Colorectal tumors are hierarchically organized and governed by populations of self-renewing cancer stem cells, representing one of the deadliest types of cancers worldwide. Emergence of cancer stemness phenotype depends on epigenetic reprogramming, associated with profound transcriptional changes. As described for pluripotent reprogramming, epigenetic modifiers play a key role in cancer stem cells by establishing embryonic stem-like transcriptional programs, thus impacting the balance between self-renewal and differentiation. We identified overexpression of histone methyltransferase G9a as a risk factor for colorectal cancer, associated with shorter relapse-free survival. Moreover, using human transformed pluripotent cells as a surrogate model for cancer stem cells, we observed that G9a activity is essential for the maintenance of embryonic-like transcriptional signature promoting self-renewal, tumorigenicity, and undifferentiated state. Such a role was also applicable to colorectal cancer, where inhibitors of G9a histone methyltransferase function induced intestinal differentiation while restricting tumor-initiating activity in patient-derived colorectal tumor samples. Finally, by integrating transcriptome profiling with G9a/H3K9me2 loci co-occupancy, we identified the canonical Wnt pathway, epithelial-to-mesenchyme transition, and extracellular matrix organization as potential targets of such a chromatin regulation mechanism in colorectal cancer stem cells. Overall, our findings provide novel insights on the role of G9a as a driver of cancer stem cell phenotype, promoting self-renewal, tumorigenicity, and undifferentiated state. Colorectal cancer (CRC) is accountable for over 860,000 deaths per year worldwide . AdvanceCompelling evidence indicates that cancer stemness is closely related to pluripotency gene expression signatures in human tumors and such dysfunctional embryonic-like transcriptional programs strongly correlate with aggressiveness, metastasis, and fatality –8. This Here, we investigated the impact of inhibiting G9a expression and H3K9me2 deposition on pluripotent-like neoplastic stemness programs. Using human primary CRC tissues and a robust transformed pluripotent stem cell model, we established a fundamental relationship between G9a/H3K9me2 and CSC-related transcriptional networks mediating self-renewal and differentiation. Moreover, pharmacological inhibition of H3K9me2 deposition significantly reduced tumor-initiating activity in patient-derived CRC stem cells, emphasizing the importance of G9a as a therapeutic target to suppress CSC activity .p = 0.0036) and DNMT3A  overexpression to be significantly associated with shorter RFS in colon adenocarcinomas (COAD)   were found within the cluster of high G9a/LSD1-expressing patient samples  compared to LSD1 inhibition (GSK-LSD1: 22.1μM)  used as a positive control in this experiment. Thus, G9a expression could contribute to the elevated risk of relapse by driving tumor stemness and establishing pluripotent-like transcriptional programs in human CRC.We sought to determine which epigenetic pathways are prominently associated with therapeutic failure and relapse in CRC. Relapse-free survival (RFS) analysis in various cohorts determined the risk factors associated with 13 key chromatin regulators across different cancer types. We identified EHMT2/G9a (Hazard ratio (HR): 2.2, AD) Fig. . In cont28) Fig. . The exp28) Fig. . Global 28) Fig. , as well28) Fig. . Cluster28) Fig. . Consideles Fig.  24, 25], 25p = 0les Fig. . This suμM) Fig. . G9a inhNext, we studied the role of G9a in the context of neoplastic stemness using a transformed variant of H9 human ES cells (t-hESC), as a surrogate model for CSC in adherent cultures , 27. t-hLong Interspersed Elements (LINE) and endogenous retroviruses (hERVs) [Thus, we conducted methylated DNA immunoprecipitation (MeDIP) experiments to further study the impact of G9a inhibition on CSC methylome. Given the drastic changes in 5meC levels observed in t-hESCs upon G9a inhibition Fig. , we focu (hERVs) . Inhibit (hERVs) . MeDIP a (hERVs) . Further (hERVs) . In addi (hERVs) . Accordi50 normal/EC50 cancer)  to recover for 24 h and subsequently assessed whether increased H3K9me2 deposition can modify normal intestinal progenitor growth rate vs. controls (H3K9me2Low). We observed that H3K9me2High HIEC retained substantially slower growth rates compared to H3K9me2Low cells at 72 h post-plating  Fig. . Accordier) Fig. . While Ger) Fig. . We let ing Fig. , while Ging Fig. . Altoget+/CD44+ cells, comparable to the CSC-like HT29 cell line .. Small molecule inhibitors were purchased from Tocris Bioscience.Human colon carcinoma tissue microarray sections, including normal colon tissues  and fixed cells were stained using anti-H3K9me2 primary antibody  over control (right) testicles.Next-generation RNA sequencing was performed at the StemCore facility of the Ottawa Hospital Research Institute (OHRI) on an Illumina NextSeq 500 platform. Quantitative PCR analysis were performed using an ABI 7500 Real-Time PCR System. Primer sequences used in this study are presented in Table-SRNA-seq alignment and specialized bioinformatics analyses were performed with the support of the Ottawa Bioinformatics Core Facility. Data were analyzed using R. For RNA-seq data, see GSE154057.Extracellular staining was performed using anti-CD133-PE and anti-CD44-APC , BIX-01294 (1 μM), or vehicle control (DMSO) for 7 days, followed by a 7-day drug-free incubation period. For secondary passages, primary organoids were dissociated, re-plated as described above, and grown for 14 days with no further drug treatment.P values < 0.05 were considered significant. “n” denotes the number of times the data were replicated.Data are presented as mean ± SEM. Supplementary files sectionFigure S1Figure S2Figure S3Figure S4Table-S1Table-S2Table-S3Table-S4Table-S5Table-S6Table-S7Table-S8Table-S9Table-S10


There are several challenges to its clinical application, including their paucity, impaired suppressive activity, and instability in vivo. Herein, we report that IL-27 pre-stimulation enhances suppressive functions of both mouse and human Tregs. In a complete MHC mismatched murine bone marrow transplant model, IL-27 pre-stimulated polyclonal iTregs diminish acute (a)GvHD lethality, while preserving the GvL effect. Allo-antigen specificity further improves suppressive functions when combined with IL-27 pre-stimulation. In a xenogeneic (human to mouse) GvHD model, IL-27 pre-stimulated human iTregs are superior in protecting recipients from GvHD. Lastly, we compared gene expression profiles of circulating Tregs isolated from HCT recipients with and without aGvHD and found that Tregs from aGvHD patients express distinct gene signatures enriched in immune activation and inflammation. Therefore, these results highlight a novel function of IL-27 in enforcing Treg functions to prevent aGvHD mediated lethality, proposing the hypothesis that dysregulated Treg functions may account for the potential mechanisms underlying GvHD development.Graft-versus-host disease (GvHD) remains a significant complication of allogeneic hematopoietic cell transplantation (HCT), associated with significant morbidity and mortality. GvHD is characterized by dysregulated immune responses and resulting tissue damage of target organs. Recent investigations have focused on Foxp3 GvHD neration , 5. Durisly lost . While Tsly lost , 8. tTresly lost , 8, altheveloped , 10; howeveloped . iTregs eveloped –14, and eveloped . Howevereveloped –18, thus−CD4 Tr1 cells  is a heterodimeric IL-12 family cytokine composed of the p28 and Ebi3 subunits, with pro- and anti-inflammatory properties that is strikingly diverse in supporting Th1 differentiation, inhibiting Th17 and Th2 responses, and inducing IL-10 production in Foxp3r1 cells –22. The r1 cells , 24. In r1 cells . IL-27 p cancers –29. ThesHerein, we sought to test whether IL-27-stimulation enhances therapeutic efficacy of Tregs in aGvHD using complete MHC-mismatched allogeneic (B6 to BALB/c) and xenogeneic (x)GvHD (human to mouse) models.d), C57BL/6 , Thy1.1 C57BL/6, C57BL/6 Il27ra−/−, NOD-SCID Il2rg−/− (NSG) mice were purchased from the Jackson Laboratory. Foxp3GFP reporter mice (BALB/c (H-2ter mice  obtained+CD4+CD25highCD127low/−) or naive Tcon (CD3+CD4+CD25−CD127+) were sorted using a FACSAria II (BD Biosciences) and the purity was consistently above 99%.Blood was collected from 4 patients who underwent HCT and developed GvHD (GP), 4 HCT recipients who did not develop GvHD by day 100 (NP), and normal healthy stem cell donors (DN) at the Taussig Cancer Center upon informed consent approved by Cleveland Clinic Institutional Review Board. Patients, diseases, and transplant characteristics of patients are detailed in the 6 T-cell-depleted bone marrow (TCD-BM) cells isolated from C57BL/6 mice and 0.7 × 106 MACS-purified CD3+ T cells from Thy1.1 mice. Sorted Tregs (from CD45.1+ Foxp3GFP mice) were stimulated with plate-bound anti-CD3 , soluble anti-CD28 , IL-2 (100 U/ml) in the presence or absence of rIL-27  for 24 h prior to transfer into BALB/c recipients on the same day of GvHD induction. Survival was monitored for 60 days. GvHD severity was assessed twice a week by scoring system that incorporates five clinical parameters: weight loss, posture (hunching), activity, fur texture, and skin integrity as previously reported  from a Cesium-137 irradiator, split into 2 doses in a 3 h interval to minimize gastrointestinal (GI) toxicity and were intravenously transferred with 6 × 10reported . Each pa4 luciferase-expressing A20 lymphoma cells were intravenously injected together with the donor graft as indicated. Tumor growth was measured on day 7 and 14 post-transfer using an IVIS SpectrumCT in vivo imaging system as previously described (For graft-versus-leukemia (GvL) experiments, 2 x 10escribed . For allex vivo with PMA  and ionomycin  for 4 h in the presence of 2 μM monensin  during the last 2 h of stimulation. The cells were immediately fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin buffer, and stained with fluorescence-conjugated Abs, including anti–IFN-γ (clone XMG1.2), anti–TNFα (clone TN3-19). All the antibodies used were purchased from BD Biosciences  or eBioscience . Stained cells were acquired using BD LSRII (BD Biosciences) and analyzed using FlowJo . Serum cytokine levels of recipient mice were quantified using a cytometric bead assay (CBA) according to the manufacturer's instructions (BD Biosciences).Single cell suspensions were prepared from recipient spleen and liver and stained with the following fluorescence-conjugated antibodies: anti-CD45.1 (clone A20), anti-CD4 (clone RM4-5), anti-CD8 (clone 53-6.7), anti-Lag3 (clone C9B7W), anti-Nrp1 (clone 3E12), anti-CD39 (clone 24DMS1), anti-CD73 (clone TY/23), anti-IL-27Rα (clone 2918), anti-CD27 (clone LG.7F9), anti-ICOS (clone C398.4A), and anti-CCR8 (clone SA214G2). Human cells were stained with the following fluorescence-conjugated antibodies: anti-CD3 (clone UTHC1), anti-CD4 (clone SK3), anti-CD25 (clone M-A251), anti-CD127 (clone HIL-7R-M21), anti-Lag3 (clone 3DS223H), anti-LAP (clone FNLAP), anti GARP (clone G14D9), anti GITR (clone AITR), and anti-ICOS (clone ISA-3). For intracellular cytokine staining, cells were stimulated +Foxp3GFP−CD44low) were sorted from lymph nodes of Foxp3GFP mice. For polyclonal iTreg generation, naive CD4 T cells were cultured with immobilized anti-CD3 , soluble anti-CD28 , rhIL-2 (100 U/ml)  and rhTGFβ1  for 3 days. For allogeneic iTreg generation, the sorted naive CD4 T cells were co-cultured with irradiated T cell-depleted splenocytes from BALB/c mice (2:1 ratio), rhIL-2 (100 U/ml), rhTGFβ1 (5 ng/ml), anti-CD3 (0.1 μg/ml) for 5 days (Naive CD4 T cells (CD4r 5 days . Tregs w+CD4+CD25−CD127+) were plated under iTreg differentiation conditions consisting of plate-bound anti-CD3 , soluble anti-CD28 , rhIL-2 (100 IU/ml), and TGF-β1 (5 ng/ml). Cells were incubated for 5 days and then rested for 24 h. iTregs were restimulated with anti-CD3/CD28 in the presence of rhIL-2 (100 IU/ml) and rhIL-27  for 3 days before used for suppression assay or transferred into xGvHD mice. In some experiments, Treg-associated molecules were determined by flow cytometry.Sorted naive CD4 T cells  NSG mice were given 10 × 10in vitro murine suppression assay, CFSE-labeled responder CD4 T cells (5 × 104/well) were mixed with Tregs at ratios 1:1–1:32 (Treg: responder) in the presence of irradiated T-cell-depleted splenocytes (5 × 104/well) and soluble anti-CD3 (1 μg/ml). For the in vitro human suppression assay, CFSE-labeled responder CD4 T cells (5 × 104/well) were mixed with human iTregs at various ratios in the presence of irradiated PBMCs (10 × 104/well) and soluble anti-CD3 . CFSE dilution was analyzed after 72 h by flow cytometry. For in vivo suppression assay, B6 mice were irradiated (450 rad) and then intravenously injected with CFSE-labeled naive CD4 T cells and Tregs (0.5 × 106 each). Spleen were harvested 14 days after cell infusion and analyzed for CFSE dilution by flow cytometry.Freshly sorted naive responder CD4 T cells were labeled with 1.25 μM CFSE (Molecular Probe) for 8 min at room temperature and then washed twice with complete RPMI to remove unbound dyes. For Recipient colons were harvested, fixed in solution containing 60% methanol, 10% acetic acid, 30% PBS, and embedded into paraffin. Cross sections were stained with H&E at the Imaging Core of the Lerner Research Institute. Images were acquired at 40X magnification.+CD4+CD25highCD127low/−) collected from 3 cohorts  were extracted by Zymo RNA kit. cDNA was produced using SuperScript VILO Master Mix (Invitrogen). Next, Multiple Target Enrichment (MTE) preamplification was carried out for 8 cycles using specific primers as recommended by manufacturer. Samples were hybridized to the human PanCancer Immune Profiling Panel at 65°C overnight, then processed on a GEN2 analysis system using the high sensitivity protocol and high resolution data capture. Data were analyzed by nSolver version 4.0 software. Background counts were removed by subtracting the geomean plus two standard deviations of the negative controls. Binding intensity was corrected using the geometric mean of the positive controls. Gene expression was normalized to the mean of three reference genes  which were expressed at stable levels in all samples. Volcano plots, Heatmaps, and Venn diagrams were analyzed using R . Functional gene enrichment was performed using the database for annotation, visualization and integrated discovery (DAVID) bioinformatics.For measurements of gene expression by Nanostring, a total of 6,000 FACS-sorted human Tregs . Log-rank (Mantel-Cox) test were used for survival curve. Pairs were compared using unpaired, 2-tailed Student's in vitro substantially enhanced suppressive function of thymus-derived Tregs (tTregs) both in vitro and in vivo (+ T cells (GvHD group) developed severe aGvHD and all recipients succumbed to death within 30 days of the reconstitution (We previously reported that pre-stimulating Tregs with recombinant IL-27 (rIL-27)  in vivo . We thustitution . Co-trantitution . By conttitution . The prolowFoxp3GFP−) CD4 T cells isolated from Foxp3GFP mice were stimulated with anti-CD3/CD28 mAbs in the presence of TGFβ1 and IL-2 for 3 days. Greater than eighty five percent of the cells expressed GFP (data not shown). The cells were then restimulated with the mAbs and various concentrations of rIL-27 for additional 48 h. IL-27 did not significantly alter Foxp3 expression in vitro ; however, median survival time was similar to that of control iTreg recipients   . This wactively)  and becactively) , which ictively) . Moreovectively) . Likewisctively) .Allogeneic donor T cells, in spite of causing GvHD, eradicate hematopoietic malignancies such as leukemia, resulting in clinical remissions and cures. It is important to ensure that suppressing GvHD does not affect graft-versus-leukemia (GvL) activity. Lymphoma A20 cells transduced with luciferase were transferred into BALB/c recipients undergoing transplantation as previously described . Tumor g+ “allogeneic” iTregs were FACS sorted. Sorted cells were then re-stimulated with anti-CD3/CD28 mAbs in the presence or absence of rIL-27 prior to transfer into recipients induced for aGvHD. As anticipated, recipients of control allogeneic iTregs were effectively protected from aGvHD, and had significantly lower GvHD score  mice were transferred with CFSE-labeled Thy1.1 naive CD4 T cells with or without iTregs (CD45.1). Indeed, donor CD4 T cell proliferation was markedly reduced by control iTreg cotransfer, and the reduction was more pronounced when rIL-27 pre-stimulated iTregs were used  mice. To compare Treg functions to prevent xGvHD, we transferred PBMCs obtained from a single donor into 3 NSG recipients, one of which were left untreated and two of which were transferred with control or IL-27 pre-treated iTregs. iTregs used were generated from naïve CD4 T cells of third-party donors that have at least 6/10 HLA matching. Without iTreg transfer, all the xGvHD recipients succumbed to death (p = 0.8465 vs. GvHD group). When IL-27 pre-treated iTregs were used, the mean survival kinetics was not different from the first two groups ; however, ~30% mice survived until day 100 post-BMT without any noticeable GvHD symptoms  were sorted and examined for gene expression using a Nanostring analysis (the human PanCancer Immune Profiling Panel). Nanostring gene analysis uncovered 166 genes with at least 2-fold significant differences in the GP group compared to the DN group, in contrast to 63 genes which were different in Tregs from the NP group (n = 57) in the NP group overlapped with the GP group, however, 109 unique genes were found only in the GP, and not in the NP group , GvHD patients (GP) who developed aGvHD after HCT, and non-GvHD patients (NP) who underwent HCT but did not develop GvHD symptoms by day 100 . Human TNP group . Most ofNP group . PathwayNP group . Therefoin vitro-generated, acquire highly suppressive capacity to prevent the development of aGvHD following IL-27 pre-stimulation prior to transfer. We compared the therapeutic potential of thymus-derived tTregs and of in vitro generated iTregs. While we confirmed that tTregs are efficient in mitigating aGvHD-associated mortality; we also found that IL-27 pre-stimulated in vitro-generated Foxp3+ Tregs with enriched allogeneic Ag specificity may substantially improve the therapeutic efficacy of iTregs in preventing aGvHD. Unlike thymus-derived tTreg subsets, in vitro-generated iTregs are generally considered ineffective in protecting mice from aGvHD  does instead enhance suppressive functions, tissue trafficking, and early survival of those Tregs in aGvHD. The molecular mechanisms underlying IL-27 effects on Tregs remain to be determined.The mechanisms by which IL-27 enhances Treg functions to mitigate GvHD development remain to be determined. Drobyski et al. previously reported that inhibiting the IL-27 signaling pathway via anti-IL-27p28 Ab treatment or genetic deletion of the IL-27R results in reduced GvHD development, and the reduction is mostly attributed to enhanced Treg reconstitution/generation . It is ig et al. . Of noteg et al. –27. In sg et al. . HoweverThe cellular mediators accounting for IL-27-dependent Treg suppression need to be tested in the future investigation. IL-10 produced by IL-27-stimulated conventional CD4 T cells is considered a key cytokine involved in IL-27-dependent immune regulation , 47, 48.Functionally dysregulated Tregs are thought to be associated with chronic inflammation, infection, and tumors –52. Geneclinicaltrials.gov). There have been attempts to boost Treg expansion/generation. For example, IL-2 infusion may enhance Treg expansion for the prevention and treatment of GVHD)  , 59. Tari et al. ]. Our reThe datasets generated for this study are available on request to the corresponding author.The studies involving human participants were reviewed and approved by Cleveland Clinic IRB. The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by Cleveland Clinic IACUC.HL designed and performed most of the experiments, analyzed the data, and wrote the manuscript. KK and QN helped with experiments. BB helped with the setup and critical reading of the manuscript. BM and BH designed the experiments, analyzed the data, and wrote the manuscript.BB receives remuneration as an advisor to Kamon Pharmaceuticals, Inc., Five Prime Therapeutics Inc., Regeneron Pharmaceuticals, Magenta Therapeutics, and BlueRock Therapeuetics; research support from Fate Therapeutics, RXi Pharmaceuticals, Alpine Immune Sciences, Inc., Abbvie Inc., Leukemia and Lymphoma Society, Childrens' Cancer Research Fund, KidsFirst Fund and is a co-founder of Tmunity. The remaining 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.


Calcium and vitamin D analogs for treating ADH1 predispose to nephrocalcinosis and renal impairment3. CaSR antagonists, known as calcilytics, represent a possible treatment2, but are clinically unavailable. However, recombinant PTH(1-34) may increase serum calcium without causing hypercalciuria in patients with forms of hypoparathyroidism5. We assessed the effectiveness of continuous subcutaneous PTH(1-34) infusion (CSPI) in a retrospective cohort of six ADH1 patients (aged 5 weeks-22 years), who were selected as they experienced hypocalcemic seizures despite using calcium and vitamin D analogs, and/or bolus PTH injections  in 5 patients, and increased mean serum magnesium by 0.09mmol/L  in 4 patients  is caused by gain-of- function calcium-sensing receptor (CaSR) mutationsjections . CalciumNPS-2143 . CSPI trNPS-2143 . This wapatients . CSPI repatients . Seriouspatients . All CSPSupplementary Appendix


Combinational use of these host-based antiviral agents with virus-based antivirals to target different processes of the SARS-CoV-2 replication cycle should be evaluated in animal models and/or clinical trials.The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) signals an urgent need for an expansion in treatment options. In this study, we investigated the anti-SARS-CoV-2 activities of 22 antiviral agents with known broad-spectrum antiviral activities against coronaviruses and/or other viruses. They were first evaluated in our primary screening in VeroE6 cells and then the most potent anti-SARS-CoV-2 antiviral agents were further evaluated using viral antigen expression, viral load reduction, and plaque reduction assays. In addition to remdesivir, lopinavir, and chloroquine, our primary screening additionally identified types I and II recombinant interferons, 25-hydroxycholesterol, and AM580 as the most potent anti-SARS-CoV-2 agents among the 22 antiviral agents. Betaferon (interferon-β1b) exhibited the most potent anti-SARS-CoV-2 activity in viral antigen expression, viral load reduction, and plaque reduction assays among the recombinant interferons. The lipogenesis modulators 25-hydroxycholesterol and AM580 exhibited EC Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel betacoronavirus that was first identified in patients with unexplained pneumonia in Wuhan, Hubei, China in December 2020 ,2. WithiEffective antivirals are essential for improving the clinical outcome of patients with severe COVID-19. As de novo development of novel antiviral agents would take years and inevitably lag behind the rapid expansion of the pandemic, repurposing of existing antiviral agents has been exploited to identify immediately available treatment options for COVID-19. Among existing antiviral agents, the most likely agents that may be active against SARS-CoV-2 would be those with known broad-spectrum antiviral activities and those with reported activities against coronaviruses. A number of such antiviral agents, such as remdesivir, lopinavir, chloroquine, and hydroxychloroquine, have been recently reported to have anti-SARS-CoV-2 activity in vitro ,9,10,11.® CRL-1586™) cells were purchased from ATCC  and maintained in Dulbecco’s modified eagle medium  culture medium supplemented with 10% heat-inactivated FBS , 50 U/mL penicillin, and 50 µg/mL streptomycin as previously described [SARS-CoV-2 HKU-001a (GenBank accession number: MT230904) was isolated from the nasopharyngeal aspirate specimen of a laboratory-confirmed COVID-19 patient in Hong Kong . The virescribed . All expTwenty-two antiviral agents with reported activities against coronaviruses and/or other viruses were included in the study. In the primary screening, 10,000 IU/mL of each recombinant IFN or 20 µM of each of the other antiviral agents was used to treat SARS-CoV-2-infected (MOI = 0.001) VeroE6 cells for 72 h. The viral loads in the cell culture supernatants were then determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) as previously described . The cut4 cells/well) were incubated with different concentrations of the individual compound for 48 h, followed by the addition of substrate and measurement of luminance 10 min later. The 50% cytotoxic concentrations (CC50) of the antiviral agents were calculated by SigmaPlot  in an Excel add-in ED50V10.The CellTiterGlo luminescent assay  was performed to detect the cytotoxicity of the selected antiviral agents as previously described . BrieflyViral load reduction assay was performed as described previously with modifications . BrieflyViral N antigen expression in SARS-CoV-2-infected VeroE6 cells was performed as we described previously using immunofluorescent staining with an in-house rabbit antiserum against SARS-CoV-2-N protein as previously described . Cell nu50) as we previously described with slight modifications [5 cells/well in 24-well tissue culture plates on the day before carrying out the assay. After 24 h of incubation, 50 plaque-forming units (PFU) of SARS-CoV-2 were added to the cell monolayer with or without the addition of antiviral agents and the plates were further incubated for 1 h at 37 °C in 5% CO2 before removal of unbound viral particles by aspiration of the media and washing once with DMEM. Monolayers were then overlaid with media containing 1% low melting agarose  in DMEM and appropriate concentrations of individual compound, inverted and incubated as above for another 72 h. The wells were then fixed with 10% formaldehyde  overnight. After removal of the agarose plugs, the monolayers were stained with 0.7% crystal violet  and the plaques counted. The percentage of plaque inhibition relative to the control  wells were determined for each antiviral agent concentration. EC50 was calculated using a sigma plot (SPSS) in an Excel add-in ED50V10. The plaque reduction assay experiments were performed in triplicate and repeated twice for confirmation.Plaque reduction assay was performed to plot the 50% maximal effective concentration (ECications . Briefly5 cells/well). The cells were inoculated with SARS-CoV-2  and then incubated for 1 h for virus internalization at 37 °C. DMSO (0.5%) was included as a negative control. The viral loads in the culture supernatants normalized by DMSO at the different phases of the assay were determined by qRT-PCR.Time-of-drug-addition assay was performed for selected compounds as previously described with slight modifications . BrieflyA total of 22 antiviral agents with reported activity against coronaviruses and/or other viruses were included in the primary screening. These included types I  and II (Immukin) IFNs, nucleoside analogues , 4-aminoquinoline (chloroquine), anthracycline (idarubicin), antihistamine (chlorcyclizine), calcineurin inhibitor (cyclosporine), flavonoid (silibinin), kinase inhibitors (erlotinib and everolimus), macrolide (azithromycin), oxysterol (25-hydroxycholesterol), polymerase inhibitor (filibuvir), protease inhibitors (lopinavir and rupintrivir), and retinoic acid receptor agonist (AM580) . In thisBased on the results from the primary screening, Avonex (IFN-β1a), Rebif (IFN-β1a), Betaferon (IFN-β1b), Pegasys (pegylated IFN-α2a), Immukin (IFN-γ1b), AM580, and 25-hydroxycholesterol were further evaluated in the SARS-CoV-2 N antigen expression assay. As shown in p < 0.05) reduction in viral load of >50% as compared to the baseline in cell culture supernatants inoculated with each of the eight antiviral agents. As controls, remdesivir and lopinavir achieved 8.3 log10 copies/mL and 3.2 log10 copies/mL reduction in viral RNA load, respectively, at a concentration of 40 µM. In comparison, 25-hydroxychloestero and AM580 achieved 4.4 log10 copies/mL and 4.2 log10 copies/mL reduction in viral RNA load, respectively, at the same antiviral agent concentration. Consistent with the primary screening results, IFN-β1a IFN-β1b demonstrated more potent anti-SARS-CoV-2 activity than IFN-α2a and IFN-γ1b in the viral load reduction assay. At a concentration of 3000 IU/mL, Avonex (IFN-β1a), Rebif (IFN-β1a), and Betaferon (IFN-β1b) respectively achieved 3.1 log10 copies/mL, 2.8 log10 copies/mL, and 3.0 log10 copies/mL reduction in viral RNA load, whereas Pegasys (pegylated IFN-α2a) and Immukin (IFN-γ1b) achieved only 1.6 log10 copies/mL and 1.5 log10 copies/mL reduction in viral RNA load. At a higher concentration of 30,000 IU/mL, Pegasys and Immukin achieved modestly higher reduction in viral RNA load .To quantify the anti-SARS-CoV-2 activity of the identified antiviral agents in the primary screening and the viral N antigen expression assay, SARS-CoV-2 viral load reduction assay by qRT-PCR was conducted to determine the SARS-CoV-2 RNA copies released in the cell culture supernatant with or without antiviral agent treatment. As show in 50 of the antiviral agents were determined (50 (31.2 IU/mL) and highest selectivity index (>1602.6). Avonex and Rebif (IFN-β1a) also demonstrated comparable EC50 (109.6 and 70.8 IU/mL) and high selectivity indices (>456.2 and >706.2). 25-hydroxycholesterol (4.2 µM) and AM580 (7.6 µM) both exhibited EC50 at low micromolar levels with selectivity indices >10.0.In addition to reduction in viral N antigen expression and RNA load, inhibition of infectious SARS-CoV-2 particles was evaluated using plaque reduction assay . Among ttermined . Among tTo explore the mode of action of the two non-IFN antiviral agents, a time-of-drug-addition assay was performed. As shown in In this study, we evaluated the anti-SARS-CoV-2 activity of antiviral agents with broad-spectrum antiviral activities against coronaviruses and/or other viruses. In our primary screening using a fixed antiviral agent concentration and virus inoculum, we identified recombinant IFNs and lipogenesis modulators to be the most potent anti-SARS-CoV-2 agents among 22 broad-spectrum antivirals. These findings have important implications for the choice of clinically available recombinant IFNs to be used in COVID-19 patients and development of lipogenesis modulators as potential anti-SARS-CoV-2 therapeutics.50 of 31.2 IU/mL and the highest selectivity index of >1602.6. Importantly, the EC50 of Betaferon against SARS-CoV is below its achievable peak serum concentration (Cmax) with standard subcutaneous dosing of 16 million units of Betaferon (40 IU/mL). Notably, Betaferon was similarly found to be the most potent recombinant IFN for the highly virulent MERS-CoV and significantly improved the clinical, virological, and histopathological parameters of MERS-CoV-infected common marmosets [IFNs are glycoproteins with strong antiviral activities that represent one of the first lines of host immune response against invading pathogens . These parmosets ,42. In Sarmosets ,58,59. Aarmosets . This trarmosets .In addition to the recombinant IFNs, we also identified antiviral agents that target the host lipogenesis pathways as potential anti-SARS-CoV-2 agents. AM580 is a selective retinoic acid receptor-α agonist which was recently identified to have broad-spectrum antiviral activities against various families of DNA and RNA viruses, including Coronaviridae, Flaviviridae, Orthomyxoviridae, Picornaviridae, and Adenoviridae . AM580 iThe oxysterol 25-hydroxycholesterol is a metabolite of cholesterol that is produced and secreted by macrophages and has multiple effects on lipid metabolism, especially lipid biosynthesis and immunity . 25-hydr50 among the 22 broad-spectrum antivirals. This might have overlooked antiviral agents that can inhibit SARS-CoV-2 at higher concentrations. For example, favipiravir has been shown to inhibit SARS-CoV-2 replication with an EC50 of 67 µM [50 of 57.7 µM [Our study had limitations. First, we used a fixed antiviral agent concentration in our primary screening in order to identify the antiviral agents with the lowest ECof 67 µM . Similar 57.7 µM . Second, 57.7 µM . Additio


In both eor-2(ju190) and eor-1(ju198) mutants, Punc-25GFP expression was almost completely abolished in RMED/V cells, whereas RMEL/R cells and the D neurons showed normal morphology  animals lost Punc-25GFP expression in RMED and 67% in RMEV (N=100). ju198 behaves as a partial loss of function mutation because 100% and 94% of eor-1(cs28) animals do not express Punc-25GFP expression in RMED and RMEV, respectively (N=100) . eor-2(ju190)animals also displayed mild Unc, low penetrant Egl and rod-like lethality. The loss of Punc-25GFP expression in eor-2(ju190) and eor-1(ju198) could be due to cell fate alterations or cell death. To distinguish between these possibilities, we first examined the cell body positions of RMED and RMEV cells under Nomarski microscope . In both eor-2(ju190) and eor-1(ju198) mutants, the RMED and RMEV cells were found in their normal locations (eor-2(ju190) and ced-3(n717), which blocks apoptosis, and found that eor-2(ju190); ced-3(n717) double mutants showed absence of Punc-25GFP expression in RMED/V, similar to eor-2(ju190) single mutants, indicating that ineor-2(ju190) and eor-1(ju198) animals, the RMED and RMEV cells are alive, but that their differentiated traits are likely altered.In a visual screen for genes that regulate the pattern of the rphology . We obseocations . We alsoavr-15GFP, which is normally expressed in both RMED and RMEV neurons  and Plim-4GFP transgenes, which is normally expressed in RMEV neuron and some other head neurons . We found that in eor-2(ju190) animals,Pavr-15GFP was not expressed in RMED/V  alters multiple differentiated aspects of RMED/V neurons.To further examine whether other properties of the RMED/V cells might be altered in these mutants, we looked at the expression of Pn RMED/V , the GFPng cells . These deor-1(ju198), juIs76; CZ2006 eor-2(ju190); juIs76.Strains are: CZ2014 unc-25GFP]juIs76juIs73[P; avr-15GFPP; lim-4GFP.PThe GFP reporters used are listed below:


Scientific Reports 10.1038/s41598-020-66093-8, published online 22 June 2020Correction to: This Article contains errors in the accompanying Supplementary Dataset, where Supplementary Tables S1 to S12 were omitted.The corrected Supplementary Dataset Supplementary Dataset 1.


HisGUG half molecules is >200-fold higher than that of the most abundant EV-microRNA (miRNA). Sequence identification of the 5′-tRNA halves using cP-RNA-seq revealed abundant and selective packaging of specific 5′-tRNA half species into EVs. The EV-5′-tRNAHisGUG half was experimentally demonstrated to be delivered into endosomes in recipient cells and to activate endosomal TLR7. Up-regulation of the 5′-tRNA half molecules was also observed in the plasma of patients infected with Mycobacterium tuberculosis. These results unveil a novel tRNA-engaged pathway in the innate immune response and assign the role of “immune activators” to 5′-tRNA half molecules.Toll-like receptors (TLRs) play a crucial role in the innate immune response. Although endosomal TLR7 recognizes single-stranded RNAs, their endogenous RNA ligands have not been fully explored. Here, we report 5′-tRNA half molecules as abundant activators of TLR7. Mycobacterial infection and accompanying surface TLR activation up-regulate the expression of 5′-tRNA half molecules in human monocyte-derived macrophages (HMDMs). The abundant accumulation of 5′-tRNA halves also occur in HMDM-secreted extracellular vehicles (EVs); the abundance of EV-5′-tRNA Although Toll-like receptors (TLRs) play a crucial role in the innate immune response, their endogenous ligands have not been fully explored. This study identifies tRNA half-molecules as abundant TLR ligands which are upregulated upon infection by mycobacteria and activate TLR7. Mycobacterium tuberculosis (Mtb), one of the greatest threats to humans, causing more than 1.2 million deaths annually  are shown in green and blue circles, respectively. (B) Indicated synthetic RNAs were synthesized by in vitro transcription, gel-purified, and analyzed with denaturing PAGE.(EPS)Click here for additional data file.S3 FigIndicated amounts of synthetic RNAs were subjected to stem-loop/TaqMan RT-qPCRs. Proportional correlations of synthetic RNA input to the Ct were observed and used as standard curves for estimation of the expression levels of respective RNAs. Ct, cycle threshold; RT-qPCR, quantitative reverse transcription PCR; tRNA, transfer tRNA.(EPS)Click here for additional data file.S4 FigCleavage sites in the tRNA anticodon loops were predicted based on the 3′-terminal positions of the 5′-tRNA halves. Anticodons are shown in green. tRNA, transfer tRNA.(EPS)Click here for additional data file.S5 Fig Florescent end-labeled, synthetic 5′-tRNAHisGUG half (A) or 5′-tRNAGluCUC half (B) was transfected into HMDMs and observed in green. Scale bar, 20 μm.  EVs produced from host HMDMs containing the labeled 5′-tRNAHisGUG half or 5′-tRNAGluCUC half were isolated and applied to recipient HMDMs. Delivery of the labeled, EV-5′-tRNAHisGUG half (C) or EV-5′-tRNAGluCUC half (D) into endosomes was observed in green. Immunofluorescence staining of TLR7 is shown in red, and DNA was counterstained with DAPI in blue. Scale bar, 100 μm. Clear co-localization of the labeled 5′-tRNA halves and TLR7 was observed. EV, extracellular vehicle; HMDM, human monocyte-derived macrophage; TLR, Toll-like receptor; tRNA, transfer tRNA.(TIF)Click here for additional data file.S6 FigHisGUG half and ssRNA40 were transfected into HMDMs. Total RNAs from the cells were subjected to RT-qPCR for the indicated mRNAs. Averages of 3 experiments with SD values are shown. HMDM, human monocyte-derived macrophage; mRNA, messenger RNA; RT-qPCR, quantitative reverse transcription PCR; SD, standard deviation; tRNA, transfer tRNA.Using RNAiMAX or Lipofectamine LTX (Thermo Fisher Scientific), the synthetic 5′-tRNA(EPS)Click here for additional data file.S7 Fig(A) The indicated amounts of the synthetic 5′-tRNAHisGUG half were transfected into HMDMs using DOTAP. Total RNAs from the cells were subjected to RT-qPCR for the indicated mRNAs. Averages of 3 experiments with SD values are shown. (B) After the RNA transfection, culture medium was subjected to ELISA for quantification of TNFα and IL-1β. DOTAP, 1,2-dioleoyloxy-3-trimethylammonium-propane; HMDM, human monocyte-derived macrophage; IL, interleukin; mRNA, messenger RNA; RT-qPCR, quantitative reverse transcription PCR; SD, standard deviation; TLR, Toll-like receptor; TNFα, tumor necrosis factor α; tRNA, transfer tRNA.(EPS)Click here for additional data file.S8 Fig(A) HMDMs were transfected with control siRNA (siControl) or siRNA targeting TLR7 (siTLR7) or TLR8 (siTLR8). To confirm the reduction of the targeted mRNA, total RNAs from the cells were subjected to RT-qPCR for TLR7 and TLR8 mRNAs (RPLP0: control). (B) Double KDs of TLR7 and TLR8 were performed by simultaneously transfecting both siTLR7 and siTLR8, and reduction of the both mRNAs was confirmed by RT-qPCR. (C) In HMDMs, the expression of both TLR7 and TLR8 was silenced by siRNAs and then DOTAP-fused 5′-tRNAHisGUG half or ssRNA40-M was transfected. Total RNAs from the cells were subjected to RT-qPCR for the indicated mRNAs. DOTAP, 1,2-dioleoyloxy-3-trimethylammonium-propane; HMDM, human monocyte-derived macrophage; KD, knockdown; mRNA, messenger RNA; RT-qPCR, quantitative reverse transcription PCR; TLR, Toll-like receptor.(EPS)Click here for additional data file.S9 Fig(A) EVs were isolated from human plasma and were analyzed by NTA. Representative size distribution profile is shown. (B) Isolated EVs were treated with RNase A and/or Triton X-100 and then subjected to TaqMan RT-qPCR for quantification of 5′-tRNA halves. Averages of 3 experiments with SD values are shown . (C) Human plasma samples (batches #2–4) were treated with RNase A and/or Triton X-100 and then subjected to TaqMan RT-qPCR for quantification of 5′-tRNA halves. Averages of 3 experiments with SD values are shown. EV, extracellular vehicle; NTA, nanoparticle tracking analysis; RT-qPCR, quantitative reverse transcription PCR; SD, standard deviation; tRNA, transfer tRNA.(EPS)Click here for additional data file.S1 MovieNTA, nanoparticle tracking analysis.(MP4)Click here for additional data file.S2 MovieEV, extracellular vehicle; NTA, nanoparticle tracking analysis.(MP4)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableRT-qPCR, quantitative reverse transcription PCR.(PDF)Click here for additional data file.S3 TableRT-qPCR, quantitative reverse transcription PCR.(PDF)Click here for additional data file.S4 TableRT-qPCR, quantitative reverse transcription PCR.(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S1 Raw Image(TIF)Click here for additional data file.S2 Raw Image(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


We tested the potential effect of ginsenoside Rh3 (GRh3) in the process. Our results show that GRh3 activated Nrf2 signaling in T-HESC cells and primary murine endometrial cells. GRh3 induced Nrf2 Ser-40 phosphorylation and Keap1-Nrf2 disassociation, causing Nrf2 protein stabilization and nuclear translocation, which led to transcription and expression of antioxidant response element-dependent genes . In T-HESC cells and primary murine endometrial cells, GRh3 potently attenuated OGDR-induced reactive oxygen species production, lipid peroxidation and mitochondrial depolarization, as well as cell viability reduction and necrosis. Activation of Nrf2 is required for GRh3-induced anti-OGDR actions in endometrial cells. Nrf2 inhibition, by Nrf2 shRNA, knockout (through CRISPR-Cas9-editing) or S40T mutation, abolished GRh3-induced endometrial cell protection against OGDR. Additionally, forced activation of Nrf2, by Keap1 knockout, mimicked and nullified GRh3-induced anti-OGDR actions in T-HESC cells. Together, we conclude that GRh3 protects endometrial cells from OGDR via activation of Nrf2 signaling.Oxygen and glucose deprivation (OGD)-reoxygenation (OGDR) induces oxidative injury to endometrial cells  It carocedure , 8, thati.e. by phosphorylation), Nrf2 will disassociate from Keap1, then allowing Nrf2 protein stabilization and cytosol accumulation [heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), γ-glutamyl cysteine ligase catalytic subunit (GCLC) [Nuclear-factor-E2-related factor 2 (Nrf2) is a well-known transcription factor, promoting the transcription of multiple antioxidant genes and detoxifying enzymes. In the resting condition, Nrf2 stays in the cytoplasm, binding to its suppressor protein Kelch-like ECH-associated protein 1 (Keap1). With the help of Cul3 ubiquitin ligase complex, Keap1 will promote ubiquitination and proteasomal degradation of inactivated Nrf2. Once activated (mulation –11. Actimulation –11. It wt (GCLC) . Activatt (GCLC) .Considering that ROS production and oxidative stress are key pathological mechanisms of clinical endometrium ischemic injury, agents that could provoke significant antioxidant response should have important therapeutic value for postpartum hemorrhage. Ginseng and its active component ginsenosides are widely-utilized and multi-functional traditional Chinese medicines (TCM) , 13. TheIn humans, Ginseng Rg5 will be metabolized into Ginseng Rh3 (GRh3). The latter shows excellent pharmacological efficiency , 19. GRhWe first tested whether GRh3 could activate Nrf2 signaling in endometrial cells. Performing the co-immunoprecipitation (“Co-IP”) assay, we show that Nrf2 associated with its suppressor protein Keap1 in the untreated T-HESC human endometrial cells . SignifiHO1, NQO1 and GCLC in T-HESC cells  reduction  and necrhuman Nrf2, were individually transfected to T-HESC cells. Following selection by puromycin two stable cell lines, namely “shNrf2-a” and “shNrf2-b”, were established. Furthermore, the lenti-CRISPR-Cas9-Nrf2-KO-GFP construct  was traAs shown, the applied Nrf2 shRNA or Nrf2-KO construct resulted in depletion of Nrf2 in T-HESC cells, even with GRh3 stimulation . FurtherTo test whether Nrf2 Ser-40 phosphorylation is the primary mechanism of Nrf2 signaling cascade activation by GRh3, the lentiviral S40T Nrf2 construct (from Dr. Jiang ) was traSignificantly, in T-HESC cells with Nrf2 S40T mutation, OGDR-induced mitochondrial depolarization  was traOur group , 8 and oHO1, NQO1 and GCLC, was significantly increased in GRh3-treated endometrial cells. Activation of Nrf2 by GRh3 potently attenuated OGDR-induced ROS production and lipid peroxidation. Significantly, Nrf2 activation is required for GRh3-induced actions in endometrial cells. Nrf2 shRNA or KO abolished GRh3-induced anti-oxidative activity and endometrial cell protection against OGDR. Furthermore, forced activation of Nrf2, by Keap1 KO, mimicked and also nullified GRh3-induced endometrial cell protection. Therefore, GRh3-induced endometrial cell protection against OGDR requires Nrf2 activation.The results of this study show that GRh3 induced significant Nrf2 signaling activation in endometrial cells. Nrf2 cascade activation was evidenced by Nrf2 Ser40 phosphorylation and Keap1-Nrf2 disassociation, as well as Nrf2 protein stabilization and nuclear translocation. Moreover, expression of Nrf2-dependent genes, Nrf2 post-transcriptional modification is one key mechanism for Nrf2 signaling cascade activation . Existinin vivo studies. Further studies will be needed to test the potential activity of GRh3 in animal models of postpartum hemorrhage. Postpartum hemorrhage is one important contributor of maternal morbidity and mortality. Current treatment options include operational and pharmacologic interventions [One weakness of the study is the lack of ventions –3. We prGRh3 protects endometrial cells from OGDR via activation of Nrf2 signaling.Puromycin and polybrene were obtained from Sigma-Aldrich Chemicals . The cell culture reagents were provided by Gibco BRL . A Nrf2 Ser-40 antibody was from Dr. Jiang , 30 at NT-HESC cells, the immortalized human endometrial cells, were cultured as described previously , 31. TheAt 3000 cells per well, T-HESC cells or the primary murine endometrial cells were seeded into 96-well plates. By using cell counting kit-8  the cell viability was measured [4 cells per well, T-HESC cells or the primary murine endometrial cells were seeded into 12-well plates. Following the applied OGDR treatment, LDH release to the medium, the quantitative measurement of cell necrosis, was tested by a two-step LDH detection kit .At 5 × 102/5% CO2) for 4h (OGD procedure). Thereafter, cells were maintained in the complete medium and re-oxygenated (OGDR) for indicated time periods.The detailed procedure of OGDR was previously described . In brie5 cells per well, T-HESC cells or the primary murine endometrial cells were seeded into six-well plates. After the applied OGDR procedure, RIPA lysis buffer  was added to obtain total cell lysates. Quantified protein lysates (40 μg per treatment of each lane) were separated by a 10% SDS-PAGE gel, then transferred to a polyvinylidene difluoride  blot [At 1.5 × 10na) blot . Westernna) blot , 33. Assna) blot .The detailed protocols were described previously , 35. AftThe protocol of Mito-IP was described previously , 8. Brie4 cells per well, T-HESC cells were seeded into six-well plates. Following the applied OGDR treatment, the mito-dye JC-1  assay was performed to test mitochondrial depolarization (“ΔΨ”), using the described protocol [At 2 × 10protocol . Under aThe endometrial cells, with or without the applied OGDR stimulation, were subjected to the superoxide colorimetric assay , examining the cellular superoxide contents. The superoxide detection reagent (50 μL/well) was added for 15 min under the dark, with the superoxide’s absorbance tested at the wavelength of 450 nm .5 cells per well, T-HESC cells were seeded into six-well plates. Following the applied OGDR treatment, total cell lysates (20 μg of each treatment) were mixed with 20% of acetic acid and TBAR solution. After heating, the mixture was centrifuged, and the red pigment dye in the supernatant was quantified [As previously described , 8, a TBantified . TBAR va5 cells per well, T-HESC cells or the primary murine endometrial cells were seeded into six-well plates. After the applied treatment, total cellular RNA was extracted by using the TRIzol reagents  [Ct−ΔΔ method was applied for the quantification of targeted mRNAs, using glyceraldehyde-3-phosphatedehydrogenase(GAPDH) as the internal control.At 1.5 × 10, China) . Using a, China) , 30, 38.As previously described , two lenAs previously described , the lenThe S40T dominant negative Nrf2 pSV2 puro Flag plasmid was provided by Dr. Jiang at Nanjing Medical University , 22, 24.The lentiviral Keap1 CRISPR-Cas9 KO construct was provided by Dr. Di from Wannan Medical College . The conp < 0.05 were considered statistically significant.Data were presented as mean ± standard deviation (SD). Analysis of variance (ANOVA) followed by Dunnett’s post hoc test for multiple comparisons (SPSS 19.0) was performed to test statistical significance. A 2-tailed unpaired T test  was utilized to test significance between two treatment groups. Values of


The nu444 allele was originally isolated from a forward genetic screen for mutants that suppressed the “Hic” (Hypersensitivity to Inhibitors of Cholinesterase) phenotype of dgk-1(nu62) mutants, which had increased acetylcholine release at the neuromuscular junction . In this screen, several genes that are important for neuropeptide secretion were recovered, including pkc-1(nu448)  and ric-7(nu447) . Sanger sequencing of the exons and exon-intron junctions of the pkc-1 locus revealed that nu444 had a nonsense mutation  in an early exon that is only present in pkc1a and pkc-1c isoforms . Thus, pkc-1(nu444) allele is probably a null allele for both pkc-1a (Q53stop) and pkc-1c (Q109stop), but presumably does not affect pkc-1b.Here, we report pkc-1(nu444) V; dgk-1 (nu62) XKP1939 pkc-1(nu444) VOJ580


Fusarium solani strain, isolated from the plant Aponogeton undulatus Roxb., was investigated. Cerevisterol (CRVS) was identified from endophytic fungi, a Fusarium solani strain, and moreover exhibited anti-inflammatory activity. However, the underlying mode of action remains poorly understood. The aim of this study is to reveal the potential mechanisms of CRVS against inflammation on a molecular level in LPS-activated RAW 264.7 peritoneal macrophage cells. CRVS was isolated from F. solani and characterized based on spectral data analysis. The MTT assay was performed to measure cell viability in CRVS-treated macrophages. Anti-inflammatory activity was assessed by measurement of nitric oxide (NO) and prostaglandin E2 (PGE2) levels, as well as the production of various cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and -6 (IL-6) in LPS-stimulated macrophages. RT-PCR and immunoblotting analyses were done to examine the expression of various inflammatory response genes. A reporter gene assay was conducted to measure the level of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and activator protein-1 (AP-1) transactivation. CRVS suppresses the LPS-induced production of NO and PGE2, which is a plausible mechanism for this effect is by reducing the expression of iNOS and COX-2. CRVS also decreases the expression of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β. CRVS halted the nuclear translocation of NF-κB by blocking the phosphorylation of inhibitory protein κBα (IκBα) and suppressing NF-κB transactivation. The mitogen-activated protein kinases (MAPK) signaling pathways are also suppressed. CRVS treatment also inhibited the transactivation of AP-1 and the phosphorylation of c-Fos. Furthermore, CRVS could induce the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) by down-regulating Kelch-like ECH-associated protein 1 (Keap-1) and up-regulating hemeoxygenases-1 (HO-1) expression. The results suggest that CRVS acts as a natural agent for treating inflammatory diseases by targeting an MAPK, NF-κB, AP-1, and Nrf2-mediated HO-1 signaling cascade.As part of our continuous effort to find potential anti-inflammatory agents from endophytic fungi, a  Inflammation is a physiological defense response of the body to tissue injury and infection caused by wounding, microbial pathogen infections, or chemical irritation . VariousVarious signaling pathways are involved in transducing the inflammatory response. Transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) regulates inducible nitric oxidase synthase (iNOS), cyclooxygenase-2 (COX-2), and other pro-inflammatory cytokines in LPS-induced macrophages by binding to their promoter regions . In restFusarium solani strain, isolated from the plant Aponogeton undulatus Roxb., was investigated. Chemical investigation of the ethyl acetate extract of the endophytic fungus led to the isolation of cerevisterol (CRVS)   along wiF. solani were described elsewhere ..20].http://www.rcsb.org). The protein structures were prepared using UCSF Chimera 1.13.1 (http://www.cgl.ucsf.edu/chimera) to remove all non-receptor atoms, including water, ions, and miscellaneous compounds. The ligand (CRVS) was prepared using ChemBio3D Ultra version 12.0 , and then an MMFF94 energy minimization was performed. AutoDock Vina  was used for the molecular docking simulations [http://www.ebi.ac.uk/thornton-srv/software/LIGPLOT).The crystal structure of the Kelch-Neh2 complex [PDB: 4L7b] was obtained from the Protein Data Bank (PDB).  was used for the analysis of data. All data are expressed as the mean ± standard deviation  and analyzed using one-way analysis of variance (ANOVA), followed by Dennett’s test. A value of 2 (PGE2) production by 10 fold and 12 fold, respectively (column 3 of 2 inhibitory activity to NS-398 (N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulfonamide), a selective cyclooxygenase 2 (COX-2) inhibitor in PGE2 generation.As shown in 2 as well as pro-inflammatory cytokines by regulating their respective genes in activated RAW 264.7 macrophages.RT-PCR analysis indicates that mRNA levels of inducible iNOS, COX-2, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) are significantly increased in LPS-stimulated RAW 264.7 cells compared with untreated cells, whereas CRVS treatment significantly lessened this trend, as shown in As shown in LPS treatment significantly increases activator protein-1 (AP-1) luciferase activity by 5 fold compared with that in untreated control cells E. TogethTo further elucidate the anti-inflammatory mechanism of CRVS, the mitogen activated protein kinases (MAPKs) , extracellular signal-regulated kinases (ERKs), and p38 mitogen-activated protein kinases (p38) phosphorylation was analyzed by Western blotting. As indicated in The anti-inflammatory effects of CRVS were also evaluated using LPS-stimulated murine peritoneal macrophages. The results show that CRVS does not exhibit cell toxicity up to a concentration of 20 µM A. As expLPS can initially increase reactive oxygen species (ROS). As shown in CRVS treatment significantly down-regulates Kelch-like ECH-associated protein 1 (Keap-1) expression by 4 fold at 10 µM in a dose-dependent manner -2-{[(1S)-1-[methyl]-3,4-dihydroisoquinolin-2(1H)-yl]carbonyl}cyclohexanecarboxylic acid) bind with Keap-1 was shown −10.0 kcal/mol. It was found to interact specifically with the P1, P3, and P5 subpocket of Keap-1 receptor , isolated from the endophytic fungal strain pression . This st


Consistently, SBF2-AS1 knockdown hindered the growth of NSCLC cell in nude mice. The following luciferase reporter gene assay and RNA immunoprecipitation (RIP) assay suggested the relationship between miR-338-3p and SBF2-AS1. The rescue experiments showed that miR-338-3p inhibitor abolished SBF2-AS1 silencing caused inhibition on the growth, migration and invasiveness of NSCLC cell. The luciferase reporter assay and immunoblotting assay validated that A Disintegrin and Metalloprotease 17 (ADAM17) was a target of miR-338-3p. In addition, SBF2-AS1 positively regulated the level of ADAM17 through sponging for miR-338-3p. Finally, we revealed that SBF2-AS1 contributed to the proliferation and metastatic phenotypes of NSCLC cell via regulating miR-338-3p/ADAM17 axis.Non-small cell lung cancer (NSCLC) is a type of refractory malignant lung cancer with a high rate of metastasis and mortality. Currently, long non-coding RNA (lncRNA) SBF2 Antisense RNA 1 (SBF2-AS1) is considered as a biomarker for a variety of tumors. However, the function of SBF2-AS1 in the growth and metastasis of NSCLC needs to be further studied. In this study, we revealed that SBF2-AS1 was overexpressed in NSCLC tissues compared with that in normal tissues. SBF2-AS1 silencing restrained the growth and aggressive phenotypes of NSCLC cell  Lung cancer is the mainly cause of cancer-related mortality worldwide. Although the great advance in the therapeutic methods, including targeted therapies and immunotherapy, the prognosis for patients with advanced non-small cell lung cancer (NSCLC) stages remain very poor. The primary reason for the poor outcomes in patients with NSCLC is the presence of systemic metastases. Hence, it is critical to investigate novel molecule biomarkers for combating the metastasis of NSCLC.Long non-coding RNAs (lncRNAs) are a class of noncoding RNAs with longer than 200 nucleotides in length. Increasing reports have proved that lncRNAs are commonly dysregulated in multiple tumor types. Simultaneously, an increasing number of reports have confirmed that lncRNAs serve as oncogenes or suppressors through controlling the progression of malignant tumors. For instance, lncRNA Colorectal Neoplasia Differentially Expressed (CRNDE) stabilized by Heterogeneous Nuclear Ribonucleoprotein U Like 2 (hnRNPUL2) facilitates the growth and metastasis of colon cancer cell by activating Ras/MAPK signaling pathway . LINC005Notably, promising evidences have implied that lncRNAs, including SBF2-AS1, serve as ceRNAs to modulate genes expressions through competitively binding with microRNAs (miRNAs) in cancers. Investigations of the precise roles and molecular mechanisms of lncRNA-miRNA-mRNA crosstalk are necessary to improve the clinical outcomes of patients with cancer. In gastric cancer, lncRNA SBF2-AS1 plays oncogenic roles via regulating the miR-302b-3p-E2F Transcription Factor 3 (E2F3) axis . SilenciMiRNAs, which are noncoding RNAs, interact with the 3’-UTR of target genes to regulate the expressions of cancer suppressors or oncogenes. Previous investigations verify that miRNAs are involved into numerous pathological processes, for example cancers. Currently, miR-338-3p has been reported to function as a cancer inhibitor in many tumor types. For example, miR-338-3p suppresses the metastasis of ovarian carcinoma cell through suppressing proliferation and epithelial-mesenchymal transition (EMT) enhanced by MET Transcriptional Regulator MACC1 (MACC1) . PreviouHerein, we revealed the precise interaction between lncRNA SBF2-AS1 and miR-338-3p, which regulated the growth and aggressiveness of NSCLC cell by modulating its target gene ADAM Metallopeptidase Domain 17 (ADAM17). Based on these results, a novel regulatory signaling axis composed of SBF2-AS1/miR-338-3p/ADAM17 was illuminated in the progression of NSCLC.www.kmplot.com) . The upregulated lncRNAs and downregulated lncRNAs were summarized in the volcano plot  and heatlot.com) . Overallin vitro . As shown in in vitro . Finallyhttp://starbase.sysu.edu.cn/) was applied to find the possible miRNAs that interact with SBF2-AS1. As show in Increasing evidences have indicated that lncRNAs acts as ceRNAs competitively share miRNAs recognition sites with mRNAs to regulate the expressions of cancer-related genes. To find the potential target miRNAs of SBF2-AS1, Starbase website  human bronchial epithelial cell BEAS-2B and NSCLC cell lines . The results of qRT-PCR indicated that miR-338-3p was significantly downregulated in NSCLC cell lines compared than in BEAS-2B cell . Furtherin vitro –4D. Meanin vitro –4F. All Increasing reports demonstrate the ceRNA hypothesis, which reveals lncRNAs are involved into lncRNAs-miRNAs-mRNAs crosstalk. We firstly verified ADAM17 as a downstream target gene of miR-338-3p by using three bioinformatics software  . The folFinally, SBF2-AS1, ADAM17 and miR-338-3p were cotransfected into A549 cell, the protein expression of ADAM17 in A549 cell was measured by using qRT-PCR assay . Then, twww.kmplot.com)  . Given tin vivo, A549 cell was stable transfected with sh-SBF2-AS1 or sh-Con and inoculated into nude mice to construct xenograft models. As showed in in vivo. The levels of SBF2-AS1 in tumor tissues were further assessed using qRT-PCR assay. As shown in in vivo. sh-Con or sh-SBF-AS1 transfected A549 cells were inoculated into nude mice through the lateral tail vein. After twelve weeks, lung metastasis was apparent in mice injected with sh-Con transfected A549 cell  promotes the development of NSCLC cell by modulating miR-204-5p/Autophagy Related 3 (ATG3) axis . LncRNA Previous investigations proved that lncRNAs act as miRNAs sponges to modulate miRNAs. For example, UICLM, LINC00657 and SNHG15 were reported to facilitate cancer development via serving as miRNAs sponges in colorectal carcinoma, esophageal squamous cell carcinoma and papillary thyroid carcinoma , 27, 28.MiRNAs are a class of non-coding RNAs that function as post-transcriptional regulators through interacting with the 3’-UTR of mRNAs. Our study demonstrated that miR-98-5p bound to the 3’-UTR of ADAM17 and reduced the expression of ADAM17 in NSCLC cell. The Pearson analysis indicated there was an inversely relationship between miR-338-3p and ADAM17 in NSCLC tissues. However, ADAM17 level was positively related with the level of SBF2-AS1 in NSCLC tissues. The expression and activity of ADAM17 increase under some pathological conditions such as breast cancer and lung cancer –33. ConsOur study demonstrated that lncRNA SBF2-AS1 acted as a ceRNA to modulate the expression of ADAM17 through sponging miR-338-3p and exerted oncogenic roles during the progression of NSCLC . This rehttp://www.ncbi.nlm.nih.gov/geo/). The R software package was used to detect the differential expression of lncRNAs and to construct the figures.GSE dataset (GSE19804) containing lncRNAs expression profiles were retrieved from the Gene Expression Omnibus (GEO) database , non-neoplastic human bronchial epithelial cell line BEAS-2B were purchased from the Chinese Academy of Sciences . Cells were cultured in RPMI-1640  supplemented with 10% FBS and 100 U/ml penicillin/streptomycin and maintained in an incubator with 5% CO4 A549 or H1975 cells were transfected with 100 μmol/L miRNAs, or 5 μg pcDNA3.1, or 50 nM siRNA using 7.5 μl of Lipofectamine 3000 (Thermo Fisher Scientific) in 125 μl of Opti-MEMTM medium combination with 5 μl of p3000 for twenty-four hours. For functional assay in vivo, sh-SBF2-AS1 and sh-Con were purchased from RiboBio and constructed into A549 cell. The cDNA sequences of SBF2-AS1 were constructed by Shanghai Jima Pharmaceutical Technology Co., Ltd.,  and were subcloned into pLKO.1 lentivirus vector (Addgene). pLKO.1 empty vector was used as negative control plasmid. Then, lentivirus plasmid was transfected into HEK-293T cell along with lentivirus packaging plasmids . 72 hours after transfection, cell supernatants were collected and A549 cell was infected with lentivirus, followed by the screening of 1.5 μg/ml puromycin (Sigma). After 7 days, stable lentivirus transfected A549 cell lines were obtained.The pcDNA3.1 vector carrying SBF2-AS1 (SBF2-AS1), pcDNA3.1 vector carrying ADAM17 (ADAM17) or empty vector (vector) were obtained from RiboBio . MiR-338-3p mimics, miRNA negative control (miR-NC), miR-338-3p inhibitor, miR-NC inhibitor, small interfering RNA (siRNA) targeting SBF2-AS1 or ADAM17  and scramble control siRNA (si-Con) were purchased from RiboBio. 2×10The fragment of SBF2-AS1 possessing the miR-338-3p binding sites was inserted into pmirGLO luciferase vector  to construct SBF2-AS1-wild-type (SBF2-AS1-Wt). The corresponding mutant of miR-338-3p binding sites was constructed to form SBF2-AS1-mutated-type (SBF2-AS1-Mut). The 3’-UTR fragment of ADAM17 or its mutant of the miR-338-3p binding sites was inserted into pmirGLO to form ADAM17-3’-UTR-Wt or ADAM17-3’-UTR-Mut, respectively. The pmirGLO vector and miR-338-3p were transfected into A549 or H1975 cell using Lipofectamine 3000. After 48 hours, the luciferase activities were measured using Luciferase Reporter Assay System (Promega).Cells were lysed using RIP lysis buffer (Thermo Fisher Scientific) and the lysates were conjugated with anti-Argonaute2 (Ago2) antibody  or negative control anti-IgG in magnetic bead. Finally, the retrieved RNA was detected using qRT-PCR analysis.4) were plated into 96-well plates for 24, 48, 72 or 96 hours. Cell viability was measured using CCK-8 kit .Cells (1 × 103) were cultured into 6 well plates. After culturing for two weeks, the cell colonies in plates were fixed using methanol and stained with 1% crystal violet solution for 15 min -ATP by using a MEGALABEL Kit (Thermo Fisher Scientific). Hybridization was conducted in hybridization buffer at 65 °C overnight. The membrane was washed in 2x SSC and 0.5% SDS for 30 min and in 0.2x SSC and 0.5% SDS at 65 °C for 30 min. Finally, the signals were detected with a BAS-3000 image-analyzer .-ΔΔCt).Total RNAs were extracted utilizing Trizol kit (Thermo Fisher Scientific). RNA (1 μg) was reversely transcribed into cDNA using the PrimeScript RT reagent kit  and a TaqMan miRNA reverse transcription kit . qRT-PCR was carried out using SYBR Premix Ex Taq™ kit (TakaraBio) and miRNA-specific TaqMan miRNA assay kit (Applied Biosystems) on the Applied Biosystems 7500 Sequence Detection system (Applied Biosystems). The primers were as follows: SBF2-AS1 , ADAM17 , U6 , GAPDH . GAPDH was used as internal control for genes and U6 was used as internal control for miR-338-3p. The comparative cycle threshold (Ct) method was used to calculate the level of miRNA or genes by calculating the 2 followed by incubation with FITC-conjugated anti-rabbit secondary antibody. Cell nuclei were visualized with DAPI (Sigma). The fluorescence signal was examined under a fluorescence microscope .4 per well) in 96-well plates were transfected with si-Con or si-SBF2-AS1. Culture medium was collected, and secreted matrix metalloproteinase-2 (MMP-2) and MMP-9 were detected with the human MMPs ELISA kit .A549 or H1975 cells (5 × 10http://kmplot.com) based on the lung cancer database by selecting the non-commercial spotted platform, and patients were split by auto select best cutoff.To analyze the overall survival of NSCLC patients with low expression or high expression of SBF2-AS1, KM Plotter was used online (7) were suspended in 100 μl of FBS-free RPMI-1640 and subcutaneously implanted into nude mice. Tumor volume (TV) was calculated each week. TV = 0.5×length×width2. Relative tumor volume (RTV)=Vt/V0. Vt is the tumor volume on each measurement and V0 is the tumor volume at the beginning. Treatment/Control (T/C) %= RTV in sh-SBF2-AS1 group/RTV in sh-Con group×100. T/C%<60% indicates the significant inhibitory role of sh-SBF2-AS1 in the growth of A549 cell in vivo. Immunohistochemical (IHC) staining of ADAM17 was conducted using the xenograft tumor tissues. For IHC scoring, weighted score was computed that represented the positive staining of ADAM17. Percent positive staining was categorized as follows: 0, <5%; 1, 5-25%; 2, 26-50%; 3, 51-75%, and 4, >75% [BALB/C nude mice were bought from Shanghai SLAC Laboratory Animal Center . SBF2-AS1 knockdown lentivirus (sh-SBF2-AS1) or empty lentivirus control (sh-Con) transfected A549 cells  and calculated using GraphPad Prism 8.0. All data are from three independent experiments. The differences were calculated using Student’s t-test or one-way ANOVA followed by Tukey’s post-hoc test. Spearman correlations among variables were calculated. Survival curves were plotted using Kaplan-Meier method and calculated using the log-rank test. The Mann-Whitney U test was used to determine the association between SBF2-AS1 expression and clinicopathological parameters in patients with NSCLC. Supplementary FiguresSupplementary Table 1


C. elegans hermaphrodites, sto-3 promoter has been previously shown to drive gene expression in RIBL/R neurons and in three unidentified non-neuronal cells in the tail . (A) We have found that in C. elegans males, in addition to RIBL/R, two pairs of bilaterally-symmetrical tail neurons show strong Psto3::wrmScarlet expression ). These neurons send their processes to rays 4 and 8 of the male tail. In the figure, right lateral aspect is shown; arrows indicate rays containing processes expressing Psto3::wrmScarlet. (B) All rays in the male tail are innervated by A and B type neurons . To identify which neuron type expresses the sto-3 reporter, we have crossed Psto3::wrmScarlet transgenic males with a Ppkd-2::GFP reporter strain MT11318, which expresses GFP in the B-type neurons of rays 1-5, 7-9 and not in the A-type neurons . In the F1 cross-progeny males, which also carried the ceh-30(n3714) mutation in the background, Psto-3::wrmScarlet expression is colocalized with Ppkd-2::GFP for both pairs of ray neurons expressing Psto-3::wrmScarlet. This indicates that sto-3 is expressed in the R4BL/R and R8BL/R ray neurons. Left lateral aspect; arrows point at the cell bodies of Psto3::wrmScarlet-expressing neurons.In zfEx898[Psto-3::wrmScarlet + lin-15(+)]; lin-15 (n765ts) X. Psto-3::wrmScarlet transcriptional fusion. The plasmid was made by cloning 971 bp promoter region of sto-3 into a wrmScarlet-unc-54 3’UTR vector with a pUC19 vector backbone.Strains: QW1876.


PADI4, plays critical roles in the immune system; however, its contribution to the pathogenesis of lupus nephritis remains controversial. The pathological roles of PAD4 were investigated in lupus model mice. An imiquimod (IMQ)-induced lupus model was analyzed in wild-type (WT) and Padi4-knockout (KO) mice. Proteinuria, serum anti-double stranded DNA (anti-dsDNA) antibody, and renal infiltrated cells were evaluated. Neutrophil migration and adhesion were assessed using adoptive transfer and adhesion assay. PAD4-regulated pathways were identified by RNA-sequencing of Padi4 KO neutrophils. Padi4 KO mice exhibited significant improvements in proteinuria progression compared with WT mice, whereas, serum anti-dsDNA antibody and immune complex deposition in the glomeruli showed no difference between both mice strains. Padi4 KO mice showed decreased neutrophil infiltration in the kidneys. Adoptively transferred Padi4 KO neutrophils showed decreased migration to the kidneys of IMQ-treated WT mice, and adhesion to ICAM-1 was impaired in Padi4 KO neutrophils. Padi4 KO neutrophils exhibited reduced upregulation of p38 mitogen-activated protein kinase (MAPK) pathways. Toll-like receptor 7 (TLR7)-primed Padi4 KO neutrophils demonstrated reduced phosphorylation of p38 MAPK and lower expression of JNK-associated leucine zipper protein (JLP), a p38 MAPK scaffold protein. Neutrophils from heterozygous Jlp KO mice showed impaired adhesion to ICAM-1 and decreased migration to the kidneys of IMQ-treated WT mice. These results indicated a pivotal role of PAD4-p38 MAPK pathway in renal neutrophil infiltration in TLR7 agonist-induced lupus nephritis, and the importance of neutrophil-mediated kidney inflammation. Inhibition of the PAD4-p38 MAPK pathway may help in formulating a novel therapeutic strategy against lupus nephritis.Peptidylarginine deiminase 4 (PAD4), encoded by  Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by multi-organ inflammation with immune complex deposition and leukocyte infiltration. The treatment of SLE remains a huge challenge for clinicians considering the unfavorable prognosis associated with the side effects of steroids and immunosuppressive drugs. Genetic studies revealed the susceptibility of genes associated with the Toll-like receptor (TLR) pathway, interferon pathway, immune complex processing, and immune signal transduction in SLE, suggesting that the pathogenesis of SLE is based on immunological abnormalities . AutoantPeptidylarginine deiminase 4 (PADI4), which encodes PAD4, was initially identified as a non-HLA susceptibility gene in rheumatoid arthritis  is an enzyme that converts arginine residues to citrulline through post-translational modifications . Peptidytrophils . Subsequrthritis , 5, myocrthritis , and deerthritis . Severalipotency , p53 taripotency , and regipotency  have beeipotency . In addiipotency . ConsidePadi4 KO background to explore the pathological roles of PAD4 in lupus nephritis.The aim of the present study was to elucidate the pathological roles of PAD4 in lupus nephritis. A TLR7 agonist, Imiquimod (IMQ)-induced SLE mouse model  was usedPadi4 KO mice were generated by deletion of Padi4 exon 1 in C57BL/6 (B6) background mice (Jlp KO mice (Jlp +/– mice) were described previously  was administered on the skin of the left ear of age-matched 8–9-week-old female B6 WT and eviously . Degree The ear skin and kidneys were excised from sacrificed IMQ-treated mice 8 weeks after the first IMQ treatment, fixed with 4% paraformaldehyde, followed by embedding in paraffin. Paraffin-embedded fragments were stained with H&E. For kidney immunohistochemistry, paraffin-embedded sections were immunostained for 1 h at 4°C with goat antiserum to mouse complement C3 primary antibody (ICN/CAPPEL), followed by staining for 1 h at room temperature with Alexa Fluor 594 Donkey anti-goat IgG secondary antibody (Invitrogen). The, slides were also immunostained with 1 h at 4°C with rabbit F (ab') 2 anti-mouse IgG primary antibody (BIO-RAD), followed by staining for 1 h at room temperature with Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (Invitrogen). DAPI (Invitrogen) was used for nuclear staining.Inflammatory cells in the ear skin were counted per high-power field. Glomerular score represents the sum of scores for glomerular inflammation, proliferation, crescent formation, and necrosis as described previously . Each scPadi4 KO mice were isolated magnetically using a Neutrophil isolation kit (Miltenyi Biotec). CD11b+ Ly6G+ neutrophils were isolated at higher than 85% purity by density gradient isolation, and > 97% by magnetic isolation.Bone marrow-derived neutrophils were isolated by density gradient centrifugation. For transcriptome analysis, bone marrow neutrophils from 8-week-old female B6 WT and Padi4 KO and Jlp +/– mice were stained with CellTracker Green CMFDA Dye (Thermo Fisher Scientific), and 2.5 × 106 neutrophils were adoptively transferred to B6 WT control mice and B6 WT mice after IMQ treatment for 4 weeks as described previously  containing 20 mM HEPES and 0.1% bovine serum albumin (BSA) were incubated with or without 1 μg/ml of R848 for 1 h. In some wells, p38 MAPK inhibitor SB203580 (Sigma-Aldrich) was added 30 min prior to R848 stimulation. Furthermore, neutrophils were plated in 96-well plates coated with recombinant mouse ICAM-1/CD54 Fc chimeric protein (R&D systems) for 30 min. Supernatants was removed, and analyzed for the neutrophil adhesion to ICAM-1 using CytoTox 96 non-radioactive cytotoxicity assay (Promega) as described previously , 1 mM CaCl2 , 10 mM HEPES (Invitrogen), and 0.2 μM SytoxGreen (Thermo Fisher Scientific) and plated at a concentration of 3 × 105/ml. Neutrophils were incubated without any stimulants overnight 37°C in 5% CO2. Supernatants were removed and plates were washed with PBS twice. SytoxGreen positive cells, which included NETs in the extracellular parts, were counted using a fluorescence microscope in a blinded fashion. The percentage of SytoxGreen positive cells were calculated as the average of 5 fields (×100) per well.Bone marrow-derived neutrophils from age-matched female B6 WT and Cell staining was performed in the presence of purified rat anti-mouse CD16/32 antibody (BD). Antibodies used for staining were as follows; anti-Ly6G-PE , anti-CD11b-APC , anti-Ly6G-APC-Cy7 , anti-Ly6C-APC-Cy7 , mouse biotin-conjugated anti-CD45 , anti-streptavidin-V500 (BD), and anti-7-AAD (BioLegend).6/ml of bone marrow-derived neutrophils were stimulated with 1 μg/ml of R848 (Enzo Life Sciences) for 30 min in RPMI 1640 (Invitrogen) containing 10% fetal bovine serum (FBS), 100 μg/ml of L-glutamine (Sigma-Aldrich), 100 U/ml of penicillin (Sigma-Aldrich), 100 μg/ml of streptomycin (Sigma-Aldrich), and 50 μM 2-mercaptoethanol (Sigma-Aldrich). Stimulated neutrophils were stained with anti-CD11b-APC  and anti-Ly6G-APC-Cy7  antibodies in the presence of purified rat anti-mouse CD16/32 antibody (BD), fixed with Lyse/Fix Buffer (BD), and permeabilized by BD Phosflow Perm Buffer II (BD). Then, neutrophils were intracellularly stained with Alexa Fluor 488 Mouse IgG1κ isotype control (BD) or Alexa Fluor 488 mouse anti-p38 MAPK (pT180/pY182) (BD).For intracellular staining for phosphorylated p38 MAPK, 1 × 10Flow cytometric analysis was performed using MoFlo XDP and data were analyzed using FlowJo Software (Tree star).Padi4 KO mice were resuspended in HBSS (with Ca2+ and Mg2+) (Wako) containing 0.25% fatty acid free, low endotoxin BSA (Sigma-Aldrich), and 14 mM HEPES at a concentration of 5 × 106/ml. Then, 300 μl of 1 nM recombinant mouse CXCL2 (carrier-free) (BioLegend) was loaded at the bottom of the 24-well plate . Neutrophils in 200 μl of the above-mentioned buffer were put on the Transwell filter  and placed at the top of the plate. The plates were incubated in 37°C, in 5% CO2. After 1 h, the Transwell filter and medium in the lower chamber were collected. Lower chambers were filled with 300 μl of HBSS (without Ca2+ and Mg2+) (Wako) containing 5 mM EDTA and put on ice for 30 min. Additionally, 300 μl of HBSS (without Ca2+ and Mg2+) containing 5 mM EDTA was added again and the cells were collected. Cell numbers were counted and relative migration was calculated for each well.Transwell assays were performed as described previously . Bone ma6/ml were incubated in 24-well plates and left unstimulated or stimulated with 1 μg/ml of R848 for 6 h in RPMI 1,640 containing 10% FBS, 100 μg/ml of L-glutamine, 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 50 μM 2-mercaptoethanol. Each sample was analyzed in duplicate. After collecting the cells and removal of the supernatant, 250 μl of PBS was added, followed by pipetting with 750 μl of TRIzol LS reagent (Life Technologies) in accordance with the manufacturer's instructions. RNA extraction was performed using an RNeasy micro kit (Qiagen) in accordance with the manufacturer's instructions. The quality of extracted RNA was confirmed using a 2,100 Bioanalyzer (Agilent) and quantified with a Qubit RNA HS assay kit (Thermo Fisher Scientific). RNA sequencing libraries were prepared using a TrueSeq RNA sample preparation kit v2 (Illumina) in accordance with the manufacturer's instructions. Paired-end sequencing was performed using a MiSeq Reagent V2 kit (Illumina) on the MiSeq system.Neutrophils at a concentration of 1 × 10https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) (v0.11.7). After trimming off the adaptors using cutadapt (6. Filtered reads were mapped to the UCSC mm10 using STAR (v2.5.3a) . We conducted comparison analysis in IPA between the results of the core analysis of WT and Padi4 KO neutrophils. Activation Z scores were calculated using IPA comparison analysis between WT and Padi4 KO neutrophils. Read counts were normalized as count per million and scaled by low Z-score and were plotted as a heatmap with hierarchical clustering based on Ward's Method.Raw read quality was evaluated using FastQC (cutadapt  (v1.16),v2.5.3a) , and thev2.5.3a) . Genes ee DESeq2  package Padi4 KO neutrophils were defined as differentially upregulated genes  in TLR 7-primed WT neutrophils but not in Padi4 KO neutrophils. Overlapping genes were selected between PAD4-binding genes and downregulated genes of TLR7-primed Padi4 KO neutrophils.PAD4-binding genes were adopted from a report that involved ChIP-chip analysis in MCF-7 . Gene naActb (5′- AGAGGGAAATCGTGCGTGAC-3′ and 5′- CAATAGTGATGACCTGGCCGT-3′), Padi4 (5′-CTCTCCAGGAGTCATCGTAG-3′ and 5′-CCAACACCAGCTGATACTTT-3′), Jlp (5′-GAGCATGTGTTTACAGATCCACTG-3′ and 5′-CATTTTCTGAGCTTCTTCTCTCGC-3′).Total RNA from neutrophils was extracted using an RNeasy Micro kit as described in the manufacturer's instructions. RNA was reverse-transcribed to cDNA with random primers (Invitrogen) and SuperScript III (Invitrogen). To measure gene expression, quantitative real-time PCR was performed by using a CFX connect real-time PCR detection system (Bio-Rad) with a QuantiTect SYBR Green PCR kit (QIAGEN). Gene expression was normalized to β-actin expression levels as a control. Sequences of the PCR primers were as follows: 6/ml) from age-matched female B6 WT, Padi4 KO mice were incubated in 24-well plates and stimulated with 1 μg/ml of R848 in RPMI 1640 containing 10% FBS, 100 μg/ml of L-glutamine, 100 U/ml of penicillin, 100 μg/ml of streptomycin, and 50 μM 2-mercaptoethanol. After collecting cells and removal of the supernatant, neutrophils were homogenized in RIPA buffer (Sigma-Aldrich) containing Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific) and Halt protease inhibitor (Thermo Fisher Scientific). The protein samples were heated to 95°C for 5 min in Laemmli sample buffer (Bio-Rad) containing 2-mercaptoethanol and loaded onto Mini-PROTEAN TGX precast gels (Bio-Rad). Following electrophoretic transfer of proteins to an Immobilon transfer membrane (Millipore) and membrane was blocked with PVDF blocking reagent for Can Get Signal® (Toyobo). The membrane was incubated overnight with primary antibodies as follows; anti-JLP  and anti-GAPDH . The membrane was then incubated with a secondary goat anti-rabbit IgG-HRP (Invitrogen) antibody, and specific proteins were visualized using ImageQuant LAS4010  with ECL Select .Bone marrow-derived neutrophils  was used for statistical analysis except in sequencing data. Two-tailed unpaired Padi4 KO mice. The spleen weight was almost equivalent between untreated WT and Padi4 KO mice (Padi4 KO (Padi4 KO-IMQ) mice showed a significant reduction in splenomegaly compared with IMQ-treated WT (WT-IMQ) mice  mice . EpidermIMQ mice . MoreoveIMQ mice . The ser KO mice . The con KO mice , in kidnIMQ mice . SpontanIMQ mice . Taken tPadi4 KO-IMQ mice .Pertaining to the immunological characteristics, there was no difference in serum anti-dsDNA IgG and anti-Sm titers between WT-IMQ and IMQ mice , S4. ImmIMQ mice . In contIMQ mice . In both+Ly6G+) and CD11b+CD11c+ cells in the kidneys of Padi4 KO-IMQ mice  decreased in the spleen of Padi4 KO-IMQ mice . In Transwell assays, there were no differences in chemotaxis between WT and Padi4 KO neutrophils toward CXCL2 in vitro , was included among these  mice lacking ICAM-1 or lymphocyte function-associated antigen-1 showed amelioration of glomerulonephritis, which was correlated with a decrease in neutrophil accumulation in the glomeruli -binding protein. When stimulated by binding GTP, various signaling pathways are activated (Rnf11 encodes RING finger protein 11 (RNF11), involved in various pathways, such as regulation of signal transduction, trafficking and modulation of the transcriptional activity (Rnf11 is involved in selenium deficiency-induced cardiac necrosis via MAPK activation (Hbp1 encodes HMG-box transcription factor 1 (HBP1), which is involved in transcriptional repressor and cell cycle inhibitor (Ptgs2 encodes cyclooxygenase 2 (COX2), and COX2 is also downstream target in p38 MAPK pathway  revealed a number of critical genes and pathways for SLE  and (P18-098).HS, TK, TO, KYa, and KF designed experiments. NH conducted the experiments and acquired data. NH, HH, YN, and AS analyzed the data. AS, IG, and KYo provided animals. NH, HS, and KF wrote the manuscript. NH, HH, and YN prepared figures. All authors contributed to the article and approved the submitted version.NH has received grants from GlaxoSmithKline. HS has received speaking fees from Takeda, BMS, Asahi Kasei, Sanofi, Eli Lilly, Janssen, Astellas, Pfizer, Chugai, UCB, and Daiichi-Sankyo. YN has received financial support or fees from BMS, Kissei, Mitsubishi Tanabe, and Pfizer. TK has received grants from GlaxoSmithKline. TO has received grants from Chugai. KYa has received fees for lecture from AbbVie, Ayumi, BMS, Chugai, Eisai, Janssen, Mitsubishi Tanabe, Ono, and UCB. KF has received grants, consulting fees, speaking fees, and/or honoraria from Mitsubishi Tanabe, BMS, Eli Lilly, Chugai, Janssen, Pfizer, Ono, AbbVie, Ayumi, Astellas, Sanofi, Novartis, Daiichi-Sankyo, Eisai, Asahi Kasei, MSD, and Japan Blood Products Organization. The remaining 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.


H234A and UL36(480)D232A, but not C98 mutants. These in vitro results suggested that the C98 and H234 are essential catalytic residues of UL36-DUB. UL36-DUB exhibited a strict substrate specificity. Inhibition assay revealed that UL36-DUB exhibits resistance to the Roche protease inhibitor cocktail and serine protease inhibitor, but not to the Solarbio protease inhibitor cocktail. (4) Conclusions: UL36-DUB exhibited a strict substrate preference, and the protocol developed in the current study for obtaining active UL36-DUB protein should promote the high-throughput screening of UL36 inhibitors and the study on the function of MDV-encoded UL36.(1) Background: Deubiquitinase (DUB) regulates various important cellular processes via reversing the protein ubiquitination. The N-terminal fragment of a giant tegument protein, UL36, encoded by the Marek’s disease (MD) virus (MDV), encompasses a putative DUB (UL36-DUB) and shares no homology with any known DUBs. The N-terminus 75 kDa fragment of UL36 exists in MD T lymphoma cells at a high level and participates in MDV pathogenicity. (2) Methods: To characterize deubiquitinating activity and substrate specificity of UL36-DUB, the UL36 N-terminal fragments, UL36(323), UL36(480), and mutants were prepared using the Bac-to-Bac system. The deubiquitinating activity and substrate specificity of these recombinant UL36-DUBs were analyzed using various ubiquitin (Ub) or ubiquitin-like (UbL) substrates and activity-based deubiquitinating enzyme probes. (3) Results: The results indicated that wild type UL36-DUBs show a different hydrolysis ability against varied types of ubiquitin chains. These wild type UL36-DUBs presented the highest activity to K11, K48, and K63 linkage Ub chains, weak activity to K6, K29, and K33 Ub chains, and no activity to K27 linkage Ub chain. UL36 has higher cleavage efficiency for K48 and K63 poly-ubiquitin than linear ubiquitin chain (M1-Ub4), but no activity on various ubiquitin-like modifiers. The mutation of C98 and H234 residues eliminated the deubiquitinating activity of UL36-DUB. D232A mutation impacted, but did not eliminated UL36(480) activity. The Ub-Br probe can bind to wild type UL36-DUB and mutants UL36(480) Ubiquitination is one of the most important regulatory machinery of post-translational modification of intracellular proteins . The modIn human, approximately 100 proteases for deconjugating Ub or Ub chains were found and classified into two main families, cysteine proteases and metalloproteases . The cysPathogens have evolved a series of molecular strategies to promote infection, proliferation, and survival during host–pathogen interactions. Many viruses encode viral DUBs to hijack host cell defense mechanisms, such as Herpes simplex virus (HSV), Kaposi’s sarcoma-associated herpesvirus (KSHV), Epstein–Barr virus (EBV), MDV, pseudorabies virus (PRV), Human cytomegalovirus (HCMV), severe acute respiratory syndrome-coronavirus (SARS-CoV), and Middle East respiratory syndrome-related coronavirus (MERS-CoV) which encode USP type DUBs ,11,12. E+ T cells, and the N-terminal 75 kDa fragment of UL36, which contains the viral DUB catalytic domain (UL36-DUB), exists in MDV-induced T lymphoma cells in high level )/([S] + KM). A Lineweaver–Burk plot was used to determine the KM and Vmax, and then the Vmax/enzyme concentration to calculate the catalytic constant kcat. The catalytic efficiency of each type of UL36 was compared using the ratio kcat/KM.The kinetics of the deubiquitinating activity of wild type UL36(323), UL36(480), and the mutants, UL36(480)C98A, UL36(480)C98S, UL36(480)D232A, and UL36(480)H234A, was characterized by testing the hydrolysis efficiency on a variety of Ub substrates. A reaction system containing 98 μM of substrate , 1× reaction buffer in a final volume of 100 μL was initiated by adding 200 nM UL36-DUBs at 37 °C for different incubation times. The hydrolyzed products were separated by SDS-PAGE and stained with Coomassie brilliant blue R-250. Standard Western blotting was performed using primary antibody against ubiquitin , and horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody  was used for ECL imaging.The substrate preference of wild type UL36(323), UL36(480), and the mutants, UL36(480)SUMO1-GST, SUMO2-GST, SUMO3-GST ,48, di-Subiquitin gene (GenBank ID M14693.1) and the NEDD8 gene (GenBank ID XP_015130268.1), the sequence of two genes were optimized using software Codon OptimWiz (Genewiz Inc.). The genes of chicken Ub(75) and NEDD8(75) proteins from which the Gly76 of chicken Ub(76) and NEDD8(76) were absent were amplified by PCR with primers, Ub-probe-F and Ub-probe-R, and NEDD8-probe-F and NEDD8-probe-R (E. coli BL21 (DE3) with the induction by 0.1 mM isopropyl-β-d-thiogalactoside (IPTG). The whole purification process was performed according to the manual of IMPACT™-CN System , except that the elution was carried out post the incubating column with cleavage buffer ) for 12 h at 25 °C to generate Strep-Ub(75)-MESNa or Strep-Nedd8(75)-MESNa. Strep-Ub(75)-MESNa or Strep-Nedd8(75)-MESNa was incubated with 400 mM of bromoethylamine at pH 8.0 for 20 min at 25 °C to generate inhibitor probe, Strep-Ub(75)-Br or Strep-Nedd8(75)-Br. Finally, the prepared probe was dialyzed in a storage buffer  glycerol, pH 7.5), aliquoted for use or stored at −80 °C.Some animal or pathogen DUBs have dual deubiquitinating/de-NEDDylating activity ,31,32,33-probe-R , and theFor the direct binding test, 5 pmol of the Strep-Ub(75)-Br or Strep-Nedd8(75)-Br probe was incubated with 4 pmol of UL36(480) for 1 h at 37 °C. The reaction products were separated with SDS-PAGE and identified by Western blotting using standard protocols  with priFor the catalytic activity assay of the probe treated UL36(480), 125 nM UL36(480) or mutants, and 4 μM Strep-Ub(75)-Br or Strep-Nedd8(75)-Br were preincubated in 1× reaction buffer for 1 h at 37 °C, in this case, a final concentration of 100 nM Ub-AMC was added to initiate the deubiquitinating reaction. The deubiquitinating activity was evaluated by measuring the fluorescence intensity of the AMC released from the reaction on a FluoroMax 4 fluorescence spectrophotometer (HORIBA Scientific) with excitation and emission wavelengths of 380 nm and 460 nm, respectively.For the assay of inhibitor interference to the binding between probes and UL36(480), UL36(480) protein was pre-incubated with 1× working concentration (1 mM) of phenylmethanesulfonyl fluoride (PMSF)  and two types of protease inhibitor cocktails, respectively, manufactured by Solarbio Lifesciences  or Roche of Millipore Sigma  for 1 h at 4 °C, and then followed by the catalytic activity assay as described above. Western blotting was carried out to assess the inhibition effect using primary antibodies against ubiquitin, as described above. Two hundred millimolar stock solution of PMSF was freshly dissolved in DMSO, and the stock solutions of both inhibitor cocktails were prepared fresh before use according to the instructions.


The pulmonary renin angiotensin (Ang) system (RAS) comprises two pathways whose balance is important for lung homeostasis. Endothelial ACE generates Ang II, acting on AT1 receptors to promote vasoconstriction and pro-inflammatory effects, whereas epithelial ACE2 cleaves Ang II into Ang1–7, acting on the Mas receptor to exert vasodilatory and anti-inflammatory effects. A shift towards predominant ACE-dependent Ang II formation has been postulated as an important pathophysiological mechanism in various forms of ARDS [+ cells and of cytotoxic IFNγ+-CD8+ T cells (Fig. + cells cytotoxicity have been proposed to be involved in the hyperinflammatory state characterizing COVID-19 ARDS [SARS-CoV-2 uses lung ACE2 as its cellular receptor, resulting in ACE2 degradation and ACE/ACE2 imbalance, which could drive Ang II-mediated vascular inflammation and lung injury in severe COVID-19 disease . MR acti-19 ARDS .Fig. 1SDysregulated RAS signaling with enhanced aldosterone-mediated MR activation could represent an important link between SARS-CoV-2/ACE2 interaction and inflammatory lung injury, suggesting an interesting therapeutic potential of RAS inhibitors  and in p