MicroRNAs (miRNAs) are post-transcriptional regulatory RNAs that can modulate cell signaling and play key roles in cell state transitions. Epstein-Barr virus (EBV) expresses >40 viral miRNAs that manipulate both viral and cellular gene expression patterns and contribute to reprogramming of the host environment during infection. Here, we identified a subset of EBV miRNAs that desensitize cells to B cell receptor (BCR) stimuli, and attenuate the downstream activation of NF-kappaB or AP1-dependent transcription. Bioinformatics and pathway analysis of Ago PAR-CLIP datasets identified multiple EBV miRNA targets related to BCR signal transduction, including GRB2, SOS1, MALT1, RAC1, and INPP5D, which we validated in reporter assays. BCR signaling is critical for B cell activation, proliferation, and differentiation, and for EBV, is linked to reactivation. In functional assays, we demonstrate that EBV miR-BHRF1-2-5p contributes to the growth of latently infected B cells through GRB2 regulation. We further determined that activities of EBV miR-BHRF1-2-5p, EBV miR-BART2-5p, and a cellular miRNA, miR-17-5p, directly regulate virus reactivation triggered by BCR engagement. Our findings provide mechanistic insight into some of the key miRNA interactions impacting the proliferation of latently infected B cells and importantly, governing the latent to lytic switch. Understanding the molecular mechanisms regulating EBV latency and entry into the lytic replication cycle is important in developing therapies for viral disease. We demonstrate here that a subset of EBV miRNAs target host factors within the BCR signaling pathway and consequently, negatively regulate cellular responses to BCR cross-linking. Disrupting activity of individual EBV miRNAs, specifically miR-BHRF-1-2-5p and miR-BART2-5p, enhanced lytic gene expression following BCR engagement, suggesting a key role for these miRNAs in attenuating viral reactivation. Our experiments establish a link between EBV miRNAs and signaling through the BCR, providing new insight into miRNA-mediated aspects of latency and reactivation. Epstein Barr virus (EBV) is a human gamma-herpesvirus that infects >90% of adults worldwide. Primary infection is often asymptomatic or presents as infectious mononucleosis. In immunocompromised individuals, the virus is linked to post-transplant lymphoproliferative disease (PTLD) and hematological malignancies including Burkitt’s (BL) lymphoma, Hodgkin lymphoma, and AIDS-related lymphoma; cancers of epithelial origin such as gastric and nasopharyngeal carcinomas are also associated with EBV infection ,2. FolloEBV encodes over 85 open reading frames (ORFs) and several non-coding RNAs (ncRNAs) which are temporally and differentially expressed throughout the viral life cycle. At least three distinct latency programs  have been described for EBV, distinguished by viral gene expression patterns . The latWhile exact functions for EBV miRNAs continue to emerge, previous studies demonstrate that these small, viral ncRNAs act akin to their cellular counterparts to post-transcriptionally regulate gene expression via interactions with both viral and cellular RNA targets. EBV and other g-herpesvirus miRNAs are known to regulate multiple cell signaling pathways, including those associated with innate and cell-mediated anti-viral immune responses such cytokine and interferon signaling –14. SpecAberrant BCR signaling is a hallmark of cancer progression; somatic mutations in cellular components that result in constitutively active or tonic BCR signaling contribute to enhanced proliferation of malignant B cells in cancers such as lymphoma ,21. BCR In the context of EBV infection, antigenic stimulation of the BCR can initiate plasma cell differentiation, which triggers the switch from latency to lytic replication ,23,24. NIn this study, we sought to determine whether EBV miRNAs could influence BCR signaling and to subsequently elucidate the molecular mechanism(s) by which this may occur by determining cellular targets involved. To understand how the identified EBV miRNA target interactions might be relevant to the EBV life cycle, we examined gain and loss of function outcomes for specific EBV miRNAs in the growth of latently infected B cells as well as consequences for lytic reactivation triggered through surface Ig cross-linking.To determine if EBV miRNAs could functionally regulate signaling through the BCR, we screened individual viral miRNAs in EBV-negative BJAB BL cells which express surface IgM and are responsive to antigenic triggering of the BCR using antibodies against IgM . BJAB ceWe have recently demonstrated that the EBV BHRF1-2 miRNAs can reduce NF-kappaB activation in response to IL-1 cytokines . There iEngagement of the BCR also activates JNK  and p38, triggering MAPK signaling, and regulating activity of AP1 (Activator protein 1) family transcription factors such as JUN and ATF2 ,30. PrevIn order to identify the molecular mechanisms by which EBV miRNAs attenuate BCR signaling, we queried published B cell Ago PAR-CLIP datasets for experimentally derived, protein-coding mRNA targets of EBV miRNAs, focusing specifically on cellular targets for EBV BART2, BART9, BART17, BART18, and BHRF1-2 miRNAs. Combined datasets included EBV B95-8 LCLs , two EBV wild-type LCLs , and one EBV+/KSHV+ PEL  . 3,501 tFrom the list of target genes related to BCR signaling, we selected several 3’UTRs for further investigation by conventional 3’UTR reporter assays; this included the GRB2 and MALT1 3’UTRs which are potentially regulated by multiple EBV miRNAs . With thTo further investigate 3’UTR interactions for the BHRF1-2 miRNAs, we then used site-directed mutagenesis to disrupt individual seed-match sites, focusing our efforts on the MALT1, GRB2, RAC1, INPP5D, and PAG1 3’UTRs. The Ago-CLIP identified interaction between miR-BHRF1-2-3p and the PRDM1 3’UTR was recently confirmed ,43. DisrTo ask if the targets of EBV miRNAs validated above exhibited changes at the protein level, we first examined Grb2 protein levels in BHRF1-2 miRNA mutant LCLs or BJAB cells that were stably transduced with pLCE control vector or pLCE-BHRF1-2 to express the BHRF1-2 miRNAs in trans . ConsistTo examine targets in the context of EBV infection, we also compared Grb2 and Malt1 protein levels in established, donor-matched EBV B95-8 wild-type LCLs versus BHRF1-2 miRNA mutant LCLs that lack both miR-BHRF1-2-3p and miR-BHRF1-2-5p ,45. MaltHaving identified several BCR signaling components as EBV miRNA targets, we then asked whether RNAi-mediated knockdown of the individual target RNAs could potentially phenocopy EBV miRNA function. We generated shRNAs (short hairpin RNAs) for individual cellular targets of the EBV BHRF1-2 miRNAs since activity of both NF-kappaB and AP1 reporters was reduced by these miRNAs in response to anti-IgM stimulation . miR-30-cis [Previous studies show that genetic ablation of the EBV BHRF1 miRNAs from the viral genome impairs but does not fully inhibit LCL outgrowth in vitro –48. Furtcis ,50, the cis ,41. We tUsing cell viability assays (trypan-blue exclusion) as well as MTT -2,5-diphenyltetrazolium bromide) assays to monitor metabolic activity, we first measured the growth of established, donor-matched LCLs (>6–8 weeks post-infection) that are latently infected with either EBV B95-8 2089 wild-type virus or a miRNA mutant virus in which both BHRF1-2 miRNAs are deleted . In agreTo examine whether cellular factors identified above might contribute to LCL proliferation, we then introduced shRNAs into BHRF1-2 miRNA mutant LCLs (LCL-D2) against GRB2, MALT1, INPP5D, SOS1, RAC1, or IL1R1 (regulated by miR-BHRF1-2-5p ). FollowThe GRB2 3’UTR is targeted by miR-BHRF1-2-5p in luciferase reporter assays . To confTo ask if we could rescue the growth phenotype conferred by miR-BHRF1-2-5p knockdown, GRB2 shRNAs were expressed in combination with the miR-BHRF1-2-5p sponge and MTT assays were performed . Consistin vitro, we next sought to determine whether miR-BHRF1-2-5p also plays a role in maintaining the growth of EBV transformed B cells that are naturally derived. Diffuse large B cell lymphoma (DLBCL) is the most common form of NHL in HIV-infected patients, and >85% of immunoblastic, AIDS-related DLBCL are EBV+ [As LCLs are artificially generated are EBV+ . Using qare EBV+ . ConsistCross-linking of surface Ig on B cells latently infected with EBV can facilitate lytic reactivation . To inveWe then tested whether EBV miRNAs could directly influence BCR-mediated EBV reactivation from latency, using sponge inhibitors to disrupt the activity of individual miRNAs in Mutu I cells. We focused on two EBV miRNAs: (i) miR-BHRF1-2-5p, for which we were able to confirm multiple targets directly linked to BCR signaling and (ii) miR-BART2-5p, which reduces BCR-mediated NF-kappaB activation  and alsoTo monitor lytic reactivation in MutuI cells, BZLF1 and BRLF1 transcripts were measured by qRT-PCR. Surface Ig cross-linking led to ~8 to 10-fold induction of EBV IE gene expression in pLCE control cells by 24 hr . We obseThe overall increase in lytic reactivation upon miR-BART2-5p inhibition could be attributed, in part, to the targeting of EBV BALF5 encoding the viral DNA polymerase catalytic subunit . To sepaIn this study, we evaluated the role of EBV miRNAs in BCR signaling, which has implications for the proliferation of latently infected B cells and importantly, for influencing virus reactivation from the latent state. Antigen stimulation of the BCR induces multiple intracellular signaling pathways, and latent viral proteins have been previously demonstrated to manipulate BCR signaling components and/or the pathways  triggered through BCR activation. As viral miRNAs are non-immunogenic viral gene products that are expressed throughout multiple stages of EBV infection, we hypothesized that the viral miRNAs would be prime candidates to coordinately modulate the signaling cascade initiated through the BCR.Through functional screens, we demonstrate that a subset of five EBV miRNAs desensitizes BL cells to BCR cross-linking, subsequently attenuating downstream transcriptional activation of NF-kappaB and/or AP1 . By analOur experiments highlight that the EBV BHRF1-2 miRNAs, in particular, act as novel and potent regulators of BCR signal transduction and consequently, can restrict entry into the lytic cycle initiated by BCR engagement. The BHRF1-2 miRNAs are evolutionarily conserved, with homologs encoded by multiple lymphocryptoviruses (LCV) that infect Old World non-human primates ,61. The Grb2 is a ubiquitously expressed signaling adaptor protein that interacts with numerous growth factor and antigen RTKs via its src-homology domains ,63. The Notably, GRB2 as well as SOS1 are regulated by non-coding RNAs in other g-herpesvirus infection systems, suggesting this may be a common strategy employed by g-herpesviruses to manipulate RTK signaling. KSHV miR-K10a, a mimic of cellular miR-142-3p, targets the SOS1 3’UTR . KSHV miThrough loss-of-function experiments, our study further reveals that miR-BHRF1-2-5p activity and optimal Grb2 levels are necessary for efficient proliferation of EBV transformed B cells. In concordance with previous reports, LCLs established with BHRF1-2 miRNA mutant viruses exhibited growth defects compared to those established with wild-type EBV B95-8 ,48 Fig . While tRecent work from Poling et. al. investigated whether growth deficiencies in LCLs generated with a virus lacking all BHRF1 pre-miRNAs could be reversed . In contLong-lived, memory B cells are thought to be a primary reservoir for persistent EBV infection in vivo as the virus is able to gain access to this compartment through viral gene expression programs that manipulate B cell growth and differentiation . While tBy suppressing signals that arise via engagement of the BCR, we propose that EBV miRNAs such as miR-BHRF1-2-5p and miR-BART2-5p protect latent cells from aberrant reactivation . We furtAn intriguing question that remains is how miR-BHRF1-2-5p physically exerts function in latency I BL cells during induction of the lytic cycle. Unlike miR-BART2-5p and cellular miR-17, the BHRF1-2 miRNAs are not detectable in latency I until cells respond to reactivation cues ; yet, inIn summary, we have shown that a subset of EBV miRNAs attenuates signaling through the BCR and governs aspects of EBV reactivation. An important goal for future studies will be to examine specific contributions from additional viral miRNAs as well as cellular miRNAs that are hijacked by the virus in order to more fully decipher the extent to which post-transcriptional regulatory mechanisms facilitate latent/lytic cell state transitions. Gaining a clear understanding of the mechanisms regulating latency and entry into the lytic replication cycle is a critical part of designing effective treatments for viral disease. Our findings thus provide valuable insight into how viral miRNAs functionally contribute to viral latency and have utility in aiding development of miRNA-focused therapeutic strategies.2-humidified atmosphere in RPMI-1640 supplemented with 15% fetal bovine serum (FBS) (unless otherwise stated) and 1% penicillin, streptomycin, and L-glutamine (P/S/G). MutuI BL cells exhibit cell surface IgM (uK+) [B cell lines  were maintained at 37°C in a 5% COgM (uK+)  and weregM (uK+) ,32,60. AgM (uK+) ,45; LCL-gM (uK+) . DLBCL cgM (uK+)  and wereHEK293T cells (originating from Dr. Bryan Cullen’s laboratory) were maintained in high glucose DMEM supplemented with 10% FBS and 1% P/S/G. For preparation of lentiviruses, HEK293T cells were plated in 15-cm plates in complete media and transfected using Polyethylenimine (PEI) with 7.5 ug pL-based lentivector, 4.5 ug pDeltaR8.75 and 3 ug pMD2G. Media was changed to complete RPMI-1640 between 8 hrs and 16 hrs post-transfection. Lentiviral particles were harvested by sterile filtration of the supernatant using a 0.45 micron filter at 48 and 96 hrs post-transfection and used to transduce ~1 to 5 x 10^6 cells. BJAB, MutuI, LCLs, and DLBCLs were transduced with one (BJAB) or two  rounds of lentivirus and monitored by fluorescent microscopy for transduction efficiency by green fluorescent protein (GFP) and/or mCherry expression. DLBCLs were further subjected to sorting by flow cytometry to obtain >95% pure populations of GFP-positive cells. To induce lytic reactivation, MutuI cells were spun down and plated at 1 x 10^6 cells in fresh media containing soluble anti-IgM (Sigma) at concentrations and times indicated in figure legends (2.5–5 ug/mL for 22–48 hrs).Renilla luciferase in the psiCheck2 dual luciferase reporter vector. When achievable, the entire 3’UTR was cloned for a given target. For longer 3’UTRs, a minimum of 1 kb containing the region predicted to be targeted by each miRNA was cloned. Mutant 3’UTR reporters, containing nucleotide changes in miRNA seed match sites as identified by PAR-CLIP, were generated by Phusion Taq site-directed mutagenesis.EBV miRNA expression vectors in pLCE are previously described ,17,32. pLentiviral vector miRNA sponge inhibitors contain six to eight imperfect, tandem decoy binding sites for a single miRNA as previously described ,17,79. SCells were maintained in log phase and split 1:2 or 1:3 one day prior to seeding into 96-well plates at 2.5 x 10^3 cells/well in complete RPMI media without phenol red. At each time point, 100 ul per well of MTT -2,5-diphenyltetrazoliumbromide) reagent  was added. Cells were incubated for 2 hrs at 37°C, lysed in MTT solvent (isopropanol containing 0.5% NP40 and 4 mM HCl), and incubated for an additional 2 hrs at 37°C. Lysates were read using an ELISA plate reader (Absorbance = 562 nm), and relative cell growth was determined by comparing absorbance values of cell lysates at time zero (T0) to the values at 24, 48, or 72 hrs post-plating (Tn) as indicated.Growth curves were performed 4 to 7 days after final transductions when LCLs were >75% GFP positive by microscopy and in the log-phase of growth. FAC-sorted DLBCLs were rested for 2 days after sorting and prior to plating for growth curves, To assay growth, cells were split 1:2 in BJAB-conditioned complete media one day prior to plating and plated in triplicate or quadruplicate at 2 x 10^5 cells per mL in 24-well plates. Viable cells were counted by trypan blue exclusion using a hemacytometer at time points indicated.Cells were lysed in NP40-lysis buffer  NP40, 0.5 mM dithiothreitol (DTT)) and protein concentrations determined using the BCA protein assay kit (Thermo Scientific). 20 ug of total protein per lane was resolved on 10% Tris-glycine SDS-PAGE and transferred onto Immobilon PVDF membranes. Following blocking in 5% milk in TBS-Tween, blots were probed with primary antibodies to Grb2 , Malt1 , Plcg1 , beta-Actin , or Gapdh , then probed with horse-radish peroxidase (HRP) conjugated secondary antibodies (anti-rabbit IgG or anti-mouse IgG). Blots were developed using enhanced chemiluminescent substrate (Pierce) and exposed to film. Protein levels were quantified from scanned films using NIH ImageJ.NF-kappaB and AP1 activity was assayed using BJAB luciferase reporter cell lines. BJAB-NFkBLuc cells are previously described . To geneHEK293T cells plated in 96-well black-well plates were co-transfected with 20 ng of 3’UTR reporter and 250 ng of control vector (pLCE) or EBV miRNA expression vector ,17,32 usTotal RNA was extracted using TRIzol (Thermofisher). For detection of cellular or EBV transcripts, RNA was DNAse-treated and reversed transcribed using MultiScribe (Thermofisher) with random hexamers. Transcripts were detected using SYBR Green qPCR and oligonucleotides designed to amplify gene specific regions of ~200 bp. Primers for amplification of EBV transcripts BZLF1, BRLF1, BALF4, BNLF2a, and BHRF1 are described in . OligonuPAR-CLIP datasets for EBV+ BC1 cells and EBV B95-8 or wild-type LCLs are previously described ,18,32,60All luciferase and PCR data are reported as mean with standard deviations (S.D.) and values are derived from at least three independent experiments, unless otherwise stated, with technical replicates. Statistical significance was determined by paired Student’s t test, performed using Microsoft Excel 2010, and values p < 0.05 were considered significant.S1 FigA. and B. Western blot analysis of Malt1 or Grb2 levels in LCLs. Established LCLs are infected with EBV B95-8 2089 (WT) or BHRF1-2 miRNA mutant virus (D2). C-E. qRT-PCR analysis of EBV BHRF1-2 targets. RNA was isolated from established LCLs infected with wild-type EBV B95-8 2089 or BHRF1-2 miRNA mutant EBV (Donors 1–3) (n = 2). F-H. RNA was isolated from LCLBACD2 (n = 2), LCL-D2 (n = 3), or BJAB (n = 2) cells stably expressing GFP (pLCE) or the BHRF1-2 miRNAs. In C-H, cellular transcript levels were assayed by qRT-PCR. Values are normalized to GAPDH and reported relative to control cells (pLCE). Average expression values and standard deviations were calculated from two or three independent experiments. I. EBV miRNA expression in 293T cells (corresponds to miRNAs tested in (EPS)Click here for additional data file.S2 FigA. shRNAs stably expressed in BJAB cells reduce target RNA levels. BJAB cells were stably transduced with mCherry or mCherry-shRNA expressing lentiviruses. RNA was isolated and cellular transcripts were assayed by qRT-PCR. Values are normalized to GAPDH and reported relative to control cells (pLmCherry). Average expression values and S.D. were calculated from two independent experiments. B. shRNA knockdown of target genes in LCL-D2 see . RNA was(EPS)Click here for additional data file.S3 FigA. Growth curves of established LCLs at > 8 weeks post-infection. LCLs (derived from same donor) were generated with either wild-type (LCL-WT) or BHRF1-2 miRNA mutant (LCL-D2) EBV and maintained in log-phase in complete media containing 15% FBS. B and C. Proliferation of wild-type or BHRF1-2 miRNA mutant LCLs as determined by MTT assay ). A = Absorbance at 562 nm, T = time, n = 24, 72, or 96 hr as indicated. Values at Tn are normalized to the absorbance values at 0 hr (A-T0). D-F. Growth curves of LCL-D2, LCLBACD2, or BJAB transduced with control vector (pLCE) or the BHRF1-2 miRNA-expression vector (BHRF1-2). LCLs were split one day prior to initiating growth curves and plated in media containing 10% or 20% FBS as indicated. BJAB cells were grown in media containing 10% FBS. Cell counts were determined at days indicated using trypan-blue exclusion. For D-F., error bars represent S.D. of two to four experiments.(EPS)Click here for additional data file.S4 FigA-C. Growth curves of EBV B95-8 (SDLCL and LCL35) and wild-type (IBL-LCL3) LCLs following sponge inhibition of miR-BHRF1-2-5p. Cells in log phase were plated in BJAB-conditioned media mixed 1:1 with fresh RPMI-1640 containing 15% FBS and viable cell counts were determined at times indicated by trypan-blue exclusion. Cell growth rates  were calculated between 2 and 5 days post-plating using the following equation: ln(N1/N1) = k(t1-t2), where t = time and N = cell number. Experiments were performed in quadruplicate. D. and E. Control (pLCE-CXCR4s) and sponged (pLCE-BHRF1-2-5ps) SDLCL cells were transduced with mCherry or indicated shRNAs. Cell growth was determined by MTT assay. A = Absorbance at 562 nm, T = time, n = 24, 48, or 72 hr as indicated. Values at Tn are normalized to the absorbance values at 0 hr (A-T0). For D. n = 12 wells and for E., n = 14 wells. *p < 0.05 by Student’s t-test. F. EBV miR-BHRF1-2-5p levels in SDLCL cells expressing miR-BHRF1-2-5p sponge and shGRB2 compared to control cells. Levels were determined by Taqman qRT-PCR and values are relative to cellular miR-16. G. GRB2 expression in SDLCL cells expressing miR-BHRF1-2-5p sponge and shGRB2. Expression levels are normalized to GAPDH and reported relative to control (mCherry) cells. H. Sponge inhibition of miR-BHRF1-1-5p or miR-BART2-5p does not significantly impact LCL proliferation. Growth of control (pLCE-CXCR4s) and sponged SDLCL cells was determined by MTT assay; values at Tn (48 hr) are normalized to absorbance values at 0 hr (A-T0). n = 8 wells. I. EBV miRNA levels in sponged SDLCL cells from (H.). Levels were determined by Taqman qRT-PCR and values are relative to cellular U6. qPCR was run in duplicate.(EPS)Click here for additional data file.S5 FigTotal RNA was isolated from LCLs, DLBCLs , and BL  cell lines and assayed for viral gene expression. In A. and B., miRNA levels were determined by Taqman qRT-PCR (performed in duplicate). Values are normalized to cellular miR-16 and reported relative to MutuI cells. For C-E., levels of target viral genes were assayed in duplicate by qRT-PCR analysis. Expression levels are normalized to GAPDH and reported relative to EBV B95-8 SDLCL cells.(EPS)Click here for additional data file.S6 FigA. Detection of EBV lytic transcripts in reactivated MutuI cells. Cells were treated with 2.5 ug/mL anti-IgM for 22 hrs. Total RNA was collected and expression of EBV immediate-early , early , and late (BALF4) genes was assayed by qRT-PCR. Values are normalized to GAPDH and reported relative to mock treated cells. Averages and S.D. are from two independent experiments; qPCR was performed in duplicate. B. and C. Endogenous EBV BHRF1 or BART miRNA expression in MutuI cells treated with 2.5 ug/mL anti-IgM for 22 hrs. Values are normalized to cellular miR-16 and reported relative to mock treated cells. Averages and S.D. are from two independent experiments; qPCR was performed in duplicate.(EPS)Click here for additional data file.S7 FigA. BZLF1 is upregulated in Mutu I cells within 5 hrs after anti-IgM treatment. Mutu I cells were treated with 5 ug/mL anti-IgM for 5 or 48 hrs. Total RNA was harvested and levels of BZLF1, BRLF1, and GAPDH were assayed in duplicate by qRT-PCR. Values are normalized to GAPDH and reported relative to 5 hr anti-IgM treatment. B. Mutu I cells transduced with pLCE or sponge inhibitors for miR-BART2-5p or miR-BHRF1-1-5p were treated with 4 ug/mL anti-IgM for 5 hrs. Total RNA was harvested and assayed for BZLF1 by qRT-PCR. Values are normalized to GAPDH and reported relative to pLCE, anti-IgM treatment. C. miR-BART2-5p expression in anti-IgM treated, BART2-5p-sponged cells from (B.). miRNA levels were determined by qRT-PCR; values are normalized to cellular miR-16 and reported related to anti-IgM treated, pLCE control cells in (B.). PCR was performed in duplicate.(EPS)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Sphingolipids (SLs) have been implicated as potent regulators of the hepatitis B virus (HBV) life cycle. We investigated the SL biomarker potential regarding virologic endpoints in a prospective subgroup of patients with HBeAg-negative chronic HBV infection.From 2009–2016 98 patients with HBeAg-negative HBV infection were prospectively followed over four years. Clinical, laboratory and imaging data were evaluated annually. SLs were assessed in available serum probes via liquid chromatography coupled to tandem mass spectrometry.Of those 98 patients, 10 (10.2%) showed HBV reactivation, 13 (13.2%) lost HBsAg and 9 (9.1%) gained status of HBsAg-/HBsAb-coexistence, whereas 66 (67.3%) had no events. Within the four-year analysis sphingosine (p = 0.020), sphinganine (p<0.001), dhS1P (p<0.001), C16DHC (p<0.01) and C20Cer (p<0.001) showed a significant upregulation in patients without virologic events, C18Cer significantly decreased (p<0.001). At baseline decreased S1P-, dhS1P- and C16Cer-levels were observed in patients with upcoming status of HBsAg-/HBsAb-coexistence. S1P and dhS1P levels were elevated HBV genotype D infected patients.In a prospective cohort of patients with a HBeAg-negative HBV infection, serum SLs associated with the virologic course and HBV genotype D. Further studies are required to elucidate SLs as potential novel predictors of the course of HBeAg-negative HBV infection. Approximately 350 million people worldwide are chronically infected with the hepatitis B virus (HBV) . HBV is Sphingolipids (SLs) are complex bioactive molecules which play an important role in the pathophysiology and pathogenesis of multiple diseases due to their substantial implication in cellular homeostasis. As in recent reviews well summarized, SL metabolic pathways are highly interconnected , 12 Fig. SLs regFibroScan at the same time. In this study, we investigated all available serum samples of patients who had either a virologic event  or at least three to four annually follow-up visits, all meeting the following criteria: Age 18–79, HBV treatment-naïve, BMI < 35, documented TE at baseline, genotypes A or D and B, C and E only with virologic event  were routinely stored. This great multicentre prospective study includes twelve participating centres  and compic event . The cliHBV DNA was tested in plasma samples using COBAS AmpliPrep/COBAS TaqMan HBV DNA assay, version 2.0 . According to manufacturer’s information the lower limit of quantification of the CAP/CTM HBV assay is 20 IU/mL. High-performance liquid chromatography tandem mass spectrometry was performed per protocol as recently described .Statistical calculations were performed by using BiAS software for Windows . Analysis for the presented box plots was performed with GraphPad Prism for Windows . Longitudinal non-parametric calculations in patients’ parameters were performed using Wilcoxon-matched pairs analyses, two-groups non-parametric calculations were done by Mann-Whitney-U test, multiple groups non-parametric calculations were performed by Kruskall-Wallis test. Rank correlations were calculated using Spearman and Kendall method. P-values <0.05 were considered statistically significant.The study was performed in accordance with the Declaration of Helsinki. All patients had signed a written informed consent from the original multicentre ALBATROS-trial (NCT01090531) before study inclusion. It included the approval for the asservation of serum samples for future analyses. The focus on SL parameters was additionally approved by the ethics committee of the principal trial centre the Ethikkommission des Fachbereichs Medizin of the Johann Wolfgang Goethe-Universität Frankfurt (file no. 4/09 with project no. SGI.05-2009). Standards of good clinical practice were followed during patients care and study conduct at all times.According to the above described criteria 98 patients with chronic, HBeAg-negative, inactive chronic HBV infection and not considered for antiviral therapy were included. Among these 64 (65%) were infected by genotype D, 15 (15%) by genotype A, 2 (2%) by each genotype B and C and 1 patient (1%) was infected by genotype E. In 14 patients (14%) no genotype was determinable due to non-quantifiable viral load. As depicted in Serum concentrations of various SL metabolites were compared between patients with and patients without virologic events. In sub-group analyses , S1P, dhS1P and C16Cer were significantly down-regulated in patients with upcoming simultaneous HBsAg- and HBsAb-positivity . These dAdditionally, S1P (p = 0.012) and dhS1P (p = 0.043) were up-regulated in patients with genotype D compared to all other HBV genotypes . In a muBy using Spearman’s rank correlations, we identified associations between serum SL levels between demographic and biochemical patient data. Significant baseline correlations were seen between nearly all serum ceramide (Cer) and dihydroceramide (DHC) parameters and cholesterol (Chol) levels and some triglyceride (TG) levels. Some SL parameters correlated with age  and BMI . No baseline correlations were found between serum SL parameters and transaminases, HBV viral load, alpha-fetoprotein (AFP) or stage of fibrosis determined by TE .We further analysed SL metabolites of the named ten patients who suffered from HBV reactivation during the trial. Using Wilcoxon-matched-pair tests we compared SL concentrations the last visit before HBV reactivation with SL concentration the first visit after HBV reactivation. We could only observe significant SL changes in C14Cer and C16Cer Click here for additional data file.S2 FigOur patients show no significant progression or regression of liver stiffness in yearly transient elastography measurements. Bars depict mean +/- standard mean error.(TIF)Click here for additional data file.S3 FigCourse of (A) sphingosine-1-phosphate, (B) C24Cer, (C) C16Cer, (D) C14Cer, (E) C24:1Cer, (F) C18DHC, (G) C24DHC and (H) C24:1DHC in patients with no virologic events from baseline (BL) over a follow-up (FU) period of four years (1–4). Here depicted are all sphingolipid parameters without significant changes over time. Bars depict mean +/- standard mean error.(TIF)Click here for additional data file.S4 FigThere are no significant differences in the here listed (A-K) sphingolipid (SL) parameters. In patients with upcoming hepatitis B antigen (HBsAg) + hepatitis B antibody (HBsAb) status, concentrations of (H) C16DHC and (I) C18DHC could not be quantified. Bars depict mean +/- standard mean error.(TIF)Click here for additional data file.S5 FigAlterations of (A) C16DHC, (B) C18DHC, (C) C24DHC and (D) C24:1DHC in all patients with hepatitis B (HBV) reactivation. Sphingolipids are compared at last visit before and next visit after HBV reactivation. There are no significant dynamics in all shown dihydroceramides (A-D). Statistically significant differences are indicated by asterisks. "*"p<0.05, "**"p<0.01, "***"p<0.001. Bars depict mean +/- standard mean error.(TIF)Click here for additional data file.S6 FigAlterations of (A) sphinosine, (B) sphinganine, (C) sphingosine-1-phosphate and (D) sphinganine-1-phosphate in all patients with hepatitis B (HBV) reactivation. Sphingolipids are compared at last visit before and next visit after HBV reactivation. There are no significant dynamics in sphingosine and sphinganine  or their phosphate derivates . Statistically significant differences are indicated by asterisks. "*"p<0.05, "**"p<0.01, "***"p<0.001. Bars depict mean +/- standard mean error.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Correction to:Cell Death Disease9, 455 (2018); 10.1038/s41419-018-0484-2; published online 19 April 2018.Since publication of this article, the authors found a mistake in the drawing Fig. The authors apologise for any inconvenience caused.


Sulfuricurvum sp. strain IAE1 was assembled and annotated from the metagenome of an anaerobically grown 4-chlorophenol-degrading consortium. The draft genome size is 2,338,235 bp with a G+C content of 52.6%.The draft genome sequence of  Sulfuricurvum sp. strain IAE1 was assembled and annotated from the metagenome of an anaerobically grown 4-chlorophenol-degrading consortium. The draft genome size is 2,338,235 bp with a G+C content of 52.6%.The draft genome sequence of  Sulfuricurvum, are abundant in various environments and have essential roles in global sulfur cycling (Sulfuricurvum strains are capable of degrading toxic organic compounds such as polycyclic aromatic hydrocarbons and polychlorinated biphenyl (Sulfuricurvum spp. involved in anaerobic 4-chlorophenol (4-CP) degradation.Sulfur-oxidizing bacteria, such as  cycling , 2. Somebiphenyl . A metaghttps://1ofdmq2n8tc36m6i46scovo2e-wpengine.netdna-ssl.com/wp-content/uploads/2014/02/JGI-Bacterial-DNA-isolation-CTAB-Protocol-2012.pdf). The library was prepared using a NovaSeq 6000 S4 kit, and sequencing was performed on a NovaSeq 6000 sequencer . The sequencing yielded a total of 158,000,142 reads at a 2 × 150-bp paired-end read length. Sequences were trimmed using Trimmomatic version 0.36 (Sulfuricurvum strain named Sulfuricurvum sp. strain IAE1 was binned and assembled at an average 109.9× coverage using the Rapid Annotations using Subsystems Technology (RAST) Binning Service (RBS) developed by the PATRIC Bacterial Bioinformatics Resource Center (http://patricbrc.org)  at the Xi River  using shovels and sealable glass containers. All sampling apparatuses were autoclaved before use. The sediment temperature and pH were 20°C and 6.6, respectively, at the sampling time. The 4-CP-degrading consortia were grown in 160-ml serum bottles filled with 100 ml defined mineral medium (Sulfuricurvum kujiense strain DSM 16994 (GenBank accession number NR_074398) and an environmental Sulfuricurvum 16S rRNA gene clone, D2CL (EU498374), with sequence similarities of 97.8% and 99.9%, respectively (soxABXYZ gene cluster encoding the subunits of a thiosulfate-oxidizing multienzyme complex (Sulfuricurvum sp. strain MLSB (JQGL00000000) and Sulfuricurvum kujiense strain DSM 16994 (CP002355). Arsenate reductase gene arsC, arsenite efflux gene arsB, and regulatory gene arsR, as well as genes encoding cobalt/zinc/cadmium resistance proteins CzcCD, were also annotated, and such features may enable strain IAE1 to tolerate and detoxify heavy metals.BLASTn analysis indicated that strain IAE1’s 16S rRNA gene is closely related to that of ectively . Strain  complex , 12 and  complex , 13, 14.Sulfuricurvum sp. strain IAE1 has been deposited under GenBank accession number SLTI00000000. The whole-genome shotgun sequencing project has been deposited at DDBJ/ENA/GenBank under BioSample accession number SAMN11066887 and BioProject accession number PRJNA525737. The raw sequences have been deposited in the Sequence Read Archive (SRA) under the accession number SRS4850623.The draft genome sequence of


We have previously reported that long-term treatment of beta cells with interleukin-6 (IL-6) is pro-apoptotic. However, little is known about the regulatory mechanisms that are involved. Therefore, we investigated pro-apoptotic changes in mRNA expression in beta cells in response to IL-6 treatment. We analyzed a microarray with RNA from INS-1 beta cells treated with IL-6, and found that TNF-α mRNA was significantly upregulated. Inhibition of TNF-α expression by neutralizing antibodies significantly decreased annexin V staining in cells compared with those treated with a control antibody. We identified three microRNAs that were differentially expressed in INS-1 cells incubated with IL-6. In particular, miR-181c was significantly downregulated in IL-6-treated cells compared with control cells and the decrease of miR-181c was attenuated by STAT-3 signaling inhibition. TNF-α mRNA was a direct target of miR-181c and upregulation of miR-181c by mimics, inhibited IL-6-induced increase in TNF-α mRNA expression. Consequently, reduction of TNF-α mRNA caused by miR-181c mimics enhanced cell viability in IL-6 treated INS-1 cells. These results demonstrated that miR-181c regulation of TNF-α expression plays a role in IL-6-induced beta cell apoptosis. Prolonged and uncontrolled activation of inflammation is a hallmark of many diseases, such as obesity-associated metabolic disorders including type 1 and type 2 diabetes [Interleukin (IL)-6 is a pleiotropic cytokine that is associated with several immunoinflammatory and autoimmune diseases such as systemic lupus erythematous, rheumatoid arthritis and diabetes ,4,5 and Previously, we found that chronic treatment of beta cells (INS-1) with IL-6 decreased cell viability. Additionally we found that signal transducer and activator of transcription-3 (STAT-3) signaling was involved in IL-6-induced beta cell apoptosis . To idenTumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine with pleiotropic effects that are mediated through cell surface receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). TNFR1 has a death domain in its cytoplasmic region . TNFR1-lBCL2 (B-cell lymphoma 2) [MicroRNAs (miRNAs) are a group of short, non-coding RNAs (18 to 25 nucleotides) that may bind to complementary target sites in the 3′-untranslated regions (UTR) of mRNAs. These can inhibit gene expression post-transcriptionally, or can induce protein degradation in both animals and humans ,14. Manyphoma 2) . TherefoTo identify mRNAs and miRNAs associated with IL-6-induced beta cell apoptosis, we investigated changes in gene expression in IL-6-treated INS-1 cells. In this study, we found that TNF-α expression was highly upregulated in IL-6-treated INS-1 cells, and that miR-181c contributed to IL-6-induced beta cell apoptosis through regulation of TNF-α expression.To investigate induction of apoptosis by chronic IL-6 treatment, cell viability and annexin V- stained INS-1 cells were measured after 48 h treatment. As shown in 2 profiler PCR array by comparing IL-6-treated cells with control, untreated cells. We observed significant upregulation or downregulation of many genes (TNF)-α, toll-like receptor 2 (Tlr2), lympotoxin beta (Ltb), baculoviral IAP repeat-containing 3 (Birc3), Suppressor of cytokine signaling 3 (Socs3), Caspase 4, apoptosis-related cysteine peptidase (Casp4), insulin-like growth factor 1 receptor (IGF1R), and plasminogen (Plg) were the highest (>four-fold difference in expression) compared with control , anti-apoptotic (IGF1R and Birc3) or involved in IL-6-mediated receptor signaling (Tlr2 and Socs3).To identify apoptotic mechanisms activated in response to IL-6 treatment, differences in mRNA levels of apoptosis-related genes were examined using a custom RTny genes . A total control . These gAs TNF-α levels showed the greatest increase (90-fold) during PCR array analysis, we tested the mRNA expression of TNF-α using qRT-PCR. We found that TNF-α mRNA levels were gradually increased by IL-6 treatment after 6, 12, and 24 h A. To inv2 miRNA PCR array and miRDB (www.mirdb.org) prediction algorithm. We found that miR-101a, -122, and -181c were significantly downregulated about two-fold in IL-6-treated cells compared with control cells. To evaluate these results, we quantified miRNA expression using qRT-PCR. Among the three miRNAs, only the level of miR-181c was significantly decreased in INS-1 cells exposed to 20 ng/mL of IL-6 compared with untreated cells  production was involved in IL-6-induced beta cell apoptosis . ChappelRecently, many miRNAs that are involved in beta cell apoptosis and gene expression were protective against cytokine-mediated cell death. In pancreatic islet cells from type 2 diabetic patients, miR-124a was highly expressed, and its silencing in MIN-6 cells resulted in increased expression of genes involved in insulin secretion, such as Mtpn, Foxa2, Sirt1, and NeuroD1 . MoreoveMiR-181c and STAT-3 expression levels were inversely correlated in liver biopsy specimens . In MCF1Although several targets of miR-181c have been identified including protein kinase C (PKC) delta, transforming growth factor beta (TGFB)-I, NAD-dependent deacetylase sirtuin (SIRT)-1, syntaxin 3, and homeobox protein Hox-A11 ,43,44, tBeside TNF-α/miR-181c, several (up- and downregulated-) genes and miRNAs were observed in IL-6 treated cells, the involvement of additional apoptotic mechanisms will be required. In conclusion, our data suggests that miR-181c regulation of TNF-α expression is an important mechanism involved in apoptotic pathways upon IL-6 treatment, and that upregulation of miR-181c expression may lead to inhibition of IL-6-induced apoptosis in beta cells . These f2 profilerTM PCR arrays were purchased from SABioscience Corp. . The following reagents were purchased from the indicated suppliers: Annexin V-PE apoptosis detection kit ; RT2 first strand kit, RT2 qPCR master-mix, and miRNA-mimics . All other chemical reagents were from Sigma Aldrich , Invitrogen , or Takara .The recombinant mouse IL-6 and TNF-α blocking antibody (MAB 510) was prepared by R&D systems . RTThe rat insulinoma cell line, INS-1 (passage ~20 to 30), was grown in Roswell Park Memorial Institute (RPMI)-1640 medium containing 11.1 mmol/L glucose. The culture medium was supplemented with 10% fetal bovine serum (FBS), 2 mmol/L: Glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin. For experiments, INS-1 cells were incubated with 20 ng/mL IL-6 for indicated time points.Cells were treated with RPMI media containing 3--2,5-diphenyltetrazolium bromide  for 4 h at 37 °C. Supernatants were discarded and isopropanol was then added. Cell viability was measured by absorbance at 570 nm using a microplate reader .2 first strand kit (Qiagen Ltd.), and cDNA sample was mixed with RT2 SYBR Green ROXTM qPCR Mastermix (Qiagen Ltd.). Real-time PCR quantification was performed on an ABI 7900 Real-time PCR system  using an RT2 profiler custom PCR array consisting of a panel of 41 genes involved in apoptotic signaling, including a house-keeping gene (β-actin). Data were analyzed using the integrated web-based automated software for RT2 profiler PCR Array Data analysis  available through SABiosciences. Fold changes in gene expression were calculated using the ΔΔCt method, and β-actin was used for normalization of the results.Total RNA extraction and purification were performed using the RNeasy MinElute kit (Qiagen Ltd.), according to the manufacturer’s instructions. DNase enzyme digestion was performed to exclude genomic DNA contamination. RNA was quantified using NanoDrop 1000 spectrophotometer  and the integrity of the sample was assessed in a 2100 Bioanalyzer . Each RNA sample was converted into cDNA using the RTTM RTase (Takara). Quantitative real-time PCR (qRT-PCR) analysis was performed using SYBR master mix  using the ABI 7900 Real-time PCR system according to the protocols provided by the manufacturer (Applied Biosystems). The sequences of the primer pairs are as follows: TNF-α (forward) 5′-CAG CCG ATT TGC CAT TTC A-3′ and (reverse) 5′-AGG GCT CTT GAT GGC AGA GA-3′; and cyclophilin (forward) 5′-GGTCTTTGGGAAGGTGAAAGAA-3′, and (reverse) 5′-GGTCTTTGGGAAGGTGAA AGAA-3′. The relative mRNA transcript levels were calculated according to the 2Δ–CT method, in which ΔCT represents the difference in threshold cycle values between the target mRNA and the cyclophilin internal control.Extracted RNA was treated with DNase I and reverse-transcribed to single-strand cDNA using oligo(dT) primer with PrimeScript2 miRNA first strand kit (Qiagen). Real-time quantitative PCR analysis  was performed using SYBR master mix using the ABI 7900 Real-time PCR system according to the protocols provided by the manufacturer (Applied Biosystems). Primer sequence was as follows: miR-101a , miR-122a , and miR-181c . The relative mRNA transcript levels were calculated according to 2Δ–CT method, in which ΔCT represents the difference in threshold cycle values between the target miRNA and Rnu6, which was used as an internal control.Total RNA from INS-1 cells treated with 20 ng/mL IL-6 was extracted using the Qiagen miRNA isolation kit. Extracted miRNA was reverse transcribed to single-strand cDNA with RTEarly apoptotic cells were determined using an Annexin V-PE apoptosis detection kit, according to the manufacturer’s instructions. Briefly, following IL-6 treatment, cells were harvested and washed twice with PBS. The cells were then suspended in 1× binding buffer, and annexin V-PE was added. After incubation for 15 min, stained cells were analyzed by flow cytometry  using CELLQuest Pro software .INS-1 cells were transfected with miR-181c  mimics or miR-control using Lipofectamine 2000 (Invitrogen) as per the manufacturer’s protocol. After 24 h transfection, the medium was replaced with fresh medium containing 20 ng/mL IL-6.HEK293 cells were transfected with pMir-TNF-α-3′-UTR , pMir-TNF-α-3′-UTR mutant , and miR-181c mimic using Lipofectamine 2000. Cells were collected 48 h after transfection and luciferase activity was measured using a luciferase reporter assay system .t-test was used for analysis of the 2 groups. p < 0.05 was considered to indicate a statistically significant difference between values.Data are presented as mean ± standard error of mean (SEM) of three independent experiments. Data were analyzed using analysis of variance (ANOVA) followed by post hoc analysis using the Tukey range test . An unpaired 2-tailed


Here, we generated mice with a specific deletion of the glucocorticoid receptor (GR) in Foxp3+ Treg cells. Remarkably, while basal Treg cell characteristics and in vitro suppression capacity were unchanged, Treg cells lacking the GR did not prevent the induction of inflammatory bowel disease in an in vivo mouse model. Under inflammatory conditions, GR-deficient Treg cells acquired Th1-like characteristics and expressed IFN-gamma, but not IL-17, and failed to inhibit pro-inflammatory CD4+ T cell expansion in situ. These findings reveal that the GR is critical for Foxp3+ Treg cell function and suggest that endogenous GC prevent Treg cell plasticity toward a Th1-like Treg cell phenotype in experimental colitis. When equally active in humans, a rationale is provided to develop GC-mimicking therapeutic strategies which specifically target Foxp3+ Treg cells for the treatment of inflammatory bowel disease.Activation of the immune system increases systemic adrenal-derived glucocorticoid (GC) levels which downregulate the immune response as part of a negative feedback loop. While CD4 Peripheral Treg cells (pTreg cells) are induced from peripheral CD4+Foxp3− T cells in the presence of TGF-beta, however, the pool size and function of these pTreg cells is not fully characterized, mainly due to the lack of useful markers to discriminate tTreg from pTreg cells (Regulatory T cells (Treg cells) expressing the transcription factor Foxp3 maintain immune homeostasis by limiting antigen-specific immune responses and sustaining tolerance to self-antigens . Most Treg cells .in vivo Treg cell adaptation are not well-understood  on dendritic cells, secretion of inhibitory cytokines or metabolic disruption of target cells. Beyond that, Treg cells seem to have the capacity to adjust their suppressive mechanism(s) to a particular immune or inflammatory context, although the signals driving derstood .The original view that tTreg cells are terminally differentiated and phenotypically stable has been recently questioned. Some Treg cells may lose Foxp3 expression in autoimmune disease (“ex-Foxp3” cells), others, while maintaining Foxp3 expression, acquire a certain degree of plasticity which is illustrated by secretion of pro-inflammatory cytokines and reduced suppressive function . The mollck promoter to drive expression of Cre recombinase for conditional deletion of the GR, CD8+ cytotoxic T cells, CD4+ T helper cells, and Foxp3+ Treg cells were GR-deficient. Treg cell development, steady-state homeostasis and function may be affected by GC, although reports are controversial. Administration of GC has been shown to increase both the proportion and number of murine CD4+CD25+Foxp3+ Treg cells in peripheral lymphoid organs (in vitro (Glucocorticoids (GC) are best-known for their successful clinical usage as anti-inflammatory and immunosuppressive agents, despite their high potential for serious side effects. While the potency of (synthetic) GC as negative regulators of immune and inflammatory effector molecules at higher doses is well-documented, the effects of endogenous GC on the immune response and T cells in particular are much less clear. GC suppress T cell activation, both indirectly by inhibiting dendritic cell function and directly by inhibiting TCR signaling . T cell-in vitro . In contin vitro , 11. Likin vitro . Human Tin vitro , systemiin vitro  and rheuin vitro .While effects of exogenous GC on Treg cells are obvious but controversial, it is not known whether endogenous GC regulate Treg cell homeostasis, both under steady state and inflammatory conditions. Lck-Cre GRfl/fl mice that lack the GR in all T cells, reportedly have reduced numbers of Treg cells in the thymus and periphery, but Treg cell function was not tested . Moreove+ Treg cells by crossing GRfl/fl . This phenotype was associated with the acquisition of Th1 cell-like features in GR-deficient Treg cells. These data suggest that endogenous GC stabilize Treg cell fate and function under inflammatory conditions and provide a rationale for the development of GC therapy for IBD that specifically targets Treg cells and expectedly reduces the strong side-effects of these hormones.We therefore generated mice with a specific deletion of the GR in Foxp3 GRfl/fl  with Fox GRfl/fl . Remarka+ Treg cells  developed normal and did not show any signs of disease. Lack of GR in Foxp3+ Treg cells was confirmed at the protein level both in spleen (Cd28), but not others (R26-RFP), has been reported , Foxp3-Cre and Foxp3-Cre GRfl/fl mice (Nr3c1 (encoding the GR) by CD4+CD25− Tcon cells and CD4+Foxp3+ Treg cells were quantified by qPCR. Splenic Treg cells from heterozygous Foxp3-Cre GRwt/fl mice expressed Nr3c1 at approximately half of control Treg cells from Foxp3-Cre mice  and 50% of Treg cells that use the Foxp3-Cre allele . In spleen, but not in thymus, we observed a moderate competitive disadvantage of Treg cells expressing the Foxp3-Cre allele  and co-cultured with different Treg cell numbers derived from Foxp3-Cre or Foxp3-Cre GRfl/fl mice. Proliferation of Tcon cells, assessed after 3 days, was potently suppressed by Treg cells, however, Foxp3-Cre and Foxp3-Cre GRfl/fl Treg cells exhibited an equal inhibitory capacity  prevented, as expected, the development of disease. Strikingly, Treg cells derived from Foxp3-Cre GRfl/fl mice were largely ineffective under these experimental conditions  or those co-injected with Tcon cells plus Treg cells from GR-deficient Treg cell mice  Treg cells, which reportedly control in vivo lymphocyte proliferation, and GITRlowPD-1lowCD25low (Triplelow) Treg cells, which have been shown to limit colitis  would clarify whether Treg cell-derived IFN-gamma is causal for the dysfunction of GR-deficient Treg cells in experimental colitis.The proportion of functionally impaired GR-deficient Treg cells producing IFN-gamma more than doubled compared to control Treg cells but retained Foxp3, GITR and CD25 expression, whereas CTLA-4 was higher. IFN-gamma was shown by others to be involved in a functional defect of IFN-gammang Foxo1 . Foxo1-deg cells . In contpression . Hence, Foxp3 mRNA in CD4+ T cells of asthmatic patients  and have been granted by the Bundesministerium für Bildung, Wissenschaft und Kultur (bm:bwk).GRfl/fl mice  were breter mice  were purCell suspensions were prepared in KDS-BSS buffer containing 10% FCS. Cells were stained with combinations of the following antibodies for 20 min at 4°C: anti-CD4-PerCP/Cy5.5 (clone RM4-5) and anti-PD-1-PE  ; anti-CD8-PECy7 or anti-CD8-AF647 (clone 53-6.7), anti-B220-APC/Cy7 (clone RA3-6B2), anti-CD25-PE (clone 3C7) or CD25-BV421 (clone PC61), GITR-PE/Cy7 (clone YGITR765), CD45Rb-AF647 (clone C363-16A), LAP-PE (clone TW7-16B4), and CD223(LAG-3)-BV421 (clone C9B7W) ; CD62L-PE (clone MEL-14) and CD44-BV510 (clone IM-7) . DAPI and Annexin-V  were used to quantify or gate out apoptotic or dead cells.For Foxp3 intracellular staining Foxp3/Transcription Factor Buffer set and anti-Foxp3-eF660 (clone FJK-16s) (both from eBiosciences) were used according to the manufacturer's instructions. For GR intracellular staining we used BD Cytofix and BD Cytoperm reagents  and stained with anti-GR (clone D6H2L) , followed by a secondary antibody ). The same buffer set was used for cytokine staining using anti-IFN-gamma-PE (clone XMG1.2), IL-17-AF647 (clone TC11-18H10.1), anti-CTLA-4-PE , anti-IL-10-PE (clone JESS-16E3) and anti-Helios-APC .+Foxp3−(GFP−)CD25−CD45RBhigh cells were sorted from spleen and/or mesenteric lymph nodes from Foxp3-GFP reporter mice. Memory T (Tmem) cells (CD4+Foxp3−(YFP−)CD44+CD62L−CD45RBhigh) were sorted from splenocytes from Foxp3-YFP-Cre mice. CD4+Foxp3+(YFP+)CD25+CD45RBlow Treg cells were isolated from Foxp3-YFP-Cre and Foxp3-YFP-Cre GRfl/fl mice. Cell sorting was performed using a FACSAria III cell sorter (Becton Dickinson) and purity of isolated cell populations was routinely at least 98%.To obtain naïve conventional T cells (Tcon), CD46cells/mL) was incubated in flat bottom 96-well plates with corticosterone  at 125 or 625 nM for 48 h and then analyzed for cell death as decribed  (eBioscience) and cultured together with irradiated (30 Gy) splenocytes (8 × 105 cells/mL) in RPMI complete medium in 96-well round bottom plates. To induce cell proliferation, anti-CD3  0.5 μg/mL was added to the medium. In the indicated cases, different Treg cell concentrations were added to obtain Treg:Tcon ratios of 1:1, 1:2, 1:5, and 1:10. Cells were incubated for 72 h at 37°C and 5% CO2 and then analyzed for cell proliferation by Flow cytometry. For cytokine staining experiments, splenocytes were stimulated with 50 ng/mL PMA (Fluka Biochemika) and 1 mg/mL Ionomycin (Sigma) for 4 h. During the last 3 h of cell culture Monensin (Biolegend) was added.For the GC sensitivity test, single cell suspension of splenocytes  and cDNA was synthesized using iScript cDNA Synthesis Kit , according to the manufacturer's instructions. Real time PCR was performed using the following TaqMan Gene Expression Assays: GR  and Actin-beta  and Luminaris Color Probe Master Mix . Quantitative RT-PCR was analyzed using the StepOnePlus system  according to the manufacturer's instructions. The results were normalized to Actb expression and evaluated using the ΔΔCt relative quantification method.Total RNA was isolated from 1 × 10For detection of ANA we used Kallestad HEp-2 cell line 12-well slides from Bio-Rad . Serum samples were diluted 1:50 and 1:100 and incubated on the slides according to the manufacturer's instructions. Fluorescence-labeled antibody AF488 donkey anti-mouse  was used as a secondary antibody and slides were analyzed using a fluorescence microscope. Serum samples from MRL/lpr mice were used as positive controls for ANA detection.+Foxp3−CD25−CD45RBhigh Tcon cells were sorted from congenic C57BL/6 Foxp3-GFP mice and injected i.p. into 6 to 15-weeks-old C57BL/6 RAG1−/− immunodeficient recipients (3 × 105 cells/mouse). 1.5 × 105 Foxp3-YFP-Cre or Foxp3-YFP-Cre GRfl/fl Treg cells (CD4+Foxp3+CD25+ CD45RBlow) were co-injected i.p. where indicated. Mice were monitored every second day for wasting disease. Mice were sacrificed either when having lost >15% of their initial body weight or 4 weeks after cell transfer.CD4Samples of mid-colon were fixed in buffered 4% formalin solution. Three millimeter paraffin-embedded sections were cut and stained with hematoxylin and eosin. Tissues were evaluated semi-quantitatively and assigned a grade of 0 to 4 in a blinded fashion. Grade 0: no changes observed, grade 1: discrete increased inflammatory cells in the lamina propria with granulocytes in the lamina epithelialis, grade 2: as grade 1 with scattered erosions of the mucosa, grade 3: increased inflammatory cells in the lamina propria and scattered crypt abscesses, grade 4: all signs of grade 3 plus more than 3 crypt abscesses per colon circumference in the scanning magnification.5 cells/subset) were resuspended in Laemmli sample buffer and heated in boiling water for 5 min. Total proteins were loaded on 10% Bis-Tris acryl-amide gels and blotted on AmershamTM HybondTM-ECL nitrocellulose membranes . Rabbit anti-mouse GR (clone D6H2L)  and rabbit anti-mouse AKT  were used for protein detection. All primary antibodies were diluted in 5% BSA in PBST and blots were incubated overnight at 4°C.Cellular subsets  according to the manufacturer's instructions.+CD25−CD45RBhigh conventional T cells as well as CD4+Foxp3+CD25+ Treg cells sorted from WT Foxp3-GFP reporter, Foxp3-YFP-Cre mice and Foxp3-YFP-Cre GRfl/fl mice using the DNeasy blood and tissue kit . Bisulfite modification was performed using the EZ DNA Methylation-Gold Kit (ZymoResearch). MethyLight PCR analysis and the calculation of the percentage of methylated reference (PMR) were done as described previously (Genomic DNA was isolated from sorted CD4eviously , 53. Twot-test or two-way ANOVA test, where appropriate. A chi-square test was used to test for differences between groups regarding prevalence of ANA. P ≤ 0.05 were considered to indicate statistically significant differences. nsp ≥ 0.05; *p < 0.05; **p ≤ 0.01; ***p < 0.001; and ****p < 0.0001.Estimation of statistical differences between groups was carried out using the unpaired Student's All datasets generated for this study are included in the manuscript and/or the supplementary files.LR-R designed and performed most experiments, statistical analysis, prepared figures, and wrote paper. ST designed and performed experiments. LvR, MT, and HF performed experiments. MD performed histopathological assessment. HR interpreted data and wrote sections of the paper. AV designed research, interpreted data and wrote sections of the paper. DT and GW designed research, interpreted data, wrote paper, and conceived study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


S)-hydroxyheptadecatrienoic acid (12-HHT) is an abundant fatty acid, it is long considered a byproduct of thromboxane A2 production. We identified a leukotriene B4 receptor 2 (BLT2)-specific agonistic activity in lipid extracts from rat small intestine, and mass spectrometric analysis of partially purified lipids containing BLT2 agonistic activity revealed that 12-HHT is an endogenous ligand of BLT2. In a dextran sulfate sodium (DSS)-induced inflammatory colitis model, BLT2-deficient mice exhibited enhanced intestinal inflammation, possibly due to impaired epithelial barrier function. In a skin wound healing model, BLT2-deficient mice exhibited delayed wound healing via dampened keratinocyte migration. BLT2 also accelerates corneal wound healing, and eye drops containing a non-steroidal anti-inflammatory drug (NSAID) inhibit the production of 12-HHT, resulting in delayed corneal wound healing. Furthermore, BLT2 is expressed in pulmonary epithelial type II cells and vascular endothelial cells in the mouse lung, and BLT2-deficient mice are more susceptible to lung damage by pneumolysin. In this review, we summarize the identification and characterization of 12-HHT as a ligand for BLT2 and discuss recent research on the physiological and pathophysiological roles of the 12-HHT-BLT2 axis. Some side effects of NSAIDs such as delayed wound healing may be caused by reduced 12-HHT production rather than diminished production of prostaglandins.Although 12( In . In 9]. + cells .Fig. 3Ro+/CD11blow splenocytes produce 12-HHT following treatment with platinum analogs, which mediates chemotherapy resistance. Interestingly, genetic loss or chemical inhibition of BLT2 prevents 12-HHT-mediated resistance [4-BLT2-dependent reactive oxygen species (ROS) promotes anti-apoptotic, invasive, and metastatic phenotypes in cancer cells [Furthermore, 12-HHT-BLT2 signaling is reported to be involved in chemotherapy resistance. F4/80sistance . Combinesistance . We and sistance –29. Geneer cells –33, suggFor a long time, 12-HHT was considered merely a byproduct of thromboxane biosynthesis, and a biomarker of COX activation. However, we discovered that 12-HHT is an endogenous ligand of BLT2 using unbiased ligand screening and, with others, revealed that the 12-HHT-BLT2 axis mediates various biological functions including the epithelial barrier, wound healing, immunosuppression, and lung protection in vivo. Some side effects of NSAIDs, such as delayed wound healing, may be caused by reduced 12-HHT production rather than diminished PG production.


Internal translational initiation of the mRNA encoding the Arf tumor suppressor yields an N-terminally truncated small Arf protein (smArf) that lacks amino acid residues required for Mdm2 binding and p53 activation. Here, we report that female, but not male, mice engineered to produce only smArf in lieu of the full-length Arf protein retain residual, sexually dimorphic tumor suppressive activity. Arf; human p14ARF) encoded by the Cdkn2A (Ink4a/Arf) tumor suppressor complex inhibits the Mdm2 E3 ligase to activate p53. A conserved in-frame methionine codon  initiates translation to produce a truncated, unstable small mitochondrial polypeptide (smArf) in addition to the nuclear full-length, protein [smArf protein lacks amino acid residues required for interacting with Mdm2 and is overtly deficient in triggering p53-dependent tumor suppressor activity, it surprisingly rescues focal developmental defects observed in Arf-null mice that synthesize neither p19Arf nor p15smArf; conversely, mice engineered to produce only a mutant p19Arf-M45A protein  retain p53-dependent tumor suppressor activity but continue to exhibit the fully penetrant developmental ocular and spermatogenesis defects seen in Arf-null animals [The alternative reading frame protein  as neonates  and observed for tumor development for one-year thereafter. Males of both strains and Arf-null females developed tumors with mean latencies of 16-17 weeks, as previously reported for Arf-null mice of either sex [smArf females were unexpectedly more resistant to tumor formation   similar to that observed in Arf-null mice [ArfM45A mice  developed tumors throughout 60 weeks of observation. Therefore, although the p53-activating N-terminal domain of p19Arf is required for robust tumor suppression, p15smArf retains a residual tumor suppressive function manifested specifically in females. In short, irradiated smArf mice exhibit sexually dimorphic tumor suppressor activity.Syngeneic C57BL/6 mice engineered to produce either p15ther sex . In star) Figure . RegardlArf-null or Trp53-null pro-B cells, pro-B cells cultured from smArf mice are capable of continuous cytokine-dependent self-renewal. When transduced with retroviral vectors encoding the Bcr-Abl kinase, the IL7-dependence of these cells is abrogated, and they initiate acute lymphoblastic leukemia (ALL) when infused intravenously into unconditioned, syngeneic wild type  recipient mice [Arf-null donor cells expand ~10-fold in vivo in syngeneic WT animals over 3-day intervals. Under these conditions, healthy C57BL/6 mice receiving only 20 Arf-null, Bcr-Abl+ donor pro-B cells succumb to lethal ALL within 30 days of infusion, whereas mice receiving 2 × 105Arf+/+, Bcr-Abl+ donor pro-B cells develop no disease [Like Interleukin-7-dependent ent mice , 4. PrevArf-null or smArf male or female donor mice were infused into healthy syngeneic WT recipients of either sex  counts (each p < 0.05) at the time of sacrifice  mitochondria , 2, woulutophagy , althougroblasts . In cont cancers , smArf fher BRCA  or smArf


Bacteroidaceae and may have a key role in acidogenesis and acetogenesis steps during anaerobic biomass digestion.Here, we present the genome sequence and annotation of HV4-6-C5C, a bacterial strain isolated from a mesophilic two-stage laboratory-scale leach bed biogas reactor system. Strain HV4-6-C5C may represent a new genus of the family  Bacteroidaceae and may have a key role in acidogenesis and acetogenesis steps during anaerobic biomass digestion.Here, we present the genome sequence and annotation of HV4-6-C5C, a bacterial strain isolated from a mesophilic two-stage laboratory-scale leach bed biogas reactor system. Strain HV4-6-C5C may represent a new genus of the family  Q], >20; minimum length, >50 nucleotides [nt]) with BBTools v37.10, yielding 26.42 million paired-end sequences with a mean Q value of 33.24. Genome assembly was conducted with SPAdes v3.13.0 . For microbial isolation, BBL Columbia agar (BD Biosciences) supplemented with 5% laked horse blood was applied. DNA was extracted and purified using the Gentra Puregene Yeast/Bact. kit (Qiagen) and the NucleoSpin genomic DNA (gDNA) clean-up kit (Macherey-Nagel). We constructed a Nextera XT library from total genomic DNA and sequenced it using the Illumina NextSeq 500 platform (150-bp paired-end reads). Raw reads were filtered  under the strain designation DSM 104071. This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession number


S-nitrosylation has been established as one major signaling mechanism underlying cancer initiation and development, but its roles in PDAC  pathogenesis still remain largely unexplored. In this study, we identified 585 unique S-nitrosylation sites among 434 proteins in PDAC patients and PANC-1 cell line by a site-specific proteomics. Larger number of S-nitrosylated proteins were identified in PDAC tissues and PANC-1 cells than adjacent non-cancerous tissues. These S-nitrosylated proteins are significantly enriched in a multitude of biological processes associated with tumorigenesis, including carbohydrate metabolism, cytoskeleton regulation, cell cycle, focal adhesion, adherent junctions, and cell migration. Components of the pancreatic cancer pathway were extensively S-nitrosylated, such as v-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) and Signal transducer and activator of transcription 3 (STAT3). Moreover, NOS (NO synthase) inhibitor significantly repressed STAT3 S-nitrosylation in PANC-1 cells, which caused significant increase of STAT3 phosphorylation and PANC-1 cell viability, suggesting important roles of protein S-nitrosylation in PDAC development. These results revealed extensive protein S-nitrosylation associated with PDAC pathogenesis, which provided a basis for protein modification-based cancer diagnosis and targeted therapy.NO (nitric oxide)-mediated protein  Globally, PDAC remains one of the major causes of cancer death, featured by poor prognosis and a five-year survival rate of less than 8%3. Although a panel of genetic mutations were established as driving forces for pancreatic adenocarcinoma development such as KRAS and P534, their application as therapeutic targets were only effective in a small percentage of PDAC patients2. Alternative roads based on transcriptome analysis, stroma depletion, and protein post-translational modifications have recently been considered as potential strategies5, but need to be further investigated.Pancreatic ductal adenocarcinoma (PDAC), originated from exocrine cells in pancreas, is the most common pancreatic cancer subtype accounting for over 85% of malignant cases in pancreas7. In PDAC pathogenesis, dysregulated NO overproduction by inducible and endothelial (iNOS and eNOS) is critically involved in tumor development and associated with poor survival, but the underlying mechanism remains largely unexplored11. NO performs its biological functions mainly through protein S-nitrosylation, a redox modification during which a NO group is covalently added onto the cysteine (Cys) thiol of a protein to form S-nitrosothiol (SNO)13. S-nitrosylation exerts various molecular effects on protein activity, conformation, stability, subcellular distribution, and interaction networks17, thus involved in tumorigenesis and other processes19. A number of S-nitrosylated proteins were extensively investigated in cancer cells, such as B-cell lymphoma-2 (Bcl-2)20, Ras21, Fas22, mitogen-activated protein kinase phosphatase-1 (MKP-1)23, tumor necrosis factor receptor associated protein 1 (TRAP1)24 and Phosphatase and tensin homolog (PTEN)25. Particularly, S-nitrosylation of wild-type Ras protein is required for the initiation and maintenance of PDAC tumor growth21. Except for this, roles of protein S-nitrosylation in PDAC pathogenesis still remain poorly understood.Nitric oxide (NO), produced by NO synthase (NOS), serves as a critical signaling molecule in tumorigenesis by regulating pleiotropic processes including cell proliferation, apoptosis, metastasis, angiogenesis, and chemoresistanceS-nitrosylation in large scale29. S-nitrosoproteomics greatly facilitated the elucidation of protein S-nitrosylation underlying multiple processes in various species, including mitochondrial fatty acid metabolism, neural signaling, neurodegeneration, and Duchenne muscular dystrophy pathogenesis33. We previously identified large number of S-nitrosylation proteins induced by NO overproduction through site-specific proteomics34, which provided clues for functional investigations of S-nitrosylation signaling36. In this study, site-specific proteomic profiling of endogenously S-nitrosylated proteins in PDAC tissues and pancreatic cells were performed for novel insights into nitric oxide and protein S-nitrosylation during pancreatic cancer pathogenesis.Mass spectrometry-based proteomics, combined with chemically selective approaches like biotin-switch and SNO trapping by triaryl phosphine, has made it feasible to identify protein n = 4), were separately analyzed by site-specific proteomics for discovery of S-nitrosylated proteins  using a FDR of <1% at both the peptide and protein group levels for control of false identification.Cancerous and adjacent non-cancerous pancreatic tissues collected from four patients with PDAC, as four biological replicates . Detailed clinical data including clinical staging of PDAC patients were listed in Supplemental Table PDAC cell lines PANC-1 and SW1990 were purchased from the Type Culture Collection of Chinese Academy of Sciences . The immortalized non-neoplastic human pancreatic ductal epithelium cell line HPDE6c7 was obtained from Kyushu University, Japan. Cells were authenticated by the short tandem repeat (STR) profiling method and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (Invitrogen) and penicillin and streptomycin at 37 °C. PANC-1 cell viability were determined by CCK-8  following manufacturer’s instructions, after being treated with 10 μM L-NAME  for 48 h.S-nitrosylated proteins in pancreatic tissues or cells using S-alkylating labeling strategy and irreversible biotinylation procedure as previously described34. Briefly, approximately 0.8 g frozen specimens were made into fine powder in liquid nitrogen. Cultured cells were lyzed using cell lysis buffer containing 250 mM HEPES, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 0.1 mM neocuproine, 1 mM PMSF and 1% protease inhibitor cocktail (Sigma-Aldrich). Tissue or cell lysates were then blocked by S-alkylation with 200 mM iodoacetamide at 37 °C for 1 h, and incubated with HENS buffer containing 1 mM sodium ascorbate and 4 mM biotin-maleimide (Sigma-Aldrich) at room temperature for 2 h in darkness. Negative control without sodium ascorbate treatment during biotinylation of S-nitrosylated proteins was included for each tissue or cell samples.Biotin-switch assay was performed to biotinylate endogenously S-nitrosylated peptides in tissues and cells were carried out using site-specific method as previously described34. Briefly, biotinylated proteins were subjected to in-solution digestion with trypsin (Sigma-Aldrich) at 37 °C for 16 h, followed by affinity chromatography enrichment using NeutrAvidin agarose resins (Thermo Scientific). Purified peptides were resuspended in 0.1% FA, desalted using C18 Zip-tip (Millipore) and then used for mass spectrometric analysis.Proteomic identification of 6 ions. Twenty ions with the highest intensities from each MS scan were isolated, fragmented and measured in linear ion trap, and CID normalized collision energy was set to 35. The mass spectrometry proteomics data, including annotated spectrum images, have been deposited to the ProteomeXchange Consortium via the PRIDE37 partner repository with the dataset identifier PXD012512.Mass spectrometric identification of biotinylated peptides was performed using a LTQ Orbitrap Elite mass spectrometer (Thermo Fisher Scientific) coupled online to an Easy-nLC 1000 (Thermo Fisher Scientific) in data-dependent mode. Peptides were first separated by reverse phase LC using 75 μm (ID) × 250 mm (length) analytical columns packed with C18 particles of 5 µm diameter. Mobile phases for liquid chromatography separation contain buffer A (0.1% FA) and buffer B (100% acetonitrile and 0.1% FA). Peptide separation was finished using a 90-min non-linear gradient  with a flow rate of 300 nl/min. The MS measurements were carried out using the positive ion mode, and precursor ions were measured in the Orbitrap analyzer at 240,000 resolution (at 400 m/z) with a target value of 10S-nitrosylated peptide lists.All raw MS files were collected and subjected to database search using the software MaxQuant (version 1.6.0.16). The UniProt proteome sequences containing 20244 entries including canonical and isoforms  were used for database searching. Trypsin was selected as the protease for protein digestion, and the maximum allowable miscleavages was set to two. The mass tolerances for precursor ions and fragments ions were set to 20 PPM. A minimum peptide length of six amino acids and a maximum peptide length of 144 amino acids were also applied for database search. Cysteine biotinylation (451.200 Da), cysteine carbamidomethylation, N-terminal acetylation and methionine oxidation were included as variable modification. A minimum score of 40 and false discovery rate (FDR) of 0.01 was used as the threshold for peptide and protein identifications. A localization probability of over 0.5 was employed as localization score threshold, and modified peptides with unambiguous site localization were listed in a separate supplemental table. Biotinylated peptides identified in corresponding negative controls (without sodium ascorbate treatment) were finally excluded from the S-nitrosylated proteins were summarized by search against the SNObase database38, combined with recent publications. Consensus sequences of S-nitrosylated peptides were predicted using the pLogo method39. Gene ontology (GO) and KEGG pathway analysis were performed using the DAVID bioinformatics resource40. Pathway diagrams with enrichment of S-nitrosylated proteins were modified from the KEGG pathway database (www.kegg.jp).Previously reported 41. Primary antibodies used in this study were listed as follows: anti-eNOS , anti-nNOS , anti-iNOS , anti-biotin , anti-Raf-1 , anti-STAT3 , anti-p-STAT3 (Y705) , and anti-β-actin antibodies .The immunoblotting assay was done as previously describedt test or ANOVA  as appropriate using the SPSS 20.0 software. Significant differences were defined by a P value of <0.05.Difference between groups were evaluated by the Student’s S-nitrosylated protein (SNO) levels in pancreatic cancer tissues from PDAC patients were also significantly higher than corresponding adjacent tissues  in PDAC tissues showed significant increase compared with paired adjacent tissues  identified in our proteomic analysis were also previously reported, strongly validating the reliability of results obtained by this proteomic analysis  and proliferating cell nuclear antigen (PCNA), which are S-nitrosylated proteins reported by previous studies  S-nitrosylated proteins in PANC-1 cells were identified in adjacent non-cancerous pancreatic tissues  was identified as S-nitrosylated site in both the PDAC tissues and PANC-1 cells  and glutamic acid (E)  Fig.  and S6.FS-nitrosylated cysteine sites identified in this study  is flanked by two lysine residues at positions +6 and −1, and several glutamic acid residues at proximal positions as well  analysis. S-nitrosylated proteins in adjacent pancreatic tissues, PDAC tissues and PANC-1 cells were separately analyzed , Rac2, v-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1), cell division cycle 42 (CDC42), Signal transducer and activator of transcription 1 (STAT1), STAT3 and retinoblastoma (RB) proteins, which are key regulators of cell apoptosis, cell cycle and other biological processes , minichromosome maintenance complex component 3/5 (MCM3/5), growth factor receptor-binding protein-2 (GRB2), Rho-associated coiled-coil kinase 2 (ROCK2), P21-activated kinase 2 (PAK2), Ras homolog gene family member A (RhoA), mitogen-activated protein kinase P38, vasodilator-stimulated phosphoprotein (VASP) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (14-3-3), all of which are widely involved in cell cycle, focal adhesion, adherent junction and cytoskeleton regulations , triosephosphate isomerase (TPI1) and aldehyde dehydrogenase family 1, member A3 (ALDH1A3), indicative of the regulation of cancer cell metabolism by S-nitrosylation during PDAC development. Proteins in other cancer-related processes, including pyruvate and propanoate metabolism, glycerolipid metabolism, cell cycle progression, focal adhesion, adherent junctions, neurotrophin signaling, and leukocyte trans-endothelial migration, were extensively S-nitrosylated as well. Large number of target proteins suggested the pleiotropic effects of protein S-nitrosylation on PDAC pathogenesis, which deserve further investigation.In addition, cellular metabolism reprogramming serves as a critical mechanism for cancer cells to maintain viability and build new biomassS-nitrosylated proteins and their modification sites in PDAC tissues and cells, involving multiple processes and signaling pathways closely associated with pancreatic tumorigenesis. NOS inhibitor treatment significantly suppressed STAT3 S-nitrosylation, promoted STAT3 phosphorylation and enhanced viability of pancreatic cancer cells, further indicating the essential roles of S-nitrosylation in PDAC pathogenesis. These findings provided novel insights into nitric oxide signaling and S-nitrosylation in pancreatic cancer development, and also a basis for protein modification-based cancer diagnosis and treatment.Taking together, our site-specific proteomics identified a large number of endogenously Supplemental Figure LegendsSupplemental Figure S1Supplemental Figure S2Supplemental Figure S3Supplemental Figure S4Supplemental Figure S5Supplemental Figure S6Supplemental Figure S7Supplemental Figure S8Supplemental Figure S9Supplemental Table S1Supplemental Table S2Supplemental Table S3Supplemental Table S4Supplemental Table S5Supplemental Table S6Supplemental Table S7Supplemental Table S8


Importantly tHSCs-induced DIgR2 expression was blocked by Nrf2 shRNA or knockout . Conversely, forced activation of Nrf2, by Keap1 shRNA or the Nrf2 activators , induced significant DIgR2 expression. tHSCs stimulation induced reactive oxygen species (ROS) production in mDCs. Conversely, ROS scavengers inhibited tHSCs-induced ROS production, Nrf2 activation and DIgR2 expression in mDCs. Significantly, tHSCs inhibited production of multiple cytokines  in mDCs, reversed by Nrf2 depletion. Moreover, Nrf2 shRNA or KO attenuated splenic T cell inhibition by tHSCs-stimulated mDCs. Together, we conclude that Nrf2 activation mediates tHSCs-induced DIgR2 expression in mDCs.Our previous studies discovered that tumor-specific hepatic stellate cells (tHSCs) induced dendritic cell-derived immunoglobulin receptor 2 (DIgR2) expression in bone marrow-derived dendritic cells (mDCs), inhibiting splenic T cell activation. The current study aims to explore the underlying mechanism of DIgR2 expression by focusing on Nrf2 (nuclear-factor-E2-related factor 2) signaling. We show that tHSCs co-culture induced significant Nrf2 signaling activation in mDCs. The latter was evidenced by Nrf2-Keap1 disassociation, Nrf2 protein stabilization, accumulation and nuclear translocation. Expression of Nrf2-dependent genes, including  Hepatocellular carcinoma (HCC) is a major health threat and a primary cause of cancer-associated human mortalities –3. HCC’sHepatic stellate cells (HSC) are the primary source of extracellular matrix in the fibrogenesis process –15. ReceUnder basal conditions, nuclear-factor-E2-related factor 2 (Nrf2) binds to its suppressor protein, Kelch-like ECH-associated protein 1 (Keap1) –21. The Our previous studies have shown that tHSCs co-culture induces DIgR2 expression in bone marrow-derived DCs (mDCs) , 17. Theheme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1)  were purchased from Sigma-Aldrich . MIND4-17 was provided by Dr. Wang . All the3), transplanted to rat livers [As previously described , 44, MRHt livers . Using aAs previously described , 43, 44,5 mDCs were incubated with 1.25×104 tHSCs .As previously described , 46, theAs described previously , 44, theAs reported , spleen Ct−ΔΔ method to quantify targeted gene expression, using glyceraldehyde-3-phosphatedehydrogenase (GAPDH) as the reference gene and internal control.Trizol reagents  were utilized for RNA extraction. A SYBR Green PCR kit  was utilized for reverse transcription. qPCR assays were performed using the previously described protocol , 17. TheLysates (30 μg proteins of each treatment in each lane) were separated by a 10% SDS-PAGE gel, transferred to a polyvinylidene difluoride (PVDF) blot  . After iAs described , followiH2DCFDA (dichlorofluorescin diacetate), a fluorogenic dye, measures hydroxyl, peroxyl and other ROS activity. In brief, following co-culture with tHSCs, mDCs were incubated with H2DCFDA  dye for 45 min under dark. H2DCFDA absorbance was detected by a fluorescence spectroscopy with excitation and emission at 495 nm and 529 nm, respectively.Tetraethylbenzimidazolylcarbocyanine iodide (JC-1) is a cationic dye that accumulates in energized mitochondria. It forms green monomers after mitochondrial depolarization. In brief, following co-culture with tHSC, mDCs were incubated with JC-1 dye  dye for 30 min under dark at the room temperature. At the test wavelength of 530 nm JC-1 green fluorescence absorbance was tested.The Nrf2 lentiviral particles or the Keap1 shRNA lentiviral particles were purchased from Shanghai Genechem , added to mDCs for 12h. Cells were then cultured in fresh complete medium (with 10% FBS), following by selection with puromycin (2.0 μg/mL) for another 48h. In the stable cells over 95% of knockdown of target protein (Nrf2 or Keap1) was detected.The single-guide RNA (sgRNA) targeting Nrf2 was sub-cloned into a lenti-CRISPR-GFP-puro construct. The Nrf2 knockout (KO) construct was transfected to mDCs by Lipofectamine 2000. mDCs were further subjected to selection with puromycin (2.0 μg/mL) for 48h. In the stable cells Nrf2 KO was verified by Western blotting and qPCR.All statistical analyses were performed by using SPSS 18.0 software. Values are expressed as the mean ± standard deviation (SD). P < 0.05 was considered to indicate a statistically significant result.


Natural dibenzo-α-pyrones are an important group of metabolites derived from fungi, mycobionts, plants and animal feces. They exhibit a variety of biological activities such as toxicity on human and animals, phytotoxicity as well as cytotoxic, antioxidant, antiallergic, antimicrobial, antinematodal, and acetylcholinesterase inhibitory properties. Dibenzo-α-pyrones are biosynthesized via the polyketide pathway in microorganisms or metabolized from plant-derived ellagitannins and ellagic acid by intestinal bacteria. At least 53 dibenzo-α-pyrones have been reported in the past few decades. This mini-review aims to briefly summarize the occurrence, biosynthesis, biotransformation, as well as their biological activities and functions. Some considerations related to synthesis, production and applications of dibenzo-α-pyrones are also discussed. H-benzo[c]chromen-6-ones, and 6H-dibenzopyran-6-ones) are an important group of heptaketide coumarin derivatives that have a fused tricyclic nucleus , alternariol 9-methyl ether (11), botrallin (16), and 2-chloro-4,6-dihydro-1,7-dihydroxy-3,9-dimethoxy-1-methyl-1H-dibenzopyran-4,6-dione . The structures of the dibenzo-α-pyrones from fungi are shown in Dibenzo-α-pyrones are mainly distributed in the  TC 1674 . From th11), was isolated from the roots of Anthocleista djalonensis (Loganiaceae). The authors postulated djalonensone to be a significant taxonomic marker of the plant species pyran-6-one (31), -3,10-dihydroxy-9-O--dibenzopyran-6-one (32), and fasciculiferol (33) were isolated from the heartwood of Umtiza listerana ,6354), w10) was first thought to be biosynthesized via norlichexanthone [10) could be biosynthesized by simple cyclization and aromatization of a polyketide precursor [13C]acetate and acetate to cultured lichen mycobionts of Graphis spp., acetate units were incorporated into the 6H-dibenzopyran-6-one derivatives including alternariol (10), AME (11) and graphislactones A-F (18–23) [10) could suffer oxidative demethylation at C-1, hydroxylation at C-4, and O-methylation at C-9 to lead formation of graphislactones E (22) and F (23). On the other hand, alternariol (10) could be transformed to graphislactones A-D (18–21) without demethylation via AME (11) [18–23) in the cultured lichen mycobionts of Graphis spp. are shown in Alternariol  and histone deacetylase (HDAC) inhibitors  are useful to induce the expression of otherwise silent biosynthetic genes under standard laboratory conditions [10), alternariol 9-methyl ether (11), 4-hydroxyalternariol 9-methyl ether (14) and altenusin (56) [10, 11, 14 and 56 involves the condensation of seven molecules of malonyl-CoA, followed by aldol-type cyclizations between C-2 and C-7, and C-8 and C-13, and the subsequent lactonization leads to alternariol (10) (10) by a methyltransferase results in 4-hydroxyalternariol 9-methyl ether (14). Furthermore, the reduction of the C-9 carbonyl group of the heptaketide intermediate by a reductase, and subsequent aldol-type cyclization would produce a biphenyl. Methylation of the C-5 hydroxyl group, and the hydroxylation of C-5' would then lead to altenusin (56). The hypothetical biosynthetic pathways [10) and its derivatives 11, 14 in an endophytic fungus from Datura stramonium are shown in Epigenetic modifiers, including DNA methyltransferase (DNMT) inhibitors (nditions . Supplemsin (56) . The proiol (10) . On the pathways  of alter55) in the acidic environment of the stomach by the action of the intestinal bacteria. The proposed transformation from ellagic acid to urolithins by the intestinal bacteria [Urolithins include a family of metabolites of dibenzo-α-pyrone structures with different phenolic hydroxylation patterns. They are produced in different animals after the intake of ellagitannins and ellagic acid (EA) ,72. Ellabacteria ,7,63 is Dibenzo-α-pyrones and their derivatives with diverse chemical properties have been clarified . Some ofAlternaria fungi with human and animal health is not a recent phenomenon. Alternaria toxins have been linked to a variety of adverse effects  on human and animal health [1), alternariol (10), and alternariol 9-methyl ether (11) were studied for their toxicity to chickens. Addition of these compounds in chicken feed from sublethal to lethal levels progressively reduced feed efficiency, suppressed weight gain and increased internal haemorrhaging [The association of mycotoxins from l health . Altenuerrhaging ,73. Alternaria metabolites on brine shrimp  [50 values of altenuene (1) and alternariol (10) were 375 and 100 µg/mL, respectively, to brine shrimp larvae by using the disk method of inoculation and an exposure period of 18 h [1) and alternariol (10) along with alternariol 9-methyl ether (11) were also verified to be toxic to brine shrimp [There were a few reports about the toxicity of lina L.) ,75. The e shrimp .Alternaria dibenzo-α-pyrones, alternariol (10) was the most active metabolite to have cytotoxic activity on L5178Y mouse lymphoma cells [10) was a topoisomerase I and II poison which might contribute to the impairment of DNA integrity in human colon carcinoma cells [10) and its 9-methyl ether (11) induced cytochrome P450 1A1 and apoptosis in murine heptatoma cells dependent on the aryl hydrocarbon receptor [11), alternariol 9-O-sulfate (13), and altenusin (56) were also screened to be cytotoxic [Among ma cells , as wellma cells . Furtherma cells ,76. It ireceptor . Other a17), isolated from A. tenuis, was found to be a specific inhibitor of eukaryotic DNA polymerase α to show its strong cytotoxic activity on tumor cells [in vitro [Dehydroaltenusin , graphislactone A (18), graphislactone G (24), and graphislactone H (25) from the endophytic fungus Cephalosporium acremonium IFB-E007 derived from Trachelospermum jasminoides showed pronounced activity against SW1116 cell with IC50 values of 14.0, 21.0, 12.0 and 8.5 μg/mL, respectively [Alternariol 9-methyl ether (ectively .55) were screened to have cytotoxic and anti-tumor activities. Urolithin A (40) inhibited cell growth of human colon cancer cell lines HT29 by inhibiting the canonical wnt signal pathway and interfere with β-catenin/TCF-dependent transcription [40), urolithin B (42), and 8-O-methylurolithin A (50) also showed antiproliferative effect on human bladder cancer T24 cells [Urolithins derived from ellagic acid , alternariol (10), alternariol 9-methyl ether (11), alternuisol (15), and dehydroaltenusin (17) [The metabolites from fungal pathogens are usually toxic to plants and are called phytotoxins which are divided into host-specific ,84 and hernariol , alternasin (17) ,39,41,4540), isourolithin A (41), and urolithin B (42) from the fruits of Trapa natans showed antioxidant activity. Among them, isourolithin A (41) showed the strongest, and urolithin B (42) showed weak antioxidative effect [i.e., urolithins A, C, and D) exhibited antioxidant activity in a cell-based assay [Urolithin A 0, isouroed assay . Howevered assay ,87. Receed assay . The anted assay .40), isourolithin A (41), and urolithin B (42), from the feces of Trogopterus xanthipes showed hyaluronidase inhibitory activities with IC50 values of 1.33, 1.07 and 2.33 mM, respectively that indicated their antiallergic activity [28) from the fungus Phoma sp. TC 1674 [28) would inhibit IL-4 signaling and would be useful in the treatment of allergic disease [Urolithin A  from endophytic Alternaria spp. exhibited inhibitory activity on the appressorium formation of Magnaporthe grisea (M. oryzae) with IC50 value of 51.0 μg/mL [11), botrallin (16) and palmariol B (27) from endophytic fungus Hyalodendriella sp. Ponipodef12 showed moderate antimicrobial activity [Other biological activities of dibenzo-α-pyrones include antimicrobial, antimalarial, antinematodal activities as well as calmodulin-dependent, estrogenic and antiestrogenic, and acetylcholinesterase (AChE) inhibitory activities. Alternariol 9-methyl ether  from the plants Chrozophora senegalensis (Euphorbiaceae) and Sebastiania chamaelea (Euphorbiaceae) showed moderate antimalarial activity (IC50 > 10 μg/mL) on Plasmodium falciparum [11), botrallin (16) and palmariol B (27) from endophytic fungus Hyalodendriella sp. Ponipodef12 also showed moderate antinematodal inhibitory activity on Caenorhabditis elegans [2,3,4,9,10-Pentahydroxy-6lciparum . Alterna17) from Penicillium verruculosum IAM-13756 inhibited the calmodulin-dependent activity of myosin light chain kinase (MLCK) with IC50 value of 0.69 μM [Dehydroaltenusin ( 0.69 μM .40) and B (42) from human feces exhibited estrogenic and antiestrogenic activities, which suggested that consumption of ellagitannin-containing foodstuffs such as pomegranate, walnuts, berries, and oak-aged wines may exert some proestrogenic/antiestrogenic effects [Both urolithins A ( effects .11), botrallin (16) and palmariol B (27) from endophytic fungus Hyalodendriella sp. Ponipodef12 showed moderate AChE inhibitory activity [16) from the endophytic fungus Microsphaeropsis olivacea was also screened to have AChE inhibitory activity with IC50 value as 6.1 μg/mL [Alternariol 9-methyl ether  [2) [5) [10) [11) [15) [17) [18), C (20), D (21) and H (25) [28) [34) [40, 42 and 43) [48) [The potential applications of dibenzo-α-pyrones as antitumor agents, antiallergics, antioxidants, and antimicrobials have attracted considerable interest within the pharmaceutical community. Chemical syntheses have been achieved for a few bioactive dibenzo-α-pyrones such as altenuene (1) , isoalte1) [2) , neoalte [2) [5) , alterna[5) [10) , alterna10) [11) , alterte11) [15) , dehydro15) [17) , graphisd H (25) , TMC-26425) [28) , lysilac and 43) , and uro43) [48) . In addi43) [48) ,17,18.i.e., phytotoxins and mycotoxins) [With comprehensive understanding of the biosynthetic pathways of some dibenzo-α-pyrones in the next few years, we may be able to effectively not only increase the yields of bioactive dibenzo-α-pyrones, but also block the biosynthesis of some toxic dibenzo-α-pyrones (otoxins) .