylation  and tetra-acetylation  relative to input  spanning K9 and K14. We found that BRD4short significantly enriched for di-acetylated H3K9acK14ac over input . Additionally we found that B4N enriches for di-acetylation at both H3 K9 and K14 by 2-fold over BRD4short  spanning K79. We found that BRD4short significantly enriched for H3K79 mono-methylation over input . We found that B4N enriched for K79me1, but not K79me2  that spans K27 and K36. The H3 data described so far in this paper could not distinguish between canonical H3 or variant H3.3, as the two proteins yield identical peptides at the K18/K23 and K9/K14 positions. However the alanine and serine substitution at position 31 allows us to use LC-MS to distinguish peptides spanning K27 and K36 from the canonical and variant proteins. In fact K27 and K36 PTM-specific antibodies likely cross-react with both canonical and variant H3 proteins.Following the K79 data, we investigated the methyl and acetyl marks enriched by B4N by focusing on the variant H3.3 27–40 peptide  and tri-methylated K27 (K27me3K36un), where B4N strongly depletes for both methyl forms. Rather than enriching for individual methyl PTMs, we found that B4N enriches for combinatorial PTMs such as mono-methylated K27 paired with di-methylated K36 (K27me1K36me2) as well as with tri-methylated K36 (K27me1K36me3). Notably K36 tri-methylation is rarely detected without the simultaneous occurrence of K27 mono-methylation in human cells.Our proteomic experiments revealed that B4N does not enrich for H3.3 containing only K27 methylation or only K36 methylation . This tracK36un) and paired with mono-methylated K36 (K27acK36me1). The K27acK36me1 combination is especially intriguing as this peptide is often below the quantifiable limit in other samples. Additionally K36 mono-methylation and K27 acetylation generally have distinct metagene profiles, with K36me1 in genic regions and K27ac in enhancers [Consistent with previous genomics data , our curnhancers . MS/MS cac  [It is more difficult though to interpret the B4N enrichment of K27as NSD3) . Hence, short  [short with pulse labeling of isotopically heavy glucose, which labels new acetyl groups. If our model for PTM generation within B4N domains were correct, we expect the acetyl labeling efficiency in B4N-associated chromatin relative to non-associated chromatin to be higher than BRD4short-associated chromatin relative to non-associated chromatin.A more direct future experiment to determine whether de novo PTM formation and turnover is occurring would be to induce B4N and BRD4With respect to the dynamics of histone PTM regulation, it is interesting to note that the rate of histone H4 acetylation turnover in human foreskin fibroblasts is nearly double when cells are actively proliferating as opposed to when cells are quiescent . As NMC One intriguing aspect of NMC is the sequestration of B4N-bound enzymes. Previous experiments have shown that B4N has lower intra-nuclear mobility than wild type BRD4, presumably reflecting the B4N complex being trapped within regions of concentrated histone acetylation . The titIn summary, B4N enriches for chromatin containing multiple acetyl and methyl groups. It would have been difficult to assay such complex isoforms with conventional antibody-based approaches. Furthermore it would have been challenging to isolate B4N-bound complexes with conventional native affinity approaches. The use of formaldehyde and sonication effectively preserves and solubilizes complexes containing DNA, RNA, and protein that could otherwise be destabilized with MNase digestion and salt extractions. In this respect, BioTAP-XL will be an invaluable method to understand how aberrant regulation of the histone code may drive other chromatin-centric diseases.S1 FigWestern Blot analysis of protein extracts from tetracycline induced 293-TREx that are non-transfected (Lane 1), expressing BioTAP-L3MBTL3 , BioTAP-BRD4short  (Lane 3), and BioTAP-B4N . PAP detects BioTAP-tagged baits. GAPDH and Ponceau staining reveal equal loading of extracts.(TIF)Click here for additional data file.S2 Figshort represents standard deviation of 4 replicates while error bar of B4N sample represents range of 2 replicates.Recovery of H2A variant peptides across genomic and immunoprecipitated chromatin spanning endogenous substitutions at position 16 (serine compared to threonine). Error bar of genomic sample and BRD4(TIF)Click here for additional data file.S3 Fig2 transformed and multiplied by -1. Note that, aside from H3K79 di- and tri-methylation, most PTMs on a genome wide scale do not significantly change upon B4N induction.Comparison of global histone PTM levels in 293-TREx cells before and 7 hours after B4N induction. Each point in the plot represents a PTM. Relative PTM levels were determined as described in the report, where the area under the peak for each modified peptide was normalized to the sum of all the areas of the unmodified and modified forms of a given peptide backbone. The relative quantification was performed for both 0hr and 7hr induction, and the values per PTM at the two timepoints were log(TIF)Click here for additional data file.S4 Figshort, and B4N). Above the diagonal are scatterplots of each comparison, with each point representing a histone PTM. For each comparison, we tested the likelihood that we would have observed the sample correlation if our null hypothesis of r being 0 were true. Thus low p-values suggest a high likelihood that there is a correlation. Statistical values are shown below the diagonal, where p-values are bolded and the lower and upper 95% confidence interval for Pearson’s coefficient are shown below. We find that within replicates, our MS-based PTM quants are tightly correlated. However, the B4N samples are not correlated (positively or negatively) with either input or BRD4short, suggesting that the B4N-associated PTM patterns as a whole are highly distinct from input and BRD4short.Pearson’s coefficient r was determined for the collective relative abundances of histone PTMs in pairwise comparisons between our samples Click here for additional data file.S5 Figshort stained with anti-HA (green), which labels the bait protein, and anti-H3K18ac (red), acquired on non-confocal widefield fluorescence microscopy. Scale bar represents 10 micrometers.Images of 293-TREx cells expressing inducible HA-B4N and HA-BRD4(TIF)Click here for additional data file.S6 FigSame confocal image as (TIF)Click here for additional data file.S7 Figshort sample represents standard deviation of 4 replicates. Error bar of B4N sample represents range of 2 replicates. Asterisk denote significant difference in H3K9K14ac2 levels between input and BRD4short (p < 0.05).Relative levels of unmodified, mono-acetylation and di-acetylation of the H3 peptide spanning K9 and K14 in genomic and immunoprecipitated histones. Error bar of genomic and BRD4(TIF)Click here for additional data file.S8 Figshort sample represents standard deviation of average of 3 replicates. Error bar of B4N sample represents range of 2 replicates. Asterisk denote significant difference in K79me1 levels between input and BRD4short (p < 0.05).Relative levels of unmodified, mono-methylated and di-methylated H3K79 in genomic and immunoprecipitated histones. Error bar of genomic and BRD4(TIF)Click here for additional data file.S9 FigAnnotated tandem mass spectrum of the H3.3 peptide containing K27 acetylation and K36 monomethylation. Numbers above and below peptide sequence correspond to nominal masses of b and y fragment ions. Bolded masses indicate those ions annotated from the spectrum. Pr = propone group from derivatization.(TIF)Click here for additional data file.S10 Figshort sample represents standard deviation of 4 replicates. Note the significant enrichment for K27 mono-methylation paired with K36 di- and tri-methylation by B4N.Relative levels of combinatorially modified forms of K27 and K36 methylation and acetylation. Error bar of genomic and BRD4(TIF)Click here for additional data file.S11 FigAnnotated tandem mass spectrum of the canonical histone H3 peptide containing K27 acetylation and K36 monomethylation. Numbers above and below peptide sequence correspond to nominal masses of b and y fragment ions. Bolded masses indicate those ions annotated from the spectrum. Pr = propone group from derivatization.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Cygnus cygnus) at Gangjin Bay in South Jeolla province, South Korea. Identification of HPAIVs in wild birds is significant as there is a potential risk of transmission of these viruses to poultry and humans.On November 20, 2016 two novel strains of H5N6 highly pathogenic avian influenza virus (HPAIVs) were isolated from three whooper swans  gene, the viruses were most closely related to A/duck/Guangdong/01.01SZSGXJK005-Y/2016 (H5N6) (98.90 ~ 99.74%). The PA genes of the two novel Gangjin H5N6 viruses were most closely related to AIV isolates previously characterized from Korea, A/hooded crane/Korea/1176/2016 (H1N1) (99.16%) and A/environment/Korea/W133/2006 (H7N7) (98.65%). The lack of more recent viruses to A/environment/Korea/W133/2006 (H7N7) indicates the need for analysis of recent wild bird AIVs isolated in Korea because they might provide further clues as to the origin of these novel reassortant H5N6 viruses.Although research on the origins and epidemiology of these infections is ongoing, the most likely route of infection for the whooper swans was through direct or indirect contact with reassortant viruses shed by migratory wild birds in Korea. As H5N6 HPAIVs can potentially be transmitted to poultry and humans, continuous monitoring of AIVs among wild birds will help to mitigate this risk. As a natural reservoir for avian influenza viruses (AIVs), wild birds do not typically exhibit clinical signs of infection . In mostCygnus cygnus) exhibiting neurological signs including torticollis and ataxia, along with the carcasses of two adults were found at Gangjin Bay in South Jeolla. Samples collected from all three birds were positive for H5 subtype AIV by RT-PCR [On 16 November, 2016 the government of South Korea reported outbreaks of H5N6 HPAI in poultry farms in South Jeolla province . On 20 Ny RT-PCR . No otheThe viruses contained conservative residues within the receptor-binding pocket of the hemagglutinin protein , which is associated with a preference for “avian” type cell surface receptors containing alpha 2,3-sialic acid residues . The dedPhylogenetic analysis of HA gene sequences from the three viral isolates classified them within Asian H5 clade 2.3.4.4 lineage Fig. , which ehttp://bioedit.software.informer.com/) and Clustal Omega  revealed that all three viruses were homologous with each other, sharing 99.03 ~ 100% nucleotide identity among seven out of eight segments (polymerase basic 2 (PB2), polymerase basic 1 (PB1), HA, nuclear protein (NP), NA, matrix (M), nonstructural (NS)). The PA subunit segments of Gangjin 48 and Gangjin 49–2 were identical to each other but differed (94.05% identity) from Gangjin 49–1, which suggests that at least two kinds of novel H5N6 HPAIV strains have circulated among wild birds in Korea. A BLAST (www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) search and phylogenetic analysis showed that all three viruses likely originated from A/duck/Guangdong/01.01SZSGXJK005-Y/2016  (98.90 ~ 99.74% identity)  Fig. . The datty) Fig. .Fig. 2ScAnas platyrhynchos, > 3,500) and spot billed ducks  were observed around Gangjin Bay.Whooper swans breed in northern Eurasia and winter in Europe and eastern Asia, including South Korea. Previous satellite data describing their southern migration to South Korea indicate the birds follow the Flyway along inner Mongolia, northeast China and the Korean peninsula during October and November . Indeed,It is possible that whooper swans introduced both H5N6 viruses into Korea. However, it is also possible that whooper swans which are highly susceptible to HPAI H5N1 infection , may havIn conclusion, we have identified the two novel HPAIV H5N6 strains from three whooper swans in Korea. Phylogenetic and genetic analyses have shown that the two strains resulted from reassortment of the clade 2.3.4.4 H5N6 viruses that circulated in Guangdong, and they are distinguishable from HPAIVs that caused previous poultry and wild bird outbreaks in Korea. Increased understanding of clade 2.3.4.4 H5N6 virus transmission and pathogenesis in wild birds would serve to increase knowledge of the risk these viruses present to wildlife, domestic animals, and humans. Furthermore, as the H5N6 HPAIVs are potentially transmissible to poultry and humans, continuous monitoring of AIVs among wild birds is warranted.


H19 knockout mouse model to determine the role of H19 in liver development, we quantified gene expression for insulin growth factor signaling, Wnt signaling, key cytochrome P450 (P450) enzymes known to change as the liver develops, and fetal and adult plasma protein produced in liver. In mice lacking H19 expression, liver weights were significantly increased immediately after birth and significant increases were found in the number of actively proliferating cells. Increases in cell proliferation may be due to increases in β-catenin protein affecting Wnt signaling, increases in insulin-like growth factor 2 (IGF2) expression, and/or increases in insulin-like growth factor 1 receptor (IGF1R) expression at the protein level. Loss of targeted inhibition of IGF1R by microRNA 675 (miR-675) may be the cause of IGF1R increases, as miR-675 expression is also abrogated with loss of H19 expression in our model. P450 expression patterns were largely unchanged. No change in the production of plasma proteins was found, indicating H19 may not be important for liver maturation despite its role in controlling cell proliferation during liver growth. H19 may be important for normal liver development, and understanding how the liver matures will assist in predicting drug efficacy and toxicity in pediatric populations.H19 RNA is highly expressed at early postnatal ages and precipitously decreases at a specific time corresponding with increases in expression of genes important for mature liver function, such as drug metabolizing enzymes. H19’s role in the regulation of liver maturation is currently unknown. Using an  Liver development requires both cell proliferation and differentiation. Cell proliferation allows the liver to achieve its proper size in the body and cell differentiation allows the liver to attain proper functions. Both events coincide concurrently during postnatal liver maturation. During this important, but understudied, phase of development, the liver undergoes a switch in functions. In mouse, the liver becomes the major hematopoietic organ in the fetus between embryonic days 10 to 15 . ThroughSrsf3 in mice results in a decline in postnatal liver growth by a prolonged expression of fetal markers including persistence of hepatic hematopoiesis [Proteins involved in signaling pathways and regulation of splicing are implicated in playing important roles in cell growth and differentiation during postnatal liver maturation. Knockout of mouse liver β-catenin, the intracellular transducer in Wnt signaling, results in a decrease in cell proliferation and a decrease in postnatal liver size . Yes-assopoiesis . Clearlycis protein-coding partner, IGF2, an important fetal growth factor, exhibits a similar temporal expression pattern. Despite being one of the most studied lncRNAs, H19’s role in liver development has not been fully determined.Long non-coding RNAs (lncRNAs) also change in identifiable expression patterns throughout ontogenesis and potentially regulate liver development. Recently, our laboratory examined the alterations of gene expression in lncRNAs during the liver’s functional transition through the prenatal stage to adult life in mice . Three mH19 knockout mouse model [H19 is involved in normal liver physiology and is implicated in liver diseases . H19 is se model .H19 is an imprinted gene uniquely expressed only from the maternal allele [H19 knockout mice to generate mice heterozygous for the null mutation strictly controlling which parental allele H19 is fully intact or fully removed. Using wild type mice (H19+/+), H19 maternal allele knockouts (H19M-/P+), and H19 paternal allele knockouts (H19M+/P-), we were able to assess the role of H19 in postnatal liver development when H19 is expressed or not expressed from a specific allele.To test the effect of H19 on postnatal liver development, we used three genetically different mouse groups. l allele . TherefoH19 knockout mice [H19 heterozygous male knockout mice  were received from Dr. Linheng Li’s laboratory at the Stowers Institute for Medical Research . Mice were housed according to the animal care guidelines provided by the American Association for Animal Laboratory Sciences and were bred under standard conditions in the Laboratory Animal Resources Facility at the University of Connecticut (Protocol Number: A15-040). The use of these mice was approved by the Institutional Animal Care and Use Committee, Office of Research Compliance. A breeding scheme to generate paternal and maternal H19 knockout mice is illustrated in H19 knockout (H19+/-) mice (F-0) carrying the H19- on one allele with undetermined parental origin were initially bred with wild type (H19+/+) mice of the same C57BL/6 background to generate F-1 male-H19+/- and female-H19+/- heterozygous. The F-1 female H19+/- were further bred with wild type mice to generate F-2 male and female maternal H19 knockout (H19M-/P+) offspring and the F-1 male-H19+/- were further bred with wild type mice to generate F-2 male and female paternal H19 knockout (H19M+/P-) offspring. F-2 wild type (H19M+/P+) mice generated were used as controls. Liver samples were collected at the following ages: day 5, 10 , 15, 20, 30 (adolescent), and 60 (adult) after birth. Livers from both males and females were collected for days 30 and 60 only, and livers from animals collected before age 30 days were not considered sexually mature and not separated by sex. The livers were immediately frozen in liquid nitrogen and stored at -80°C or fixed in formalin.Animals used to generate preliminary RNA-sequencing (RNA-Seq) data were previously described . In thisout mice  was rece5’-CTGTTCATACTCCGTGGGATAG-3’, forward CAGACATTCATCCCGGTTACTT-3’, reverse 5’-CCTACCCATTACGAGCCTTAC-3’, and reverse 5’-GGGACCCATCTGTGTCTTGT-3’.Ear or tail snips were collected from mice for DNA extraction. PCR reactions were performed for identification of H19 deletion using a REDExtract-N-Amp Tissue PCR Kit purchased from Sigma-Aldrich . Primers were designed using a PrimerQuest Tool from Integrated DNA Technologies . Sequences for the primers are forward Total RNAs were isolated from livers without gallbladders using TRIzol reagent from Life Technologies  according to the manufacturer's protocol. RNA concentrations were determined using a Nano Drop spectrophotometer from Nano Drop Technologies  at a wavelength of 260 nm. The integrity of the total RNAs was evaluated on an Agilent 2200 Tape Station from Agilent Technologies . Gene expression at the RNA level of H19, miR-675-3p, miR-675-5p, IGF2, IGF1, IGF1R, β-catenin, cyclin D1, CYP3A16, CYP3A11, CYP2B10, CYP2C29, and GAPDH was determined by TaqMan assays from Life Technologies  according to the manufacturer's protocol. Data were analyzed by generation of cycling time (Ct) and delta Ct (ΔCt) values for all genes against GAPDH.Fresh liver tissues were fixed in 10% buffered formalin, embedded in paraffin, and sections were prepared and stained by the Connecticut Veterinary Medical Diagnostic Laboratory at the University of Connecticut . Immunohistochemistry was performed using antibodies against Ki-67  and proliferating cell nuclear antigen (PCNA)  from Abcam . A secondary antibody conjugated to peroxidase allowed for color precipitation using a VECTOR NovaRED Peroxidase (HRP) Substrate Kit from Vector Laboratories . Images were captured using an EVOS XL Core Cell Imaging System from Thermo Fisher Scientific . Positively stained nuclei were identified by using the ImageJ image processing program, version 1.50i, from National Institutes of Health  using a color brightness threshold with a signal intensity greater than or equal to 200.Total proteins in liver lysates were isolated in RIPA Buffer and protein concentrations were determined by using a Qubit 2.0 Fluorometer by Invitrogen  and the Lowry protein assay from Bio-Rad  with absorbance at 750 nm using a Spectra MAX 190 from Molecular Devices . Proteins were run on polyacrylamide gels using the Mini-PROTEAN Tetra System by Bio-Rad  and transferred onto PVDF membranes. Primary antibodies purchased from Cell Signaling Technology  include IGF1R , active β-catenin , and total β-catenin , and albumin . Primary antibodies against Wnt6  were purchased from Abcam , α-fetoprotein  from Thermo Fisher Scientific , and GAPDH  from Sigma-Aldrich . Secondary antibodies, anti-rabbit IgG  and anti-mouse IgG , were purchased from Cell Signaling Technology . Primary antibodies were conjugated to secondary antibodies conjugated to Horseradish peroxidase for detection using a ChemiDoc MP Imaging System from Bio-Rad .t tests when two groups were compared. ANOVA was used when means were compared between more than two groups. The Kruskal-Wallis test was used when data were not normally distributed. Statistical analyses were performed using Prism7, version 7.01 from GraphPad Software, Inc. . Differences were considered to be significant if p<0.05.The data are shown as the mean ± standard deviation. The significance of differences between means was determined using two-tailed unpaired Student’s Previously generated RNA-Seq data  were useH19 knockout, and paternal H19 knockout mice was determined by RT-PCR at ages of 5, 10, 15, 20, 30, and 60 days after birth (H19M-/P+). Wild type mice (H19+/+) and heterozygous mice with H19 knockout on the paternal allele (H19M+/P-) exhibited similar expression of H19 at all ages.Expression of H19 in livers of wild type, maternal er birth . In all H19 [H19 was intact at the maternal allele (H19M+/P+ and H19M+/P-) while knockout on the paternal allele (H19M+/P-) was inconsequential to miR-675 expression. Essentially, H19 maternal allele knockout mice (H19M-/P+) are also miR-675 knockout mice.Two different conserved microRNAs, miR-675-3p and miR-675-5p, are produced from the first exon of H19 . Using tp<0.05) after birth in mice without H19 expression. No significant changes in liver weight were observed between wild type mice and mice not expressing H19 for ages 15, 20, or 30 days after birth. Adult males, but not adult females, have significantly higher (p<0.01) liver weights when measured 60 days after birth with H19 knockout on the maternal allele (H19M-/P+).H19 impacts liver weight in the developing and adult male livers . Liver wp<0.05) and 60 (p<0.001) days after birth. Liver/body weight ratios were largely unaffected indicating weight changes were not specific to liver (p<0.001) and 20 days after birth (p<0.001). Both males and females had significant (p<0.001) increases in body weight at 60 days after birth when H19 was not expressed. Females, but not males, were significantly heavier at 30 days after birth (p<0.01) with no H19 expression through life.H19 impacts total body weight in developing and adult mice in both males and females . Despiteto liver . H19 alsH19 maternal allele knockouts (H19M-/P+) show significantly more Ki-67 positive nuclei in livers of mice at ages 5 (p<0.001), 10 (p<0.001), 20 (p<0.001), 30 in both males and females (p<0.001), and 60 only in males (p<0.001) days after birth. PCNA was also used to measure cell proliferation in an independent experiment. These data closely resemble Ki-67 staining results, indicating livers without H19 expression proliferate more rapidly during development when compared to wild type. In the maternal allele knockout mice, there were significantly more cells stained positive for PCNA at ages 5 (p<0.01), 10 (p<0.001), 15 (p<0.001), and 20 (p<0.001) days after birth. Similar to Ki-67 results, significant differences were observed throughout postnatal liver development using PCNA staining. Significant cell proliferation was only observed utilizing Ki-67 staining at later ages 30 and 60 days after birth indicating significant changes for H19 maternal allele knockout mice (H19M-/P+) while PCNA did not stain significantly different at either of these ages. Measurements for heterozygous paternal allele H19 mutants (H19M+/P-) resemble wild type (H19+/+).Corresponding data indicates liver weights are increased due to increases in cell proliferation throughout mouse development when H19 is not expressed. Two markers of cell proliferation, Ki-67 and PCNA, were used to measure proliferating cells in the tissue sections . When stp<0.01) in IGF2 mRNA was found in H19 maternal allele knockout mice (H19M-/P+) compared to wild type (H19+/+) at 20 days after birth, indicating H19 affects IGF2 expression. However, at all other ages measured, no significant differences were found. IGF1 mRNA was not impacted by the absence of H19 expression.Gene expression patterns for IGF2 and IGF1 were determined using RT-PCR for mice at ages 5, 10, 15, 20, 30, and 60 days after birth  compared to wild type (H19+/+). Although no significant differences were observed in mRNA expression at all ages measured  in β-catenin was observed at 10 days after birth at the mRNA level, and no changes were observed for cyclin D1 (p<0.05) was observed at the protein level for active β-catenin in mice at age 5 days after birth. Total β-catenin in mice at age 10 days after birth was also significantly increased (p<0.05), and this measurement coincides with the increase observed at the RNA level. Despite these changes, no difference in active/total β-catenin was observed , H19 maternal allele knockouts (H19M-/P+), and wild type mice (H19+/+). There were no significant differences in mRNA expression for CYP3A16 (p<0.01) was found in CYP2B10 in H19 maternal allele mutants (H19M-/P+) compared to wild type (H19+/+) for male mice aged 30 days (H19+/+) and in paternal H19 allele knockouts (H19M+/P-), CYP2C29 begins to be slightly expressed at 15 days after birth. However, when H19 is not expressed (H19M-/P+), CYP2C29 is not expressed until later in development and resumes its full normal expression at 20 days after birth.Gene expression was determined for P450 enzymes in  CYP3A16 , CYP3A11 CYP3A16 , or CYP2 CYP3A16 . A signi 30 days . Signifi 30 days . NormallH19 maternal allele knockouts (H19M-/P+) and wild type (H19+/+)  . In miceLiver postnatal protein expression for albumin was examined in mice at the early ages of development. Western blots indicate no significant difference in protein expression of albumin in livers of mice at ages 5 or 10 days after birth  for eithet al. initially characterized H19 expression in mouse from before birth through the postnatal age until expression is terminated, by their observation, around 28 days after birth [Research on H19 in normal liver development has primarily focused on fetal development with little regard to H19’s role after birth despite this being a time when the liver is continually growing and is dynamically changing its function from being a hematopoietic organ to a metabolic organ. When discovered, Pachnis er birth . We haveH19-null allele from their mother [1, S, G2, and M phases of the cell cycle, but not in the resting G0 phase [Liver growth is accelerated early in postnatal liver development when H19 is not expressed. The overgrowth phenotype has been previously characterized for mice inheriting the r mother . HeterozG0 phase . PCNA isG0 phase . Both maH19 maternal allele knockout mice. Liver weights are only significantly increased immediately in early life and in adult males while increases in proliferation are observed throughout most of liver development at postnatal ages. Interestingly, Ki-67 staining indicates no significant increases in cell proliferation at 15 days after birth despite showing significant increases before and after this age in the absence of H19 expression, suggesting H19 does not influence proliferation at this specific time of development, or changes were only detectable when assaying for PCNA expression.Increases in cell proliferation do not directly result in increases in liver mass. There is a discordance with observations in significant liver weight increases and increases in the markers indicating cell proliferation in H19 in developing mice induces changes in liver that persist even at ages when H19 is not normally expressed (ages 30 and 60 days after birth). An increase in adult male liver weight can be explained by the accumulation of organ mass at earlier ages too subtle to have been detected at prior ages. Increases in proliferation as observed by Ki-67 at ages 30 and 60 days after birth in H19 maternal allele knockout mice may be the result of more complex pathway changes that are initiated at the time of normal H19 expression and persist after H19 is no longer expressed. These late age changes were not observed in the PCNA stain  between H19 and Igf2 resulting in disruption of imprinting of Igf2, which authors have concluded leads to biallelic expression of IGF2 and the overgrowth phenotype [H19 and a portion of its promoter containing an Sp1 site and TATA box removed, leaving the ICR intact allowing us to study only the effects of H19 on postnatal liver development [ICR, causing bialleleic expression of IGF2, is not needed to induce the overgrowth phenotype.We then sought to determine how the loss of H19 expression results in an increase in liver mass and an increase in proliferating cells throughout postnatal development by first examining IGF signaling. There is a strong correlation in expression patterns between lncRNAs and protein coding genes within the same loci, suggesting  partner . Others  partner . H19 andhenotype . Howeverelopment . Our resH19 loss-of-function.Our measurements indicate H19 has a minimal effect on IGF2 expression, with significant increases in IGF2 expression observed only at 20 days after birth . However1 to S phase transition, is a target for regulation by β-catenin [The Wnt signaling pathway has been shown to be important in the development of many different tissue types and organs. The canonical intracellular transducer, β-catenin, is activated after the Wnt ligand binds to a Frizzled family receptor. This activation causes β-catenin accumulation in the cytoplasm and its eventual localization into the nucleus where it acts as a coactivator of transcription factors, affecting gene transcription . Cyclin -catenin  in many -catenin . Wnt sig-catenin , and Wnt-catenin . A signi-catenin . No sign-catenin . It is nH19 knockout on the maternal allele abolishes expression of both H19 and miR-675 despite the status of the paternal allele Click here for additional data file.


ARID1A) was frequently mutated in cancers. The modulation mechanism of ARID1A for PI3K/AKT signaling in gastric cancer (GC) remains elusive. Here, we found that depletion of endogenous ARID1A enhanced the in vitro proliferation, colony formation, cellular growth, nutrient uptake and in vivo xenograft tumor growth of GC cells. PI3K/AKT activation by ARID1A-silencing was profiled using a phospho-protein antibody array. The phosphorylation of PDK1, AKT, GSK3β and 70S6K, and the protein and mRNA expressions of PI3K and PDK1, were upregulated by ARID1A-silencing. Chromatin immunoprecipitation and luciferase reporter assay revealed that ARID1A-involved SWI/SNF complex inhibited PIK3CA and PDK1 transcription by direct binding to their promoters. Serial deletion mutation analyses revealed that the ARID1A central region containing the HIC1-binding domain, but not the ARID DNA-binding domain and the C-terminal domain, was essential for the inhibition of GC cell growth, PI3K/AKT pathway phosphorylation and its transcriptional modulation activity of PIK3CA and PDK1. The proliferation, cellular growth and glucose consumption of ARID1A-deficient GC cells were efficiently prohibited by allosteric inhibitors mk2206 and LY294002, which targeting AKT and PI3K, respectively. Both inhibitors also downregulated the phosphorylation of PI3K/AKT pathway in ARID1A-deficient GC cells. Such cells were sensitized to the treatment of LY294002, and AT7867, another inhibitor of AKT and p70S6K. The administration of LY294002 alone inhibited the in vivo growth of ARID1A- deficient GC cells in mouse xenograft model. Our study provides a novel insight into the modulatory function and mechanism of ARID1A in PI3K/AKT signaling in GC.The tumor suppressor gene AT-rich interactive domain-containing protein 1A ( Recently, a novel tumor suppressor AT-rich interactive domain-containing protein 1A (ARID1A) has been found to be frequently mutated in human cancers, including 8–27% of GCs [ARID1A encodes BRG1-associated factor 250 a (BAF250a), a noncatalytic subunit of the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin-remodeling complex [Gastric cancer (GC) is the fourth and sixth most common cancer in men and women, respectively. The estimated incidence of GC is around 952,000 cases worldwide, along with 723,000 deaths, according to GLOCOCAN statistics in 2012  and SMAD3 transcription and tumor growth in gynecologic cancers [E2F1 [CCNE1 [c-MYC [ARID1A silencing increased the migration and invasion abilities of liver cancer cells [oft agar . Silencie effect , 21. ARI cancers . ARID1A rs [E2F1 , CCNE1 [1 [CCNE1  and c-MY1 [c-MYC , 28. ARIer cells . We founer cells .ARID1A mutation in cancer tended to occur in a synergistic fashion with PIK3CA [ARID1A in glioma, ovarian and colon cancer cells upregulated the phosphorylation of AKT and P70S6K [h PIK3CA , 29–32. d P70S6K –35. Despin vitro and in vivo. We performed a phosphorylation profiling of PI3K/AKT signaling in GC cells with ARID1A depletion and identified the direct transcriptional targets of ARID1A in PI3K/AKT pathway. We also mapped the essential region of ARID1A protein in the transcriptional regulation of its target genes. We analyzed the in vitro and in vivo drug responses of GC cells with ARID1A depletion.In the present study, we analyzed the ARID1A functions in GC cell proliferation, cellular growth and nutrient consumption ARID1A in GC cell lines MGC-803, AGS, HGC-27 and/or SGC-7901 using a siRNA or shRNAs. The siRNA remained as effective till 5 days post-transfection  and heavier (p = 0.04) tumors than the controls with luciferase silencing  of dy array . The phody array . These rt Figure . InteresARID1A increased p-AKTS473 in Hela cells  and SMAD3, were decreased in HGC-27 with ARID1A silencing  significantly downregulated the proliferation of 293FT cells  (aa 1017–1108), a HIC1-binding domain (aa 1355–1424)/Gln domain (aa 1327–1404) , a glucon Figure . Overexps Figure  and the s Figure . PTEN haS Figure  and 4E aS Figure , suggesty Figure . Overexp Figures . We failARID1A deficiency. Given that PIK3CA mutation occurs frequently in a synergistic fashion with ARID1A, we focused on the drug effectiveness of small inhibitors against PIK3CA, as well as p-AKT, which is a “hub” target of diverse tumorigenic signaling. Although the drug sensitivity of ARID1A-deficiency has been analyzed in ovarian cancer cell lines [in vivo effectiveness of PI3K/AKT inhibitors for GC cells with ARID1A depletion has been addressed.The above findings suggested that PI3K, PDK1 or p-AKT might be the therapeutic targets of GCs with ll lines , no in vARID1A was silenced in HGC-27 and SGC-7901 cells, which were treated with mk2206, an allosteric inhibitor that inhibits Akt phosphorylation. p-AKTS473 and cell growth were obviously upregulated by ARID1A depletion but were suppressed by mk2206 treatment   than in ARID1A-intact cells (1.615)   than the control cells with native ARID1A (LogIC50 = 1.772)   and HGC-27  to AT7867, an inhibitor against AKT and p70S6K  Figure . In HGC-) Figure . ARID1A K Figure  and 6D.ARID1A-depletion were significantly shrunk by LY294002 treatment comparing with mock controls after 8 days of administration (p = 0.041 at 8th day and p = 0.043 at 9th day)  Figure . HoweverARID1A-deficiency.These observations suggested that mk2206 and LY294002 targeting PI3K/AKT signaling could effectively suppress the proliferation of GC cells with ARID1A gene emerges as a bona fide tumor suppressor, while its function and molecular mechanism in GC remains elusive. Although ARID1A has been observed to regulate GC cell proliferation by in vitro experiments [in vivo evidence was established to date. Here we verified the tumorigenesis role of ARID1A knockdown in GC by in vivo xenograft mouse model and in vitro proliferation analyses. Another interesting finding was that ARID1A depletion enhanced cellular nutrient uptake and increased average cell size, suggesting ARID1A deficiency enhanced tumorous metabolism processes other than accelerating cell cycle, suppressing apoptosis, triggering EMT and increasing cancer cell migration/invasion [eriments , 21, no invasion , 27, 40.ARID1A depletion using an antibody array and Western blot. The increased phosphorylations of AKT, PDK1, TSC2, mTORC1 and p70S6K were in line with the enhanced cellular growth of GC cells. The increased phosphorylation of GSK3β was in agreement with the upregulation of glucose consumption, while the phosphorylation of p27 Kip1 and p21 Cip1, two cell cycle inhibitors, were related with accelerated cell cycle. The upregulated phosphorylation of BCL2 were associated with an inhibition of apoptosis [S166 indicated a suppression of p53 function [S134/S136 and 14-3-3 contributed to GC cell survival. These results suggested that the activation of PI3K/AKT pathway by ARID1A depletion contributed to multiple cellular alterations including uncontrolled cell cycle, increased survival ability and enhanced nutrient uptake, which had been addressed with additional experiments in current study.In current study, we profiled and verified the phosphorylation of PI3K/AKT pathway in GC cells with poptosis . Increasfunction . The incARID1A deficiency has been observed, no direct link was established between ARID1A, a transcriptional regulator, and PI3K/AKT pathway, a typical pathway by phosphorylation cascade [PIK3CA and PDK1 were the direct transcriptional targets of the ARID1A-involved SWI/SNF complex. Furthermore, we found that the negative regulation function of ARID1A for PIK3CA and PDK1 was independent of its N-terminus containing the ARID domain, the LXXLL motifs and its C-terminal fragment containing the GR-binding domain, but interestingly relied on the central region containing the HIC1-interaction domain. ARID domain was known to mediate the binding of ARID1A to DNA sequence in a non-specific fashion [et al identified that GR-dependent transcriptional activation was reduced slightly by the deletion of the ARID DNA-binding domain but was dramatically reduced by the deletion of its C-terminal GR-binding domain (aa 1637 to 2285) [et al reported that the C-terminus (aa 1759 to 2285) of ARID1A, the same fragment as ARID1A-C3 in our study, interacted with p53 to regulate the transcription of p21, a downstream target gene of p53 [et al [E2F1 transcription through the recruitment of SWI/SNF complex by a direct interaction with ARID1A [ARID1A is one of the 9 to 12 subunits of SWI/SNF chromatin remodeling complexes which contributed to nucleosome remodeling and transcriptional regulation of a broad profile of target genes , 36. Alt cascade , 39, 43. fashion . Nie Z eain aa 16 to 2285 e of p53 . Althoug3 [et al , however3 [et al , which ih ARID1A . ARID1A/ARID1A-deficiency showed some in vitro sensitivities to PI3K/AKT inhibitors [in vivo responsiveness of GC cells with ARID1A-deficiency effectiveness of PI3K/AKT inhibitors has been addressed. We analyzed the effects of chemical inhibitors against PI3K/AKT signaling in vitro and in vivo. Mk2206 and LY294002, which respectively targeting p-AKTS473 and PI3K, were effective to downregulate GC cell proliferation, glucose consumption and cellular growth (cell size) conferred by ARID1A depletion. Accordingly, the PI3K/AKT signaling was downregulated by mk2206 or LY294002. The drug sensitivity of ARID1A-deficient GC cells was increased for the treatment of LY294002 and AT7867, another inhibitor against AKT and p70S6K. The administration of LY294002 alone inhibited the in vivo growth of the xenograft tumors with ARID1A silencing, comparing with the cells with native ARID1A.As described above, the transcriptional mechanism of ARID1A would be addressed to deal with the heterogeneity of different cancers and physiological and pathological variations. Although ovarian cancer cell lines with hibitors , no in vARID1A confers an increased proliferation, cellular growth and nutrient consumption of GC cells, companied with an activation of PI3K/AKT signaling. PIK3CA and PDK1 were the direct transcriptional targets of the ARID1A-involved SWI/SNF complex. The central region containing the HIC1-binding domain of ARID1A was essential for its transcriptional regulation of downstream targets. ARID1A deficiency and the activation of PI3K/AKT were hazard factors of GC patients and associated with poor prognosis. MK2206 and LY294002 were effective to suppress the proliferation of GC cells with ARID1A deficiency by downregulating activated PI3K/AKT signaling. Our study provides a novel insight into the function and mechanism of ARID1A in the modulation of PI3K/AKT signaling in GC.Taken together, our study revealed that deficient 2. Hela and HEK293FT were cultured in DMEM supplemented with 10% FBS. The cell lines were authenticated on August, 2014.GC cell lines HGC-27 and MGC-803 were purchased from the Cell Bank of Shanghai Institutes for Biological Sciences, China. SGC-7901 was generously provided by Dr. Jian-Jun Du at Huashan Hospital, Fudan University. AGS was a gift by Dr. Qing-Hua Zhang at the Shanghai University of Traditional Chinese Medicine. GC cells were cultured in RPM1640 media supplemented with 10% FBS in a humidified incubator at 37°C and 5% COARID1A gene was cloned as described previously [The eviously . The shReviously . Lentivieviously . The priImmunoblotting analysis was performed as previously described . All antARID1A silencing according to the procedure established previously [Immunofluorescence was performed to monitor the proliferation of GC cells after eviously .ARID1A) using the Extraction Buffer . Next, 25 μg proteins of each sample were labeled with 30 μg biotin in 4% N,N-Dimethylformamide solution. The reaction was stopped after 2 hours of incubation at room temperature. The labeled proteins were diluted with 6 mL Coupling Solution . The antibody arrays were blocked with the Blocking Reagent  at room temperature for 45 min and then rinsed 10 times with Milli-Q water. The diluted labeling products were incubated with the antibody arrays at 4°C overnight. The array slides were washed with Milli-Q water as above. The conjugated proteins on the array slides were detected with Cy3-streptavidin (0.5 μg/mL) solution. The slides were scanned using a microarray scanner GenePix 4000B with software GenePix Pro 6.0 . The raw data was treated with the Grubbs' algorism and outliers in each six-repeat data point of an antibody were excluded. The data was average for each antibody and all data were normalized using the average against beta-actin. The phosphorylation ratio = phospho/unphospho. The phosphorylation fold change = (phosphorylation sh2-ARID1A)/(phosphorylation sh-Luciferase). The proteins with a fold change ≥ 1.8 were reported in t test and a p value < 0.05 was considered as statistically significant.The experiment was performed by Wayen Biotechnologies (Shanghai), Inc., using the Full Moon AKT/PKB phospho antibody array containing 216 antibodies against the corresponding proteins of AKT/PKB pathway . There are 93 paired antibodies, which react with a phosphorylated site and a paired non-phosphorylated site. Proteins were extracted from SGC-7901 or HGC-27 cells stably transfected with sh-Luciferase or sh2  with a Beckman Flow Cytometer . Cells in suspension were fixed by adding 2.5 mL 70% alcohol and incubated at 4°C overnight. Alcohol was aspirated with centrifugation and the cells were washed with PBS/1% BSA triple times and suspended with 50 μg/mL propidium iodide and 0.1 mg/mL RNase. After incubation at 37°C for 40 min, the cells were washed by adding 3 mL PBS/1% BSA. Finally, the cells were re-suspended with 1 mL PBS and analyzed by a cytometer.Cell cycle alteration by Total RNA isolation and qPCR were performed as previously described . PrimersColony formation assay was performed as describe previously .ARID1A siRNAs were synthesized as previously reported [Plasmid or siRNA transfection using lipofectamine (Life Technologies - Invitrogen) and OPTI-MEM reduced serum media (Life Technologies - Invitrogen) was performed as described previously . The celreported . The siRCell proliferation was analyzed using a BrdU Cell Proliferation ELISA Kit  as previously described .6). First, 1 mL of culture medium of each sample was added into a test tube and a standard sample in another tube was prepared by adding 950 μL of water and 50 μL of the Glucose Standard solution into the tube. The reactions were started at zero time by adding 2 mL of the Assay Reagent to each tube at an interval of 30–60 seconds. The tubes were incubated at 37°C for 30 min. The reactions were stopped by adding 2 mL of 12 N H2SO4 into each tube at 30–60 seconds intervals. The absorbance of each tube against the reagent blank at 540 nm was measured using a microplate spectrophotometer. The glucose concentration was calculated as: mg glucose = (ΔA540 of Test) (mg glucose in Standard)/ΔA540 of Standard. The glucose consumption was:  – (glucose concentration at test).Cells were seeded in culture dishes and the media were changed after 6 h. The cells were further incubated for 24 h, and the culture media were collected and used for glucose measurement using a Glucose Assay kit . The assay was performed according to the manufacturer's instructions. The glucose consumption and lactate production were normalized by cell counts (per 10Cell proliferation was measured using methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay as previously described . AlternaPIK3CA and PDK1 was amplified using normal human genomic DNA, and inserted into pGL3 luciferase reporter vector  [PIK3CA were cloned, including −1889 to +1 , −1276 to +1 and −516 to +1. The promoter region from −1325 to +1  of PDK1 was cloned for analysis. The primers were deposited in The promoter regions of , China) . Three pChIP assay was performed using an antibody against BRG1 or IgG as a negative control with an EZ-ChIP kit (Upstate Biotechnology) according to the manufacturer's instruction and previous reports , 48. The4 per well) were seeded on a 96-well plate and treated with different concentrations of drug chemicals for 72 hrs at 37°C. The concentrations of LY294002 and AT7867  were 0, 0.01, 0.1, 1, 10 and 100 μM. Twenty μL of MTT solution (5 mg/mL)  was added to each well and the cells were incubated for 4 hrs at 37°C. Finally, 150 μL DMSO was added and allowed to dissolve for 10 min. The absorbance was monitored at 490 nm using a microplate spectrophotometer.Gastric cancer cells (1–2 × 106) mixed with equal volume of Matrigel  were injected subcutaneously into both flanks of each mouse. When the tumor volume reached 100 mm3, the mice were injected intraperitoneally of LY294002  every day with a dosage of 30 mg/kg (body weight). The control group was injected with 1 × PBS (pH 7.4). Tumor diameters were measured using a caliper every two days and the tumor volume was calculated using the formula: V (mm3) = (width)2 × length/2. The mice were executed 30 days after cell inoculation. The tumors were isolated from the bodies and weighed, followed by fixation with 4% neutral paraformaldehyde. All experimental procedures were conducted according to the guidelines of the Animal Experimental Ethics Committee of Shanghai Medical College of Fudan University.The BK nude mice  were 4–5 weeks old at the beginning of the study. These mice were purchased from Shanghai Laboratory Animal Center, Shanghai, China, and allowed to acclimate for 5 days in the animal facility before further intervention. Cells (5 × 10t test. Drug sensitivity statistics was performed using GraphPad Prism 6 and the concentration was Log transformed and the divergence of LogIC50 was evaluated with an F-test. A p value < 0.05 was considered as statistically significant. All statistical tests were two-sided.Differential significance was calculated using unpaired Student's


Histone 3.3 (H3.3) hotspot mutations in bone tumors occur in the vast majority of giant cell tumors of bone , chondroblastomas (95%) and in a few cases of osteosarcomas. However, clinical presentation, histopathological features, and additional molecular characteristics of H3.3 mutant osteosarcomas are largely unknown.H3F3A and H3F3B. H3.3 mutant osteosarcomas were re-evaluated in a multidisciplinary manner and analyzed for genome-wide DNA-methylation patterns and DNA copy number aberrations alongside H3.3 wild-type osteosarcomas and H3F3A G34W/L mutant GCTBs.In this multicentre, retrospective study, a total of 106 conventional high-grade osteosarcomas, across all age groups were re-examined for hotspot mutations in the H3.3 coding genes H3F3A hotspot mutations. No mutations were found in H3F3B. All patients with H3F3A mutant osteosarcoma were older than 30 years with a median age of 65 years. Copy number aberrations that are commonly encountered in high-grade osteosarcomas also occurred in H3F3A mutant osteosarcomas. Unlike a single osteosarcoma with a H3F3A K27M mutation, the DNA methylation profiles of H3F3A G34W/R mutant osteosarcomas were clearly different from H3.3 wild-type osteosarcomas, but more closely related to GCTBs. The most differentially methylated promoters between H3F3A G34W/R mutant and H3.3 wild-type osteosarcomas were in KLLN/PTEN (p < 0.00005) and HIST1H2BB (p < 0.0005).Six osteosarcomas (6/106) carried H3F3A at mutational hotspots. They are overall rare, but become more frequent in osteosarcoma patients older than 30 years. Osteosarcomas carrying H3F3A G34W/R mutations are associated with epigenetic dysregulation of KLLN/PTEN and HIST1H2BB.H3.3 mutations in osteosarcomas may occur in The online version of this article (doi:10.1186/s13569-017-0075-5) contains supplementary material, which is available to authorized users. Osteosarcoma is a malignant bone forming neoplasm with a broad spectrum of morphologies . ConventThe etiology of osteosarcomas is still largely unknown. However, several genetic syndromes (e.g. Li-Fraumeni and hereditary retinoblastoma) greatly increase the risk of developing osteosarcomas, especially in younger patients , 4. OsteTP53, RB1, or c-MYC [Chromothripsis, a single cellular catastrophic event resulting in extensive DNA rearrangements and DNA copy number changes, has been observed in roughly one-third of osteosarcomas [The genomic landscape of osteosarcomas is complex . A remaror c-MYC , 10. Seqor c-MYC . Howeversarcomas .ATRX  mutations, which result in a loss of its nuclear expression, were found in osteosarcomas at a frequency of 20–30% [H3F3A at codon 34, whereas chondroblastomas are characterized by a high frequency of H3F3B mutations at codon 36 [Alternative lengthening of telomeres (ALT), a telomere maintaining mechanism compensating progressive telomere attrition by homologous recombination of telomere sequences, is associated with chromosomal instability in osteosarcomas . Almost f 20–30% , 16. Losf 20–30% . Howevercodon 36 –20. Littcodon 36 , 18–21.The present study addresses these issues by determining the frequency of H3.3 mutations across the entire age spectrum in de novo high-grade osteosarcomas of the conventional type. Genome-wide DNA methylation and DNA copy number profiling was applied to gain further molecular insight into osteosarcomas carrying H3.3 mutations.Tissue samples of 106 de novo high-grade osteosarcomas of the conventional type  and 14 GCTBs, one of them with signs of malignancy in the local relapse two years after surgical treatment, serving as H3.3 G34 mutant bone tumor control group were collected from the Institutes of Pathology of the University Hospital Heidelberg, Cologne, Jena (Germany), and Nijemegen (the Netherlands). Patients’ characteristics are outlined in Additional file ® 16FFPE Plus LEV DNA Kit was applied on the automated Maxwell device  according to the manufacturer’s instructions. Polymerase chain reaction was performed with 20 ng of DNA under conditions as previously described [Source material for DNA extraction was formalin-fixed and paraffin-embedded (FFPE) tumor tissue. Representative tumor tissue with highest available tumor content was chosen for genomic DNA isolation, as microscopically documented on H&E stained paraffin sections. In GCTBs, which are known for their prominent non-tumoral giant cell component, areas with predominant tumor cells (at least 70%) were selected for DNA extraction. The Maxwellescribed . Two micThe Illumina Infinium HumanMethylation450 (450 k) array was used to assess the DNA methylation status of 482,421 CpG sites , according to the manufacturer’s instructions at the Genomics and Proteomics Core Facility of the German Cancer Research Center (DKFZ) Heidelberg. DNA methylation data were normalized by performing background correction and dye bias correction . Probes targeting sex chromosomes, probes containing multiple single nucleotide polymorphism and those that could not be uniquely mapped were removed. In total, 438,370 probes were kept for analysis.For unsupervised hierarchical clustering, we selected 20,000 probes that showed the highest standard deviation (SD) of beta values across the entire dataset. Samples were hierarchically clustered using the 1-Pearson correlation coefficient as distance measure and average linkage. DNA methylation probes were reordered using Euclidian distance and complete linkage.http://www.bioconductor.org).Copy number profiles were generated from the 450 k raw data using the ‘conumee’ R package and assessed manually. The dmpFinder method of the R package minfi was used for the identification of differentially methylated regions . Both paA two-sided t test was used to test for differences between the mean values for continuous variables. P values of less than 0.05 were considered significant.H3F3A and H3F3B) in 106 de novo high-grade osteosarcomas revealed six H3F3A mutant cases  and were significantly older (p < 0.05) compared to the control group of H3F3A mutant GCTBs . Hence, the incidence increased from overall 5% (6/106) to 15% (6/40) in osteosarcoma patients over 30 years of age  and a distinct deletion of 3q13.31 involving LSAMP (2/6). However, c-MYC amplification, 6p12–21 amplification or chromothripsis, both hallmark copy number alterations in osteosarcomas, were absent in all six H3F3A mutant osteosarcomas. GCTBs lacked any copy number alterations except for the single GCTB with a secondary malignant transformation  and KLLN/PTEN (p < 0.0005) showed significantly higher methylation levels in H3F3A G34 mutant osteosarcomas .Interestingly, the DNA methylation analysis revealed several differing aspects in H3F3A G34 mutant and wild-type osteosarcomas. DNA hypomethylation is generally more pronounced with increased aggressiveness, which might explain this pattern [H3F3A G34 mutant osteosarcomas compared to their wild-type counterparts. The most differentially methylated promoter regions involved the HIST1H2BB and KLNN/PTEN genes. Both showed significantly higher methylation in H3F3A G34 mutant osteosarcomas. The role of HIST1H2BB in cancer is largely unknown. HIST1H2BB is an integral element of the nucleosome complex and hence, impaired expression could disrupt the physiological status and affect nucleosome remodelling [KLNN is located juxtaposed to PTEN on chromosome 10q23 and both genes share the same promoter site [H3F3A G34 mutant osteosarcomas. An important role of PTEN in the development of osteosarcomas has not been described yet, although PTEN alteration has been observed [KLLN functions at the pericentric region of chromosomes. KLLN organizes the heterochromatin by maintaining the H3K9 trimethylation and thereby contributes to chromosomal stability. Loss of KLLN resulted in the dysregulation of pericentric heterochromatin with consequent chromosomal instability and numerical chromosomal aberrations in vitro [H3F3A G34 mutant osteosarcomas compared with GCTBs. Whether the hypomethylation phenotype may be a surrogate for pericentric instability and the role of KLLN in context of H3.3 mutations will be subject of future projects.In-depth analysis of the methylome data revealed widespread DNA hypomethylation in both, odelling . KLNN ister site . Accordiobserved , 10, 37.in vitro . InteresH3F3A mutations in bone tumors does not exclude malignancy in individual cases, especially in older patients. H3F3A mutant conventional high-grade osteosarcomas may radiologically, histologically and karyotypically be indistinguishable from conventional high-grade osteosarcomas, but present with distinct epigenetic features.Taken together, our data confirm that detection of Additional file 1: Table S1. Clinical information of the entire study cohort.Additional file 2: Figure S1. Radiological data of H3F3A mutant osteosarcomas. (A) Case 77896: anteroposterior and lateral radiograph of the left wrist. Arrows indicate toward the lesion with permeated growth pattern (moth-eaten) and periosteal reaction. (B) Case 84676: anteroposterior and lateral radiograph (upper panel) with arrows indicating a large soft tissue mass with prominent mineralization and corresponding sagittal T1- and fat-saturated proton-weighted MR image (lower panel) of the right knee. The tumor involves the adjacent epiphyses through the epiphyseal growth plate (arrow). (C) Case 94316: anteroposterior and lateral radiograph (upper panel) and corresponding sagittal T1-weighted MR image (lower panel left) of the right knee. The MR image shows the intact bone cortex in the cranial part of the lesion (white arrows), but cortical disruption with “wrap-around” sign in the caudal part of the lesion. Anteroposterior radiograph (lower panel right) shows the local recurrence in the soft tissue adjacent to the knee joint prosthesis. (D) Case 84712: anteroposterior and lateral radiograph (upper panel) and corresponding axial T1-weighted and sagittal fat-saturated proton-weighted MR image (lower panel) of the right knee. Arrows indicate periosteal reaction and cortical disruption. (E) Case 94314: anteroposterior radiograph , coronal fat-saturated T1-weighted MR image after administration of gadolinium contrast agent  and coronal T1-weighted MR image  of the right hip. Thickened bone cortex  is wrapped by the lesion. Anteroposterior radiograph  after hip joint replacement.Additional file 3: Figure S2. Histological data of the six H3F3A mutant osteosarcomas (A–F) and the single giant cell tumor of bone with malignant transformation (G).Additional file 4: Figure S3. Copy number plots of the six H3F3A mutant osteosarcomas (A–F), one prototypic H3.3 wild-type osteosarcoma with an scattered chromosome arm 2q and the entire chromosome 3 indicating chromothripsis (G), one prototypical giant cell tumor of bone with a flat profile, (H) and one malignant giant cell tumor of bone with hints for a segmental loss of chromosome arm 2q and segmental gain of 7q (I). Abbreviations: OS = osteosarcoma; GCTB = giant cell tumor of bone.


S100A8, S100A9 and S100A12) play key immune response roles in inflammatory disorders, including cardiovascular disease. Long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFA) may have systemic and adipose tissue-specific anti-inflammatory and cardio-protective action. Interactions between calgranulins and the unsaturated fatty acid arachidonic acid (AA) have been reported, yet little is known about the relationship between calgranulins and the LC n-3 PUFA eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). We explored tissue-specific action of calgranulins in the setting of evoked endotoxemia and n-3 PUFA supplementation. Expression of calgranulins in adipose tissue in vivo was assessed by RNA sequencing (RNASeq) before and after n-3 PUFA supplementation and evoked endotoxemia in the fenofibrate and omega-3 fatty acid modulation of endotoxemia (FFAME) Study. Subjects received n-3 PUFA  or matched placebo (n = 6) for 6–8 weeks, before completing an endotoxin challenge (LPS 0.6 ng/kg). Calgranulin genes were up-regulated post-LPS, with greater increase in n-3 PUFA  compared to placebo . In an independent evoked endotoxemia study, calgranulin gene expression correlated with the systemic inflammatory response. Through in vivo and in vitro interrogation we highlight differential responses in adipocytes and mononuclear cells during inflammation, with n-3 PUFA leading to increased calgranulin expression in adipose, but decreased expression in circulating cells. In conclusion, we present a novel relationship between n-3 PUFA anti-inflammatory action in vivo and cell-specific modulation of calgranulin expression during innate immune activation.Calgranulin genes ( S100A8, S100A9 and S100A12, are members of the S100 gene family of calcium binding proteins. These proteins have known functions in immune response, and act as damage-associated molecular pattern (DAMP) proteins, signaling through the receptor for advanced glycation end products (RAGE), and acting as endogenous toll-like receptor 4 (TLR4) ligands.[S100A12 preferentially forms a homodimer,[S100A8 and S100A9  form calprotectin, present as a heterodimer or heterotetramer; [S100A8 and S100A9 may also form homodimers and other higher complexes, subject to post-translational modification[Calgranulin genes, which include  ligands. S100A12 omodimer, while S1etramer; , 7 S100A2+ concentrations within the cell compared with the extracellular environment, and may exert anti-microbial action through sequestration of metal ions, thus depriving pathogens of essential nutrients.[in vitro in the presence of the n-6 polyunsaturated fatty acid arachidonic acid when compared with saturated and monounsaturated fatty acids.[The specific function and disease relevance of calgranulin genes is complex. Expression and protein levels are increased during immune activation and inflammation, in diverse disease settings including diabetes, obesity, cardiovascular disease, arthritis, cancer, pancreatitis, psoriasis, inflammatory bowel disease, and pathogenic infection.–14 Serumutrients. As a hetutrients., 25 S100ty acids. S100A8/Aty acids. However,in vivo lipopolysaccharide (LPS) challenge in the Fenofibrate and omega-3 fatty acid modulation of endotoxemia (FFAME) Study.[S100A8, S100A9 and S100A12 in response to LPS in vivo, with evidence of markedly enhanced induction in the setting of high-dose (3600mg/day EPA/DHA) supplemental n-3 PUFA. In an independent human endotoxemia study (GENE), we found a tissue-specific relationship between calgranulin expression and the systemic inflammatory response, with higher adipose tissue expression associated with lower systemic inflammation, while higher expression in circulating mononuclear cells is associated with increased systemic inflammation. Finally, we provide integrative genomic and in vitro evidence for these genes as cell-specific, novel nutrient-dependent regulators of inflammatory responsiveness.We examined calgranulin gene expression in adipose tissue after n-3 PUFA supplementation and E) Study. We foundAs described, the FFAME coli-derived LPS; U.S. standard reference, lot No. CCRE-LOT-1+2, Clinical Center, Pharmacy Department at the National Institutes of Health, Bethesda MD).[® CD14, ThermoFisher, Waltham MA), and frozen in TRIzol reagent  for subsequent RNA extraction via standard protocol.[The GENE Study recruited healthy individuals  ancestry to a University of Pennsylvania (UPenn) inpatient Clinical and Translational Research Center (CTRC) protocol as previously described . Participesda MD)., 31 Multprotocol.. The GENin vitro experiments, RNA was subjected to RT-PCR and subsequent quantitative PCR (qPCR) using pre-designed primers and probes  for measurement of candidate gene mRNA . Gene expression levels were normalized to GAPDH and analyzed using the relative quantitation 2-(ΔΔCt) method to determine relative-change.RNA from FFAME Study adipose tissue biopsies (3 samples per subject) was extracted using the RNeasy total RNA kit . RNA concBlood samples were collected into EDTA tubes, mixed by inversion, and immediately placed on ice. Plasma was obtained through centrifugation, and immediately frozen and stored at -80°C for subsequent analysis. Plasma S100A9 protein was measured in a subset of individuals from the GENE study before and after LPS (n = 16) using the SOMALogic SOMAscan assay. This assin vitro experiments, pre-adipocytes from the stromal vascular fraction (SVF) were isolated, cultured and differentiated to adipocytes as previously described.[For escribed. Briefly,escribed., 36 AdipAs described, periphehttp://www.ncbi.nlm.nih.gov/geo/), accession number GSE87426. GENE Study data are deposited under accession number GSE39118. Publicly available expression data (GSE5099 and GSE28070) were queried from datasets deposited in GEO and analyzed using GEO2R.[In vitro and protein data were assessed for normality of distribution, and analyzed by 1-way ANOVA, 2-way ANOVA and by Spearman correlation. Data analysis was carried out using IBM SPSS Statistics 22 and GraphPad Prism 6. All p values quoted for array-wide experiments  are after adjustment for platform-specific multiple testing.RNASeq data were aligned to the hg19 reference genome using STAR 2.3.0e with defS100A8, S100A9 and S100A12 expression was increased in adipose tissue post-LPS in both the placebo and the n-3 PUFA group, however this was markedly enhanced following n-3 PUFA supplementation. Expression levels of S100A8 and S100A9 were higher than that of S100A12 . All three calgranulin genes were highly up-regulated post-LPS in the n-3 PUFA group , while they were only moderately increased post-LPS in the placebo group  (S100A9 (p = 0.04). As previously described, individuals in the n-3 PUFA group displayed a lower systemic inflammatory response to LPS compared with placebo.[In the FFAME Study endotoxin challenge,  = 0.06) . This inS100A8 and S100A9 expression in adipose tissue from individuals with low habitual n-3 PUFA intake  vs. high habitual intake . There was ~4-fold higher expression in individuals with n-3 PUFA intake >2g/day compared with those consuming <1g/day  consistent with a role for n-3 PUFA in enhancing expression in adipose tissue in the chronic inflammatory state of obesity. There were no significant differences between the groups in other known inflammatory genes, e.g. IL6, IL10, CX3CL1, CCL2,  indicating that n-3 PUFA modulated S100A8 and S100A9 specifically, rather than broadly modifying the overall inflammatory state.We hypothesized that this n-3 PUFA and inflammation-dependent difference in calgranulin expression may be more pronounced in a disease setting. In a cross-sectional study of obese metabolic syndrome patients (Gene Expression Omnibus (GEO) dataset GSE28070 analyzed using GEO2R) we obserin vivo. In an independent human endotoxemia protocol, the GENE Study,[S100A8, S100A9 and S100A12 pre- and post-LPS in adipose tissue (n = 25), and CD14+ monocytes (n = 15) of healthy individuals (n = 11 overlapped both tissues). There was a significant increase in all three genes in adipose tissue post-LPS: S100A8 , S100A9 , and S100A12 . Further, expression of all three genes was increased significantly 2 hours post-LPS in CD14+ monocytes . Because these individuals had previously been chosen for RNASeq based on their extreme phenotypic response to LPS (top and bottom 5% of the distribution for plasma cytokine and systemic febrile response), we examined the stratified gene expression changes in these “high” or “low” responders to LPS. We hypothesized that calgranulin gene expression in response to LPS would differ in individuals with strongly divergent clinical responses to LPS. Indeed, in adipose tissue, there was an approximately 3-fold greater increase in calgranulin gene up-regulation in “low” responders vs. “high” responders . In CD14+ monocytes, baseline S100A8 expression was positively correlated with post-LPS S100A8 expression . Strikingly, baseline CD14+ monocyte S100A8 and S100A9 expression was significantly negatively correlated with post-LPS adipose expression of all 3 genes, e.g. for S100A9 baseline in CD14+ monocytes, the relationship with post-LPS adipose expression was as follows: S100A8 Spearman’s rho = -0.8, P = 0.003; S100A9 Spearman’s rho = -0.75, P = 0.007; S100A12 Spearman’s rho = -0.67, P = 0.02). Thus, high calgranulin expression in circulating cells at baseline may predict low adipose tissue calgranulin induction during inflammatory stress.Our discovery sample was focused on adipose tissue. However, we hypothesized that there would also be an increase in gene expression in circulating mononuclear cells post-LPS NE Study,, 32 whicsponders . This coponders. , suggestWe probed whether the observed tissue increase in gene expression resulted in an increase in secreted protein levels. While plasma protein could not be measured in the FFAME Study , we measured plasma S100A9 levels in a subset of the individuals (n = 16) whose adipose and CD14+ monocyte gene expression was measured in the GENE Study. S100A9 pin vitro to n-3 PUFA and LPS in the THP-1 monocyte cell line and adipocytes. In THP-1 cells, expression of S100A8, S100A9 and S100A12 increased significantly following treatment with EPA + DHA  (S100A8 and S100A9 decreased significantly with DHA + EPA treatment (p<0.001). We could not detect expression of S100A12 in adipocytes, indicating that expression of this gene in adipose tissue may derive from other adipose cell sources .To further examine the divergent adipose and monocyte-specific responses, we examined cell-specific responses of calgranulin expression S100A12) . In contS100A9 and S100A12 were significantly up-regulated by LPS, while there was no significant change in S100A8. However, pre-treatment with n-3 PUFA prior to LPS resulted in significant attenuation of the LPS-induced increase in expression, with significantly lower expression following EPA+DHA treatment compared with control in all 3 genes , S100A9  and S100A12  decreased during maturation of monocytes to mature macrophages. In our own expression data from primary human macrophages and M1 or M2 polarized macrophages (available at GEO data series GSE55536),[S100A8  and S100A9  during polarization to M1, and similarly during polarization to M2: S100A8 down ~5-fold p = 0.005; S100A9 down ~3-fold p = 0.005, with a greater decrease in S100A9 in M1 compared with M2 (p = 0.0004). There was no significant change in S100A12 expression in these cells.We queried expression of calgranulins in freshly isolated monocytes and during maturation to macrophages (GEO data series GSE5099). ExpressiSE55536), there waS100A8, S100A9 and S100A12, however to our knowledge these genes have not been studied for potential interaction with n-3 PUFA. We present evidence that consumption of n-3 PUFA is associated with enhanced expression of calgranulins within adipose tissue in response to an acute inflammatory challenge, as well as in the setting of chronic low-grade inflammation. We report novel differences in the response in adipocytes and adipose tissue compared with circulating mononuclear cells. Through integration of these data, we hypothesize that calgranulins mediatecell-specific n-3 PUFA-dependent regulation of systemic inflammation.As important immune-mediating genes, calgranulins are of particular interest to chronic inflammatory diseases including cardiovascular disease. Dietary n-3 PUFA consumption may protect against a diverse range of immune-related diseases, although conflicting data on mechanisms and outcomes have hindered translational progress. Previous studies have examined the specific pro- or anti-inflammatory actions of There was no difference in adipose calgranulin expression after n-3 PUFA treatment prior to endotoxemia in the FFAME study, consistent with the expectation that basal tissue inflammation in healthy individuals is at negligible levels. External endotoxemic inflammatory stress was required to observe expression differences in this lean and healthy sample. However in adipose tissue from obese individuals there wain vivo, there was greater induction of calgranulins concurrent with lower systemic inflammatory response. This was observed in the FFAME Study, where the n-3 PUFA supplemented group had lower febrile and cytokine response to LPS than the placebo group. Similarly, in the GENE Study, where individuals were designated as “high” or “low” responders based on their febrile and cytokine response to LPS, the individuals with “low” response had higher post-LPS up-regulation of calgranulins in adipose tissue. Remarkably, within a subset of the same individuals in the GENE Study, “low” responders had lower post-LPS up-regulation of calgranulins in CD14+ monocytes. This striking difference raised the possibility of tissue-localization as crucially important in downstream consequences of calgranulin gene expression. The tissue-specific difference was recapitulated in vitro, where the effects of n-3 PUFA alone, as well as n-3 PUFA and LPS had opposing effects in adipocytes compared with THP-1 monocytes. Our data highlight that n-3 PUFA treatment has different effects on calgranulin expression in both adipocytes and monocytes when it is used alone, compared with the combination of n-3 PUFA and LPS. This could indicate that the relationship between n-3 PUFA and calgranulin expression is fundamentally altered during acute inflammatory stress compared with an unstressed state. Plasma responses of S100A9 protein were broadly consistent with CD14+ monocyte expression, indicating that plasma S100A9 protein may be primarily derived from secretion by circulating inflammatory cells rather than from adipose tissue.Strikingly, we observed tissue-specific differences in calgranulin gene expression in individuals with high or low systemic inflammatory responses (as assessed by fever and circulating cytokines). In adipose tissue S100A8 and S100A9 are involved in the recruitment of adipose tissue macrophages from bone marrow through a positive feedback loop,[S100A8 and S100A9 lead to inflammasome activation in a TLR4- and MYD88-dependent manner,[s100a8 adipose expression, as well as S100A8 protein was shown to stimulate macrophage migration as an early mediator of adipose inflammation in response to a pro-inflammatory obesogenic diet.[S100A8, S100A9 and S100A12 correlated positively with visceral obesity.[circulating calgranulins are associated with increased chronic inflammation. Conversely, high adipose expression may be associated with reduced inflammation. Indeed, subjects with high baseline expression of calgranulins in CD14+ monocytes had lower induction of calgranulin expression in adipose tissue post-LPS. In the acute inflammatory setting, our novel findings suggest that the presence of LC n-3 PUFA may facilitate rapid up-regulation of calgranulin expression in adipose. This may lead to a more rapid resolution of systemic inflammation, potentially through an increase in macrophage recruitment and inflammasome activation. While intriguing, the correlative associations in our data do not demonstrate causality, and the mechanisms remains to be determined in future studies.Recent reports suggest that that ack loop, and moduack loop, S100A8 at manner, and stimt manner, In our dnic diet.. In obes obesity. Our datain vivo adipose tissue and CD14+ monocyte expression data reflects expression from a mixed cell population. In vitro expression data indicate higher S100A8, S100A9 and S100A12 expression in monocytes compared with mature macrophages, with further decreases during polarization to M1 or M2 macrophages. Thus, observed differences in expression within adipose tissue and in the circulation may relate to the proportion of monocytes compared with activated macrophages, rather than the number or activity of resident and infiltrating cells. The relative contribution of macrophage recruitment vs. macrophage activation status remains to be determined. Circulating or adipose tissue protein could not be measured in the FFAME Study due to limited sample availability, and thus the degree to which gene expression relates to translated protein could not be verified. However in the GENE Study expression of S100A9 in adipose tissue post-LPS was significantly correlated with plasma protein. The specific functions of the calgranulin genes and interaction with n-3 PUFA may depend on formation of multimers, as well as intracellular vs. extracellular localization, which our data do not resolve. Given previous studies showing direct interaction between calgranulins and fatty acids[Our study has a number of strengths, but also some limitations. Our tty acids, 25, 27,in vivo and in vitro interrogation, we highlight calgranulins S100A8, S100A9 and S100A12 as n-3 PUFA-dependent regulators of systemic and adipose tissue inflammatory responses, and highlight a potential novel mechanism for n-3 PUFA modulation of inflammation.Given the increasing prevalence of complex inflammatory cardiometabolic diseases, strategies aimed at understanding specific mechanisms of disease development, and interaction with environmental factors, are of particular importance. We used a combined model of n-3 PUFA supplementation and evoked endotoxemia in healthy volunteers to understand the interplay between nutritional status and inflammatory responses prior to development of cardiometabolic disease. Through S1 File(XLSX)Click here for additional data file.


We report here the genome sequence of the influenza A virus strain A/swine/Siberia/1sw/2016, isolated from a swine in Russia. On the basis of sequence analysis, A/swine/Siberia/1sw/2016 is characterized by unusual surface glycoproteins phylogenetically distinct from those of swine A(H1N1)pdm09 influenza virus. Swine influenza virus causes an acute and highly contagious respiratory disease in swine. The pig can be a mixing vessel for generating new genetic variants of influenza virus –3 becaus–Here, we report the genome sequence of influenza A virus strain A/swine/Siberia/1sw/2016, initially isolated from a swine pulmonary tissue sample. Sectional material from pigs was collected on a private pig farm in Russia in 2016. Influenza virus A was detected by real-time PCR. The strain was isolated on Madin-Darby canine kidney (MDCK) cells. Complete genome sequencing was performed in the SB RAS Genomics Core Facility . Viral RNA was extracted using TRIzol LS . cDNA was synthesized using a random hexamer primer with RevertAid Premium . The second DNA chain was synthesized using the NEBNext mRNA second-strand synthesis module , in accordance with the manufacturer’s instructions. DNA libraries were made using TruSeq DNA sample prep kits (version 2), as specified by the manufacturer . The DNA sequence of the libraries was made using the MiSeq system  with version 2 reagent kits (300 paired-end), according to the manufacturer’s instructions. Sequence assembling was conducted using the CLC Genomics Workbench version 5.5 software .The genome of the A/swine/Siberia/1sw/2016 strain consisted of the following eight gene segments: polymerase , hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix protein (M), and nonstructural protein (NS) genes. The sequence of the PB2 gene consisted of 2,195 nucleotides (nt), the PB1 gene 2,328 nt, the PA gene 1,965 nt, the HA gene 1,776 nt, the NP gene 1,551 nt, the NA gene 1,405 nt, the M gene 1,015 nt, and the NS gene 890 nt.Phylogenetic analysis showed that genes encoding surface glycoproteins (HA and NA) of the A/swine/Siberia/1sw/2016 strain are significantly different from the HA and NA gene sequences represented in databases . The closest (90%) sequences of HA and NA belong to the influenza A (H1N1) virus isolated from humans in the United States in the 1980s. Thus, the phylogenetic strain A/swine/Siberia/1sw/2016 refers to the subtype H1N1, but it is not a modern pandemic variant of A(H1N1)pdm09. However, the nucleotide sequences of the genes encoding the internal proteins of the virus  are similar to the genes of the influenza A(H1N1)pdm09 virus isolated from humans.Thus, the A/swine/Siberia/1sw/2016 influenza virus strain is a previously unregistered locally circulating reassortant variant of the H1N1 subtype in the pig population.MF370340 to MF370347.The genome sequence of A/swine/Siberia/1sw/2016(H1N1) was deposited in GenBank under the accession numbers