Bacteroides vulgatus and strong immunogenic Escherichia coli, we found that the induction and longevity of Bregs depend on strong Toll-like receptor activation mediated by antigens of strong immunogenic commensals. The potent B cell stimulation via E. coli led to a pronounced expression of suppressive molecules on the B cell surface and an increased production of anti-inflammatory cytokines like interleukin-10. These bacteria-primed Bregs were capable of efficiently inhibiting the maturation and function of dendritic cells (DCs), preventing the proliferation and polarization of T helper (Th)1 and Th17 cells while simultaneously promoting Th2 cell differentiation in vitro. In addition, Bregs facilitated the development of regulatory T cells (Tregs) resulting in a possible feedback cooperation to establish immune homeostasis. Moreover, the colonization of germfree wild type mice with E. coli but not B. vulgatus significantly reduced intestinal inflammatory processes in dextran sulfate sodium (DSS)-induced colitis associated with an increase induction of immune suppressive Bregs. The quantity of Bregs directly correlated with the severity of inflammation. These findings may provide new insights and therapeutic approaches for B cell-controlled treatments of microbiota-driven autoimmune disease.B cells fulfill multifaceted functions that influence immune responses during health and disease. In autoimmune diseases, such as inflammatory bowel disease, multiple sclerosis and rheumatoid arthritis, depletion of functional B cells results in an aggravation of disease in humans and respective mouse models. This could be due to a lack of a pivotal B cell subpopulation: regulatory B cells (Bregs). Although Bregs represent only a small proportion of all immune cells, they exhibit critical properties in regulating immune responses, thus contributing to the maintenance of immune homeostasis in healthy individuals. In this study, we report that the induction of Bregs is differentially triggered by the immunogenicity of the host microbiota. In comparative experiments with low immunogenic  The fact that B cells play a critical role during the onset and course of inflammatory processes is indisputable and has been demonstrated in many studies in both mice and humans. However, these studies focused on the production of autoantibodies with B cells being understood as the cause for the development of inflammation, e.g., multiple sclerosis (MS) –3. Never+CD21hiCD23hiCD24hi), B10 cells (CD19+CD5+CD1d+IL10+), and Tim-1+ B cells (CD19+Tim-1+)  . Hithert+Tim-1+) – 21. A c+Tim-1+) –25. IL-1+Tim-1+) , 27. The+Tim-1+) –31. Depe+Tim-1+) –34; (II)+Tim-1+) . Moreove+Tim-1+) . The inf+Tim-1+) –38.In addition to the production of IL-10, Bregs express and secrete suppressive molecules and thus possess further mechanisms to regulate immune responses in an IL-10-independent manner: CD73 is a cell-surface enzyme that converts adenosine monophosphate to adenosine with potential immunosuppressive effects ; PD-L1 –43; FasLin vitro and in vivo. It has thus far been shown that several bacterial and viral pathogens, as well as parasites, manipulate B cell function directly to modulate host immune responses as part of an immune evasion strategy facilitating their survival and prolonging infection (Bacteroides vulgatus (weak immunogenic) and Escherichia coli (strong immunogenic) to modulate and regulate the immune system of the host via B cells. In this context, we could already show that a weak immunogenic signal provided by B. vulgatus is beneficial in genetically predisposed host (deficient for Rag1 or IL-2) in the course of inflammation. In contrast, the administration of strong immunogenic E. coli aggravates the disease progression due to the lack of a functional B cell immunity which can restore immune tolerance in a healthy host by counter-regulating the induced pro-inflammatory immune response  this regulation mechanism may serve as a feedback loop to maintain immune homeostasis and even attenuate inflammatory processes in autoimmune disease.In this study we demonstrated the following: (I) B cells can be activated directly by commensal members of the host microbiota and, depending on the immunogenic potential of the encountered bacterial species, B cells can mint strong regulatory cell phenotypes to promote immune tolerance; (II) the intensified induction of Bregs by E. coli mpk was grown in Luria-Bertani (LB) medium under aerobic conditions at 37°C. B. vulgatus mpk was grown in Brain-Heart-Infusion (BHI) medium and anaerobic conditions at 37°C.Tlr2−/−, Tlr4−/−) were provided by Jackson Laboratory. All animals were kept and bred under SPF conditions. For isolation of B cells, T cells and bone marrow, only female mice aged 8–10 weeks were used. Germfree C57BL/6J mice were bred and housed in our own gnotobiotic facility. Animal experiments were reviewed and approved by the responsible institutional review committee and the local authorities.C57BL/6NCrl mice and C57BL/6-Tg(TcraTcrb)425Cbn/Crl (OT-II) mice were purchased from Charles River Laboratories. Toll-like receptor 2 and 4 double KO mice (−/− × TLR4−/− mice by magnetic isolation using negative selection with microbeads (Miltenyi Biotec) according to the manufacturer's instruction. The purity of CD19+CD43−CD4−Ter119− B cell population was >95%. B cells were cultured in complete medium  at a density of 1 × 106 cells/mL in flat-bottom plates for subsequent stimulation experiments. The purity of naïve CD4+CD44−CD8a−CD11b−CD11c−CD19−CD25−CD45R−CD49b−CD105−MHCII−Ter-119−TCRγ/δ− T cell population was >94%. After purification, T cells were directly used for co-culture experiments. For proliferation assays, isolated splenic B cells or T cells were adjusted to a concentration of 106 cells/mL in PBS/1% FCS, resuspended in 10 μM CFSE and incubated at 37°C for 20 min.B cells were purified from spleens of WT or TLR2+ bone marrow-derived dendritic cells (BMDCs) were harvested and used for co-culture stimulation experiments.Bone marrow cells were isolated from femurs and tibias of WT mice and cultivated for differentiation in GM-CSF supplemented media as described previously . Cells w6/mL splenic B cells or BMDCs were stimulated with PBS, B. vulgatus mpk or E. coli mpk at a Multiplicity of infection (MOI) of 1 at 37°C (“-stimulated B cells/BMDCs”). One μL/mL gentamicin was added to prevent bacterial overgrowth. According to the experimental setting, cells were harvested following stimulation at different time points.1 × 10+ T cells, B cells were incubated with 10 μg/mL Ova-Peptide  for 2 h at 37°C. B cells were washed and supernatant was exchanged with fresh media (“-pulsed B cells”). Primed and pulsed B cells and naïve T cells were co-cultured at different ratios for 72 h at 37°C and 100 ng/mL purified anti-mouse IL-10 antibody  was added to certain samples.For B-T cell co-culture experiments B cells served as APCs and were stimulated for 24 h with commensals as described previously (“-primed B cells”). Prior to co-cultivation with naïve CFSE-labeled OT-II CD4+ dendritic cells maturation assay, naïve B cells and differentiated immature BMDCs were simultaneously stimulated with bacteria at MOI 1 and co-cultured at a ratio of 5:1 (B cells/BMDCs). To show the effect of indirect cell-cell interaction, naïve B cells and BMDCs were additionally co-cultured in Transwells (pore-size 0.4 μm) and stimulated with bacteria at MOI 1. To mimic the influence of soluble IL-10 produced by B cells on the maturation of BMDCs, 10 μg/mL recombinant mouse IL-10 (BioLegend) were added to BMDC mono-cultures.For CD11c8B. vulgatus or E. coli per mL sterile drinking water. After day 8, mice received sterile drinking water without bacteria. Colonization was checked weekly by collecting fresh feces and determining CFU on selective growth agars (Germfree (GF) C57BL/6J mice were colonized for 7 days with 1 × 10th agars . After 4Viable cells were counted and cultured for 4 h with 2 μl/mL Leukocyte Activation Cocktail with Brefeldin A (BD Bioscience). After incubation, cells were prepared for flow cytometrical analysis.Harvested cells were washed and FC-receptors were blocked to avoid non-specific binding. Cell death was measured by LIVE/DEAD Cell Viability kit (ThermoFisher) staining for 20 min at 4°C. Intracellular staining was performed using Cytofix/Cytoperm (BD Biosciences) according to the manufacturer's description. For intracellular and cell-surface staining, cells were labeled for 25 min at 4°C with specific fluorophore-conjugated antibodies, washed, and resuspended in staining buffer (PBS/1% FCS). Multi-color FCM analyses were performed on a FACS LSRII (BD Biosciences). For compensation, fluorescence minus one (FMO) samples served as controls. Doublets were excluded using FSC-A/FSC-H gating. Data was analyzed using FlowJo software .Antibodies: CD11c-APC, CD19-BV421, CD1d-AF647, CD21/CD35-BV605, CD23-BB515, CD24 AF700, CD365(TIM-1)-PE, CD4-APC, CD4-Bv605, CD40-APC, CD40-Bv421, CD45R(B220)-FITC, CD5-Bv605, CD80-PerCP-Cy5.5, CD86-PE-Cy7, CD178(Fas-L)-PE, CD274(PD1-L)-PE, CD73-PE, CD210(IL10R)-PE, FoxP3-AF647, I-A/I-E(MHC-II)-FITC, IFNγ-PE-Cy7, IgM-APC, IL10-PE, IL17A-APC-Cy7, IL4-PE, Vβ5.1,5.2-Bv605, GITR-L-PE, Ebi3-PE. All fluorophore-coupled antibodies were purchased from BD Biosciences.For determination of TNF and IL-10 concentrations in cell culture supernatants, ELISA Kits purchased from BD Bioscience were used according to the manufacturer's instructions.t-test or ANOVA for data with Gaussian distribution and via Mann-Whitney or Kruskal-Wallis test for non-parametric statistics. Correlation analyses were performed via Spearman's rank correlation coefficient (r). Statistical significance: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Error bars represent ± standard deviation (SD).Data have been tested for normality using the Shapiro-Wilk normality test. Statistical analyses were then performed via unpaired student's B cells are mainly associated with the production of antibodies against invaders during adaptive immune responses . Activat−/− × TLR4−/− (KO) mice were stimulated with two different immunogenic bacteria: B. vulgatus mpk and E. coli mpk  and TLR2coli mpk –69.+ B cells was investigated in vitro after 0, 24, 48, and 72 h of stimulation with B. vulgatus or E. coli. The viability of B cells decreased significantly 24 h after stimulation with PBS (Mock) or B. vulgatus in comparison to E. coli-stimulated B cells, indicating that the survival of B cells is strongly dependent on a potent stimulus with strong immunogenic E. coli . We determined the percentage of the best characterized Breg subsets: B10 cells, T2-MZP cells, and Tim-1+ B cells, at several time points after stimulation with strong immunogenic E. coli or low immunogenic B. vulgatus in comparison to naïve, unstimulated B cells (+CD5+CD1d+IL10+ (B10 cells) and CD19+CD21hiCD23hiCD24hi (T2-MZP cells) were significantly enhanced after being challenged with E. coli in comparison to B. vulgatus or PBS stimulation, reaching the maximum proportion after 48–72 h of incubation (E. coli-stimulated B cells differentiated to a higher proportion into CD19+ Tim-1+ cells (Tim-1+ B cells), an additional regulatory B cell phenotype compared to PBS and B. vulgatus stimulation . As. AsE. cocubation , 3. Furtmulation . Tim-1+ vulgatus . In contvulgatus . Furthermulation .E. coli-activated B cells featuring regulatory properties via the intensified production and upregulated expression of suppressive molecules directly influence the function of other immune cells, e.g., dendritic cells (DCs). We primarily focused on the maturation of differentiated CD11c+ bone marrow-derived dendritic cells co-cultured with bacteria-stimulated B cells  for 72 h with naïve CFSE-labeled CD4+CD44− T cells isolated from OT-II mice, which express an Ova peptide-specific TCR , Th2 (CD4+IL4+), Th17 (CD4+IL17A+IL4−IFNγ−) cell, and Treg (CD4+FoxP3+) differentiation in B-T cell co-culture was analyzed by flow cytometry. PBS-primed naïve B cells appeared to favor the differentiation of naïve T cells to Th1 and Th17 cells, whereas E. coli-primed B cells led to a significantly enhanced T cell polarization toward Tregs. In addition, co-culture of E. coli-primed B cells with naïve T cells resulted in a polarization of Th2 cells at a T cell-B cell ratio of 1:5 -induced colitis model in gnotobiotic C57BL/6 mice which have a physiological B and T cell response. We colonized germfree WT mice with B. vulgatus or E. coli for 4 weeks via drinking water  in the spleen and mLN, whereas the proportion of Th1 (CD19−CD3+CD4+IFNγ+) and Th17 (CD19−CD3+CD4+IL-17+) cells was significantly reduced in the mLN in comparison to germfree DSS-treated mice  and ulcerative colitis (UC) –79. FurtBeside pathogens which are typically acquired from the environment, often provoking acute infections and are the target of immunity, also members of the resident microbiota, found in most healthy hosts, may be the cause for several inflammatory disorders in a genetic or environmental predisposed organism –94. The However, it is conceivable that pathobionts do not actively use specific mechanisms to harm the host under certain circumstances; rather, mechanisms, signaling pathways or cell functions are malfunctioning or are lacking in an immune-compromised host, which pathobionts usually activate in a healthy host to counter-regulate inflammatory processes.But which immune response mechanisms are consulted by commensals in a healthy host to maintain immune homeostasis? One possible mechanism could be the induction of regulatory B cells.Most studies investigate the interaction of pathogenic bacteria with B cells during infection. Alternatively, we wanted to demonstrate that the induction of regulatory B cell populations is a mechanism which commensals use to suppress self-caused pro-inflammatory immune responses in a healthy host , 95. We Primarily, naïve B cells are activated via the help of Th cells which present microbial antigens to the B cell receptor (BCR) followed by the uptake of the antigen through receptor-mediated endocytosis, degradation and presentation of peptide in complex with MHC-class-II molecules on the B cell membrane . The recFurther, we suggest that not only the presence of pattern recognition receptors (PRRs), such as TLRs on the B cell surface is crucial for B cell activation. Additionally, the antigen origin and its immune-stimulatory level might be decisive for the strength of activation and thus the differentiation and survival of Breg subsets.B. vulgatus (low immunogenic) and E. coli (strong immunogenic), we were able to show that the potent stimulation of naïve B cells with E. coli resulted in a strong activation and differentiation of Breg phenotypes characterized by a significantly elevated secretion of anti-inflammatory IL-10 and higher production of suppressive molecules like EBI3, CD73, GITRL, FasL, and PD-L1. In addition, the secretion of the pleiotropic modulatory cytokine IL-6, that exerts either pro-inflammatory or anti-inflammatory properties, was upregulated past stimulation of B cells with E. coli whereas only low levels of pro-inflammatory TNFα were detectable -labeled bacteria 4 h after oral gavage , or B cell-deficient (μMT) mice (The inflammation-suppressive role of Breg subsets in autoimmune disease has been demonstrated previously in MT) mice , 127–132+ DCs), concluding that a potent TLR activation provided by strong immunogenic bacteria leads to enhanced immune responses, aggravating the course of disease (−/−), the disturbed proliferation and induction of Bregs (IL-2−/−), or the dysfunction of Bregs (IL-10−/−) in all three colitis mouse models. Hence in these colitis models, a potent immune-stimulatory signal mediated by strong immunogenic E. coli exacerbated inflammation, since important counter-regulation mechanisms (such as the induction of Bregs) for the compensation of overshooting immune responses malfunctioning. Similar observation could be made with the bacterium Helicobacter hepaticus. H. hepaticus is a member of the mouse microbiota colonizing the lower intestine and activating innate immunity via Toll-like receptors without inducing immune pathology in a healthy host (H. hepaticus thus induces an anti-inflammatory immune response through the activation of regulatory macrophages to maintain immune homeostasis (−/− or Rag2−/− mice, H. hepaticus triggered exacerbated intestinal inflammation as a result of aberrant regulatory T cell function (In earlier studies, we could demonstrate the effect of different immunogenic commensals on the differentiation and maturation of certain immune cells (e.g., CD11c disease , 134. Th disease , 61, 135thy host . H. hepaeostasis . Howeverfunction –140. In E. coli Nissle or Helicobacter pylori have inflammation-suppressive properties and can even prevent the onset of disease, on the condition that the host provides a functional immune system (However it has already been published in mouse models for IBD and T1D that strong immunogenic bacteria, such as e system –143. In B. vulgatus- or strong immunogenic E. coli-associated mice emphasizes the importance of a potent immune stimulus in DSS-induced inflammation since the colonization with E. coli significantly prevented intense weight loss, alleviated disease symptoms and reduced inflammation in the affected tissue. In contrast, colonization with B. vulgatus alleviated disease symptoms slightly, but not significantly, compared to germfree mice. Reason for that could be the weak immunogenic properties of B. vulgatus still provoking a low activation of B cells, minor induction of Bregs and modest inhibition of T cell proliferation and polarization, as observed in in vitro experiments. In addition, other bacteria-associated host immune modulating effects which are B cell- and T cell-independent, such as the restoration and education of the immature immune system and mucosal barrier present in germfree mice could be causal for the attenuated inflammation development shown in B. vulgatus-colonized mice. However, these potential immunogenic-independent regulatory mechanisms may be insufficient to significantly reduce inflammation processes in DSS-induced colitis. Though, these findings could explain the non-significant differences between B. vulgatus- and E. coli-colonized mice in some read outs (The comparison of germfree, low immunogenic ead outs , 145.in vitro experiments, the colonization of germfree mice with E. coli prior to DSS-administration, and the accompanying induction of Bregs, resulted in a significantly enhanced development of Tregs and inhibition of a Th1 and Th17 polarization (B. vulgatus-colonization resulted in a slightly and non-significant increased induction of Bregs, the differentiation of T helper cells is not altered in comparison to germfree mice. In contrast, the significant enhanced induction of Bregs in vitro and in vivo in E. coli-associated mice leading to an anti-inflammatory balance of Th1/Th2/Th17/Treg cells, marked by a pronounced differentiation of Tregs, which is decisive for the development of inflammation and consequently the possible reason for the extenuated inflammation in E. coli-colonized and DSS treated mice (Moreover, we provide evidence that the induction of Bregs directly correlated with the severity of colonic inflammation and therefore negatively correlated with the immunogenicity of the colonizing bacteria. These findings are in line with other studies reporting a decrease of intestinal regulatory B cells in colonic inflammation in mice and humans –148. In rization . Even thted mice –123.in vivo. In combination, these two crucial regulatory cell populations might develop a powerful anti-inflammatory milieu capable of suppressing inflammation in various autoimmune diseases (Thus, the increased accumulation of Bregs and Tregs established an immune-homoeostatic state diseases .In conclusion, Bregs are an important interface in microbiota-driven immune regulation. They contribute to maintenance of immune homeostasis in a healthy host and counter-act emerging inflammatory processes in immune-compromised hosts via the production of suppressive molecules and the interaction with other immune cell populations. The induction and longevity of Bregs specifically depend and correlate with the strength of TLR-ligation and subsequent cell activation, provided by commensal antigens. The presented results directly link different characteristics of commensal bacteria with the immune response of the host and thus provide new insights in the inter-kingdom communication between commensals and their hosts.All datasets generated for this study are included in the article/This study was carried out in accordance with the principles of the Basel Declaration. Protocols and experiments involving mice were reviewed and approved by the responsible Institutional Review Committee and the local authorities within H1/15, H1/17, §4 09.01.2015, §4 14.06.2017.JM and J-SF conceived and designed the experiments. JM, CT, AL, RP, LM, AS, HY, and H-CL performed the experiments. JM, CT, and J-SF analyzed the data. JM, AL, and J-SF wrote the manuscript. All authors gave final approval to publish the article.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.


Arabidopsis thaliana reduces DNA rearrangements and mitigates plastid genome instability phenotypes. This effect can be observed on a genome-wide scale, as the loss of the plastid sigma transcription factor SIG6 prevents DNA rearrangements by favoring conservative repair in the presence of ciprofloxacin-induced DNA damage or in the absence of plastid genome maintenance actors such as WHY1/WHY3, RECA1 and POLIB. Additionally, resolving R-loops by the expression of a plastid-targeted exogenous RNAse H1 produces similar results. We also show that highly-transcribed genes are more susceptible to DNA rearrangements, as increased transcription of the psbD operon by SIG5 correlates with more locus-specific rearrangements. The effect of transcription is not specific to Sigma factors, as decreased global transcription levels by mutation of heat-stress-induced factor HSP21, mutation of nuclear-encoded polymerase RPOTp, or treatment with transcription-inhibitor rifampicin all prevent the formation of plastid genome rearrangements, especially under induced DNA damage conditions.Though it is an essential process, transcription can be a source of genomic instability. For instance, it may generate RNA:DNA hybrids as the nascent transcript hybridizes with the complementary DNA template. These hybrids, called R-loops, act as a major cause of replication fork stalling and DNA breaks. In this study, we show that lowering transcription and R-loop levels in plastids of  Plastids form a large family of cellular organelles in plants and algae that includes chloroplasts, which are responsible for photosynthesis. Plastids of vascular plants originated from an ancestral cyanobacterial endosymbiont, and therefore possess their own genome . The prenucleus-encoded polymerases (NEP: RPOTp and RPOTmp) and one bacterial-type plastid-encoded polymerase (PEP), composed of four basic core subunits encoded by the plastid genes rpoA, rpoB, rpoC1, and rpoC2 , s_049021)  and sig5_141383) , sig6-1 893_C09) ,29,31, aulfonate  were obt SALK_0096 and sigescribed ,20,35. TChlamydomonas reinhardtii upon β-estradiol induction, was kindly provided by Dr. David L. Herrin . This line was used to cause a DSB at the psbA plastid gene intron according to the author’s protocol -labeled transcripts were isolated and hybridized to ~500 bp plastid gene probes blotted on a Nylon membrane. RNAse A treatment was performed as negative control. Experiments were performed in triplicate, and one representative experiment is presented. (B) Histogram showing the average radioactive signal intensity ± standard error (n = 3) of each DNA probe. Chloroplast genes psbA, rbcL, psaA, rrn16S and accD are represented in light blue, green, grey, white and orange, respectively. One-way ANOVA, Tuckey t-test .(PDF)Click here for additional data file.S4 Fig(A) Relative ptDNA levels (mean ± standard error) measured at three sites of the genome by qPCR in WT, sig6-1, why1why3 and why1why3sig6-1 plants grown for 14 days on soil, normalized to the nuclear genome. Experiments were performed in duplicate. Kruskal-Wallis and Dunns test (ns: not-significant). (B-D) Plastid DNA sequencing coverage curves for Arabidopsis WT and mutant lines indicated. Positions were rounded down to 1 kb. All reads mapping to the plastid large inverted repeats (IRs) were only assigned to the first IR. Y axis represents the number of reads per 1,000,000 total plastid reads. The plastid large-single copy region (LSC), the first IR, and the small-single copy region (SSC) are depicted as a long blue bar, a red bar and a short blue bar, respectively.(PDF)Click here for additional data file.S5 Fig(A-C) Plastid rearrangements per 100 nucleotide (nt) window overlapping by 50 nt along the plastid genome, normalized to the coverage for 1 million reads, in (A) wild type (WT) and sig6-1, (B)why1why3 and why1why3sig6-1, (C) WT and WT RNAseH1 lines treated with Estradiol (EST) and (D) why1why3 and why1why3 RNAseH1 EST-treated lines. All rearrangements mapping to the plastid large inverted repeats (IRs) were only assigned to the first IR. Peaks accumulating higher levels of rearrangements are indicated. A graphical representation of plastid genome of Arabidopsis thaliana from 1 to 128,214 nt (without the second IR) is shown above each graph. A zoom-in region spanning the rRNA operon is represented to the right.(PDF)Click here for additional data file.S6 Fig(A) PCR analysis of ptDNA rearrangements in 14-d-old wild type (WT) and rpotp plants on MS or with 0.5 μM CIP. (B) The bar graph represents the fold change intensity (mean ± standard error) of the PCR bands in A respect to WT on MS for each primer pair (n = 5). All PCR experiments were performed at least three times with duplicate samples. Low cycle amplification of the atpB plastid gene was used as a loading control. Primers pairs used for PCR reactions are indicated on the left side of the gels. Arrows and numbers on the right side of the gels represent the position and size of the DNA ladder bands in kilobases. P-values were generated by a two-tailed paired t-test, . (C) Representative photographs of the plants described in A.(PDF)Click here for additional data file.S7 Fig(A) Diagram of the expression of RNAse H1 induced with β-estradiol. Expression of the inducible cassette is driven by promoter UBQ10. Sequence of the XVE fusion protein is followed by a 3’UTR, pea rbcS E9, and LexA operator sequence. The minimal 35S promoter drives the expression of E. coli RNAse H1 protein fused to rbcS1 target peptide in N-terminal and StrepII tag in C-terminal. The transcription is stopped by the Nos-terminator. (B) Relative RNAse H1 expression levels (mean ± standard error) measured by qRT-PCR in control  and RNAse H1_A, B and C transgenic lines grown for 14 days on solid basal media alone (MS) or with 50 μM β-estradiol (EST), normalized to the expression of the nuclear gene β-Tubulin. The values were acquired from three independent experiments, and the expression level of RNAse H1_A EST-induced was adjusted to 1. One-way ANOVA and Tukey t-test . (C) Representative Western blot performed on total Ctl_A/B and RNAse H1_A chloroplast proteins obtained from seedlings grown on soil and spray-induced with 100 μM EST for 14 days. Antibody against StrepII was used to visualize expression of RNAse H1 and antibody against RBCL was used as a loading control. (D) Dot-blot showing the effect of exogenous RNAse H1 expression on the accumulation of plastid R-loops. Plastid DNA was extracted from control  Histograms showing the fold change intensity (mean ± standard error) of the dot-blot assays described in D (n = 3). Two-tailed paired t-test, . (F) Representative PCR analysis carried out on total DNA from control (Ctl_A), why1why3, RNAse H1_A, B, C and why1why3 RNAse H1_A lines grown 14 days on solid media supplemented with 0.5 μM ciprofloxacin alone  or with 50 μM β-estradiol . PCR results shown are representative of at least three independent experiments. Low cycle amplification of the atpB plastid gene was used as a loading control. Primer pairs used for each PCR reaction are shown next to the gels. Arrows and numbers on the right side of the gels represent the position and size of the DNA ladder bands in kilobases. (G) Representative photograph of 14-d-old plants used in F grown on CIP+EST. (H) The bar graphs represent the fold change intensity (mean + standard error) of the PCR bands in F for each plant line with respect to Ctl_A grown on CIP without EST treatment. Two-tailed paired t-test, .(PDF)Click here for additional data file.S8 Fig(A) Representative PCR analysis carried out on total DNA from control (Ctl_A and B), RNAse H1_A, B, C, why1why3, and why1why3 RNAse H1_A lines grown 14 days on solid media supplemented with 50 μM β-estradiol alone (EST) or in combination with 0.5 μM ciprofloxacin (EST+CIP). PCR results shown are representative of at least three independent experiments. Low cycle amplification of the atpB plastid gene was used as a loading control. Primer pairs used for each PCR reaction are shown next to the gels. Arrows and numbers on the right side of the gels represent the position and size of the DNA ladder bands in kilobases. (B) The bar graphs represent the fold change intensity (mean + standard error) of the PCR bands in A for each plant line with respect to Ctl_A grown on EST. Two-tailed paired t-test, .(PDF)Click here for additional data file.


E. coli podovirus HK620. Fluorescence-based monitoring of DNA release showed that HK620 phage particles in vitro ejected their genome at velocities comparable to those found for other podoviruses. Moreover, we found that HK620 irreversibly adsorbed to the LPS receptor via its TSP at restrictive low temperatures, without opening the particle but could eject its DNA at permissive temperatures. DNA ejection was solely stimulated by LPS, however, the composition of the O-antigen dictated whether the LPS receptor could start the DNA release from E. coli phage HK620 in vitro. This finding can be significant when optimizing bacteriophage mixtures for therapy, where in natural environments O-antigen structures may rapidly change.Gram-negative bacteria protect themselves with an outermost layer containing lipopolysaccharide (LPS). O-antigen-specific bacteriophages use tailspike proteins (TSP) to recognize and cleave the O-polysaccharide part of LPS. However, O-antigen composition and structure can be highly variable depending on the environmental conditions. It is important to understand how these changes may influence the early steps of the bacteriophage infection cycle because they can be linked to changes in host range or the occurrence of phage resistance. In this work, we have analyzed how LPS preparations in vitro trigger particle opening and DNA ejection from the  Bacteriophages have come back into research focus in recent years because new antimicrobial tools are needed due to the global rise of antibiotic resistance . HoweverE. coli are an important bacteriophage therapy target and reductionist laboratory model systems are required to develop efficient strategies for employing bacteriophages as part of the more complex gut microbiota [However, many phages use non-protein receptors. Especially for those that come across diderm, i.e., gram-negative, cell wall architectures only limited data is available on interactions with outer membrane components other than proteins. Here, pathogenic crobiota ,11. Gramcrobiota ,12,13,14crobiota ,16. It wcrobiota . HoweverSalmonella phage P22. P22 is an O-antigen-specific podovirus with an icosahedral head filled with double stranded DNA and a short, non-contractile tail [How LPS initiates particle opening in vitro has been investigated for ile tail ,19. P22 ile tail . In the ile tail . This stile tail . As a coile tail . In vitrile tail ,8.E. coli podovirus HK620. HK620 is a temperate lambdoid podovirus that infects E. coli with a serogroup O18A1 O-antigen [E. coli under the control of the transcription termination factor Rho [E. coli host [In this work we have analyzed in vitro particle opening in -antigen ,23,24. Tctor Rho . HK620 hctor Rho . Virion ctor Rho , with a ctor Rho  and a stoli host . We founE. coli H TD2158 as described elsewhere [E. coli strains were purchased from the German collection of microorganisms and cell cultures (DSMZ): DSM 10809 (O18A:K1:H-), DSM 10797 (O18A1: K5:H7), DSM 10837 (O18B:K?:H14), DSM 10922 (O18B1:K?:H14). E. coli H TD2158 [E. coli IHE3042 (O18A1:K5:H7) was kindly provided by Anja Siitonen from the National Institute for Health and Welfare, Finland. If not indicated otherwise, 50 mM Tris/HCl, pH 7.6, 4 mM MgCl2 was used as a standard buffer. The truncated HK620TSPΔN without an N-terminal particle-binding domain was used for all experiments, as the latter does not participate in O-antigen binding [Phage HK620 was kindly provided by Mireille Ansaldi from University of Marseille and was propagated on lsewhere . E. coli binding .ompA and ompC genes of E. coli H TD2158 were replaced by tetracycline and kanamycin resistance gene cassettes, respectively, using homologous recombination as described before for Shigella [ompA replacement was constructed by amplifying the TetRA module from strain UB-1766 [ompA in E. coli: 5′-CGGGG TTTTTCTACCAGACGAT AACTTAAGCCTGCGGCTGAGTTACAACGCACCAAACACCCCCCAAAACC and 5′-GTGCTC GGCATAAGCCGAAGATATCGGTAGAGTTAATATTGAGCAGATCCACACAACCACACCACACCAC, transforming TD2158 with the amplified DNA and selecting for tetracycline resistance. The ompC replacement was constructed by using primers 5′-GGCCCGACGGTAATAT and 5′-TTGCTGGAAATTATGC to amplify an approximately 2 kbp DNA fragment that includes the kanamycin cassette that replaces the ompC gene in E. coli K-12 strain JM2203 DNA [ompC gene in strain TD2158.The Shigella . The omp UB-1766  with the2203 DNA , and usiE. coli cells of strain H TD2158 were grown overnight at 37 °C in LB medium. Specific staining showed that all other E. coli strains used in this study expressed capsules described earlier as K1 or K5. These were cultured at 18 °C in order to minimize capsule synthesis. LPS purification, as well as TSP-digested LPS, have been described [escribed .−1 LPS and about 5000 phage HK620 plaque forming units (pfu) per mL in 50 mM Tris pH 7.6, 4 mM MgCl2 were incubated in glass tubes at 37 °C or 45 °C for indicated times. Then, 100 µL of the solutions were plated on E. coli H TD2158.In vivo inhibition of phage HK620 by LPS preparations was determined in a plaque forming assay. 25 µg mL® fluorescence as described [9 pfu mL−1 HK620 phages with LPS from E. coli H TD2158, at different temperatures. At the end of each experiment, DNase I (10 µg/mL) was added as a control for DNA accessibility. Kinetic traces were fitted to Equation (1) describing two sequential first-order processes [In vitro DNA ejection was monitored via Yo-Proescribed  by incubrocesses   in a 3 mm quartz cell in standard buffer. To determine Stokes radii, 50 or 100 correlation functions were measured in 8.4 s intervals at 37 °C ± 0.1 °C. Average concentration independent Stokes radii were calculated with CONTIN [LPS was analyzed by dynamic light scattering , a high quantum yield avalanche photo diode and an ALV 7002–25 ns correlator ). Different LPS concentrations were investigated (0.01 mg mLh CONTIN  using dyE. coli H TD2158 (O18A1), emptied particles attached to the LPS and appeared dark in the negative stain, indicating that the virions were empty and devoid of DNA  A. After d of DNA B,C. In ad of DNA . We therE. coli H TD 2158 (O18A1) host we observed an increasing fluorescence signal . In the control, the number of pfu was stable within the experimental time frame. We also incubated phage HK620 with lipid A or an O18A1 O-antigen polysaccharide preparation lacking the lipid part; however, no significant decrease in infectivity could be detected. This is in agreement with the fact that these LPS components alone were also unable to trigger DNA ejection in vitro (cf. E. coli H TD2158 (O18A1) by homologous recombination. HK620 had the same infectivity on these two mutant strains as wild types and cleared liquid cultures of these strains within the same time frame  on host bacteria to 35% of the initial value A. t-Testitro cf. . Thus, ime frame B. Hence,E. coli outer membrane was observed. This is in contrast to Shigella phage Sf6 that copurified with Omps [It is important to note here that all LPS preparations used in the inhibition tests or in the in vitro experiments were free from outer membrane proteins (Omps). Moreover, during HK620 preparation no binding of phage particles to any protein component from the ith Omps . Taken tE. coli LPS preparations both for TSP interactions and their capacity to provoke DNA release. Purified HK620TSP recognizes and enzymatically cleaves the O-antigen of the immunological serogroup O18A1, which contains a hexasaccharide repeat structure [E. coli strain DSM10809, while different glycosidic linkages that occur in the O18B serogroups prohibited binding. HK620TSP rapidly cleaved the O-antigen from different strains containing the O18A1 type O-polysaccharide (E. coli IHE3042 [E. coli H TD2158 (Tailspike proteins (TSP) on LPS recognizing phages confer O-antigen specificity to the infection process ,36,37. Ttructure ,24. We ptructure  and analccharide B,C. Long IHE3042  in the sH TD2158 C.E. coli strains were tested for their ability to inactivate HK620 phage particles. They only differ in a single α-d-Glcp-(1→6)-branch linked to the reducing end GlcNAc that is lacking in the O-polysaccharide repeat unit of O18A strains [E. coli DSM10809 (O18A) contained shorter filaments, for LPS from E. coli IHE3042 (O18A1) even smaller and only rod-like structures were found  had Stokes radii of about 95 nm. For E. coli DSM10809 LPS (O18A), slightly reduced radii of about 70 nm were found, similar to E. coli IHE3042 LPS (O18A1) with a smaller Stokes radius of 65 nm. The E. coli IHE3042 LPS (O18A1) showed small structures on the EM grid and rarely formed rods (cf. E. coli H TD2158 LPS (O18A1) (95 nm)  .E. coli DSM10809 LPS (O18A) as a receptor, although with a lower efficiency than found for the host LPS (E. coli IHE3042 (O18A1) did not provoke DNA release. It is important to note here that HK620 was unable to successfully infect both E. coli DSM10809 (O18A) and E. coli IHE3042 (O18A1) strains tested and only formed plaques on its host strain E. coli H TD2158 (O18A1). However, when plaque formation on this strain was monitored after incubation with LPS from the two non-host strains, E. coli DSM10809 LPS (O18A) was found to significantly (α < 0.05) decrease HK620 pfu to 16% of the initial value (E. coli IHE3042 LPS (O18A) was unable to inactivate HK620 phage particles and could not provoke a decrease in the number of pfu on host cells  LPS via DNA fluorescence as described above  LPS triggered DNA release in an in vitro fluorescence assay, (ii) in electron microscopy where LPS incubation showed particles that had released their DNA and (iii) in LPS-mediated inhibition of infective particles. Like bacteriophage P22, HK620 needed no additional protein receptor for in vitro DNA release , this isE. coli H TD2158, with long O-antigen chains forming ribbons and filaments able to initiate phage HK620 ejection. Similar morphologies were also observed for S. Typhimurium LPS preparations that triggered DNA release from phage P22 [E. coli DSM10809 that could in part provoke DNA release from phage HK620, or (iii) a high concentration of rather short O-antigen chains dominated LPS preparations from E. coli IHE3042, that were unable to act as a receptor for HK620.For podovirus HK620 we observed that the LPS preparation must contain enough molecules equipped with long O-antigen chains to trigger DNA egress in vitro. If the LPS receptor did not contain enough long chains or was depleted from the O-antigen by enzymatic cleavage with HK620TSP, no ejection could occur. The proportion of molecules in the preparation with long O-antigen chains is also typically reflected in different LPS aggregate morphologies in electron microscopy . LPS isohage P22 . (ii) A S. typhimurium, which transiently blocked infection by phages P22, 9NA and Det7 [E. coli are most probably the reason why the phage was not able to infect alternative O18A strains. Both strains, E. coli IHE3042 and E. coli DSM10809, were protected with capsules at the permissive 37 °C replication temperatures of HK620. However, both strains produced sufficient amounts of smooth LPS at 18 °C , a temperature where HK620 particles could not open.These results illustrate that even if the correct O-antigen receptor is present and can be addressed by the TSP, this will not ultimately lead to DNA ejection from an O-antigen-specific phage. Rather, this emphasizes that infection is not only O-antigen specific but is also tightly linked to the LPS composition that is dynamic with bacterial growth conditions and metabolic state ,41,42. Hand Det7 . In the In vitro experiments are useful in testing how well an LPS preparation can mimic the membrane environment that the phage encounters in vivo. Although they cannot show how phage proteins and genetic material enter the cytosol of a living bacterial cell, they provide a reductionist approach to the mechanisms in the bacteriophage infection machine. In vivo, to transport its DNA over all parts of the gram-negative cell envelope, P22 particles extend their tails to form tubular structures, as found in other gram-negative-specific phages ,46,47. TWe monitored kinetics of in vitro HK620 particle opening with a DNA-sensitive fluorescence assay. The DNA egress can be described by two sequential first-order equations, suggesting that our bulk manual mixing experimental setup resolved two steps of the process. Remarkably, similar kinetics of in vitro DNA ejection were found for other O-antigen-specific podoviruses like P22 and Sf6 . These kA very important finding for O-antigen-specific bacteriophages is that in order to act as a functional receptor the O-antigen must be part of a membrane and that the isolated O-polysaccharide will not trigger particle opening in vitro ,21. HereShigella podophage Sf6 could only be inactivated in vitro by a mixture containing both LPS and OmpA or C [S. flexneri host lacking Omp A or Omp C [Shigella host requires a different particle interaction strategy. This is illustrated on the one hand by the special structure of the Sf6 gp26 needle with a C-terminal knob that is lacking in P22 and HK620 [Also, upon in vivo infection the phage might need to encounter specifical dynamic regions of the bacterial membrane. Especially Omps might play a key role as experimental and theoretical studies showed that they may influence the LPS membrane thickness and structure ,58. The mpA or C . Sf6 wasor Omp C . Apparennd HK620 . Future nd HK620 . It remaE. coli LPS showed that smooth LPS was less densely packed and more dynamic than rough LPS [In principle, the thermal barriers that we observed for the HK620 DNA ejection process in vitro could have two origins: First, a temperature-dependent destabilization of the tail complex leading to an ejection-competent particle at a permissive temperature. Recently, it was shown for the podovirus T7 that tapping the capsid wall with an oscillating atomic force microscope cantilever could trigger rapid DNA ejection via the portal, illustrating that a conformational switch can be mechanically activated by external forces . Second,ough LPS . O-antig


POLR3A, encoding the largest subunit of Pol III, cause POLR3-related hypomyelinating leukodystrophy (POLR3–HLD), characterized by deficient central nervous system myelination. Identification of the downstream effectors of pathogenic POLR3A mutations has so far been elusive. Here, we used CRISPR-Cas9 to introduce the POLR3A mutation c.2554A→G (p.M852V) into human cell lines and assessed its impact on Pol III biogenesis, nuclear import, DNA occupancy, transcription, and protein levels. Transcriptomic profiling uncovered a subset of transcripts vulnerable to Pol III hypofunction, including a global reduction in tRNA levels. The brain cytoplasmic BC200 RNA (BCYRN1), involved in translation regulation, was consistently affected in all our cellular models, including patient-derived fibroblasts. Genomic BC200 deletion in an oligodendroglial cell line led to major transcriptomic and proteomic changes, having a larger impact than those of POLR3A mutations. Upon differentiation, mRNA levels of the MBP gene, encoding myelin basic protein, were significantly decreased in POLR3A-mutant cells. Our findings provide the first evidence for impaired Pol III transcription in cellular models of POLR3–HLD and identify several candidate effectors, including BC200 RNA, having a potential role in oligodendrocyte biology and involvement in the disease.RNA polymerase III (Pol III) is an essential enzyme responsible for the synthesis of several small noncoding RNAs, a number of which are involved in mRNA translation. Recessive mutations in  POLR3A, POLR3B, and POLR1C, encoding subunits of the ubiquitous RNA polymerase III (Pol III),1–POLR3A mutations were also recently linked to hereditary spastic ataxia  is specifically affected in POLR3–HLD remains puzzling, as most Pol III transcripts are highly abundant and expressed ubiquitously. Indeed, the CNS appears to be particularly vulnerable to impaired Pol III and/or tRNA function, because mutations in a basal Pol III transcription factor also cause a rare cerebellar disorder , 10, whe11–23–POLR3A, POLR3B, and POLR1C encode subunits of Pol III, one of the three essential eukaryotic nuclear RNA polymerases. POLR3A and POLR3B are the two largest subunits of Pol III and form the active center of the enzyme  exclusively possess upstream promoter elements  mutation, located in the cleft domain of Pol III harboring the template DNA during transcription  of BC200 in an oligodendroglial-derived cell line results in major transcriptomic and proteomic changes, suggesting an important functional role of BC200 RNA in this cell type. Altogether, our results indicate that tRNAs, BC200 RNA, and its downstream targets may be important effectors of POLR3–HLD pathogenesis.In this study, we performed comprehensive profiling of the Pol III transcriptome in human cells carrying the Fig. S1), indicating that this mutation does not impair Pol III complex assembly or nuclear import as did the previously reported POLR1C mutations  and mutant M852V) POLR3A in HeLa cells and performed anti-FLAG affinity purification followed by MS as well as immunofluorescence. POLR3A–M852V pulled down similar levels of Pol III complex subunits compared with the WT and did not accumulate in the cytoplasm  mutation in the endogenous POLR3A gene in HEK293 cells using the CRISPR-Cas9 system . These clones mimic the genotypes observed in POLR3–HLD cases carrying the c.2554A→G (p.M852V) mutation, because four out of the five reported patients that are compound heterozygous for this mutation have a premature stop codon on the other allele  in patient-derived fibroblasts, resulting in decreased levels of POLR3A protein (b and c). POLR3A mRNA levels were reduced in clones M1 and M2 compared with WT clones but not in the homozygous mutant clone M3 . Upon inspection of the cDNA, we observed partial (∼40%) in-frame skipping of exon 19, carrying the M852V mutation, in clone M3 . It was previously shown that in-frame skipping of exons carrying an indel sometimes occurs following CRISPR-Cas9 editing . ChIP followed by quantitative PCR at three Pol III-transcribed genes also showed comparable POLR3A occupancy in the two groups . Although a direct correlation generally exists between Pol III occupancy and nascent RNA levels . We synthesized exogenous spike-in RNAs to confirm small RNA enrichment  and focused on precursor tRNAs to (i) overcome the quantification challenges associated with the strong secondary structure and RNA modifications of mature tRNA, and (ii) to more directly assess Pol III transcription levels, without the confounding effects of tRNA stability . In contrast to the drops in coverage expected at highly-modified positions in mature tRNAs , indicating that most reads originate from precursor tRNA molecules that have not yet acquired post-transcriptional modifications. For Pol III transcripts larger than 200 nucleotides, we performed standard rRNA-depleted RNA-seq. Pol III transcripts showed a global trend toward decreased expression in mutants compared with controls in both RNA-seq datasets (a). Furthermore, stratifying according to their promoter type revealed that this decrease was mostly attributable to Pol III transcripts possessing type 2 internal elements, whereas type 3 transcripts were less affected (b).Pol III occupancy levels measured by ChIP-seq have traditionally been used as a proxy for Pol III transcription levels , 39, becA levels , this core tRNAs , 42, we datasets a. Furtheaffected b.p < 0.0001, Wilcoxon rank-sum test) (a). However, although the majority of expressed precursor tRNAs showed a trend toward a decrease , at the individual gene level only four tRNA genes demonstrated a statistically significant decrease (FDR <0.05), due to the variability in the genotypes of the three mutants. To reduce variability and analyze the effects of the potentially most detrimental POLR3A mutations on tRNA expression, we also analyzed biological triplicates of mutant M2, the clone with the lowest POLR3A expression (c and d). Indeed, the large majority of nuclear-encoded tRNAs showed decreased expression in M2 compared with control C3 (c and d), with 77 tRNAs reaching statistical significance at the individual gene level . In contrast, levels of other categories of small RNAs (snRNAs and snoRNAs), the majority of which are not synthesized by Pol III, were unaffected, both in individual clones  and in replicates of clone M2 . Decreased tRNAs belonged to most tRNA isoacceptor families (a), suggesting a widespread effect on transcription of tRNA genes and arguing against a specific impact on tRNAs cognate for certain codons or amino acids. We next confirmed the decreased precursor tRNA levels by an independent method, measuring the expression levels of three tRNAs by qRT-PCR with primers specific to the precursor form for two of them (b). However, the down-regulation of precursor levels did not result in a decrease in levels of three mature tRNAs detectable by Northern blots (c), suggesting that the stability of tRNAs is sufficient to overcome the impaired tRNA synthesis in HEK293 cells. Altogether, these results provide the first evidence that a leukodystrophy-causing POLR3A mutation causes a global reduction in transcription levels of nuclear-encoded tRNA genes.Because nuclear-encoded tRNAs represent the majority of transcripts with a type 2 promoter, we examined their expression in the small RNA transcriptome data, consisting mostly of precursor tRNAs. Indeed, we observed a global reduction of precursor tRNA levels in the three mutants compared with controls (um test) a. Howevepression , c and dfamilies a, sugges of them b. Howevern blots c, suggesBC200  and genes encoding 7SL RNA were among the most significantly down-regulated genes in the large RNA transcriptome . In contrast, vault RNA levels were comparable across both groups, making them the only transcripts with type 2 internal elements that were not decreased in mutants, which could be due to their vault RNA-specific distal regulatory sequences . Taken together, these results strongly suggest that transcripts possessing type 2 internal promoter elements are the most vulnerable to the studied disease-causing POLR3A mutation.We next examined the other transcripts with type 2 internal promoter elements, which include BC200 RNA, 7SL RNAs, and vault RNAs , 29, 31.a and b), suggesting a particular sensitivity to Pol III hypofunction. To validate this finding, we performed Northern blotting for BC200 RNA in four controls and four POLR3A mutants  carrying the F849L mutation in POLR3A (see supporting Methods)). This confirmed that BC200 RNA levels are significantly decreased in mutants compared with controls (c). To determine whether this transcript was also affected in patient cells, we examined two independent datasets of patient-derived fibroblasts. First, in RNA-seq from four French Canadian POLR3A patients (see genotypes in Table S5), BC200 RNA was the top affected Pol III transcript in patients compared with controls , although not reaching statistical significance after multiple testing correction . Other large Pol III transcripts did not display significant differences between patients and controls . Second, using a custom microarray for all known Pol III transcripts in four American POLR3A cases , we found that BC200 RNA displayed a significant reduction in patients compared with controls . Altogether, we show that BC200 RNA is the top down-regulated large Pol III transcript in three separate datasets of POLR3A mutant cells, including carriers of several distinct mutations, emphasizing its vulnerability to Pol III hypofunction and suggesting a possible role in the pathophysiology of POLR3–HLD.Among the large RNA transcriptome, BC200 RNA, the only Pol III transcript observed to have a predominant CNS expression, was the most down-regulated transcript in HEK293 mutant clones , a and bcontrols c. To detPOLR3A mutant cells and patient-derived fibroblasts, we next investigated the effects of decreasing BC200 RNA expression. Because oligodendrocytes are likely to be one of the main dysfunctional cell types in POLR3–HLD , resulting in absent expression . Next, using the same approach as above, we generated MO3.13 cells carrying the POLR3A c.2554A→G (p.M852V) mutation in compound heterozygosity with a null allele . We verified that POLR3A mutant MO3.13 cells recapitulate the effects observed in HEK293 cells. Indeed, they display reduced levels of POLR3A protein . They also show significantly decreased levels of BC200 RNA . Consistent with our above results, type 3 Pol III transcripts RN7SK, RPPH1, and RMRP were unaffected in POLR3AM852V cells . However, contrary to what we observed in HEK293 cells, 7SL RNA levels were not decreased in mutant MO3.13 cells , suggesting cell type–specific effects that could be related to upstream regulatory sequences specific to 7SL genes (M852V and absent in BC200KO), which was confirmed by Northern blotting (a).Given the expression of BC200 RNA in the CNS and its consistent down-regulation in OLR3–HLD , 8 for tOLR3–HLD , 44. FirSL genes . In sum,blotting a.b), detecting a total of 1,272 proteins. We found that, compared with WT cells, BC200 KO had a more potent effect on the transcriptome and proteome than the POLR3A M852V mutation did, leading to a higher number of dysregulated mRNAs and proteins and to more of them showing a large effect size  . In particular, more proteins were up-regulated than down-regulated in BC200KO cells  . These observations point to a clear functional role for BC200 RNA in MO3.13 cells, because its inactivation leads to significant changes for more than 20% of detected proteins  and expressed mRNAs  (Tables S7 and S8). Gene Ontology analysis showed an over-representation of proteins located in the plasma membrane, endoplasmic reticulum, and cytoskeleton among the up-regulated proteins. In contrast, down-regulated proteins were enriched for ones involved in the G1/S transition, including members of the condensin and minichromosome maintenance complexes, suggesting slower cell growth or cell cycle defects.Given the potential role of BC200 RNA in mRNA stability, transport, and translation regulation , 45, 46,Fig. 6n = 1,200)  and searched for common effects in BC200KO and POLR3AM852V. Although only 29 proteins were consistently significantly dysregulated (log2 fold change >0.5) in both mutants (d), we observed that proteins affected in BC200KO  tended to be affected in the same direction if they were also significant in POLR3AM852V (FDR <0.05), albeit with a lower fold change (e). We also observed a similar effect at the mRNA level . This suggests that the changes in mRNA and protein levels observed in POLR3A mutant cells may be partially explained by reduced expression of BC200 RNA and further support a contribution of this ncRNA to POLR3–HLD pathogenesis.For the remainder of our analyses, we focused on mRNA/protein pairs that were properly detected in both the RNA-seq and SILAC datasets  that were significantly increased or decreased in BC200KO compared with WT but that did not display significant differences at the mRNA level , implying protein-level regulation. Furthermore, we found an additional 54 proteins  that had substantially greater fold changes compared with their mRNA counterparts . Thus, although the majority of deregulated proteins (63.0%) have similar fold changes in SILAC and RNA-seq, suggesting that their regulation occurs at the transcriptional/mRNA level, the remainder (37.0%) appears to undergo changes at the protein level only as a result of BC200 KO (f), perhaps through the direct action of this ncRNA on translation initiation (n = 65) than down-regulated ones (n = 37) in this category (f), which is consistent with the putative role of BC200 as a negative regulator of translation . This type of promoter is composed of control elements located within the transcribed sequence, called A- and B-boxes  with significant phenotype overlap with POLR3–HLD . For differentiation of MO3.13 cells, cells were switched to serum-free 1× DMEM supplemented with 100 nm phorbol 12-myristate 13-acetate (PMA), as described previously (Stable HeLa cell lines expressing FLAG-tagged POLR3A variants (WT or M852V) were produced by transfection with Lipofectamine according to the manufacturer's instructions. MO3.13 cells were purchased from Cedarlane CELLutions Biosystems (no. CLU301). Unless otherwise specified, all cells were grown in 1× DMEM (Wisent no. 319-005-CL) supplemented with 10% fetal bovine serum (Wisent no. 080-150) and 1% penicillin–streptomycin–eviously , and colAffinity purification, MS, and immunofluorescence in cell lines expressing FLAG-tagged POLR3A subunits were performed as described previously , 61.BC200 KO cell lines were generated using an approach adapted from Ref. BC200 gene . Detailed methods are described in the supporting Methods.POLR3A mutant cell lines carrying the c.2554A→G (p.M852V) or c.2547C→G (p.F849L) mutations were generated using CRISPR-Cas9 coupled with homology-directed repair, as described previously . BC200 KPOLR3A were amplified (see Table S10 for primers) and Sanger-sequenced (McGill University and Génome Québec Innovation Centre).For RT-PCR, 1 μg of RNA was reverse-transcribed using the Superscript III reverse transcriptase (Thermo Fisher Scientific). For detection of the M852V mutation, exons 17–21 of m Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, and protease inhibitors (Roche Applied Science)) and incubated on ice for 30 min, followed by centrifugation at 14,000 rpm for 10 min and collection of the supernatant. Protein concentration was determined using DCTM protein assay (Bio-Rad). Protein samples were separated onto a 4–12% NuPAGE BisTris gel (Thermo Fisher Scientific) and transferred onto a nitrocellulose membrane (Bio-Rad). Immunoblots were probed with anti-POLR3A (Abcam no. ab96328), anti-GAPDH (GeneTex no. GTX627408), and anti-actin (Abcam no. ab3280).Cells were homogenized in lysis buffer  were enriched from total RNA using Qiagen miRNeasy (no. 217004), directly followed by random priming, cDNA synthesis, and next-generation sequencing. To monitor the level of small RNA enrichment in each sample, we synthesized three spike-in RNAs  and added them at the beginning of the procedure. Libraries were prepared with the KAPA-stranded RNA-seq library preparation and sequenced on an Illumina HiSeq 2500 with 100-bp single-end reads at the McGill University and Génome Québec Innovation Centre, resulting in an average of 25 million reads/sample. Quality control and trimming were performed as described previously (supporting Methods).Detailed protocols and data analysis are described in eviously . Trimmedeviously , includieviously  and UCSCeviously  version p < 0.05 and a mean normalized expression of >100. Because Pol III transcripts are highly repetitive, alignment and subsequent analyses were repeated with the inclusion of all multimapping reads to confirm consistency in results. Only results that replicated when using either uniquely mapped or multimapping reads were reported.Total RNA was extracted using miRNeasy (Qiagen) or TRIzol (Thermo Fisher Scientific), treated with the RNase-free DNase set (Qiagen), and quantified on Nanodrop. RNA Integrity Number (RIN) was assessed on an Agilent Bioanalyzer and was routinely above 8. RNA extractions were submitted to Illumina TruSeq rRNA-depleted stranded library preparation (HEK293 cells and patient fibroblasts) or NEBNext rRNA-depleted stranded library preparation (MO3.13 cells). HEK293 libraries were sequenced on Illumina HiSeq 2500 with 125-bp paired-end reads at an average of 42 million reads/sample. Fibroblast libraries were sequenced on Illumina HiSeq 2000 with 50-bp paired-end reads at an average of 60 million reads/sample. MO3.13 libraries were sequenced on an Illumina HiSeq 4000 with 100-bp paired-end reads at an average of 61 million reads/sample. Quality control and trimming were performed as described previously . TrimmedPOLR3A-mutated cells were cultured to 80% confluence and cross-linked with 1% formaldehyde directly in the cell medium for 5 min followed by a 5-min quenching in 125 mm glycine. For ChIP experiments, nuclei from 6 × 106 cells were lysed and resuspended in sonication buffer . Chromatin was prepared by sonicating the nuclei in a Covaris E-Series E22 for 8 min at Duty 2. This generated chromatin fragments of 500 bp on average. Sonicated chromatin was immunoprecipitated using rabbit POLR3A antibody (Abcam no. ab96328) bound to magnetic beads (protein A and protein G Dynabeads) (Sigma) for 4 h. The eluates were loaded to a PCR purification kit (Qiagen). Sequencing libraries were prepared from input and ChIP eluates using the TruSeq DNA library preparation kit and sequenced on an Illumina HiSeq 2000 with 50-bp paired-end reads at an average of 18 million reads/sample for input libraries and 56 million reads/sample for ChIP libraries. Quality control and trimming were performed as described previously (read_count/inputread_count), where IP is immunoprecipitation, as described in Orioli et al.  according to the manufacturer's instructions. Real-time PCR was performed in technical duplicates with a 1:25 dilution (tRNAs and BC200 RNA) or 1:2 dilution (MBP) using the SYBR GreenERTM qPCR SuperMix (Thermo Fisher Scientific) on a Bio-Rad CFX96. The ΔΔCt method was used to calculate relative RNA expression, with normalization to genes PSMB6, SDHA, and COPS7A (HEK293 cells) or NDUSF2 and PMM1 (MO3.13 cells). All primers used are indicated in Table S10.Total RNA was extracted using miRNeasy (Qiagen), treated with the RNase-free DNase set (Qiagen), and quantified on Nanodrop. RIN was assessed on an Agilent Bioanalyzer and was routinely above 8. For qRT-PCR of tRNAs, BC200 RNA and m urea denaturing 8% PAGE and transferred to positively charged nylon transfer membranes . The resulting blots were hybridized with 32P-5′ end–labeled probes detecting BC200 RNA, tRNA–Ala–TGC, tRNA–Lys–TTT, tRNA–Leu–CAA and 5.8S rRNA in ExpressHyb hybridization solution (TaKaRa). All probes used are indicated in Table S10 and are complementary to the last 24 nucleotides in 3′ of mature tRNAs, including the CCA trinucleotide. 5.8S rRNA was used for normalization of BC200 RNA and tRNA levels.Total RNA was extracted using miRNeasy (Qiagen), treated with the RNase-free DNase set (Qiagen), and quantified on Nanodrop. RIN was assessed on an Agilent Bioanalyzer and was routinely above 8. RNA samples (3 μg) were separated on a 7 supporting Methods. Briefly, cells were grown in DMEM for SILAC  supplemented with 10% dialyzed fetal bovine serum  and light, medium, or heavy amino acids for MO3.13 WT, BC200KO, and POLR3AM852V, respectively (b). For each of the six independent replicate experiments, equal protein quantities from the three conditions were mixed. Samples were digested with trypsin and analyzed via LC-MS/MS. Data analysis was performed with MaxQuant (Detailed reagents, protocols, and data analysis are included in the ectively b. For eaMaxQuant  version K. C., G. B., A. V., R. S., M. T., B. C., B. B., and C. L. K. conceptualization; K. C. and D. F. data curation; K. C., D. F., E. M., M. T., and C. L. K. formal analysis; K. C., D. F., E. M., and M.-J. D. validation; K. C., D. F., E. M., M.-J. D., M. R. F., B. B., and C. L. K. investigation; K. C., D. F., M. R. F., and C. L. K. methodology; K. C., B. B., and C. L. K. writing-original draft; K. C. project administration; K. C., D. F., E. M., M.-J. D., G. B., A. V., R. S., M. R. F., M. T., B. C., B. B., and C. L. K. writing-review and editing; G. B., B. C., B. B., and C. L. K. funding acquisition; B. C., B. B., and C. L. K. supervision; C. L. K. resources.


Type IV lupus nephritis (LNIV) is a severe disease characterized by diffuse proliferative lesions, and its prognosis is worse with cellular crescent (LNIV-CC) involvement. Urinary exosomes have been shown to reflect the degree of kidney injury. This study was aimed to identify non-invasive diagnostic markers for LNIV-CC. We analysed the expression profile of microRNAs (miRNAs) isolated from urinary exosomes in patients with LNIV-CC and LNIV, and healthy individuals using high-throughput sequencing.A total of 66 differentially expressed miRNAs were identified, which were significantly enriched in 15 signalling pathways. Bioinformatic analysis revealed a co-expression network of miRNAs, predicted transcription factors and target mRNAs. Expression of three miRNAs including miR-3135b, miR-654-5p, and miR-146a-5p were further analysed and validated by reverse transcription-quantitative polymerase chain reaction. ROC analysis suggested these as candidate biomarkers for LNIV-CC.LNIV-CC has a unique miRNA expression profile of urinary exosome and complex regulatory network. miR-3135b, miR-654-5p and miR-146a-5p in urinary exosomes could be used as novel non-invasive diagnostic markers for LNIV-CC. Type IV lupus nephritis (LNIV) is a severe disease characterized by diffuse proliferative lesions. The treatment regimens of LNIV are determined based on pathological features in accordance to the guidelines of “the Kidney Disease: Improving Global Outcomes” (KDIGO) . RecentlExosomes are small vesicles (diameter of 30–120 nm) containing microRNAs (miRNAs), mRNAs, and proteins , 6. TheyIn the present study, we investigated the expression profiles of miRNAs isolated from urinary exosomes in patients with LNIV-CC and LNIV (without CC), we validated three selected differentially expressed miRNAs by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and performed bioinformatic analysis on the differentially expressed miRNAs and receiver operating characteristic (ROC) analysis for the validated miRNAs.−1 with stable renal function without active urinary sediments. The study was approved by the Ethics Committee of the First Hospital Affiliated to Army Medical University  in accordance with the Helsinki Declaration amended in 2008. All participants signed informed consents.A total of 44 patients were enrolled at the Department of Nephrology, the First Hospital Affiliated to Army Medical University from February 2017 to January 2018, including 15 active LNIV, 15 active LNIV-CC and 14 inactive LNIV. A total of 13 healthy volunteers without systemic diseases and normal renal function were also recruited from the health centre of the hospital from February 2017 to January 2018. miRNA sequencing was performed on 5 cases of active LNIV, 5 cases of active LNIV-CC, 4 cases of inactive LNIV, and 3 cases of healthy volunteers. The qRT-PCR validation was performed on additional patients  and healthy volunteers (10 cases). All patients with LNIV were diagnosed according to the KDIGO Clinical Practice Guidelines [g for 30 min to remove the cells and debris, and then centrifuged at 17,000×g for 30 min at 4 °C to remove large membrane vesicles. The resulting supernatant was centrifuged  at 200,000×g for 70 min at 4 °C to pellet the exosomes. The resulting pellets were resuspended in phosphate-buffered saline (PBS) and centrifuged at 200,000×g for 70 min at 4 °C. Finally, the putative exosome pellets were resuspended in PBS and stored at − 80 °C.Fresh first-morning urine samples (100 ml) were collected in sterile containers and processed within 1 h after collection. Exosomes were isolated from the urine specimens using differential centrifugation. Briefly, the urine sample was centrifuged at 2500×Caenorhabditis elegans miRNA cel-miR-39 , lacking the endogenous human urinary miRNA. The RNA concentration was determined using the Qubit™ 3.0 Fluorometer .RNA was extracted from the exosomes using Trizol  according to the manufacturer’s recommended protocol. Denatured exosomes were spiked with the normalization control, the synthetic To verify the characteristics of exosomes, we examined the morphology of exosomes using transmission electron microscopy. A drop (~ 30 μl) of exosomes was resuspended in PBS and placed on parafilm. A formvar/carbon-coated copper grid was positioned on top of each drop for 15 min. Finally, a drop of 2% uranyl acetate was placed on the parafilm and incubated the exosomes for 5 min in the dark. The grids were negatively stained, air-dried, and examined with transmission electron microscopy .g for 15 min. The supernatant was collected and transferred to a new EP tube. An equal amount of total soluble protein was subjected to 8% sodium dodecyl sulphate–polyacrylamide gel electrophoresis  and transferred to a polyvinylidene fluoride membrane . The membranes were incubated with primary antibodies of CD9 and CD81  and a primary antibody of non-exosomal protein calnexin mouse polyclonal . Then, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies. Blots were visualized with chemiluminescence reagents .The urinary exosomes were lysed in RIPA lysis buffer on ice, and then centrifuged at 12,000×Total RNA was extracted from exosomes and used for miRNA high-throughput sequencing. Library preparation and miRNA sequencing were performed by Ribobio . Briefly, total RNA samples were fractionated and only small RNAs were used for library preparation. After PCR amplification, the products were sequenced using the Illumina HiSeq 2500 platform  following the manufacturer’s instruction on running the instrument. Raw sequencing reads were obtained by using related Illumina’s analysis software.http://www.targetscan.org/vert_72/), miRDB (http://mirdb.org/), miRTarBase , and miRWalk  software tools were used to predict the target gene of the identified differentially expressed miRNAs. Cytoscape  was used to establish miRNA-based regulatory network. The visualized network diagram of TFs, miRNAs, mRNAs, and their interactions was performed [Differentially expressed exosomal miRNAs were screened out from the sequencing results. To predict TFs of the identified 66 differentially expressed miRNAs, BioPython  of JASPAerformed . The enrerformed .(−ΔΔCt) after normalization to cel-miR-39 as an exogenous control.The differentially expressed miRNAs were further validated by RT-qPCR. The expression of selected miRNAs was performed using the Stem-loop TaqMan miRNA RT Kit  and TaqMan RT assay according to the manufacturer protocols. A fixed volume of cDNA (1.2 μl) was combined with TaqMan universal PCR master mix II no UNG. The stem-loop sequences were UGCCCAGGCUGGAGCGAGUGCAGUGGUGCAGUCAGUCCUAGCUCACUGCAGCCUCGAACUCCUGGGCU for miR-3135b, GGGUAAGUGGAAAGAUGGUGGGCCGCAGAACAUGUGCUGAGUUCGUGCCAUAUGUCUGCUGACCAUCACCUUUAGAAGCCC for miR-654a-5p, and CCGAUGUGUAUCCUCAGCUUUGAGAACUGAAUUCCAUGGGUUGUGUCAGUGUCAGACCUCUGAAAUUCAGUUCUUCAGCUGGGAUAUCUCUGUCAUCGU for miR-146a-5p. The relative expression levels of target miRNAs were compared between samples using the comparative cycle threshold (Ct) method 2t test. We measured the area under the curve (AUC) of the receiver operating characteristic (ROC) curve for the validated miRNAs. The discrimination capability of the simple risk score was also presented by ROC curve. p < 0.05 was considered statistically significant.Statistical analysis was performed by SPSS Statistics for Windows software version 19.0  with student’s p < 0.01). Moreover, the 24-h proteinuria was lower in the active LNIV-CC group than the active LNIV group (p < 0.05)  , and creThe extracted microvesicles were cup-shaped, < 100 nm in diameter, and scattered , whereas the miR-654-5p expression level in the LNIV-CC group was significantly higher than LNIV and control groups (p < 0.001). The expression levels of miR-146a-5p in the inactive LNIV (p < 0.05) and LNIV-CC (p < 0.05) groups were significantly higher compared to that of the control group   was usedThe network diagram of TFs, miRNAs, mRNAs, and their interactions are shown in Fig. To evaluate the prediction value of miR-3135b, miR-654-5p, and miR-146a-5p, receiver operating characteristic (ROC) analysis was performed Fig. . In LNIVmiRNAs play an important role in biological function and renal pathology , 22. CirWe demonstrated that the miRNA expression profile of urinary exosome in LNIV-CC is distinct from other groups, and miR-3135b and miR-654-5p are candidate biomarkers of LNIV-CC. The specificity of prediction of them was as high as 83.33–96.67%, which indicates the value of these miRNAs for further research and potential clinical application. miR-146a expression levels were also higher in the LNIV-CC group than the control group. miR-146a has been reported as a marker of glomerular and tubular interstitial tissue injury , 26. ThemiR-3135b is a biomarker of coronary artery calcification, heart failure, and non-ST segment elevation acute coronary syndromes, which is closely related to a poor cardiovascular prognosis , 31. ThumiR-654-5p has been reported to promote carcinoma proliferation in oral squamous cell through the miR-654-5p/GRAP/Ras/Erk signalling pathway , and dowThere are several limitations in the present study. First, the number of subjects was relatively small. We only performed miRNA sequencing on only 17 patients, and validated the data on additional 40 patients. Therefore, large-scale cohort studies are needed to further analyse and validate these results, especially for diagnosis. Second, this is a cross-sectional study, and thus follow-up after treatment of lupus nephritis is needed to confirm the findings. Third, recent reports suggest that exosomes may cross the blood–brain barrier through receptor-mediated transcellular transport , 35; howOverall, we demonstrate that LNIV with crescents has a unique miRNA expression profile of urinary exosome and complex regulatory network, and found that miR-3135b, miR-654-5p and miR-146a-5p in urinary exosomes could be used as novel non-invasive diagnostic markers for LNIV with crescents. Moreover, we identified 15 signalling pathways potentially associated with the formation of crescents in LNIV and established a visualized network of TF–miRNA–mRNA interactions, which lays the foundation for further studies on the pathology of lupus nephritis.


Coxsackievirus group A (CV-A) strains are important pathogens of hand, foot, and mouth disease and herpangina. We report here the near-complete genome sequences of 12 CV-A strains isolated from infants and children with different clinical diseases. Coxsackievirus group A (CV-A) strains are important pathogens of hand, foot, and mouth disease and herpangina. We report here the near-complete genome sequences of 12 CV-A strains isolated from infants and children with different clinical diseases. The presented data will be very useful for future genome-based epidemiological studies. Enterovirus in the family Picornaviridae (http://www.picornaviridae.com/enterovirus/enterovirus.htm). Enteroviruses (EVs) are a group of naked positive single-stranded RNA viruses, and their genome comprises a 5′ untranslated region (UTR), four capsid proteins (VP1 to VP4), seven nonstructural proteins (2A to 2C and 3A to 3D), and a 3′ UTR (3Coxsackievirus group A (CV-A) strains belong to the genus oteins VP to VP4,  3′ UTR 37.In recent years, emerging CV-A2, CV-A6, and CV-A10 strains were frequently detected in HFMD and HA patients in Shenzhen, China. In order to investigate the genetic characteristics of these strains, a total of 12 CV-A strains associated with different clinical diseases, collected between 2012 and 2015, were selected for amplification of the genome sequences. Detailed epidemiological data for these strains (listed in The genome length, GC content, and most closely related strain in GenBank of the 12 CV-A strains of this study are summarized in The near-complete genome sequences of 12 coxsackievirus group A strains from the present study have been deposited in DDBJ/ENA/GenBank under the accession numbers listed in


O-6-methylguanine-DNA methyltransferase (MGMT) gene in the occurrence of cancer. Based on the available data from the online database, we performed an updated meta-analysis. We retrieved 537 articles from our database research and finally selected a total of 54 case–control studies (21010 cases and 34018 controls) for a series of pooling analyses. We observed an enhanced risk in cancer cases compared with controls, using the genetic models T/T compared with C/C  = 1.29) and T/T compared with C/C+C/T . We detected similar positive results in the subgroups ‘Caucasian’, and ‘glioma’ . However, we detected negative results in our analyses of most of the other subgroups (P>0.05). Begg’s and Egger’s tests indicated that the results were free of potential publication bias, and sensitivity analysis suggested the stability of the pooling results. In summary, the T/T genotype of MGMT rs12917 is likely to be linked to an enhanced susceptibility to cancer overall, especially glioma, in the Caucasian population.In the present study, we aimed at determining the potential role of rs12917 polymorphism of the  O-6-methylguanine-DNA methyltransferase (MGMT) protein, encoded by the MGMT gene located on chromosome 10 (10q26) . . O-6-met2917 Leu8Phe and re143Val) . In addiMGMT rs12917 polymorphism on its susceptibility to cancer, but the results were inconclusive. Before 2013, only three relative meta-analyses investigated the potential role of this polymorphism in the overall risk for cancer ) OR Methylated-DNA-Protein-Cysteine S-Methyltransferase) OR Methylated DNA Protein Cysteine S Methyltransferase) OR S-Methyltransferase, Methylated-DNA-Protein- Cysteine) OR O(6)-Methylguanine Methyltransferase) OR O(6)-Alkylguanine-DNA Alkyltransferase) OR O(6)-MeG-DNA Methyltransferase) OR O(6)-Methylguanine DNA Transmethylase) OR Guanine-O(6)-Alkyltransferase) OR O(6)-AGT) OR DNA Repair Methyltransferase II) OR DNA Repair Methyltransferase I) OR MGMT)) AND  OR Polymorphisms, Genetic) OR Genetic Polymorphisms) OR Genetic Polymorphism) OR Polymorphism (Genetics)) OR Polymorphisms (Genetics)) OR Polymorphism) OR Polymorphisms)) AND ((((((((((((((((((Neoplasms [MeSH Terms]) OR Neoplasia) OR Neoplasias) OR Neoplasm) OR Tumors) OR Tumor) OR Cancer) OR Cancers) OR Malignant Neoplasms) OR Malignant Neoplasm) OR Neoplasm, Malignant) OR Neoplasms, Malignant) OR Malignancy) OR Malignancies) OR Benign Neoplasms) OR Neoplasms, Benign) OR Benign Neoplasm) OR Neoplasm, Benign).P-value of Hardy–Weinberg equilibrium (HWE) <0.05; (O), lack of full genotype frequency data; (S), review, meta, poster, or meeting abstract. Eligible articles had to be designed as case–control studies, targetting the genetic relationship between MGMT rs12917 and cancer risk and containing the full genotype  frequencies in both cancer cases and negative controls.We designed our inclusion and exclusion criteria according to Patient, Intervention, Comparison and Outcome and Study design (PICOS) principles. We ruled out duplicates and screened improper articles. Exclusion criteria were as follows: (P), non-cancer patients; (I), other variants, gene expression or methylation; (C), lack of study controls or After extracting usable data, we listed the basic information in tables. We assessed methodological quality via the Newcastle–Ottawa Scale (NOS) . High-qu2 test. A P-value of Cochran’s Q statistic < 0.1 or 2I value > 50% was considered to show a high level of heterogeneity. We thus used the DerSimonian–Laird association test with a random-effects model. Otherwise, we used the Mantel–Haenszel association test with a fixed-effects model. The P-value of association test, summary odds ratio (OR), along with the corresponding 95% confidence interval (CI) could be obtained for the allele (T compared with C), homozygous (T/T compared with C/C), recessive (T/T compared with C/C+C/T), heterozygous (C/T compared with C/C), dominant (C/T+T/T compared with C/C), and carrier (T compared with C) models.We used STATA software version 12.0-SE  to perform our analyses. We first assessed the inter-study heterogeneity using Cochran’s Q statistic and the IWe performed subgroup analyses by race, cancer type, and control source. Additionally, we assessed possible publication bias by means of Begg’s and Egger’s tests and evaluated the robustness of the results through sensitivity analysis.MGMT rs12917 polymorphism and cancer risk via an overall meta-analysis. As shown in P-values of heterogeneity > 0.1; 2I < 50%). For other models , we performed a DerSimonian–Laird association test with a random-effects model. Pooling data  and T/T compared with C/C+C/T  genetic models. Nevertheless, we failed to detect any statistical difference between cancer cases and negative controls under other genetic models (P>0.05). Forest plot data are shown in MGMT rs12917 polymorphism was likely to be associated with an increased susceptibility to cancer.First, we studied the association between the ing data  indicateP<0.001; OR = 1.35), ‘glioma’ , ‘population-based control (PB)’  and ‘hospital-based control (HB)’ . Next, we carried out four subgroup analyses by race, cancer type, and control source. For the T/T compared with C/C model , the assP<0.001; OR = 1.37), ‘Asian’ , ‘glioma’ , ‘PB’ , and ‘HB’ . Supplementary Figures S8–S10 present the forest plot data.For the T/T compared with C/C+C/T model , we alsoP<0.05) except for the subgroups ‘colorectal cancer’ , ‘HB’  under the C/T compared with C/C model; and the subgroup ‘head and neck cancer’  under the carrier T compared with C model. Thus, the T/T genotype of MGMT rs12917 may have been associated with an increased risk of cancer in cases, especially the glioma cases, in the Caucasian population.We did not detect positive results for the other genetic models . A Begg’s funnel plot with pseudo–95% confidence limits under the T/T compared with C/C model is shown in Begg’s and Egger’s tests indicated that results were free of possible publication bias , genotype data were very limited in many subgroup analyses. For instance, we used only three case–control studies in our analyses of the subgroups for gastric ,44,47, bMGMT rs12917 polymorphism in combination with other variants, such as rs2308321 of MGMT, rs25487 of X-ray cross-complementing group 1 (XRCC1), and rs13181 of xeroderma pigmentosum complementation group D (XPD), in certain specific cancers.We did not investigate the genetic effects of the We extracted certain demographic information such as the mean age at diagnosis and the sex of subject, but not other confounding factors such as lifestyle and clinical features. Moreover, we did not perform the relevant stratified meta-analyses due to lack of sufficient usable data.We detected significant heterogeneity amongst studies under the allele T compared with C, C/T compared with C/C, C/T+T/T compared with C/C, and carrier T compared with C genetic models. Complicating factors such as race and cancer type may be sources of inter-study heterogeneity. For instance, we detected decreased levels of heterogeneity in the ‘Caucasian’ and ‘esophageal cancer’ subgroups. Although we observed a positive conclusion in the ‘glioma’ subgroup, we failed to detect reduced inter-study heterogeneity. Only five case–control studies ,30,42,48There may be other undetected or unpublished articles containing potential eligible case–controls in other geographical locations or languages; in other words, our study may suffer from selection bias.MGMT rs12917 and the risk of cancer in general for the T/T compared with C/C and T/T compared with C/C+C/T models. Considering the distinct etiopathogenesis or pathogenesis of different kinds of cancers, more studies of large-scale populations of different ethnicities are required for a more scientific elucidation of MGMT rs12917’s functional role in each particular cancer type.Last but most important, our meta-analysis found a positive conclusion between Some of the limitations to our meta-analysis are as follows:MGMT rs12917 polymorphism is likely to be associated with an enhanced susceptibility to cancer overall, especially glioma, in the Caucasian population.To sum up, our updated pooling analysis offered additional evidence that


H9N2 avian influenza viruses are present in poultry worldwide. These viruses are considered to have pandemic potential, because recent isolates can recognize human-type receptor and several sporadic human infections have been reported. In this study, we aimed to identify mutations related to mammalian adaptation of H9N2 influenza virus. We found that mouse-adapted viruses had several mutations in hemagglutinin (HA), PB2, PA, and PB1. Among the detected mutations, PB1-K577E was a novel mutation that had not been previously reported to involve mammalian adaptation. A recombinant H9N2 virus bearing only the PB1-K577E mutation showed enhanced pathogenicity in mice, with increased virus titers in nasal turbinates compared to that in mice infected with the wild-type virus. In addition, the PB1-K577E mutation increased virus polymerase activity in human cell culture at a lower temperature. These data suggest that the PB1-K577E mutation is a novel pathogenicity determinant of H9N2 virus in mice and could be a signature for mammalian adaptation. The natural reservoirs of influenza A viruses are aquatic birds; however, these viruses are occasionally transmitted to fowl or mammals. Although most cases are transient, in rare cases, an influenza A virus can cause a pandemic by jumping species barriers and establishing a new lineage in a new host. To date, the H1, H2, H3, H5, H6, H7, H9, and H10 subtypes of influenza A viruses have been shown to infect humans ,5. AmongH9N2 avian influenza virus was first isolated from a turkey in the United States in 1966 , and sinIn 1999, the first human H9N2 avian influenza virus infection was reported in Hong Kong ,15. Furt2 at 37 °C. A/Hong Kong/1073/99 (H9N2) virus (HK1073) was propagated in 10-day-old embryonated chicken eggs at 37 °C, and the allantoic fluids containing viruses were collected and stored at −80 °C.Madin–Darby canine kidney (MDCK) cells, obtained from the American Type Culture Collection , were maintained in minimal-essential medium (MEM) supplemented with 5% newborn calf serum (NCS) and antibiotics. Human embryonic kidney 293T cells, obtained from RIKEN BioResource Research Center (RCB2202), and DF-1 chicken fibroblasts, obtained from ATCC (CRL-12203), were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% and 5% fetal bovine serum (FBS), respectively. All cells were incubated in 5% COThree 6-week-old female BALB/c mice  were anesthetized with sevoflurane  and inoculated intranasally with 50 μL of 10-fold-diluted allantoic fluids containing HK1073 virus in MEM supplemented with 0.3% bovine serum albumin (MEM/BSA). At 3 days post-inoculation, mice were euthanized, and their lungs were collected. The lungs were suspended in phosphate-buffered saline (PBS) containing 2% penicillin and streptomycin and homogenized with a TissueLyser II . The homogenates were centrifuged, and the supernatant was collected. Three mice were prepared for the next passage and inoculated intranasally with 50 μL of a 20% lung homogenate. Viruses obtained after 10 passages in mice were utilized as the MA viruses for subsequent experiments.The RNA of wild-type (HK1073) and MA viruses was extracted from the allantoic fluid of chicken eggs or mouse lung emulsions produced after the tenth passage, respectively. Reverse transcription of viral RNA was performed using primers containing the conserved sequences at the 3′ ends of the viral segments. Then, polymerase chain reaction (PCR) was conducted using specific primer pairs for each gene segment . PCR proThe cDNAs of the viral segments obtained from HK1073 or MA viruses were cloned into the genomic RNA expression plasmid pHH21. Escherichia coli DH5α cells transformed with the cDNA-inserted plasmids were incubated at room temperature or 37 °C on LB agar plates supplemented with ampicillin. The coding sequences of the PB2, PB1, PA, and NP segments of the HK1073 and MA viruses were amplified by reverse transcription PCR (RT-PCR) using specific primer pairs and cloned into the protein expression plasmid pCAGGS. All plasmids were sequenced to ensure the absence of unexpected mutations. The primer sequences used in this study will be provided upon request.Wild-type and mutant HK1703 viruses were generated by reverse genetics as described previously . Briefly5 plaque-forming units (PFU) of wild-type or mutant HK1703 virus in 50 μL of MEM/BSA. The body weights and mortality of the mice were monitored for 14 days. Mice that lost more than 25% of their body weight were euthanized. To assess the replication ability of the mutant viruses in vivo, mice were anesthetized and inoculated intranasally with 1 × 105 PFU of wild-type or mutant HK1703 virus. At 3 days post-inoculation, lungs and nasal turbinates were harvested and homogenized in PBS containing 2% penicillin and streptomycin with a TissueLyser II. Subsequently, the homogenates were centrifuged, and the supernatants were titrated on MDCK cells by plaque assay.Six-week-old female BALB/c mice were anesthetized with sevoflurane and inoculated intranasally with 1 × 10Confluent monolayers of MDCK cells were washed with MEM/BSA, infected with diluted virus, and incubated for 60 min at 37 °C. After the virus inoculum was removed, the cells were washed and overlaid with MEM/BSA containing 1% agarose and 1 μg/mL TPCK-trypsin. The plates were incubated at 37 °C for 48 h, and then the virus plaques were counted.We prepared plasmids that encode a firefly luciferase gene inserted between the 3’ and 5’ noncoding regions of the NP segment of A/Puerto Rico/8/34 (PR8) under the control of the human PolI promoter (for human 293T cells) or chicken PolI promoter (for chicken DF-1 cells) (named pPolI-NP(0)Fluc(0) and pPolGG-NP(0)Fluc(0), respectively). The pRL-null plasmid  encodes Renilla luciferase and was utilized as an internal control to normalize transfection efficiency. Human 293T and chicken DF-1 cells were transfected with pPolI-NP(0)Fluc(0) or pPolGG-NP(0)Fluc(0) , togetheMDCK cells were infected with viruses at a multiplicity of infection (MOI) of 1 or mock-infected as control. At 12 h post-infection, the cells were lysed on ice for 10 min in lysis buffer . Deglycosylation of cell lysates was achieved using PNGase F  according to the manufacturer’s instructions. The cell lysates were mixed with 0.05 M dithiothreitol and 2× sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer  and boiled for 5 min. Subsequently, the samples were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in 5% skim milk, incubated with anti-HK1073 polyclonal mouse serum obtained by challenging BALB/c mice with HK1073 virus, and then incubated with horseradish peroxidase-linked sheep anti-mouse IgG . The blots were reacted with ECL Prime Western Blotting Detection Reagent  and detected with an ImageQuant LAS 4000 mini .t-test with two-tailed analysis or the Log-rank test to determine the statistical significance of differences. Infectivity titers, luciferase activities, body weight changes, or mortalities in virus-infected mice were compared by Student’s Our animal study protocol was conducted at the ABSL2 containment facility in accordance with the Regulations for Animal Care at the University of Tokyo and was approved by the Animal Experiment Committee of the Graduate School of Agricultural and Life Sciences at the University of Tokyo .To explore the molecular basis of mammalian adaptation of H9N2 virus, we intranasally inoculated wild-type A/Hong Kong/1073/99 (HK1073) virus into three BALB/c mice and collected the lungs at 3 days post-infection. The organs were homogenized, inoculated into another BALB/c mouse, and serially passaged 10 times. Then, we obtained three lines of MA H9N2 viruses. After the sixth passage of the virus, the mice in each line showed clinical signs of infection, including decreased activity, huddling, hunched posture, and ruffled fur. We used three virus lines from the tenth passage, referred as HK-MA1, HK-MA2, and HK-MA3, for further analyses.To identify the amino acid changes in the MA viruses, the complete genomes of the wild-type and HK-MA1, -2, and -3 viruses were sequenced . The PB25 PFU of each virus and monitored mortality (p < 0.01 vs. rWT by Log-rank test) and 75% (p < 0.05) mortality, respectively. Only one rPB2-E627K-infected mouse recovered after a 20% body weight loss. To assess the replication properties of the recombinant viruses, we determined the virus titers in the lungs . The plaque sizes were similar in all of the three generated viruses in the infected MDCK cells. Then, we intranasally inoculated mice with 10ortality A and theortality B for 14 he lungs C and nashe lungs D of mice5 PFU of each mutant or rWT virus, which were monitored for 14 days . Then, we inoculated mice with 10 14 days . All mic 14 days A, signif 14 days B. These We also generated a mutant virus containing both HA-N198S and PB1-K577E (referred as rHA-N198S/PB1-K577E), which has the same genotype as HK-MA3, and tested its pathogenicity A,B. All To examine whether the PB1-K577E mutation affects the polymerase activity of the H9N2 virus, we performed a mini-replicon luciferase assay in mammalian and avian cells at 37 °C and 33 °C, which are the temperatures corresponding to the mammalian lower and upper respiratory tracts, respectively ,29,30,31N-glycosylation site.We hypothesized that the HA-N132D and HA-N198S/T mutations found in the MA viruses abolished N-linked glycosylation sites. To confirm that these mutations caused deglycosylation of HA, a Western blot analysis was performed. Since the growth of the rHA-N132D and rHA-N198S viruses in cell culture was suboptimal, leading to unclear resolution of the assay, we generated viruses bearing HA-N132D or HA-N198S mutation together with the PB2-E627K mutation by reverse genetics . Then, we inoculated these mutants as well as rPB2-E627K, which possesses wild-type HA, into MDCK cells. After incubation at 37 °C for 12 h, infected cells were lysed and treated with or without PNGase F to remove N-linked glycans. The cell lysates were subjected to Western blot analysis using mouse anti-H9N2 antiserum . FollowiWorldwide expansion of H9N2 avian influenza virus infection in poultry and sporadic infections in humans are a public health concern, as the virus has the potential to become a pandemic virus. For a new pandemic to emerge, the virus must obtain certain mutations that allow it to adapt to humans. However, the molecular basis of mammalian adaptation of H9N2 virus is not fully understood. Here, we identified a novel PB1 mutation, K577E, in H9N2 viruses serially passaged in mice that could increase pathogenicity in mice, suggesting that the PB1-K577E mutation could be considered to be one of the signatures for mammalian adaptation of avian influenza viruses.Most avian influenza viruses do not show pathogenicity in mice without initial adaptation. However, the rWT HK1073 virus showed pathogenicity to some extent by inducing a decrease in the body weights upon its infection . This miHere, we selected H9N2 MA viruses (HK-MA1 and -MA2) harboring the PB2-E627K mutation. Many studies have demonstrated that avian influenza viruses bearing the PB2-E627K mutant show increased replication ability in mammalian hosts and higher virulence in mammals ,29,30,31https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go=database) and found out that not only are there no naturally occurring H9N2 isolates possessing a Glu (E) at position 577 of PB1 but also that viruses possessing an E at this position are very rare, even among other subtype mammalian viruses  in mice . However viruses . These dThe molecular mechanism of the enhanced polymerase activity in mammalian cells due to the PB1-K577E mutation is unknown. A previous study indicated that the PB1 region, comprising residues 506–659, was involved in its interaction with PB2 . The thrAnother possible mechanism of PB1-K577E-mediated enhancement of polymerase activity is the involvement of a host factor that interacts with the viral polymerase and supports its activity. In the 3D structure, PB1 residues 564–577 appear to form a pocket-like structure, which may form a specific binding site for a host factor. The charge environment in this region may be altered by the K577E mutation due to the change from a negatively-charged K to a positively-charged E, possibly resulting in different binding properties for a host-specific factor. It is well known that the PB2-E627K and PB2-D701N mutations are determinants for mammalian adaptation of avian influenza viruses through their interaction with host species-specific factors, such as importin-α isoforms . InteresA PA-T97I mutation detected in a HK-MA2 virus was previously shown to increase the polymerase activity and mouse pathogenicity of H5N1 avian influenza viruses . In addiN-glycosylation at residue 198 and exhibit higher affinity for the human-type receptor and higher pathogenicity in mice than the parent virus with 198N [The HA-N132D or HA-N198S mutations detected in the HK-MA viruses abolished N-linked glycosylation . Previouith 198N ,44. Our ith 198N  (Figure In conclusion, a novel PB1-K577E mutation emerges during mouse adaptation of an H9N2 virus, thus we propose PB1-K577E as an additional signature of mammalian adaptation of avian influenza viruses. This finding may contribute not only to the molecular dissection of RNA polymerase but also avian influenza virus surveillance for pandemic risk assessment.