D-type cyclins bind and activate CDK4 and CDK6 thereby contributing to G1–S cell-cycle progression. In addition to the nucleus, herein cyclin D1 was also located in the cytoplasmic membrane. In contrast with the nuclear-localized form of cyclin D1 (cyclin D1NL), the cytoplasmic membrane-localized form of cyclin D1 (cyclin D1MEM) induced transwell migration and the velocity of cellular migration. The cyclin D1MEM was sufficient to induce G1–S cell-cycle progression, cellular proliferation, and colony formation. The cyclin D1MEM was sufficient to induce phosphorylation of the serine threonine kinase Akt (Ser473) and augmented extranuclear localized 17β-estradiol dendrimer conjugate (EDC)-mediated phosphorylation of Akt (Ser473). These studies suggest distinct subcellular compartments of cell cycle proteins may convey distinct functions.The essential G CCND1) gene, encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein (pRB), in order to promote cell cycle progression3. Newly synthesized cyclin D1 associates with CDK4/6 to form the holoenzyme that phosphorylates pRB, releasing E2F family transcription factors and inducing a gene expression network contributing to G1/S entry. Early studies demonstrated that cyclin D1 functions as a nuclear collaborative oncogene4. In this regard a cyclin D1 cDNA clone contributed to cellular transformation by complementing a transformation defective adenovirus E1A oncogene4. The requirement for cyclin D1 in oncogenic transformation has been established through cyclin D1 anti-sense6 and genetic deletion studies in the mouse9. Furthermore, cyclin D1 targeted to the mammary gland was sufficient for the induction of mammary tumorigenesis11. Clinical studies have shown a correlation between cyclin D1 expression and tumorigenesis and increased cyclin D1 expression is associated with tumor invasion and metastasis15.The cyclin D1 23 also known as syndapin), Filamin A24 and paxillin21.A growing body of evidence provides support for an extranuclear function of cyclin D1. Cyclin D1 is actively synthesized and located exclusively in an extranuclear location in hibernating hematopoietic stem cells (HSC)24 is consistent with prior studies demonstrating other components of the cell-cycle control apparatus are located in the cytoplasmic membrane including p27Kip1 and p16INK4a25. Although the physiological function of cytoplasmic membrane-associated cell-cycle components was previously not well understood, p16INK4a and CDK6 colocalize in membrane ruffles of spreading cells and functioned upstream of αvβ3-dependent activation of PKC to regulate matrix-dependent cell migration25. Cyclin D1-deficient mouse embryo fibroblasts (MEFs) and mammary epithelial cells exhibit increased adhesion and decreased motility compared with wild-type MEFs28. Transduction of cyclin D1−/− cells with a human or murine cyclin D1 cDNA, reversed this adhesive phenotype, promoting cell migration26. The induction of cell migration by cyclin D1 correlated with the reduction of Rho GTPase activity26. Mutational analysis demonstrated that cyclin D1 reduction of cellular adhesion and induction of cellular migration were independent of the pRB- and p160 coactivator-binding domains26. Cyclin E knockin of cyclin D1−/− MEFs rescued the DNA synthetic defect of cyclin D1−/− MEFs but did not rescue the migration defect26 suggesting the pRB binding of cyclins and the promigratory function may be dissociable.The association of cyclin D1 with cytoplasmic membrane proteinsvs. cytoplasmic cyclin D1 pools. The current studies were conducted in order to determine the function of cyclin D1 when localized to either the cellular membrane or the nucleus.Although cyclin D1 binds cytoplasmic membrane-associated proteins and correlative studies have suggested that cyclin D1 may promote cellular migration, no studies have selectively uncoupled the functional activity of the nuclear 23 and cyclin D1 bound to PACSIN II and paxillin (Pxn) in 3T3 cells23. In order to characterize the function of membrane-associated cyclin D1, studies were conducted in the human diploid fibroblast cell line (MRC-5) and human breast cancer samples. Using immunohistochemistry, endogenous cyclin D1 was identified at the MRC-5 cellular leading edge, in proximity with PACSIN II  is very aggressive breast cancer linked to poor prognosis. In order to assess the location of cyclin D1 in human breast cancer we compared membrane-associated cyclin D1 in patients with IBC and other breast cancers. Samples from 6 IBC patients and 17 non-IBC patients were stained for cyclin D1 and analyzed by a clinical pathologist Fig. . The sub28. In order to further characterize the molecular mechanisms by which cyclin D1 governs the induction of cellular migration we conducted subfractionation of nuclear and cytoplasmic cellular fractions from cyclin D1WTvs cyclin D1−/− 3T3 cells , which encodes a fusion protein consisting of the N-terminal 20 amino acids of neuromodulin, also called GAP-43, and a cyan fluorescent variant of the enhanced green fluorescent protein. The neuromodulin fragment contains a signal for posttranslational palmitoylation of cysteines 3 and 4 that targets ECFP to cellular membranes. Expression of ECFP-Mem in mammalian cells results in strong labeling of the plasma membrane and had been used to target proteins including ERα to the plasma membrane34. Using electroporation the transfection efficiency was >90%. Cyclin D1WT enhanced transwell migration twofold  analysis was conducted. Comparison was made to the empty control vector because of the potential impact of fluorescent proteins on apoptotic and cell-cycle control proteins35. The distribution of cells in each phase of the cell cycle assessed by FACS demonstrated cyclin D1WT, cyclin D1NUC, and cyclin D1MEM enhanced the proportion of cells in the DNA synthetic (S) phase with a doubling of the proportion of cells in S phase by cyclin D1WT (7.2 vs. 18.2%)    Fig. , an 80% 7%) Fig.  and a do2%) Fig. .Fig. 4MeWT (2.4 vs. 3.5-fold), cyclin D1NUC (2.4 vs. 3.1-fold) and cyclin D1MEM (1.6 vs. 2.7-fold) 3-LUC reporter gene  Fig. . Colony MEM Fig.  with a tMEM Fig. . In ordenes Fig. . Consistene Fig. . The immNUC Fig. . These s37. The extranuclear estrogen signaling pathway is thought to involve a membrane-associated ERα, which activates PI3-kinase and thereby Akt signaling38. Maximal activation of Akt requires phosphorylation on the carboxy-terminal site, S473, by mTORC240. In recent studies, membrane-associated estrogen signaling was shown to occur via cyclin D132. We investigated the impact of expressing cyclin D1 as either total, nuclear, or membrane-tethered forms of cyclin D1 , to the cytoplasmic membrane (PECFP-CD1MEM) or expressed in both cytoplasmic and nuclear compartments (MSCV-CD1TOT). Increased expression of cyclin D1 via an MSCV expression vector (cyclin D1WT), resulted in increased cyclin D1 abundance and increased phosphorylation of Akt1 at Ser473 compared with vector control  increased phosphorylation of Akt1 at Ser473 compared with vehicle control  is known to convey both genomic and extra genomic activities D1 Fig. . The hum2-induced signaling pathways can be distinguished using 17β-estradiol linked to a dendrimer conjugate (EDC), which excludes estradiol from the nucleus42. In order to define the residues of cyclin D1 that participate in Akt activation, mutational analysis of cyclin D1 was conducted. Breast cancer epithelial cells (MCF-7 cells) were treated with either EDC or dendrimer control. Expression of a membrane-associated cyclin D1 under control of the MSCV promoter (cyclin D1MEM) induced phosphorylation of Akt1 at Serine473 ) demonstrated an approximately 90% reduction in EDC-mediated induction of Akt1 Serine473 phosphorylation compared with empty vector control cells  showed the characteristic enrichment of membranous GFP staining  did not induce p-Ser473-Akt1 significantly , but not the nuclear localized form (cherry-CD1NUC), augmented Akt1 phosphorylation at Serine473.We conducted IF to assess the relative abundance and subcellular distribution of Akt1 Serine 473 phosphorylation upon EDC treatment in cells transduced with the distinct located forms of cyclin D1. MCF-7 cells expressing the membrane-associated cyclin D1 46. The binding of insulin to its cell surface receptor stimulates phosphoinositide-3 kinase (PI3K), which then induces the second messenger, phosphotidylinositol-3, 4, 5-triphosphate (PIP3). PIP3 activates Akt and additional downstream effectors. As LS456 was shown to serve as a specific substrate for Akt1 in response to 150 nM insulin, we examined the kinetics of insulin-mediated activation of LS456 in cyclin D1−/− MEF compared with wild-type MEFs. Insulin stimulation of Akt1 activity assessed by LS456 was delayed with reduced induction in cyclin D1−/− cells compared with the cyclin D1WT rescued cells  dye substrate for cellular Akt1 activitylls Fig. .cyclin D1−/− MEFs reduced RhoA activity26. Although these prior studies suggested that cyclin D1 may augment cellular migration by restraining RhoA activity, Rac1 and Cdc42 can also participate in cellular migration47. In order to examine the functional interactions with cyclin D1 and Rho GTPases we deployed the FRET based fluorescent probes for RhoA, Rac, and Cdc42 /FB × 100%, where FB is the intensity of the donor (CFP) after photobleaching and F0 is the intensity of the donor before photobleaching, see “Methods”). FRET efficiency was reduced 40% by cyclin D1WT but was not significantly reduced by expression of the cyclin D1KE  with higher levels of tyrosine-phosphorylated paxillin44. Herein, using cyclin D1−/− cells, we demonstrated the membrane-associated pool of cyclin D1 is sufficient to augment transwell migration. We identified cyclin D1 at the plasma membrane in inflammatory breast cancer, and cyclin D1 colocalized to the cytoplasmic membrane with PACSIN II and Paxillin (Y118) in MRC-5 cells. Cyclin D1 was previously shown by mass spectrometry to bind the membrane-associated proteins PACSIN II23, Filamin A24, Paxillin21, and several additional proteins50. PACSIN II is involved in cell spreading51, as well as endocytosis of cell–surface receptors like the EGF receptor52 and in caveolae-mediated endocytosis54. In view of clinical analyses showing a correlation between total cyclin D1 expression and tumor invasiveness and metastasis15, our studies suggest further studies assessing membrane-associated cyclin D1 may be warranted.Prior studies had shown the induction of cellular migration by cyclin D155. Akt hyperactivation contributes to human cancer correlating with poor prognosis and therapy resistance and genetic deletion demonstrated Akt1 is required for ErbB2-induced breast cancer progression and tumor metastases in vivo56. Herein the acute nongenomic E2 activation of Akt1, was augmented by the membrane-associated cyclin D1 pool. Estradiol acutely activates Akt58 in part through the association of ERα at the plasma membrane associated with the p85 regulatory subunit of PI3-kinase and other proteins including the scaffold protein caveolin-1, G proteins, Src kinase, Ras, and Shc61. ERα regulates nuclear gene expression via genomic and extranuclear non-genomic signals59. Extranuclear pools of ERα reside in the plasma membranes62 and the ability to distinguish nuclear from extranuclear ERα signaling has been enabled through the generation of a 17β-estradiol dendrimer conjugate (EDC) which is localized to the extranuclear compartment42. Herein, using nuclear excluded E2 dendrimers, cyclin D1 was shown to participate in the acute non-genomic E2 response. Genetic deletion studies in the mouse demonstrated E2-dependent induction of genes governing growth factors, growth factor receptor and promigratory processes in the mammary gland requires cyclin D163. The biological effects of estrogen, are critically dependent upon cyclin D1 in vivo64, with the current studies suggesting an important component is mediated via membrane-associated cyclin D1.Herein, cytoplasmic membrane-associated cyclin D1 augmented phosphorylation of Akt1 at Ser473. Akt, also known as Protein Kinase B, promotes cellular survival, proliferation, growth, and migration47. In the current studies, cyclin D1 restrained RhoA activity, requiring K112. Cyclin D1−/− cells have increased RhoA activity, increased ROCK II kinase and increased LIM kinase activation (threonine 505/508). LIM kinase phosphorylation at threonine 505/508 in turn phosphorylates the actin-depolymerizing protein cofilin at serine 3 and MLC2 at Thr18/Ser1926. Herein, FRET analysis evidenced cyclin D1 restrained Rho GTPase activity. In contrast, neither Rac-GTPase nor Cdc42 activity was influenced by cyclin D1. The reduction in RhoA GTPase FRET by cyclin D1 was abolished by mutation of cyclin D1 residue K112. Cyclin D1 participates in multiple functions via K112 including CDK4/6-mediated pRB phosphorylation65 and binding to p27KIP144. Rho GTPase is an important modulator of ERα activity67, and E2 enhances ERα association with the p85 subunit of PI3 kinase thereby inducing Akt phosphorylation57. An increase in ERα/PI3K interactions in patient-derived xenografts (PDXs) correlates with acquired resistance to tamoxifen68. RhoA represses Akt Ser473 phosphorylation49 and the repression of RhoA activity by cyclin D1 may have contributed to the induction of pAkt1–Ser473. The role of cyclin D1 in restraining RhoA, thereby inducing ERα activity and tamoxifen resistance, warrants further investigation.RhoA, Rac1, and Cdc42 are the best characterized members of the Rho GTPase branch of the Ras superfamily and are known to regulate cellular morphology and migration70. Furthermore, transgenic mice that overexpress the identical mutant cyclin D1 driven by the MMTV promoter (MMTV-D1T286A) developed mammary adenocarcinoma with a shorter latency relative to mice over-expressing the wild-type cyclin D1 (MMTV-D1)71. That said, the current studies suggest that in addition to the nuclear function of cyclin D1, a membrane-associated pool of cyclin D1 contributes to cellular migration, induction of Akt1 activity and the induction of a signaling pathway, defined through transcriptional reporters, that activates the immediate early gene c-Fos and cyclin D1. c-Fos is a target of Akt1 induction and Fos family members induce cell-cycle entry though the induction of cyclin D172, suggesting a mechanism by which membrane associated cyclin D1 may augment cellular growth. The major adjuvant therapy for the ~70% of ERα expressing human breast cancer involves anti-estrogen therapy. The ERα/PI3K/Akt complex pathway is hyperactivated in aggressive breast tumors73. The non-genomic actions of E2/ERα, mediated via cytoplasmic membrane-associated cyclin D1, may provide an important additional target58. As membrane-associated cyclin D1 augments activity of the ERα/PI3K/Akt complex pathway, the cytoplasmic membrane pool of cyclin D1 may be a new target for ERα expressing breast cancer treatments75.Several lines of evidence support the importance of cyclin D1 nuclear location in aberrant growth including elegant studies showing that a mutant of cyclin D1 (D1T286A), that is defective in phosphorylation-mediated nuclear export, induces cell transformation in cell culture assays and triggers B-cell lymphoma in a mouse model of mantle cell lymphomaA detailed description is provided in the Supplementary Materials.cyclin D1+/+ and cyclin D1−/− MEFs10 were prepared as described previously76.The 77, migratory velocity, and migratory distance26 were conducted as previously described.The assessment of transwell migrationKE mutant and FRET reporters , were cultured in a four-well chamber and imaged using a Zeiss laser-scanning microscope, LSM510META, with a 40× oil immersion Doc Plan-Neofluar lens objective . To detect FRET between CFP and YFP, we used time-lapse and lambda stack acquisition linked with the photobleaching command80.HEK293T cells, co-transfected with 3×FLAG vector, cyclin D1 wild-type or cyclin D177. Chromogen immunostaining of human breast cancer samples was conducted on the breast tissue with the Ventana Benchmark autostainer using deintified archival tissue which are exempt from review by the Thomas Jefferson University Institutional Review Board. Fluorescence-based immunohistochemistry for cyclin D1 multiplexed with pan-cytokeratin and DAPI counterstain was performed as previously described83 on a tissue microarray containing cores of 50 de-identified ER-positive breast cancer specimens provided by the Medical College of Wisconsin Tissue Bank under IRB-approved protocol.IF staining and confocal microscopy of cultured cells was conducted as described previously46.Live cell imaging studies were conducted as describedSupplemental Methods and Figure LegendsSupplemental Figures


Pseudomonas sp. strain NID84 and Acinetobacter sp. strain N2D84 from D84 and Enterococcus sp. strain N4D85, Enterococcus sp. strain N5D85, Lactobacillus sp. strain N6D85, and Leuconostoc sp. strain N7D85 from D85. Acinetobacter sp. strain N2D84 was identified as a human pathogen with a probability of 92%.This study reports two feces metagenomes (D84 and D85) and six metagenome-assembled genomes (MAGs). The assembled MAGs include  Pseudomonas sp. strain NID84 and Acinetobacter sp. strain N2D84 from D84 and Enterococcus sp. strain N4D85, Enterococcus sp. strain N5D85, Lactobacillus sp. strain N6D85, and Leuconostoc sp. strain N7D85 from D85. Acinetobacter sp. N2D84 was identified as a human pathogen with a probability of 92%.This study reports two feces metagenomes (D84 and D85) and six metagenome-assembled genomes (MAGs). The assembled MAGs include  Gastroenteritis is a common disease in dogs caused by a number of microorganisms, including bacteria and viruses. Its symptoms vary from diarrhea and abdominal pain to acute hemorrhagic syndrome, and it can be fatal .Canis lupus familiaris) that were housed by the Society for the Prevention of Cruelty to Animals (SPCA) in Mafikeng, North West Province, South Africa. The samples were collected directly from the rectum with sterile gloves and immediately placed in sterile containers. Metagenomic DNA was extracted from 150 mg of the fecal sample using a Quick-DNA fecal/soil microbe miniprep kit  following the manufacturer’s instruction. The library was prepared with a Nextera DNA Flex library prep kit using Nextera DNA CD index adapters (96 indexes plated) and sequenced on an Illumina NovaSeq instrument. A total of 12,512,020 reads were generated from sample D84 and 19,928,158 from sample D85, both with 1 × 150-bp paired-end read lengths, while the depth of sequencing for D84 was 156× and that for D85 was 153×.Fecal samples were separately collected from two diarrhea-affected dogs  v4.12 . The extN50 value of 52,139 bp, and an L50 value of 37. The entire assembly, which contains 7,693 coding sequences, was binned into two MAGs, which were identified as Pseudomonas sp. strain NID84 and Acinetobacter sp. strain N2D84 .PRJNA655841. These whole-genome shotgun projects have been deposited in DDBJ/ENA/GenBank under the accession numbers JACRYN000000000 and JACRYO000000000. The versions described in this paper are the first versions, JACRYN010000000 and JACRYO010000000. The SRA accession numbers for the raw reads are SRX8905271 and SRX8949278 for samples D84 and D85, respectively.All data were deposited under the GenBank BioProject number


Cytokine release syndrome (CRS) is a systemic inflammatory response characterized by the overexpression of inflammatory genes. Controlling CRS is essential for improving the therapeutic effects of chimeric antigen receptor (CAR) engineered T cells. However, current treatment options are limited given the complexity of cytokine interactions so it is important to seek a mild strategy with broad-spectrum inhibition to overcome this challenge.Using THZ1, a covalent inhibitor of cyclin-dependent kinase 7 (CDK7), we demonstrated the transcriptional suppression of inflammatory genes in activated macrophages. RNA sequencing and ChIP sequencing were conducted to identify the key target genes of the inflammatory response. Pathogen- and CAR T cell-induced CRS models were also established to assess the efficacy and safety of targeting CDK7.CDK7 blockade attenuated cytokine release, mitigated hyperinflammatory states and rescued mice from lethal CRS. Targeting CDK7 preferentially suppressed a set of inflammatory genes, of which STAT1 and IL1 were the key targets associated with super enhancers. Furthermore, we confirmed the potent efficacy of THZ1 in alleviating the CRS induced by CAR T cell infusion without causing tissue injury or impairing antitumor effects.Our work indicates the CDK7-dependent transcription addiction of inflammatory genes. Targeting CDK7 is a promising strategy for treating CRS by inhibiting multiple cytokines.The online version contains supplementary material available at 10.1186/s12943-020-01301-7. Cytokine release syndrome (CRS) is a life-threatening inflammatory disorder that can be triggered by infections or immunotherapies . Severe Despite the controversy over the immunopathological basis, macrophage overactivation is essential to maintain the hyperinflammatory state in CRS. From innate to adaptive immunity, macrophages respond to stimuli by promoting multi-cytokine release and interacting with different cell types . IL-6 anTHZ1 is a covalent inhibitor of cyclin-dependent kinase 7 (CDK7) and suppresses gene transcription by dephosphorylating the C-terminal domain of RNA polymerase II (RNA Pol II) . DifferiHere, we demonstrate that pretreatment with THZ1 significantly reduces the massive release of multi-cytokines and alleviates hyperinflammatory symptoms. The efficacy and safety of THZ1 are confirmed in bacteria- or virus-induced inflammatory responses and reduplicated successfully in CRS triggered by CAR T infusion without dampening antitumor capability. Mechanistic exploration further reveals a key role of STAT1/IL1 in the inflammatory response, which is vulnerable to THZ1 treatment under the control of SEs. Our work provides a promising strategy with broad-spectrum inhibition for current CRS treatment.Peripheral blood mononuclear cells (PBMCs) were separated from the blood samples of 4 healthy volunteers after gradient centrifugation, and cultured in DMEM for adhesion purification. After 2–4 h, suspended lymphocytes were pooled and activated with anti-CD3/CD28 for 1 day and then expanded in DMEM 10% FBS supplemented with 2 U/ml IL-7/IL-15 to obtain proliferative T cells.Two forms of macrophages were used in our experiments: Adherent fractions of PBMCs were cultured in DMEM 10% FBS supplemented with 20 ng/ml rhGM-CSF for 7 days to induce primary monocyte-derived macrophages (MDMs); PMA-induced macrophages (noted as mTHP-1) were obtained from human monocyte THP-1 cells via a 24-h incubation with 100 ng/ml PMA and cultured in PMA-free medium overnight to reduce their inflammatory baseline.Burkitt lymphoma Raji cells and human fibroblast HFF-1 cells were respectively cultured in RPMI-1640 and DMEM containing 10% FBS and 50 U/ml penicillin-streptomycin. Human umbilical vein endothelial cells (HUVEC) were cultured in endothelial basal medium supplemented with EGM-2 MV Single-Quots .The influenza virus H1N1  was used as the viral stimulus to activate macrophages.MDMs derived from 3 healthy individuals or mTHP-1 cells were treated with THZ1 for 0.5–1 h before or after stimulation. Total RNA was extracted from cell lysates and reverse transcribed to cDNA using a reverse-transcriptional kit (TaKaRa). RT-PCR was performed in a triplicate with the SYBR Prime Script RT-PCR kit (TaKaRa) on the 7300 plus Real-Time PCR system (Applied Biosystems). The primer sequences are described in the MDMs derived from 1 healthy individual were divided into three parts: the control cultured with medium, the stimulated cells cultured with LPS, and the rescued cells pretreated with 30 nM THZ1 for 0.5 h prior to LPS stimulation. Total RNA was prepared as described in the quantitative real-time PCR experiments. The isolated RNA was used to prepare RNA-seq libraries using TruSeq RNA Library Prep Kit v2 (Illumina) following the manufacturer’s instruction. The libraries were sequenced on an Illumina HiSeq X-ten sequencing System with a read length of 150 base pairs.https://asia.ensembl.org/info/data/ftp/index.html) using HISAT2 (v2.1.0) . In. In9]. ISevere CRS is an intractable event that requires a novel strategy with better efficacy and safety. After entering the era of immunotherapy, protection against the cytokine storm is indispensable for achieving the therapeutic purpose and controlling the related toxicity. In this study, we assessed the therapeutic potential of the CDK7 inhibitor THZ1 in pathogen-induced CRS models and reconfirmed its protective effects on CAR T cell-induced CRS. Preemptive inhibition of CDK7 significantly suppressed the magnitude of cytokine release and diminished the severity of CRS. Moreover, neither cytotoxicity nor tissue injury was observed in our CRS models with the proper concentrations of THZ1. These findings support the use of THZ1 as a relatively broad-spectrum inhibitor to disrupt excessive inflammatory activity.Unlike ATP-competitive CDK inhibitors with transient effects, THZ1 leads to a potent and time-dependent transcriptional inhibition because of the prolonged and irreversible CDK7 inactivation caused by the formation of covalent bonds . Due to Further insight into the CDK7-associated transcriptional addiction revealed the key targets of THZ1 treatment. Combining RNA- and ChIP-seq data, we showed the genes encoding inflammatory components such as effectors, receptors, or regulators, could be inhibited by CDK7 blockade through RNA Pol II-dependent transcription, and we depicted the landscape of SEs in activated macrophages. Our work examines the priority of STAT1/IL1 in the inflammatory response and suggests that THZ1 blunts the inflammatory cascade by decreased transcription of central STAT1/IL1 and consequent reduction of downstream TFs/genes  Effects of CDK7 inhibitors on cell proliferation. Cells were treated with the indicated concentrations for the indicated times, and detected using CCK8. (B) Effects of CDK7 inhibitors on cell apoptosis. Cells were treated with the indicated concentrations for 48 hours, and detected using Annexin V-FITC/PI flow cytometry. (C) Transcriptional levels of inflammatory genes in response to low-dose (100 ng/ml) or high-dose (500 ng/ml) LPS in mTHP-1 cells with 30 nM THZ1 pretreatment or posttreatment at 6 and 24 hours. (D) Transcriptional levels of inflammatory genes in response to 500 ng/ml LPS in mTHP-1 cells pretreated with 30 nM THZ1 at 4, 8 and 24 hours. (E) Transcriptional levels of inflammatory genes in mTHP-1 cells pretreated with 30 nM SY-1365 or 10 μM BS-181 at 6 hours after LPS stimulation. Data are the mean ± SD, n = 3-5 in (A) to (E). ***P < 0.001, **P < 0.01, and *P < 0.05 by one-way ANOVA in (C), unpaired t test in (D).Additional file 3: Figure S2 Supplementary data related to Fig. A) Survival of mice receiving different doses of LPS. The dose of 40 mg/kg was chosen to induce rapid and severe CRS. (B) Tissue sections were obtained from mice after THZ1 pretreatment and stained with H&E. (C) The gating strategy to phenotype and FACS sort myeloid populations in cells obtained from peritoneal lavage. Data are the mean ± SD, n = 5 in (A) and (B). A log-rank Mantel-Cox was performed for statistical analysis in (A).Additional file 4: Figure S3 Supplementary data related to Fig. A) Transcriptional levels of TFs in response to H1N1 infection in mTHP-1 cells pretreated with 30 nM THZ1 at 24 hours. (B) Peak plot and heatmap of RNA Pol II ChIP-seq density of 11408 genes in control mTHP-1 and LPS-stimulated mTHP-1 pretreated with THZ1 or not. (C) Boxplots of RNA Pol II levels in the ± 1kb around the transcription start sites (TSS) of the inflammatory genes under different conditions. The RNA Pol II signals at most inflammatory genes significantly increased in response to LPS stimulation and decreased with THZ1 pretreatment. ***P < 0.001, **P < 0.01, and *P < 0.05 by the paired t test in (C).Additional file 5: Figure S4 Supplementary data related to Fig. A) Boxplots of H3K27ac ChIP-seq density for all typical enhancers and SE domains. (B) The top 5 enriched GO biological processes of 1280 SE-associated genes or 58 THZ1-sensitive SE-associated genes. (C) Boxplots of the H3K27ac signals at 58 THZ1-sensitive SE-associated genes and GAPDH. (D) Analysis of the gene expression level, RNA Pol II density, and H3K27ac density at SE regions associated with STAT family. (E) H3K27ac density distribution for STAT1-proximal super enhancer in the control, stimulated and rescued cells based on 1000 bins (left). Boxplot for Pol II density at promoter-proximal bins for STAT1 . Expression change of STAT1 were presented by RNA-seq and quantitative PCR (low right). (F) Western blot analysis of STAT1 and RNA Pol II phosphorylation in mTHP-1 cells treated with 100 ng/ml IFN-γ for 30 minutes following inhibiting CDK7. ***P < 0.001 by the paired t test in (A), (C) to (E).Additional file 6: Figure S5 Supplementary data related to Fig. A) Schematic of CAR T cell generation. CD25 and CD69 were detected on day 2 to verify the T cell activation. CD3, CD4, and CD8 were examined weekly to monitor the distribution of T subsets. (B) Western blot analysis of STAT1 and RNA Pol II phosphorylation in mTHP-1 cells stimulated by the supernatant of coculture with Raji and CAR T cells following 30 nM THZ1 pretreatment for 4 hours.  Effects of THZ1 on cell proliferation. CAR T or NCT cells were treated with indicated concentrations for the indicated times, and detected using the CCK8 kit.  Effects of THZ1 on cell apoptosis. CAR T or NCT cells were treated with indicated concentrations for 48 hours, and detected using flow cytometry. (G) Transcriptional levels of inflammatory genes in NCT or CAR T cells treated with 20 nM THZ1 at 24 hours. (H) The residual Raji cells were detected in coculture systems with E/T ratio increases from CAR T: Raji = 1: 10 to CAR T: Raji = 1: 2 at 24 hours. (I) The residual Raji cells were detected in coculture systems with the E/T ratio NCT: Raji = 1: 2 at 24 hours. Coculture of NCT and Raji cells was set as the control to calculate the elimination rate. (J) The residual Raji cells were detected in coculture systems with the E/T ratio CAR T: THP-1: Raji = 1: 1: 4 at 24 hours. (K) Proliferation of CAR T or NCT cells in the presence of THZ1 was measured by CFSE dilution after 24, 48 and 72 hours. (L) Human cytokines were detected in the supernatant of coculture of Raji and CAR T cells at 24 hours with 20 nM THZ1 or not. n.s.: no statistical significance. Data are the mean ± SD, n = 3 in (A) to (L).Additional file 7: Figure S6 Supplementary data related to Fig. n = 3.Additional file 8. Supplementary Table. Lists of genes related to RNA-seq, RNA Pol II ChIP-seq and H3K27ac ChIP-seq.


Escherichia coli revealed that M4Lys alone caused rapid cell lysis. Treatment of E. coli with a Sec inhibitor did not inhibit the lysis activity of M4Lys, indicating that the Sec pathway is not involved in M4Lys-mediated cell lysis. Truncation of the TMD eliminated the cell lysis phenomenon, while production of the TMD alone did not induce the cell lysis. All these findings demonstrate that M4Lys is a novel endolysin that has a unique mosaic structure distinct from other canonical endolysins and the TMD plays a critical role in M4Lys-mediated in vitro cell lysis.Most double-stranded (ds) DNA phages utilize holin proteins to secrete endolysin for host peptidoglycan lysis. In contrast, several holin-independent endolysins with secretion sequences or signal-arrest-release (SAR) sequences are secreted via the Sec pathway. In this study, we characterized a novel lysis protein (M4Lys) encoded by the dsDNA phage BSPM4, whose lysis function is not dependent on either holin or the Sec pathway in vitro. In silico analysis of M4Lys revealed that it contains a putative virion protein domain and an unusual C-terminal transmembrane domain (TMD). Turbidity reduction assays and liquid chromatography-mass spectrometry using purified peptidoglycan showed that the virion protein domain of M4Lys has peptidoglycan lysis activity. In vitro overproduction of M4Lys in  Bacteriophages rely on diverse cell lysis mechanisms to release their progeny from host bacteria. Bacterial lysis by phages can be accomplished in at least two different ways, i.e., peptidoglycan degradation by endolysins or the inhibition of peptidoglycan synthesis ,2,3. TheEndolysins are peptidoglycan hydrolases that disintegrate integrity of the cell wall by cleaving the peptidoglycan. It is known that endolysins generally cleave glycosidic bonds, peptide bonds, or amide bonds of the peptidoglycan. Holins are small membrane proteins that dissipate membrane potential or make holes in the membrane to allow endolysins to access the peptidoglycan target ,6. In thOenococcus oeni phage fOg44 (Lys44) contains a typical signal sequence that is converted into the active form after cleavage of the N-terminal signal peptide. The N-terminal region of Lys44 functions as an export signal for the endolysin to translocate to the periplasmic region  were used if necessary. When indicated, isopropyl-β-thiogalactopyranoside (IPTG) and sodium azide (NaN3) were added at 0.1 mM and 1-10 mM, respectively.The bacterial strains and plasmids used in this study are listed in E. coli BL21 (DE3) strain and induced to examine cell lysis kinetics. The putative holin gene  was PCR-amplified with the BSPM4Holin-F-NcoI and BSPM4Holin-R-HindIII primers and cloned into the pETDuet-1 as described above. To coexpress M4Lys and M4Holin, both genes were cloned into pETDuet-1 using the primers above. To produce the proteins, 0.1 mM of IPTG was added 3 h after incubation and the lysis kinetics of each strain was observed as previously described [E. coli BL21 (DE3) strain was used as a control. All the primers used for PCR amplification are listed in The putative endolysin gene ORF_38, M4Lys) was amplified by polymerase chain reaction (PCR) with the BSPM4Lys-F-NdeI and BSPM4Lys-R-XhoI primers using phage BSPM4 DNA as template. The PCR product was digested with the respective restriction enzymes and cloned into the pETDuet-1 vector  and sequenced. The recombinant plasmids were, then, used to transform , M4Lys wE. coli BL21 (DE3) strain was incubated for 2 h until an optical density at 600 nm (OD600) of 0.5 was reached and was, then, supplemented with 0.5 mM IPTG to produce the M4LysΔTMD protein. After incubation for 3 h, cells were suspended in lysis buffer  and sonicated  to break bacterial cell walls. Sonicated cells were centrifuged at 15,000× g for 20 min to obtain the supernatant containing soluble proteins. His-tagged M4LysΔTMD was purified using a Ni-nitrilotriacetic acid Superflow column  according to the manufacturer’s instructions. The purified protein was visualized with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and stored in storage buffer  after exchanging buffers using a PD Miditrap G-25 column  [To purify a soluble form of M4Lys protein lacking the C-terminal TMD (designated as M4LysΔTMD), the M4LysΔTMD coding gene was cloned into the pET28a vector using the BSPM4Lys-F-NdeI and BSPM4Lys2-R-XhoI primers. In the cloning experiments to generate recombinant plasmids, DNA fragments encoding the putative endolysin gene were inserted between the NdeI and XhoI restriction sites of the pET28a plasmid resulting in an N-terminal His-tag. The recombinant plasmid-harboring cks, UK) .E. coli peptidoglycan (muropeptides) as substrates. Crude muropeptides of E. coli BL21 (DE3) were extracted as described previously with slight modifications [E. coli cells were harvested and the cells were disrupted by sonication. The cell suspension was then centrifuged at 1400× g for 10 min to obtain the crude cell wall lysate. Muropeptides were pelleted by centrifugation at 27,000× g for 5 min, resuspended in 4% SDS solution, and boiled for 10 min. Muropeptides were washed using cold distilled water at least 3 times, suspended in Tris-HCl buffer (pH 8.0), and stored at 4 °C until use. Peptidoglycan lysis activity of M4LysΔTMD was examined by measuring a reduction in OD600 of the muropeptide solution [600 was measured using a SpectraMax i3x  microplate reader. The reduction of turbidity was calculated by comparing with that of the respective control group without enzyme treatment. The bacterial strains used for host range analysis are listed in S. Typhimurium LT2C [Lytic activity of purified M4LysΔTMD was determined using purified ications ,21. In bium LT2C .E. coli MG1655 cells were grown exponentially (OD578 = 0.6) and harvested by centrifugation at 12,000× g for 15 min at 4 °C. The cell pellet was resuspended in 3 mL of ice-cold deionized water (D.W.) and 3 mL of boiling 8% SDS was added dropwise with vigorous stirring within 10 min. The mixture was boiled for 30 min and incubated overnight at room temperature. Muropeptides were collected by ultracentrifugation . The pellet was washed 4 times with 8 mL of D.W. to remove residual SDS. High molecular weight glycogen and covalently bound lipoprotein were cleaved by adding α-amylase  and pronase , respectively. Samples were mixed with an equal volume (8 mL) of 8% SDS and boiled for 15 min to inactivate the enzymes. Muropeptides were collected by ultracentrifugation and washed 3 times with D.W. to remove SDS from the pellet. The pellet was suspended in 20 mM Tris-HCl buffer (pH 8.0) and stored at 4 °C until use.Muropeptides for HPLC analysis was prepared as described above with slight modifications . Brieflyg, 15 min at 20 °C) and treated with purified M4LysΔTMD at 25 °C overnight. Samples were heat-inactivated for 10 min and reduced with 5 mg sodium borohydride in 500 mM sodium borate buffer (pH 9.0) for 30 min at room temperature. Borohydride was inactivated by adding 20% phosphoric acid, the pH adjusted to 4, and the sample filtrated using a 0.2 μM filter. Prepared samples were loaded onto a Kinetex C18 column  and a UHPLC C18 column  was used as a guard column. RP-HPLC was performed using the Agilent Technologies HPLC 1260 system at the Korea Basic Science Institute . Samples were injected onto a column preheated to 50 °C and allowed to bind at a flow rate of 0.5 mL/min with a solution of 1% acetonitrile and 0.1% trifluoroacetic acid (TFA) for 10 min. A gradient of 1% to 10% acetonitrile in 0.1% TFA solution was used for elution at a flow rate of 0.5 mL/min for the next 60 min. Absorbance was detected at 205 nm and the fractionated muropeptides were identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis was performed as previously described  with a f1–224, BSPM4Lys-F-NdeI and BSPM4Lys5_4-R-XhoI for M4Lys1–225, and BSPM4Lys-F-NdeI and BSPM4Lys6-R-XhoI for M4Lys1–226, respectively. All three PCR-amplified constructs were cloned into the pET28a vector and were transformed into E. coli BL21 (DE3) cells. Then, recombinant plasmid-harboring E. coli cells were used for cell lysis tests with 0.1 mM IPTG induction. Primers used for PCR amplification are listed in To understand the role of M4LysΔTMD and the TMD region, each recombinant gene was PCR-amplified, cloned into the pETDuet-1 dual-expression vector, and expressed with 0.1 mM of IPTG, as described above. The TMD region was truncated from the gene encoding the M4Lys protein with various primers. Primers BSPM4Lys-F-NdeI and BSPM4Lys2-R-XhoI were used to generate the TMD-truncated construct M4LysΔTMD. The following three primer sets were used to generate the partially truncated TMD: BSPM4Lys-F-NdeI and BSPM4Lys5_3-R-XhoI for M4LysP distance values [Phylogenetic trees were constructed based on alignment of the M4Lys protein sequence with the amino acid sequences from 37 Gram-negative bacteriophage endolysins available on the NCBI database. Protein sequences were aligned with the Clustal X2 program  and the e values . Amino ae values ,27.Salmonella phages such as iEPS5 and FSLSP099 [The phage BSPM4 genome sequence includes two putative lysis genes in the predicted lysis cassette, putative lysis protein B (ORF_37) and putative lysis protein A (ORF_38). A hypothetical gene (ORF_39) was identified downstream of the two putative lysis genes A. ArrangFSLSP099 ,29. HoweE. coli expression system. Endolysin is synthesized and accumulated in the cytosol until the holin creates pores in the inner membrane, thus allowing the endolysin to reach its peptidoglycan substrate target [E. coli cells were considerably lysed between 20 and 60 min after putative lysis protein A (M4Lys hereafter) alone was produced by IPTG induction  and the putative lysis protein A (ORF_38) using an e target ,30. Typie target ,31. Howenduction B. Furthenduction B. Coexprnduction B. Overalnduction B. We spenduction B. HoweveMost holin-independent endolysins contain N-terminal signal peptide sequences or SAR sequences essential for their secretion to the periplasm. These sequences often function as secretion sequences for Sec translocase ,10,11. H3, a well-known inhibitor of E. coli Sec translocase [3. Increasing concentrations of NaN3 delayed cell lysis by M4LysA but did not inhibit cell lysis  (data not shown). Since production of the full-length M4Lys protein caused rapid cell lysis  have a globular structure with a single catalytic domain . The NCBll lysis B, we speA putative TMD was predicted in the C-terminus of M4Lys using Phobius, Phyre2, and TMHMM algorithms ,35,36 FA. AnothePeptidoglycan lytic activity of the virion protein domain was assessed by deleting the C-terminal transmembrane domain of M4Lys (M4LysΔTMD). The lytic activity of the full-length M4Lys could not be measured since we failed to purify the protein in spite of repeated attempts, presumably due to the rapid cell lysis that occurred upon its induction. M4LysΔTMD of the predicted size (24.5 kDa) was purified  and incuN-acetylmuramidases (lysozymes), endo-β-N-acetylglucosaminidases (glycosidases), lytic transglycosylases, endopeptidases, and N-acetylmuramoyl L-alanine amidases [E. coli peptidoglycan (muropeptides) was predigested with a muramidase (mutanolysin) and incubated with purified M4LysΔTMD. Then, the muropeptides were fractionated by RP-HPLC and analyzed by MS (N-acetylglucosaminidase (glycosidase) activity of M4LysΔTMD. This result again supports the Phyre2 prediction that displayed structural homology between the N-terminus of M4Lys and the glycoside hydrolase domain of the flagella protein FlgJ from Salmonella. Interestingly, endopeptidase activity of M4Lys could also be inferred from the observation that a crosslinked dimer of disaccharide-tetrapeptide (peak 3) was reduced and a D-Glu-mDAP crosslink cleaved disaccharide  was generated   B.Our current data suggest that the ORF_37 of BSPM4 phage is not required for M4Lys-mediated cell lysis in vitro. However, we cannot currently rule out the possibility that ORF_37 is necessary for optimum cell lysis during BSPM4 phage infection in vivo.Salmonella. Subsequent BLASTp homology searches and amino acid sequence alignments revealed that the sequence of M4Lys is highly conserved (97% to 99% identity) among Chi-like phages  along with those originating from flagella-targeting (Chi-like) phages SPN19, iEPS5, FSLSP088, and Chi A 28,43,,43,44,45e phages B. These bacteria A.Salmonella phages. However, since this study provided indirect, circumstantial evidence on the potential role of TMD in M4Lys function, further studies are warranted to investigate its specific role in cells. Additionally, we hypothesize that the unique structure of M4Lys could be due to the recombination of two domains and further studies are being conducted in our laboratory to examine the interactions of the two M4Lys domains in an attempt to investigate the mechanism behind the formation of the observed mosaic domain.In this study, we identified and characterized the cell lysis characteristics of M4Lys, a novel endolysin that causes cell lysis when overproduced in vitro. Of particular note is the unusual structure of M4Lys that contains a putative virion protein domain and a TMD. A series of cell lysis kinetic analyses suggested that M4Lys has peptidoglycan degradation activity and that TMD is indispensable for host cell lysis by M4Lys when M4Lys is overexpressed in vitro. This finding enhances our understanding of the host cell lysis mechanism, especially by the flagella-targeting


Changes in chromatin structure, especially in histone modifications (HMs), linked with chromatin accessibility for transcription machinery, are considered to play significant roles in transcriptional regulation. Alveolar macrophages (AM) are important immune cells for protection against pulmonary pathogens, and must readily respond to bacteria and viruses that enter the airways. Mechanism(s) controlling AM innate response to different pathogen-associated molecular patterns (PAMPs) are not well defined in pigs. By combining RNA sequencing (RNA-seq) with chromatin immunoprecipitation and sequencing (ChIP-seq) for four histone marks , we established a chromatin state map for AM stimulated with two different PAMPs, lipopolysaccharide (LPS) and Poly(I:C), and investigated the potential effect of identified histone modifications on transcription factor binding motif (TFBM) prediction and RNA abundance changes in these AM. The integrative analysis suggests that the differential gene expression between non-stimulated and stimulated AM is significantly associated with changes in the H3K27ac level at active regulatory regions. Although global changes in chromatin states were minor after stimulation, we detected chromatin state changes for differentially expressed genes involved in the TLR4, TLR3 and RIG-I signaling pathways. We found that regions marked by H3K27ac genome-wide were enriched for TFBMs of TF that are involved in the inflammatory response. We further documented that TF whose expression was induced by these stimuli had TFBMs enriched within H3K27ac-marked regions whose chromatin state changed by these same stimuli. Given that the dramatic transcriptomic changes and minor chromatin state changes occurred in response to both stimuli, we conclude that regulatory elements (i.e. active promoters) that contain transcription factor binding motifs were already active/poised in AM for immediate inflammatory response to PAMPs. In summary, our data provides the first chromatin state map of porcine AM in response to bacterial and viral PAMPs, contributing to the Functional Annotation of Animal Genomes (FAANG) project, and demonstrates the role of HMs, especially H3K27ac, in regulating transcription in AM in response to LPS and Poly(I:C). Innate immune responses have a fundamental role in protecting the host from infection . Immune PRR activation and signaling also triggers epigenetic modulators that modify chromatin structure and as consequence, DNA accessibility . CombinaMycoplasma hyopneumoniae (Mhp) resulting in significant economic losses in the swine industry worldwide and, for influenza, increasing the risk of zoonotic disease spread , as well as better understand and utilize the pig as a human disease model, a comprehensive genome-wide annotation of HMs to construct a chromatin state map of the immune response is needed . In thisCells were isolated from eight crossbred (predominantly Large White and Landrace heritage) pigs approximately 8–13 weeks of age. Eight pigs  were housed in BLS2 rooms at the National Animal Disease Center  and all animal procedures were performed in compliance and approval by Institutional Animal Care and Use Committee.g for 10 min at 4°C. Cells were resuspended in supplemented medium  and seeded into 150 × 15 mm petri dishes for overnight incubation at 39°C and 5% CO2. Non-adherent cells were then removed by gently pipetting and discarding the media. Adherent AM were released with a cell scraper, collected and washed once. Cell count and viability data were obtained using the MUSE cell analyzer system (Millipore). AM from each of the eight pigs were aliquoted and seeded in six 100 x 15 mm petri dishes with a final volume of 5 mL and rested for 2 h. AM culture in each petri dish were stimulated for 2 or 6 h at 39°C and 5% CO2 with 0.5 μg/mL Poly (I:C) (LMW) / LyoVec™ (InvivoGen) or 1 μg/mL LPS from Escherichia coli O55:B5 (Sigma). Non-stimulated cells were included at each time point as controls. At indicated times after stimulation, AM were gently scraped and collected to determine number and cell viability for subsequent molecular assays. For chromatin immunoprecipitation, aliquots of AM from each animal were fixed for 10 min by adding 16% paraformaldehyde (Electron Microscopy Science) to a final concentration of 1%. Fixation was stopped using 2.5 M glycine (Boston Bioproducts) to a final concentration of 150 mM, and cells were thoroughly washed in cold PBS. Cell pellets were snap-frozen in liquid nitrogen and stored at −80°C. For RNA work, cell aliquots were stored in RLT buffer and stored at −80°C. A flowchart of the methods is shown in the After necropsy, lungs with trachea attached were removed from the body for collection of alveolar macrophages as previously described . Lungs wIL6, IL8, IL1β, TNF, and CASP1) involved in inflammatory response were assayed to confirm the LPS and Poly (I:C) stimulation in AM from all animals. cDNA was synthesized using IScript™ Reverse Transcription Supermix from 400 ng of RNA according to manufacturer’s recommendation (BIO-RAD). The final 10 μl PCR reaction included 1 μl of 1:3 diluted cDNA as template, 5 μl of iTaq Universal SYBR® Green Supermix (Bio-Rad), and gene specific forward and reverse primers at 20 μM to final concentration of 1 μM. QPCR was performed on a QuantStudio 5 system (Applied Biosystems) under following conditions: 95°C for 30 s; 95°C for 15 s followed by 60°C for 30 s (40 cycles), and a final dissociation step. Melting curve analysis was performed, to check specificity of each PCR product. Levels of mRNA were calculated according to the 2 CT–ΔΔ method . Tests with P-value < 0.05 were considered statistically significant. Primers sequences are listed in P < 0.05.RNA extraction from 1,000,000 cells was performed using the RNeasy Mini Kit (QIAGEN) following manufacturer’s instructions. Eluted RNA was treated with DNase Max Kit (QIAGEN) to break down and remove traces of DNA. RNA quantity and integrity were assessed with the Agilent 2200 TapeStation system (Agilent Technologies) and only samples with high RNA integrity numbers (RIN ≥ 7.6) were used for further analysis. For qPCR, a panel of five selected genes  were used for library preparation. The RNA was fragmented and prepared into sequencing libraries using the TruSeq Stranded Total RNA Sample Preparation Kit (Illumina). The 24 libraries were diluted and pooled together in approximately equimolar amounts. This pool of libraries was sequenced on an Illumina HiSeq 3000 sequencer in a 100 cycle, paired-end mode across three lanes.RNA from non-stimulated and stimulated AMs at 2 and 6 h from all animals   using the aligner STAR (version 2.5.3a) on 0.36)  with the 2.5.3a)  with the 2.5.3a)  with theDifferential gene expression analysis was performed using the R/Bioconductor package DESeq2 (version 1.24.0) . To acconbinomWaldTest function was used to estimate and test the significance of regression coefficients with the following explicit parameter settings: betaPrior = FALSE, maxit = 5000, useOptim = TRUE, useT = FALSE, useQR = TRUE. Differentially expressed genes between conditions were identified by testing the significance of relevant contrasts and using the results function with the following explicit parameters: alpha = 0.01, lfcThreshold = log2(1.5), altHypothesis = “greaterAbs”. Nominal p values were adjusted for multiple testing using the BH method (2(fold change) | > log2(1.5) and adjusted p-value < 0.05 were considered to be differentially expressed genes (DEGs).A generalized linear model was fitted for each gene in the count table, with a negative binomial response and a log link that included a DEseq2 normalization offset and the effects of pig individuals, time, treatment and time-by-treatment interaction, and the four surrogate variables as estimated above. The H method . Genes wP < 0.05 for Spearman test and P < 0.05 for Wilcoxon test (since the null hypothesis is that the gene lists were not different).Additionally, a Spearman correlation analysis was performed to validate gene expression and a Wilcoxon rank-sum test was used validate the gene expression similarity across species. The significant level was set at p-value < 0.05, κ score > 0.5 and at least 3 genes per term. All Ensembl Gene IDs with detectable expression level in alveolar macrophages  were used as the background reference and the gene ontology annotation3 for Sus scrofa was used as reference.Functional enrichment analysis based on a hypergeometric distribution was performed using ClueGO , a Cytos® 2S Plus DNA Library kit was used, following the manufacturer’s instructions. The libraries were pooled together and sequenced on an Illumina HiSeq 3000 sequencer to generate 100-bp paired-end reads across five lanes.The AM used for ChIP-seq experiment were from the same four pigs used in the RNA-seq experiment. H3K4me3 (active promoter regions) and H3K27me3 (associated with Polycomb repression) were performed using AM from two biological samples , and the other two histone marks H3K27ac (enhancer and promoter regions) and H3K4me1 (enhancer and promoter regions) were performed on the other two biological samples . While on ice, pellets of 200,000 fixed cells (for H3K4me3 and H3K27me3) or 400,000 fixed cells (for H3K4me1 and H3K27ac) were resuspended in 1 mL of cell lysis buffer  supplemented with Pierce EDTA-Free protease inhibitor mini tablets (ThermoFisher Scientific). Eosin staining was used to determine the viability and number of nuclei in a hemocytometer. The nuclei pellet was resuspended in 140 μL of nuclear lysis buffer  supplemented with Pierce protease inhibitor tablets (ThermoFisher Scientific), and chromatin was sheared using a focused ultrasonicator (Covaris ME220) with the following parameters: target size 350 bp, 90 seconds of shearing, 70 peak power, 20% duty factor, 1000 cycles per burst. ChIP was performed using iDeal ChIP-seq kit for histones (Diagenode) following manufacturer’s protocol. The following amounts of antibodies from Diagenode were used for ChIP-seq: 1.5 μg H3K4me3 (C15410195), 1 μg H3K4me1 (C15410194), 2 μg H3K27ac (C15410196) and 1 μg H3K27me3 (C15410003). Sequencing libraries for H3K4me3 and H3K27me3 ChIP and the corresponding input samples were prepared using the NEBNext Ultra II DNA library Prep Kit (Illumina). For H3K27ac and H3K4me1 ChIP and the corresponding input samples, the ACCEL-NGS4. Illumina sequencing adapters and low-quality bases were trimmed using Trimmomatic (version 0.36) on 0.36)  with theon 0.36)  with theon 0.36) . Duplicaeeptools  and the eeptools  were use6. SNP variants were filtered using the following settings: “QD < 2.0 || FS > 60.0 || MQ < 40.0 || MQRankSum < −12.5 || ReadPosRankSum < −8.0 || SOR > 3.0”. The genetic similarity scores between the four pigs were calculated using the snpgdsIBS function of the SNPRelate package (version 1.16.0)   by follo 1.16.0) , with 12Genome-wide chromatin states for each sample were predicted using the software ChromHMM . Given tPunctate  and broad (H3K27me3) histone modification enriched regions (HMERs) were identified using the software MUSIC  with the7 with the minOverlap parameter set to 1. The RPKM fold enrichment, that is, log2 (RPKMChIP/RPKMInput), were calculated for each genomic regions of the super union set for each sample using the dba.count function of the DiffBind package and used for principal component analysis using the princomp function in the stats (version 3.6.2) package8. A two-dimension PCA plot showing the relationship of the ChIP-seq samples were generated using the autoplot function of the ggfortify package9.Histone modification enriched regions identified by MUSIC were merged into a super union set of enriched regions using the DiffBind package (version 2.10.0)Expression level of each transcript was determined using the quasi-mapping tool Salmon (version 0.9.1)  based onDifferentially expressed genes with DHMRs were identified from the intersection of the list of genes with assigned DHMRs and the list of DEGs for each compared condition. Hypergeometric tests were performed to check the significance of overlapping between the two lists of interest.P-values. The protein-protein interactions were extracted from STRING  . Known mm STRING  and the m STRING  algorithIL6, IL1B, TNF, CASP1 and IL8 were confirmed for both stimulations at each timepoint  by TLR4 (LPS) or TLR3 and RIG-I/MDA-5 (Poly (I:C) agonists or media-alone as control, and collected for multiple analyses. To confirm the induction of an inflammatory response, we isolated total RNA and performed RT-qPCR to quantify differential expression of genes involved in inflammatory response. An increase in RNA levels of imepoint .Sus scrofa genome version 11.1, and an average of 41,029,762 (90.50%) were uniquely mapped. We used Principal Component Analysis (PCA) to obtain an overview of the similarities and differences between treatments as well as the time effect of culture. The first principal component (PC)-1 explained the variance and clearly separated stimulated from non-stimulated AM (2(CPM) revealed samples were clustered by time and treatment.To investigate global induction of the RNA response in AM stimulated with LPS and Poly (I:C), we performed genome-wide expression analysis using RNA-seq. An average of 45,330,687 clean reads were produced from three independent treatments  and two timepoints, each with four biological replicates . Clean slated AM  demonstrlated AM . PCA andR2 = 0.79, P < 2.2x10–16) the most correlated LPS-gene response followed by HMDM  and MBMM   treatments compared to the non-stimulated AM at the corresponding time point. In total, we identified 5,760 genes that were differentially expressed (DE) between non-stimulated and stimulated AM . The num2x10–16) . Then, w2x10–16) .R2 = 0.73, P < 0.0014)  that were enriched for “protein deacetylation” function in both treatments at 2h. Histone deacetylases (HDACs) had an opposite expression patterns: genes such as HDAC5 (down- LPS 6h) and HDAC7 (down- LPS 6h) had decreased RNA levels, meanwhile genes such as HDAC6 (up- Poly (I:C) 6h), HDAC9 (up- LPS and Poly (I:C) 6h) and SIRT1 (up- LPS and Poly (I:C) 2h and 6h) were found with increased levels of RNA. Contrary to the biological function enrichment observed, genes with decreased RNA levels were for just a few KEGG pathway terms such as “Herpes simplex virus 1 infection”  2h) and “cysteine and methionine metabolism” (Poly (I:C) 6h). In summary, DEG and GO analysis indicated a similar biological response to the two stimulations, as genes with increased RNA levels upon either LPS or Poly (I:C) were enriched for GO terms related to the activation of the immune response. However, some specific responses to stimulation of TLR4 or TLR3 and RIG-I like receptor were detected that have not previously been annotated as specific for bacterial and viral infection in swine.As an initial validation of the RNAseq data, the estimate of Fold change ratio due to treatment of the five inflammatory genes at both 2 and 6 h was compared to the values reported above for qPCR. The RNAseq data was significantly correlated with that seen for the q-PCR data ( 0.0014) . The DEG 0.0014) . As expe 0.0014) . In addi 0.0014) . MoreoveSLCO4A1 and VDR, which were higher in LPS and lower in Poly (I:C) treatment at 6h. The full description of the number of common, unique genes and their GO terms is available in the To identify the common RNA transcriptional responses via TLR4 and TLR3, RIG-I/MDA-5, we compared the DEG (treatments/controls) lists between treatments at 2h and 6h. All genes with similar response to the two treatments were also concordant between time of treatments except for To better understand the differences in RNA expression induced by the LPS versus Poly (I:C) treatment, we performed a pairwise comparison between treatments with the aim of identifying genes specifically induced by LPS versus Poly (I:C), and vice versa . Next, wSus scrofa reference genome (range 66–99%). The marks H3K27me3 and H3K4me3 had the highest number of sequencing reads across all samples analyzed  as well as the highest alignment rate to the genome , followed by narrow mark and H3K27ac (85%) (To explore regulatory mechanisms associated with modified histones on AM gene expression in response to LPS or Poly (I:C) at 2h and 6h, we performed ChIP-seq. The four histone marks analyzed in this study included all recommended FAANG HMs : H3K4me3ac (85%) . As expeac (85%)  or correac (85%)  and thisac (85%) .10. ChIPQC package was used to assess the quality of the ChIP-seq  within replicates of H3K4me1, H3K4me3, H3K27ac, and H3K27me3, respectively . EnricheChIP-seq , and theChIP-seq . AnotherChIP-seq  was calcChIP-seq , which iChIP-seq . OverallACTB), and no enrichment at the Myotilin gene (MYOT) which was not expressed in AM. These results demonstrate that ChIP-seq data consistently detected biologically relevant histone marks across different sample treatments as well as replicates  (−/+5.0 Kb) genome wide for all samples. High enrichment of active histone marks H3K4me3, H3K4me1 and H3K27ac was observed at TSS regions . In addiplicates .CD40, CD14, RELA, CCL3L1 and TNF as well as the control (non-expressed) F9 gene  was as expected. Regions around 2 Kb of TSS were enriched specifically for chromatin states 1, 4, 5, 6 and 8 corresponding to promoter regions and CpG islands (promoter regions)  stimulated cells was created. Implementation of the Hidden Markov Model (HMM) uses histone mark data to represent different hidden states of the “chromatin states” based on the presence/absence of multiple histone marks (emission parameters) and the special constraints of how these histone mark combinations occur relative to each other across the genome . Accordiregions) . Notablyregions) .2 FC| > 1, FDR < 0.05). The H3K27ac was the histone mark with more DHMRs after LPS or Poly (I:C) stimulation, followed by H3K4me3. In contrast, H3K4me1 and H3K27me3 enrichment levels were not modified by stimuli. In total, 10,290 DHMRs in all comparisons; 6,944 DHMRs between non-stimulated and stimulated AM, 1,097 between treatments type and 2,249 between timepoints  are associated strongly with gene expression changes . Therefomepoints . Enrichmmepoints , followeP < 0.05). After integration, the increase of H3K27ac and H3K4me3 at the promoter region of upregulated genes  was higher than the decrease of H3K27ac and H3K4me3 at the promoter region of downregulated genes . Furthermore, the integration showed a few H3K27ac and H3K4me changes over time and across treatments . We assigned the DHMRs to the nearest known transcripts (5kb flanking TSS) in the current porcine genome annotation, and then compared these regions with DEG abundance  2h, R2 = 0.45, P < 7x10–16; Poly(I:C) 6h, R2 = 0.68, P < 2.2x10–16) . The second group was shaped by DEG that responded to only one treatment, yet showed DHMRs for the same treatment  2h; FOSL1, LPS 6h; AMCF-II LPS 6h), Finally, the third group included genes that were DE and showed DHMRs for both treatments .Although an association between H3K4me3 and some DEG was found, only the list of genes with an increase of H3K27ac at promoter regions and higher levels of RNA included sufficient genes to be considered for further analysis. H3K27ac was significantly positively correlated with gene expression changes, although the correlation observed was weak at 2h and moderate at 6h for both treatments  . GO term2x10–16)  revealed2x10–16) . AlthougTNF and CCL3L1 after stimulation.As DHMRs-H3K27ac were observed near the promoter regions (5kb flanking TSS) of DEG involved in immune system activation, we anticipated observing predicted chromatin state changes of promoter regions associated with immune activation after stimulation. For this Integrative Genomics Viewer (IGV) analysis, we chose the increase of DHMRs-H3K27ac 1kb up and downstream of the TSS for upregulated genes involved in immune KEGG pathways mentioned above (56 genes) . A totalJun, JUNB/D, FOS, FOSL2, BATF, ATF2/3/4/7 and MAFB/F/K) interferon regulatory factors (IRF1/2/3/8) and NFκB-65 (RELA), were identified. Interestingly, the motif for NFκB p50/p52 (NFκB1) was enriched only in LPS and Poly(I:C) at 2h using the H3K27ac peaks  using HOMER . We testac peaks . Group  increased at 2h for LPS and at 6h for both treatments. CREB5, IRF4 and STAT1 were increased in response to LPS, while EHF, KLF5 and ELF1 responded only to Poly(I:C). IRF8, RELA and PRDM1 responded at 2h, and IRF2 only at 6h, for both treatments. No downregulated TF with motif enrichment in decreased H3K27ac peak regions was found. Using this combinatorial approach of HMs, TFBM and gene expression analyses, we were able to find TFs that were induced by the treatments whose TFBM was enriched in regions with a gain of H3K27ac modification. For example, pro-inflammatory  and anti-inflammatory TFs  were identified, as well as an antiviral TF (ELF1). ELF1 inhibits the infection of eight diverse RNA and DNA viruses independent from the action of the type I interferons  treatments. We identified 8, 6, 7 and 5 upregulated RNAs for transcription factors with enriched motifs in H3K27ac peak regions increased by LPS-2h, LPS-6h, Poly(I:C)-2h and Poly(I:C)-6h treatments, respectively . These uerferons . CollectAlveolar macrophages have broad effector functions to maintain lung health, and have the ability to respond to a variety of PAMPs, responding relative to the stimulus . Immune NFκB were also observed in our porcine AM data set, demonstrating consistency with these previous studies  exposure. Our results show similarities to transcriptional responses in human, mice and swine in different types of macrophages . The inc studies . It was ZNF174, a zinc finger protein  and Poly (I:C) (downregulated) treatment at 6h   exposure were common between stimuli , which ctivation . This cant at 6h . Both SLacterial  or viralacterial . In addiPHAOMEGA . All thrPHAOMEGA . The RNA3 (ATF3) . ATF3, Il mimics .STAT3, NFκBID, NFκBIE AMCF-II and IL12B, whose RNA levels increased in AM only in response only to LPS, which agrees with STAT3 and IL12B transcription observed in human and murine macrophages (NOS2) (NFκBID and NFκBIE)   which plays an important role in B cell receptor signaling, B cell survival and antigen presentation  were also found. The expression of swine leukocyte antigen (SLA) class II genes increased only in response to Poly (I:C). SLA molecules are involved in the adaptive immune response by presenting antigens to cognate T cells for effector functions, which is one of the functions of macrophages . Althougrophages . The funs (NOS2)  and inhi NFκBIE) . Interes NFκBIE) . Poly (Ied BAFF)  and DAPPentation , as wellentation .NFκB and other transcription factor in macrophages  stimulation. As AM are highly differentiated cells poised for effector function, perhaps this is not surprising, contrary to what is observed during hematopoiesis (Genome-wide histone modification profiling at 2h and 6h after LPS or Poly (I:C) stimulation showed few large changes in predicted chromatin states due to treatments in spite of the differential expression of genes involved in protein deacetylation and potential modification of the chromatin state and subsequent transcriptional response. The chromatin state map demonstrates a consistency of the chromatin structure in response to LPS and Poly (I:C) at 2h and 6h post stimulation. However, we could observe in non-stimulated AM  that expression of primary response genes was allowed by a permissive chromatin structure according to histone mark distribution , as suchopoiesis .Active HMs (H3K27ac and H3K4me3) at promoter regions are typically associated with highly expressed genes . The H3KIRF, NFκB and AP-1 factors necessary for response to these stimuli  stimulations and decreased expression at 6h LPS treatment and return to baseline for the 6h Poly (I:C) treatment. A similar pattern of H3K27ac modification near the promoter region of TNF have been observed in human nasopharyngeal epithelial cells in response to LPS: enrichment of binding sites of RELA and AP-1 members in H3K27ac peak regions in non-stimulated cells; and the increase of HM enrichment in the promoter region after LPS treatment, all contributing to the induction of the early response gene TNF  treatment. NRF2 is a TF that suppresses macrophage LPS inflammatory response by blocking proinflammatory cytokine transcription through inhibiting RNA pol II recruitment  at 2h and 6h, especially in genes activated by TLR4, TLR3 and RIG-I receptor-mediated signaling. In spite of the H3K27ac changes in respond to treatments, the H3K27ac-DHMRs between timepoints did not show large changes at promoter regions of DEG. This could indicate that the induction of secondary responses did not require dramatic changes of H3K27ac from 2h to 6h, because response to stimuli from 2h and 6h are considered a transition between early and secondary transcriptional response in macrophages . Howevergene TNF . In addiruitment . Thus, oTaken together, the RNA-seq and ChIP-seq data suggests that the differential gene expression between non-stimulated and stimulated macrophages is determined at least partly by changes in chromatin accessibility to transcription factor motifs at active regulatory regions in the genome, and that these changes are mediated primarily through H3K27ac modifications. Although we found some individual genes that responded only for one treatment and were regulated at the epigenetic level, changes to chromatin states were relatively minor after stimulations at 2h and 6h using bacterial and viral mimics. This could suggest that regulatory elements  are already active/poised and the chromatin is already open for immediate inflammatory response in porcine AM. In summary, our data reported here provides the first chromatin state of AM in response to bacterial and viral mimics, contributing to the Functional Annotation of Animal Genomes (FAANG) project . Furtherhttps://www.ebi.ac.uk/ena/browser/view/PRJEB31483.Raw sequencing data from RNA-seq and ChIP-seq are available through the European Nucleotide Archive (project: PRJEB31483), The animal study was reviewed and approved by Institutional Animal Care and Use Committee – National Animal Disease Center .JH-U was responsible for collecting data for the whole study, and for drafting the manuscript. HL performed to the bioinformatic analysis and contributed to writing of the manuscript and the biological interpretation of the data. KB and ZB participated in the macrophage isolation, stimulation, and preservation for assays. CL performed the procedures for lung lavage collection of cells. CT and CL conceived and designed the project and participated in the interpretation and discussion of the results, as well as in the writing of the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The COVID-19 pandemic negatively impacted the 2019/20 WHO European Region influenza surveillance. Compared with previous 4-year averages, antigenic and genetic characterisations decreased by 17%  and 24% . Of subtyped influenza A viruses, 56%  were A(H1)pdm09, 44%  A(H3). Of characterised B viruses, 98%  were B/Victoria. Considerable numbers of viruses antigenically differed from northern hemisphere vaccine components. In 2020/21, maintaining influenza virological surveillance, while supporting SARS-CoV-2 surveillance is crucial. The ending of the 2019/20 influenza season in the World Health Organization (WHO) European Region coincided with the start of the first wave of the coronavirus disease (COVID-19) pandemic. This study assesses potential impacts of the pandemic on influenza surveillance and presents characteristics of influenza viruses detected in the Region in 2019/20, relative to contemporary components of influenza vaccines for the northern hemisphere (NH).In the WHO European Region, the 2019/20 influenza season started in week 47 2019, peaked for 2 weeks, weeks 05 and 06 2020, and returned to baseline levels (< 10% positivity in sentinel samples) very rapidly in week 13 2020, following widespread public health and social measures implemented to control COVID-19 . InfluenNational Influenza Centres (NICs) in the Region collect influenza virological surveillance data, conduct genetic and antigenic characterisation of viruses and report to The European Surveillance System (TESSy) on a weekly basis. The WHO Collaborating Centres (WHO CC) in London and in Atlanta (at the Centers for Disease Control and Prevention (CDC)) provide NICs with post-infection ferret antisera or other antisera raised against egg and/or cell culture-propagated vaccine/reference viruses for antigenic characterisation or typing/subtyping using haemagglutination inhibition (HAI) assays. WHO CC London also provides a list of reference sequences for the assignment of viruses to haemagglutinin (HA) gene clades/subclades following Sanger or next generation sequencing (NGS) . NICs shFifty Member States of the WHO European Region reported 165,267 influenza virus detections between week 40 2019 through week 20 2020. Relative proportions of circulating influenza A(H3), A(H1)pdm09 and B/Victoria lineage viruses varied between countries [The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) occurred in March 2020, relatively late in the course of the influenza season in the Region, and the total number of influenza virus detections was comparable to previous seasons. However, the COVID-19 pandemic adversely affected the generation and reporting of virus characterisation data.Compared with the previous 4-year averages, a lower number of countries contributed antigenic and genetic data in 2019/20  , and theFor specimens collected from week 40 2019 to 20 2020, genetic characterisation data of 3,403 viruses were reported to TESSy by 21 countries and antigenic characterisation data of 2,601 viruses by 13 countries. Among A(H1)pdm09 viruses, of the 1,246 that were genetically characterised, 1,121 (90%) belonged to the 6B.1A5A group, moreover, of the 1,032 antigenically characterised, the majority  were similar to the A/Brisbane/02/2018 vaccine virus. However, 173 A(H1)pdm09 viruses were not attributed to any predefined antigenic category, indicative of possible antigenic drift; of these viruses, genetic information was reported for only 48, nine of which had the HA1 N156K amino-acid substitution in antigenic site Sa. Overall, 16%  of A(H1)pdm09 viruses with genetic sequence information were 6B.1A5A-156K.Of the 1,240 genetically characterised AH3) viruses, the majority n = 679; 55%) belonged to clade 3C.3a and were antigenically similar to the NH 2019/20 vaccine virus A/Kansas/14/2017. The remainder belonged to subclade 3C.2a1b and were antigenically distinct [5% belong viruses,Of 917 genetically characterised type B viruses, the B/Victoria-lineage accounted for 887 (97%), with 819 (92%) of these belonging to clade 1A(Δ162–164-B) and being antigenically distinct from the clade 1A(Δ162–163) vaccine virus B/Colorado/06/2017. Only 30/917 (3%) of type B viruses were assigned to the B/Yamagata-lineage, and 28 of these were assigned to clade 3, remaining antigenically similar to the B/Phuket/3073/2013 vaccine virus.An ethical approval was not needed for this study, as data are not identifiable back to the patients from whom they originated.Based on the data, influenza activity in the European Region appears to have ended abruptly in week 13 2020, earlier than previous seasons ,9. RespoDuring the 2019/20 influenza season, the vast majority of influenza A(H3) viruses fell in genetic clade 3C.3a and subclade 3C.2a1b. The 3C.3a viruses were antigenically similar to the recommended 2019/20 NH vaccine virus, while 3C.2a1b viruses were antigenically distinct ,6. Most Despite circulation of viruses antigenically distinct from vaccine components (i.e. A(H3) subclade 3C.2a1b, B/Victoria-lineage Δ162–164-B and A(H1)pdm09 6B.1A5A-156K viruses) during 2019/20, a moderately good overall level of vaccine effectiveness was observed, notably for type B and A(H1)pdm09 viruses . The issData from the 2020 SH influenza season show that circulation of influenza viruses was extremely limited in the SH winter and also elsewhere for the NH inter-seasonal period ,9,12. SiIn terms of way forward, NICs play crucial roles in surveillance of seasonal influenza and zoonotic events and are responsible for arranging the essential shipments of representative specimens to the WHO CC to ensure there are sufficient data for making VCM recommendations. With increasing number of avian influenza outbreaks and continued evolution of influenza viruses in swine, there is also need for maintained vigilance in public health laboratories to ensure detection of zoonotic events for pandemic preparedness purposes ,15. In t


Hepatitis C virus (HCV) infection is a worldwide health problem. Nowadays,direct-acting antiviral agents (DAAs) are the main treatment for HCV; however,the high level of virus variability leads to the development ofresistance-associated variants (RAVs). Thus, assessing RAVs in infected patientsis important for monitoring treatment efficacy. The aim of our study was toinvestigate the presence of naturally occurring resistance mutations in HCV NS3and NS5 regions in treatment-naïve patients. Ninety-six anti-HCV positive serumsamples from blood donors at the Center of Hematology and Hemotherapy of SantaCatarina State (HEMOSC) were collected retrospectively in 2013 and evaluated inthis study. HCV 1a (37.9%), 1b (25.3%), and 3a (36.8%) subtypes were found. Thefrequency of patients with RAVs in our study was 6.9%. The HCV NS5b sequencingreveled 1 sample with L320F mutation and 4 samples with the C316N/Rpolymorphism. The analysis of the NS3 region revealed the D168A/G/T (3.45%),S122G (1.15%), and V55A (2.3%) mutations. All samples from genotype 3a (36.8%)presented the V170 I/V non-synonymous mutation. In conclusion, we have shownthat mutations in NS3 and NS5b genes are present in Brazilian isolates fromtherapy-naïve HCV patients. Hepatitis C virus (HCV) infection is a worldwide health problem. According to theGlobal Hepatitis Report, from the World Health Organization (WHO), approximately 71million people have chronic HCV infection, and nearly 399,000 people die each year,mostly due to cirrhosis or hepatocellular carcinoma . HCV hasThere is no vaccine available for preventing HCV infections. The main antiviraltreatment until 2011, was PEGylated interferon-alfa (αPeg-IFN) alone or incombination with ribavirin, leading to a sustained virological response (SVR) in 50%of treated patients, depending on the virus genotype causing the HCV infection. In BrazThe primary targets of DAAs are nonstructural proteins essentials for HCVreplication, which include the NS3 protease, NS5B polymerase, and NS5A protein(The presence of RAVs in patients not yet under treatment has been reported previouslyin different countries (The Center of Hematology and Hemotherapy of Santa Catarina State (HEMOSC) iscurrently responsible for the nucleic acid testing for HIV, HCV, and HBV in samplesfrom blood donors from Santa Catarina and Rio Grande do Sul states. Annually, HEMOSCreceives around 300 thousand blood donations. A total of 96 samples that werepositive for HCV in 2013 were used for this study, retrospectively.HCV RNA was extracted from plasma previously conserved at -80 °C using a molecularbiology workstation , with the Qiamp one-for-all nucleic acidkit (Qiagen), according to the manufacturer’s instructions. Plasma HCV RNA wasquantified using COBAS/Taqman HCV Test v2.0 (Roche).[HCV] .The statistical program SPSS (IBM SPSS Statistics Base 22.0) was used. Multivariateanalysis of variance (ANOVA) was applied to compare means of continuous variableswith normal distribution From the 96 HCV-positive samples collected, nine did not have enough material toperform the assays and were excluded from the analysis. Eighty-seven samples wereused for genotyping and analysis of NS3 and NS5b regions.From the 87 samples evaluated, 33 (37.9%) were of genotype 1a, 22 (25.3%) were ofgenotype 1b, and 32 (36.8%) were of genotype 3a. Genotype 1 (1a plus 1b) was themost frequent, followed by genotype 3, a result that is in agreement with what waspreviously reported in Brazil (p=0.6). The detected HCV genotypes and viral loads are bothimportant predictors for therapeutic outcomes. It has been reported that patientsinfected with genotype 1 are more likely to have higher viral loads than thoseinfected with genotype 2 and 3 (p=0.0002) and genotype 1b (6.35 log10 IU/mL)  was intermediate whencompared with other Brazilian studies among HCV chronic carriers not treated withprotease inhibitors (3.2% - 18.9%)  of genotype 1a. L320F is known to confer low resistanceto sofosbuvir and sofosbuvir associated with mericitabine (The polymorphism C316N/R was present in 4 samples (6.35%) of genotype 1b. C316N isreported to confer low level of resistance to sofosbuvir . Twenty samples (26.98%) presented Q309R, and three (1.58%) A333E.Seventeen were Q309R and D244N, two were Q309R and A333E, and two were triplepositive. In a previous work, the most frequent mutation observed in Brazil wasQ309R, present in all HCV subtypes , S122G, and V55A  that confer resistance to asunaprevir,boceprevir, grazoprevir, simeprevir, and paritaprevir  and decreasedsusceptibility to gazoprevir (Y56F). This shows the importance of studying both NS3and NS5 proteins when evaluating or choosing the therapy strategy for HCV-positivepatients. Patients carrying combinations of resistance mutations are of particularinterest, since they may increase the possibility of failure in the treatment withDAAs.The frequency of resistance mutations and genotypes was twice as high among patientswith subtype 1a compared to those with subtype 1b. A similar result was found in astudy with blood donor’s samples from São Paulo (At last, several polymorphisms not associated with resistance to DAAs were observedin our study , and preIn conclusion, we have shown that mutations in NS3 and NS5b domains are present inBrazilian isolates from therapy-naïve patients, in this case, blood donors withunknown HCV infection. Monitoring the presence of RAVs is important for predictingthe response to antiviral therapy, and regional discernment can help determine localpolicies for treatment. The results presented here will help ensure therapystrategies that are more successful for HCV-infected patients in Santa Catarina andRio Grande do Sul states in Brazil.


Hepatocellular carcinoma (HCC) is a major global health burden and its treatment options are limited. Spermatogenesis associated serine rich 2(SPATS2), a recent defined oncogene, was found to be a prognostic biomarker in HCC. However, the explicit mechanism underlying SPATS2 was urged to be elucidated. In vitro, knockdown of SPATS2 hampered the proliferation, invasion and migration of HCC cells. Moreover, phosphorylation of signal transducer and activator of transcription 3 (STAT3) and its downstream oncogenes were dramatically suppressed by SPATS2 knockdown. In addition, tripartite motif containing 44 (TRIM44) was found to be positively associated with SPATS2 in TCGA and declined after SPATS2 knockdown in HCC cells. Overexpression of TRIM44 rescued the effect of SPATS2 silencing on p-STAT3 and its downstream oncogenes. In vivo, SPATS2 silencing was confirmed to impede HCC tumor development in nude mice. In our own cohort containing 112 HCC patients, high SPATS2 protein level is indicative of an unfavorable clinicopathological feature and poor prognosis and could serve as an independent risk factor. Collectively, the present study is the first to propose the mechanism of significance of SPATS2-TRIM44-p-STAT3 in HCC and provide a new theoretical basis for targeted therapy. Hepatocellular carcinoma (HCC) predominantly accounts for 80%-90% of liver cancer, which is estimated to be the second leading killer of cancer-associated death in the worldSpermatogenesis associated serine rich 2 (SPATS2) locates in cytoplasm and is expressed in 217 organs, highest in testis and developmentally regulated during spermatogenesisTripartite motif containing 44 (TRIM44) belongs to TRIM families containing a zinc finger ubiquitin protease domain in the N-terminal domainsThe present study provides the first investigation into the significant mechanism of SPATS2 in HCC, which helps clarify the meaning of SPATS2-TRIM44-STAT3 axis in HCC diagnosis and treatment.Cancer tissue specimens and paraffin sections of adjacent tissues were collected from 112 patients with HCC who received surgical treatment at Huashan Hospital, Fudan University. Diagnosis of HCC for all patients was pathologically confirmed. The usage of patients' tissue specimens was approved by the Ethics Committee of Huashan Hospital, Fudan University and informed consents were acquired from all patients. The work described has been carried out in accordance with the principles of Declaration of Helsinki for experiments involving humans.7) stably transfected with shSPATS2 or control vectors were subcutaneously injected into nude mice  . Tumors were removed after 4 weeks. All animal experiments followed the Interdisciplinary Principles and Guidelines for the Use of Animals in Research, Testing, and Education by the New York Academy of Sciences, Ad Hoc Animal Research Committee.All experimental procedures involving animals were approved by the Animal Care and Use Committee of Fudan University, China. MHCC-97H cells (1×10The Cancer Genome Atlas (TCGA) database was used to analyze the expression of SPATS2 in patient HCC tissues. LIHC patients were divided into low- and high-expression groups and the values of SPATS2 related to prognostic analysis were examined.2 at 37 °C. Then SPATS2 shRNA and TRIM44 overexpression plasmid were constructed. HEK293T cells were used for lentivirus production. Both MHCC-97H and HCC-LM3 cells with stable SPATS2 knockdowns and TRIM44 overexpression were generated by lentiviral infection and then followed by selection with 2 μg/mL puromycin for 2 weeks. The sequences of the primers of SPATS2 shRNA were listed as follows: sh-SPATS2-1-F CCGGGCACTTTGTTAGTGAACGTAACTCGAGTTACGTTCACTAACAAAGTGCTTTTTTG, sh-SPATS2-1-R AATTCAAAAAGCACTTTGTTAGTGAACGTAACTCGAGTTACGTTCACTAACAAAGTGCT; sh-SPATS2-2-F CCGGGCACGGTATCGAGTTGTAGTTCTCGAGAACTACAACTCGATACCGTGCTTTTTTG; sh-SPATS2-2-R AATTCAAAAAGCACGGTATCGAGTTGTAGTTCTCGAGAACTACAACTCGATACCGTGCT.L02 and six HCC cell lines of huh-7, Hep-3B, PLC, Hep-G2, MHCC-97H and HCC-LM3 were used in the subsequent experimental investigations. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum  and 100 mg/mL penicillin-streptomycin in a humidified atmosphere containing 5% COTM Western Chemiluminescent HRP substrate . The primary antibodies used were: Alpha Tubulin , BCL2 , HIF-1α , MMP9 , PIM1 , STAT3 , p-STAT3 , TRIM44  and SPATS2 .RIPA  was used to lyse harvested cells. After electrophoresis and transfer, the PVDF membranes were incubated and shake overnight with primary antibodies at 4°C. Then, the imprints were washed with TBST for 3 times and cultured with incubated with an HRP-conjugated secondaey antibody (diluted at 1:5000) at room temperature for 1 hour. The proteins were visualized using a Immobilon-△△Ct method. The sequences of the primers were listed as follows: β-Tubulin-F CTGCTCATCAGCAAAGTGCG, β-Tubulin-R TGCGGAAGCAGATGTCGTAG; SPATS2-F AAGAGAAGATAAATGCGGTACG, SPATS2-R TACTTCACTGGCACTACCTTCC; STAT3-F ATCACGCCTTCTACAGACTGC, STAT3-R CATCCTGGAGATTCTCTACCACT; TRIM44-F AGTAACTCGGGACCAAATGAAGA, TRIM44-R CATGTGGGATTGGATGTCTGC; BCL2-F GGTGGGGTCATGTGTGTGG, BCL2-R CGGTTCAGGTACTCAGTCATCC; MMP9-F TGTACCGCTATGGTTACACTCG, MMP9-R GGCAGGGACAGTTGCTTCT; HIF-1α-F GAACGTCGAAAAGAAAAGTCTCG, HIF-1α-R CCTTATCAAGATGCGAACTCACA; PIM1-F GAGAAGGACCGGATTTCCGAC, PIM1-R CAGTCCAGGAGCCTAATGACG.Total RNA was extracted from cultured cells using Trizol , and reversely transcribed to cDNA. Next, the q-PCR assays were performed using TB Green  with a q-PCR machine  according to manufacturer's protocol. The relative expression of mRNA was calculated by 2Cells were plated in 6-well plates at a density of 1000 cells per well. The DMEM containing 10% FBS was changed every 3 days. After culture for 2 weeks, colonies were fixed with 4% methanol and stained with Jimsa dye for 20 min. Clones were counted directly with the naked eye or under a microscope (low magnification).3 cells per well in 96-well plates and incubated at 37°C. Proliferation rates were determined at 24, 48, 72 and 96h, and the absorbance at 450nm was performed using the CloneSelect Imager System (Genetix).Cell proliferation was measured with the Cell Counting Kit-8 (CCK-8)  according to the manufacturer's recommendation. Cells were plated at a density of 2×10Cells were seeded in 6-well plates and cultured until confluent. A 200μl pipette tip was used to make a straight scratch simulating a wound. Then, cells were cultured in DMEM containing 10% FBS. The wound width was observed under a microscope  and measured by ImageJ software at 0, 24, 48 and 72h after scratching to calculate the rate of closure of the open wounds.5 cells were suspended in serum-free medium and seeded in the upper chambers, then incubated at 37°C for 36h. The lower chambers were filled with 600 μl DMEM containing 20% FBS as chemoattractant. Then, the non-migrated cells on the upper surface of the filters were removed using cotton wool swabs. The migrated cells on the lower side of the membrane were fixed in 4% methanol and stained with Jimsa dye, washed with PBS for 3 times, air-dried, then counted under a microscope .Invasion assays were performed using transwell chambers  coated with Marigel . In brief, 1×10The sections were incubated with 3% hydrogen peroxide for 10 minutes at room temperature to block endogenous peroxidase activity. To block non-specific binding of antibodies, slides were incubated with 5% normal goat serum in PBS containing 0.1% Tween 20 (PBST) for 1 hour at room temperature. Slides were then incubated with primary antibodies against SPATS2 at 4 ° C overnight. After washing, slides were incubated with HRP-labeled secondary antibody. Before counterstaining with hematoxylin, signals were developed with DAB solution. The intensity of immunohistochemical staining was analyzed by ImageJ software.2. Cox multivariate regression analysis was used to determine the independent prognostic factors of HCC. P-value<0.05 was considered statistically significant.Statistical analyses were performed using SPSS V20 and GraphPad Prism V8 statistical software. All data were presented as mean ± SEM based on at least three independent experiments. The experimental data were analyzed by Student's t-test (two-sided). Kaplan-Meier method was used to evaluate the cumulative survival, and the significance of the differences was using the log-rank test. The correlation of SPATS2 expression and clinical characteristics were analyzed by Pearson χTo determine the function of SPATS2, we profiled its transcript level in a panel of HCC cell lines. The transcript level was observed to be higher in HCC cell lines than that in L02 Figure A. Then, Since SPATS2 has been implicated in cancer-related processes, to further explore the underlying mechanism, we focused on STAT3 and its downstream pathways. Q-PCR was used to reveal that the transcriptional levels of STAT3-supporting cancer cell-intrinsic hallmarks, including BCL2, MMP9, HIF-1α and PIM1, were all decreased with SPATS2 knockdown in both MHCC-97H and HCC-LM3 cell lines , which determined RNA fate from synthesis to decay. We searched Gene section in NCBI's databases for candidate interacting genes of SPATS2. Among these genes, we found a strong positive linear correlation between TRIM44 and SPATS2 by Pearson correlation coefficient  and UCSC Xena platform (https://xena.ucsc.edu/)To clarify the role of SPATS2 in HCC, we explored its expression profile and clinical relevance based on the well-established TCGA database using GEPIA online tool  proteins control a plethora of human malignancy processesTRIM44 plays a vital role in tumorigenesis and could serve as a prognostic biomarker and therapeutic target. In HCC, previous study showed that TRIM44 could strengthen resistance of HCC cells to doxorubicin by activating NF-κBIn addition, the in-vivo data provided support for positive correlation with SPATS2 and tumor growth. Consistently, the protein levels of TRIM44, p-STAT3, BCL2, MMP9, HIF-1α and PIM1 were all decreased in SPATS2 knockdown tumor tissues, which underpinned the contribution of SPATS2-TRIM44-p-STAT3 axis to tumorigenic events in HCC.In this study, we analyzed SPATS2 gene expression in LIHC from the TCGA database. Significantly, SPATS2 was higher in tumor tissue than in normal tissue. Next, the above findings were consistent with our own cohort, which confirmed that high SPATS2 protein level is indicative of an unfavorable clinicopathological feature and poor prognosis and could serve as an independent risk factor for both OS and DFS of 112 HCC patients.Taken together, we highlight the mechanism of SPATS2 in HCC and further investigations suggest the involvement of TRIM44-p-STAT3 signaling pathway mediated by SPATS2. The present study help elucidate the significance of SPATS2-TRIM44-p-STAT3 in HCC and provide a new theoretical basis for targeted therapy.