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Protein knockdown with an inducible degradation system is a powerful tool for studying proteins of interest in living cells. Here, we adopted the auxin-inducible degron (AID) approach to detail Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) function in latency maintenance and inducible viral lytic gene expression. We fused the mini-auxin-inducible degron (mAID) tag at the LANA N-terminus with KSHV bacterial artificial chromosome 16 recombination, and iSLK cells were stably infected with the recombinant KSHV encoding mAID-LANA. Incubation with 5-phenyl-indole-3-acetic acid, a derivative of natural auxin, rapidly degraded LANA within 1.5 h. In contrast to our hypothesis, depletion of LANA alone did not trigger lytic reactivation but rather decreased inducible lytic gene expression when we stimulated reactivation with a combination of ORF50 protein expression and sodium butyrate. Decreased overall lytic gene induction seemed to be associated with a rapid loss of KSHV genomes in the absence of LANA. The rapid loss of viral genomic DNA was blocked by a lysosomal inhibitor, chloroquine. Furthermore, siRNA-mediated knockdown of cellular innate immune proteins, cyclic AMP-GMP synthase (cGAS) and simulator of interferon genes (STING), and other autophagy-related genes rescued the degradation of viral genomic DNA upon LANA depletion. Reduction of the viral genome was not observed in 293FT cells that lack the expression of cGAS. These results suggest that LANA actively prevents viral genomic DNA from sensing by cGAS-STING signaling axis, adding novel insights into the role of LANA in latent genome maintenance.IMPORTANCESensing of pathogens' components is a fundamental cellular immune response. Pathogens have therefore evolved strategies to evade such cellular immune responses. KSHV LANA is a multifunctional protein and plays an essential role in maintaining the latent infection by tethering viral genomic DNA to the host chromosome. We adopted the inducible protein knockdown approach and found that depletion of LANA induced rapid degradation of viral genomic DNA, which is mediated by innate immune DNA sensors and autophagy pathway. These observations suggest that LANA may play a role in hiding KSHV episome from innate immune DNA sensors. Our study thus provides new insights into the role of LANA in latency maintenance.

Terminal nucleotidyltransferases add nucleotides to the 3′ end of RNA to modify their stability and function. In Caenorhabditis elegans , the terminal uridyltransferases/poly(U) polymerases PUP-1 (aka CID-1, CDE-1), PUP-2, and PUP-3 affect germline identity, survival, and development. Here, we identify small RNA (sRNA) and mRNA targets of these PUPs and of a fourth predicted poly(U) polymerase, F43E2.1/PUP-4. Using genetic and RNA sequencing approaches, we identify RNA targets of each PUP and the U-tail frequency and length of those targets. At the whole organism level, PUP-1 is responsible for most sRNA U-tailing, and other PUPs contribute to modifying discrete subsets of sRNAs. Moreover, the expression of PUP-2, PUP-3, and especially PUP-4 limits uridylation on some sRNAs. The relationship between uridylation status and sRNA abundance suggests that U-tailing can have a negative or positive effect on abundance depending on context. sRNAs modified by PUP activity primarily target mRNAs that are ubiquitously expressed or most highly expressed in the germline. mRNA data obtained with a Nanopore-based method reveal that the addition of U-tails to nonadenylated mRNA is substantially reduced in the absence of PUP-3. Overall, this work identifies PUP RNA targets, defines the effect of uridylation loss on RNA abundance, and reveals the complexity of PUP regulation in C. elegans development. [ABSTRACT FROM AUTHOR]

Objective Anti-melanoma differentiation–associated gene 5 (Anti-MDA5)–positive DM is a rare but life-threatening autoimmune disorder that is associated with a high risk of developing rapidly progressive interstitial lung disease. Current empirical therapies offer limited benefit in terms of patient survival, as little is known about the aetiology of anti-MDA5 DM. To best understand its immune landscape, we applied single-cell RNA sequencing (scRNA-seq) to peripheral blood samples from DM patients and healthy controls. Methods Peripheral blood mononuclear cells (PBMCs) from eight DM patients (including three distinct subtypes of DM) and two healthy donors were sequenced using the 10X Genomics platform. Additional scRNA-seq data for four healthy donors were incorporated for further bioinformatic analysis. Results Aberrantly increased proportions of CD14+ monocytes and plasma cells were observed in anti-MDA5 DM PBMC samples. Moreover, we found an overactivated type I IFN response and antiviral immunity in both innate and adaptive immune cells derived from anti-MDA5 DM patients that was positively correlated with disease severity. Importantly, a unique subset of CD14+ monocytes that highly expressed IFN alpha–inducible protein 27 (IFI27), a biomarker for viral infection, and IFN induced with helicase C domain 1 (IFIH1 , which encodes MDA5) was specifically identified in anti-MDA5 DM samples for the first time. Conclusion Our study has illustrated the peripheral immune cell atlas of a number of DM subtypes, has provided compelling evidence for a viral infection–derived origin for anti-MDA5 DM, and has indicated potential targets for innovative therapeutic interventions. [ABSTRACT FROM AUTHOR]

In sporadic amyotrophic lateral sclerosis (sALS), IL-17A- and granzyme-positive cytotoxic T lymphocytes (CTL), IL-17A-positive mast cells, and inflammatory macrophages invade the brain and spinal cord. In some patients, the disease starts following a trauma or a severe infection. We examined cytokines and cytokine regulators over the disease course and found that, since the early stages, peripheral blood mononuclear cells (PBMC) exhibit increased expression of inflammatory cytokines IL-12A, IFN-γ, and TNF-α, as well as granzymes and the transcription factors STAT3 and STAT4. In later stages, PBMCs upregulated the autoimmunity-associated cytokines IL-23A and IL-17B, and the chemokines CXCL9 and CXCL10, which attract CTL and monocytes into the central nervous system. The inflammation is fueled by the downregulation of IL-10, TGFβ, and the inhibitory T-cell co-receptors CTLA4, LAG3, and PD-1, and, in vitro, by stimulation with the ligand PD-L1. We investigated in two sALS patients the regulation of the macrophage transcriptome by dimethyl fumarate (DMF), a drug approved against multiple sclerosis and psoriasis, and the cyclic GMP-AMP synthase/stimulator of interferon genes (cGAS/STING) pathway inhibitor H-151. Both DMF and H-151 downregulated the expression of granzymes and the pro-inflammatory cytokines IL-1β, IL-6, IL-15, IL-23A, and IFN-γ, and induced a pro-resolution macrophage phenotype. The eicosanoid epoxyeicosatrienoic acids (EET) from arachidonic acid was anti-inflammatory in synergy with DMF. H-151 and DMF are thus candidate drugs targeting the inflammation and autoimmunity in sALS via modulation of the NFκB and cGAS/STING pathways.

UNLABELLED: Glioblastoma (GBM) is an immunologically cold tumor that does not respond to current immunotherapy. Here, we demonstrate a fundamental role for the α-isoform of the catalytic subunit of protein phosphatase-2A (PP2Ac) in regulating glioma immunogenicity. Genetic ablation of PP2Ac in glioma cells enhanced double-stranded DNA (dsDNA) production and cGAS-type I IFN signaling, MHC-I expression, and tumor mutational burden. In coculture experiments, PP2Ac deficiency in glioma cells promoted dendritic cell (DC) cross-presentation and clonal expansion of CD8+ T cells. In vivo, PP2Ac depletion sensitized tumors to immune-checkpoint blockade and radiotherapy treatment. Single-cell analysis demonstrated that PP2Ac deficiency increased CD8+ T-cell, natural killer cell, and DC accumulation and reduced immunosuppressive tumor-associated macrophages. Furthermore, loss of PP2Ac increased IFN signaling in myeloid and tumor cells and reduced expression of a tumor gene signature associated with worse patient survival in The Cancer Genome Atlas. Collectively, this study establishes a novel role for PP2Ac in inhibiting dsDNA-cGAS-STING signaling to suppress antitumor immunity in glioma. SIGNIFICANCE: PP2Ac deficiency promotes cGAS-STING signaling in glioma to induce a tumor-suppressive immune microenvironment, highlighting PP2Ac as a potential therapeutic target to enhance tumor immunogenicity and improve response to immunotherapy.

Tissue-resident immune cells are critical to the initiation and potentiation of inflammation. However, the tissue-protective cellular communication networks initiated by resident immunity during sterile inflammation are not well understood. Using single-cell transcriptomic analysis, we show the liver-resident cell connectome and signalome during acute liver injury. These analyses identify Il12b as a central regulator of liver injury-associated changes in gene expression. Interleukin (IL)-12 produced by conventional type 1 dendritic cells (cDC1s) is required for protection during acute injury through activation of interferon (IFN)-γ production by liver-resident type 1 innate lymphoid cells (ILC1s). Using a targeted in vivo CRISPR-Cas9 screen of innate immune sensing pathways, we find that cDC1-intrinsic cGAS-STING signaling acts upstream of IL-12 production to initiate early protective immune responses. Our study identifies the core communication hubs initiated by tissue-resident innate immune cells during sterile inflammation in vivo and implicates cDC1-derived IL-12 as an important regulator of this process.

A fundamental strategy of eukaryotic antiviral immunity involves the cGAS enzyme, which synthesizes 2',3'-cGAMP and activates the effector STING. Diverse bacteria contain cGAS-like enzymes that produce cyclic oligonucleotides and induce anti-phage activity, known as CBASS. However, this activity has only been demonstrated through heterologous expression. Whether bacteria harboring CBASS antagonize and co-evolve with phages is unknown. Here, we identified an endogenous cGAS-like enzyme in Pseudomonas aeruginosa that generates 3',3'-cGAMP during phage infection, signals to a phospholipase effector, and limits phage replication. In response, phages express an anti-CBASS protein ("Acb2") that forms a hexamer with three 3',3'-cGAMP molecules and reduces phospholipase activity. Acb2 also binds to molecules produced by other bacterial cGAS-like enzymes (3',3'-cUU/UA/UG/AA) and mammalian cGAS (2',3'-cGAMP), suggesting broad inhibition of cGAS-based immunity. Upon Acb2 deletion, CBASS blocks lytic phage replication and lysogenic induction, but rare phages evade CBASS through major capsid gene mutations. Altogether, we demonstrate endogenous CBASS anti-phage function and strategies of CBASS inhibition and evasion.

High rates of new cervical cancer cases and deaths occur in low- and middle-income countries yearly, and one reason was found related to limitation of regular cervical cancer screening in local and low-resource settings. HPV has over 150 types, yet certain 14-20 high-risk and 13-14 low-risk types are common, and, thus, most conventional HPV nucleic acid assays, for examples, Cobas 4800 HPV test (Roche Diagnostics, New Jersey, USA) and REBA HPV-ID (Molecules and Diagnostics, Wonju, Republic of Korea) were developed to cover these types. We thereby utilized bioinformatics combined with recent isothermal amplification technique at 35-42 °C to firstly describe multiplex recombinase polymerase amplification assay that is specific to these common 20 high-risk and 14 low-risk types, and also L1 and E6/E7 genes that target different stages of cervical cancer development. Multiplex primer concentrations and reaction incubation conditions were optimized to allow simultaneous two gene detections at limit of detection of 1000 copies (equivalent to 2.01 fg) for L1 and 100 copies (0.0125 fg) for E6/E7, respectively. The assay was validated against urogenital and other pathogens, normal flora, and human control. In 130 real clinical sample tests, the assay demonstrated 100% specificity, 78% diagnostic accuracy, and 75% sensitivity compared with REBA HPV-ID test, and is much more rapid (15-40 min), less expensive (~ 3-4 USD/reaction) and does not require instrumentation (35-42 °C reaction condition so hand holding or tropical temperature is possible). Hence, the developed novel assay provides alternative screening tool for potential local screening. Furthermore, as this assay uses safe chemical reagents, it is safe for users.

Cyclic nucleotides are the most diversified category of second messengers and are found in all organisms modulating diverse pathways. While cAMP and cGMP have been studied over 50 years, cyclic di‐nucleotide signaling in eukaryotes emerged only recently with the anti‐viral molecule 2´3´cGAMP. Recent breakthrough discoveries have revealed not only the astonishing chemical diversity of cyclic nucleotides but also surprisingly deep‐rooted evolutionary origins of cyclic oligo‐nucleotide signaling pathways and structural conservation of the proteins involved in their synthesis and signaling. Here we discuss how enzyme‐centered approaches have paved the way for the identification of several cyclic nucleotide signals, focusing on the advantages and challenges associated with deciphering the activation mechanisms of such enzymes. [ABSTRACT FROM AUTHOR]

Xeno-nucleic acids (XNAs) are synthetic genetic polymers with backbone structures composed of non-ribose or non-deoxyribose sugars. Phosphonomethylthreosyl nucleic acid (pTNA), a type of XNA that does not base pair with DNA or RNA, has been suggested as a possible genetic material for storing synthetic biology information in cells. A critical step in this process is the synthesis of XNA episomes using laboratory-evolved polymerases to copy DNA information into XNA. Here, we investigate the polymerase recognition of pTNA nucleotides using X-ray crystallography to capture the post-catalytic complex of engineered polymerases following the sequential addition of two pTNA nucleotides onto the 3'-end of a DNA primer. High-resolution crystal structures reveal that the polymerase mediates Watson-Crick base pairing between the extended pTNA adducts and the DNA template. Comparative analysis studies demonstrate that the sugar conformation and backbone position of pTNA are structurally more similar to threose nucleic acid than DNA even though pTNA and DNA share the same six-atom backbone repeat length. Collectively, these findings provide new insight into the structural determinants that guide the enzymatic synthesis of an orthogonal genetic polymer, and may lead to the discovery of new variants that function with enhanced activity.

Mitochondrial DNA (mtDNA) escaping stressed mitochondria provokes inflammation via cGAS-STING pathway activation and, when oxidized (Ox-mtDNA), it binds cytosolic NLRP3, thereby triggering inflammasome activation. However, it is unknown how and in which form Ox-mtDNA exits stressed mitochondria in non-apoptotic macrophages. We found that diverse NLRP3 inflammasome activators rapidly stimulated uniporter-mediated calcium uptake to open mitochondrial permeability transition pores (mPTP) and trigger VDAC oligomerization. This occurred independently of mtDNA or reactive oxygen species, which induce Ox-mtDNA generation. Within mitochondria, Ox-mtDNA was either repaired by DNA glycosylase OGG1 or cleaved by the endonuclease FEN1 to 500-650 bp fragments that exited mitochondria via mPTP- and VDAC-dependent channels to initiate cytosolic NLRP3 inflammasome activation. Ox-mtDNA fragments also activated cGAS-STING signaling and gave rise to pro-inflammatory extracellular DNA. Understanding this process will advance the development of potential treatments for chronic inflammatory diseases, exemplified by FEN1 inhibitors that suppressed interleukin-1β (IL-1β) production and mtDNA release in mice.

AimLow-grade inflammation is the hallmark of non-alcoholic fatty liver diseases (NAFLD) and non-alcoholic steatohepatitis (NASH). The leakage of microbiota-derived products can contribute to liver inflammation during NAFLD/NASH development. Here, we assessed the roles of gut microbial DNA-containing extracellular vesicles (mEVs) in regulating liver cellular abnormalities in the course of NAFLD/NASH.MethodsWe performed studies with Vsig4-/- , C3-/- , cGAS-/- , and their wild-type littermate mice. Vsig4+ macrophage population and bacterial DNA abundance were examined in both mouse and human liver by either flow cytometric or immunohistochemistry analysis. Gut mEVs were adoptively transferred into Vsig4-/- , C3-/- , cGAS-/- , or littermate WT mice, and hepatocyte inflammation and HSC fibrogenic activation were measured in these mice.ResultsNon-alcoholic fatty liver diseases and non-alcoholic steatohepatitis development was concomitant with a diminished liver Vsig4+ macrophage population and a marked bacterial DNA enrichment in both hepatocytes and HSCs. In the absence of Vsig4+ macrophages, gut mEVs translocation led to microbial DNA accumulation in hepatocytes and HSCs, resulting elevated hepatocyte inflammation and HSC fibrogenic activation. In contrast, in lean WT mice, Vsig4+ macrophages remove gut mEVs from bloodstream through a C3-dependent opsonization mechanism and prevent the infiltration of gut mEVs into hepatic cells. Additionally, Vsig4-/- mice more quickly developed significant liver steatosis and fibrosis than WT mice after Western diet feeding. In vitro treatment with NASH mEVs triggered hepatocyte inflammation and HSC fibrogenic activation. Microbial DNAs are key cargo for the effects of gut mEVs by activating cGAS/STING.ConclusionAccumulation of microbial DNAs fuels the development of NAFLD/NASH-associated liver abnormalities.

Paramutation is a transfer of heritable silencing states between interacting endogenous alleles or between endogenous alleles and homologous transgenes. Prior results demonstrated that paramutation occurs at the P1-rr (r ed pericarp and r ed cob) allele of the maize p1 (pericarp color 1) gene when exposed to a transgene containing a 1.2-kb enhancer fragment (P1.2) of P1-rr. The paramutable P1-rr allele undergoes transcriptional silencing resulting in a paramutant light-pigmented P1-rr′ state. To define more precisely the sequences required to elicit paramutation, the P1.2 fragment was further subdivided, and the fragments transformed into maize plants and crossed with P1-rr. Analysis of the progeny plants showed that the sequences required for paramutation are located within a ∼600-bp segment of P1.2 and that this segment overlaps with a previously identified enhancer that is present in 4 direct repeats in P1-rr. The paramutagenic segment is transcribed in both the expressed P1-rr and the silenced P1-rr′. Transcription is sensitive to α-amanitin, indicating that RNA polymerase II mediates most of the transcription of this sequence. Although transcription within the paramutagenic sequence was similar in all tested genotypes, small RNAs were more abundant in the silenced P1-rr′ epiallele relative to the expressed P1-rr allele. In agreement with prior results indicating the association of RNA-mediated DNA methylation in p1 paramutation, DNA blot analyses detected increased cytosine methylation of the paramutant P1-rr′ sequences homologous to the transgenic P1.2 subfragments. Together these results demonstrate that the P1-rr enhancer repeats mediate p1 paramutation. [ABSTRACT FROM AUTHOR]

The aim of this study was to determine the role of retrograde signaling (mitochondria to nucleus) in MCF7 breast cancer cells. Therefore, in the present study, MCF7-H and MCF7-J cybrids were produced using the mitochondria from the same H and J individuals that were already used in our non-diseased retinal pigment epithelium (ARPE19) cybrids. MCF7 cybrids were treated with cisplatin and analyzed for cell viability, mitochondrial membrane potential, ROS, and expression levels of genes associated with the cGAS-STING and cancer-related pathways. Results showed that unlike the ARPE19-H and ARPE19-J cybrids, the untreated MCF7-H and MCF7-J cybrids had similar levels of ATP, lactate, and OCR: ECAR ratios. After cisplatin treatment, MCF7-H and MCF7-J cybrids showed similar (a) decreases in cell viability and ROS levels; (b) upregulation of ABCC1, BRCA1 and CDKN1A/P21; and (c) downregulation of EGFR. Cisplatin-treated ARPE19-H and ARPE19-J cybrids showed increased expression of six cGAS-STING pathway genes, while two were increased for MCF7-J cybrids. In summary, the ARPE19-H and ARPE19-J cybrids behave differentially from each other with or without cisplatin. In contrast, the MCF7-H and MCF7-J cybrids had identical metabolic/bioenergetic profiles and cisplatin responses. Our findings suggest that cancer cell nuclei might have a diminished ability to respond to the modulating signaling of the mtDNA that occurs via the cGAS-STING pathway.

Various microbial products leaked from gut lumen exacerbate tissue inflammation and metabolic disorders in obesity. Vsig4+ macrophages are key players preventing infiltration of bacteria and their products into host tissues. However, roles of islet Vsig4+ macrophages in the communication between microbiota and β cells in pathogenesis of obesity-associated islet abnormalities are unknown. Here, we find that bacterial DNAs are enriched in β cells of individuals with obesity. Intestinal microbial DNA-containing extracellular vesicles (mEVs) readily pass through obese gut barrier and deliver microbial DNAs into β cells, resulting in elevated inflammation and impaired insulin secretion by triggering cGAS/STING activation. Vsig4+ macrophages prevent mEV infiltration into β cells through a C3-dependent opsonization, whereas loss of Vsig4 leads to microbial DNA enrichment in β cells after mEV treatment. Removal of microbial DNAs blunts mEV effects. Loss of Vsig4+ macrophages leads to microbial DNA accumulation in β cells and subsequently obesity-associated islet abnormalities.

Cannabis (Cannabis sativa) is a widely used drug in the United States and the frequency of cannabis use is particularly high among people living with HIV (PLWH). One key component of cannabis, the non-psychotropic (-)-cannabidiol (CBD) exerts a wide variety of biological actions, including anticonvulsive, analgesic, and anti-inflammatory effects. However, the exact mechanism of action through which CBD affects the immune cell signaling remains poorly understood. Here we report that CBD modulates type I interferon responses in human macrophages. Transcriptomics analysis shows that CBD treatment significantly attenuates cGAS-STING-mediated activation of type I Interferon response genes (ISGs) in monocytic THP-1 cells. We further showed that CBD treatment effectively attenuates 2'3-cGAMP stimulation of ISGs in both THP-1 cells and primary human macrophages. Interestingly, CBD significantly upregulates expression of autophagy receptor p62/SQSTM1. p62 is critical for autophagy-mediated degradation of stimulated STING. We observed that CBD treated THP-1 cells have elevated autophagy activity. Upon 2'3'-cGAMP stimulation, CBD treated cells have rapid downregulation of phosphorylated-STING, leading to attenuated expression of ISGs. The CBD attenuation of ISGs is reduced in autophagy deficient THP-1 cells, suggesting that the effects of CBD on ISGs is partially mediated by autophagy induction. Lastly, CBD decreases ISGs expression upon HIV infection in THP-1 cells and human primary macrophages, leading to increased HIV RNA expression 24 hours after infection. However, long term culture with CBD in infected primary macrophages reduced HIV viral spread, suggesting potential dichotomous roles of CBD in HIV replication. Our study highlights the immune modulatory effects of CBD and the needs for additional studies on its effect on viral infection and inflammation.

Understanding transcriptomes requires documenting the structures, modifications, and abundances of RNAs as well as their proximity to other molecules. The methods that make this possible depend critically on enzymes (including mutant derivatives) that act on nucleic acids for capturing and sequencing RNA. We tested two 3' nucleotidyl transferases, Saccharomyces cerevisiae poly(A) polymerase and Schizosaccharomyces pombe Cid1, for the ability to add base and sugar modified rNTPs to free RNA 3' ends, eventually focusing on Cid1. Although unable to polymerize ΨTP or 1meΨTP, Cid1 can use 5meUTP and 4thioUTP. Surprisingly, Cid1 can use inosine triphosphate to add poly(I) to the 3' ends of a wide variety of RNA molecules. Most poly(A) mRNAs efficiently acquire a uniform tract of about 50 inosine residues from Cid1, whereas non-poly(A) RNAs acquire longer, more heterogeneous tails. Here we test these activities for use in direct RNA sequencing on nanopores, and find that Cid1-mediated poly(I)-tailing permits detection and quantification of both mRNAs and non-poly(A) RNAs simultaneously, as well as enabling the analysis of nascent RNAs associated with RNA polymerase II. Poly(I) produces a different current trace than poly(A), enabling recognition of native RNA 3' end sequence lost by in vitro poly(A) addition. Addition of poly(I) by Cid1 offers a broadly useful alternative to poly(A) capture for direct RNA sequencing on nanopores.

RNA-specific nucleotidyltransferases (rNTrs) add nontemplated nucleotides to the 3′ end of RNA. Two noncanonical rNTRs that are thought to be poly(A) polymerases (PAPs) have been identified in the mitochondria of trypanosomes – KPAP1 and KPAP2. KPAP1 is the primary polymerase that adds adenines (As) to trypanosome mitochondrial mRNA 3′ tails, while KPAP2 is a non-essential putative polymerase whose role in the mitochondria is ambiguous. Here, we elucidate the effects of manipulations of KPAP1 and KPAP2 on the 5′ and 3′ termini of transcripts and their 3′ tails. Using glycerol gradients followed by immunoblotting, we present evidence that KPAP2 is found in protein complexes of up to about 1600 kDa. High-throughput sequencing of mRNA termini showed that KPAP2 overexpression subtly changes an edited transcript’s 3′ tails, though not in a way consistent with general PAP activity. Next, to identify possible roles of posttranslational modifications on KPAP1 regulation, we mutated two KPAP1 arginine methylation sites to either mimic methylation or hypomethylation. We assessed their effect on 3′ mRNA tail characteristics and found that the two mutants generally had opposing effects, though some of these were transcript-specific. We present results suggesting that while methylation increases KPAP1 substrate binding and/or initial nucleotide additions, unmethylated KPAP1is more processive. We also present a comprehensive review of UTR termini, and evidence that tail addition activity may change as mRNA editing is initiated. Together, this work furthers our understanding of the role of KPAP1 and KPAP2 on trypanosome mitochondrial mRNA 3′ tail addition, as well as provides more information on mRNA termini processing in general.

Background & aimsLiver CRIg+ (complement receptor of the immunoglobulin superfamily) macrophages play a critical role in filtering bacteria and their products from circulation. Translocation of microbiota-derived products from an impaired gut barrier contributes to the development of obesity-associated tissue inflammation and insulin resistance. However, the critical role of CRIg+ macrophages in clearing microbiota-derived products from the bloodstream in the context of obesity is largely unknown.MethodsWe performed studies with CRIg-/-, C3-/-, cGAS-/-, and their wild-type littermate mice. The CRIg+ macrophage population and bacterial DNA abundance were examined in both mouse and human liver by either flow cytometric or immunohistochemistry analysis. Gut microbial DNA-containing extracellular vesicles (mEVs) were adoptively transferred into CRIg-/-, C3-/-, or wild-type mice, and tissue inflammation and insulin sensitivity were measured in these mice. After coculture with gut mEVs, cellular insulin responses and cGAS/STING-mediated inflammatory responses were evaluated.ResultsGut mEVs can reach metabolic tissues in obesity. Liver CRIg+ macrophages efficiently clear mEVs from the bloodstream through a C3-dependent opsonization mechanism, whereas obesity elicits a marked reduction in the CRIg+ macrophage population. Depletion of CRIg+ cells results in the spread of mEVs into distant metabolic tissues, subsequently exacerbating tissue inflammation and metabolic disorders. Additionally, in vitro treatment of obese mEVs directly triggers inflammation and insulin resistance of insulin target cells. Depletion of microbial DNA blunts the pathogenic effects of intestinal EVs. Furthermore, the cGAS/STING pathway is crucial for microbial DNA-mediated inflammatory responses.ConclusionsDeficiency of CRIg+ macrophages and leakage of intestinal EVs containing microbial DNA contribute to the development of obesity-associated tissue inflammation and metabolic diseases.

Rationale: One of the most common metabolic defects in cancers is the deficiency in arginine synthesis, which has been exploited therapeutically. Yet, challenges remain, and the mechanisms of arginine-starvation induced killing are largely unclear. Here, we sought to demonstrate the underlying mechanisms by which arginine starvation-induced cell death and to develop a dietary arginine-restriction xenograft model to study the in vivo effects. Methods: Multiple castration-resistant prostate cancer cell lines were treated with arginine starvation followed by comprehensive analysis of microarray, RNA-seq and ChIP-seq were to identify the molecular and epigenetic pathways affected by arginine starvation. Metabolomics and Seahorse Flux analyses were used to determine the metabolic profiles. A dietary arginine-restriction xenograft mouse model was developed to assess the effects of arginine starvation on tumor growth and inflammatory responses. Results: We showed that arginine starvation coordinately and epigenetically suppressed gene expressions, including those involved in oxidative phosphorylation and DNA repair, resulting in DNA damage, chromatin-leakage and cGAS-STING activation, accompanied by the upregulation of type I interferon response. We further demonstrated that arginine starvation-caused depletion of α-ketoglutarate and inactivation of histone demethylases are the underlying causes of epigenetic silencing. Significantly, our dietary arginine-restriction model showed that arginine starvation suppressed prostate cancer growth in vivo, with evidence of enhanced interferon responses and recruitment of immune cells. Conclusions: Arginine-starvation induces tumor cell killing by metabolite depletion and epigenetic silencing of metabolic genes, leading to DNA damage and chromatin leakage. The resulting cGAS-STING activation may further enhance these killing effects.

Liver diseases represent a major global health burden accounting for approximately 2 million deaths per year worldwide. The liver functions as a primary immune organ that is largely enriched with various innate immune cells, including macrophages, dendritic cells, neutrophils, NK cells, and NKT cells. Activation of these cells orchestrates the innate immune response and initiates liver inflammation in response to the danger signal from pathogens or injured cells and tissues. The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway is a crucial signaling cascade of the innate immune system activated by cytosol DNA. Recognizing DNA as an immune-stimulatory molecule is an evolutionarily preserved mechanism in initiating rapid innate immune responses against microbial pathogens. The cGAS is a cytosolic DNA sensor eliciting robust immunity via the production of cyclic GMP-AMPs that bind and activate STING. Although the cGAS-STING pathway has been previously considered to have essential roles in innate immunity and host defense, recent advances have extended the role of the cGAS-STING pathway to liver diseases. Emerging evidence indicates that overactivation of cGAS-STING may contribute to the development of liver disorders, implying that the cGAS-STING pathway is a promising therapeutic target. Here, we review and discuss the role of the cGAS-STING DNA-sensing signaling pathway in a variety of liver diseases, including viral hepatitis, nonalcoholic fatty liver disease (NAFLD), alcoholic liver disease (ALD), primary hepatocellular cancer (HCC), and hepatic ischemia-reperfusion injury (IRI), with highlights on currently available therapeutic options.

O-Acetylated sialic acid has been found in the Neisseria meningitidis serogroup W (NmW) capsular polysaccharide (CPS) and is a required structural component of clinically used NmW CPS-based polysaccharide and polysaccharide-conjugate vaccines. The role of sialic acid O-acetylation in NmW CPS, however, is not clearly understood. This is partially due to the lack of a precise control of the percentage and the location of O-acetylation which is labile and susceptible to migration. We explore chemoenzymatic synthetic strategies for preparing N-acetylated analogues of O-acetylated NmW CPS oligosaccharides which can serve as structurally stable probe mimics. Substrate specificity studies of NmW CPS polymerase (NmSiaDW) identified 4-azido-4-deoxy-N-acetylmannosamine (ManNAc4N3) and 6-azido-6-deoxy-N-acetylmannosamine (ManNAc6N3) as suitable chemoenzymatic synthons for synthesizing N-acetyl analogues of NmW CPS oligosaccharides containing 7-O-acetyl-N-acetylneuraminic acid (Neu5,7Ac2) and/or 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2). The synthesis was achieved by NmSiaDW-dependent sequential one-pot multienzyme (OPME) strategy with in situ generation of the corresponding sugar nucleotides from simple monosaccharides or derivatives to form N3-oligosaccharides which were converted to the desired NAc-oligosaccharides by an efficient one-step chemical transformation.

cGAS/DncV-like nucleotidyltransferase (CD-NTase) enzymes are immune sensors that synthesize nucleotide second messengers and initiate antiviral responses in bacterial and animal cells. Here, we discover Enterobacter cloacae CD-NTase-associated protein 4 (Cap4) as a founding member of a diverse family of >2,000 bacterial receptors that respond to CD-NTase signals. Structures of Cap4 reveal a promiscuous DNA endonuclease domain activated through ligand-induced oligomerization. Oligonucleotide recognition occurs through an appended SAVED domain that is an unexpected fusion of two CRISPR-associated Rossman fold (CARF) subunits co-opted from type III CRISPR immunity. Like a lock and key, SAVED effectors exquisitely discriminate 2'-5'- and 3'-5'-linked bacterial cyclic oligonucleotide signals and enable specific recognition of at least 180 potential nucleotide second messenger species. Our results reveal SAVED CARF family proteins as major nucleotide second messenger receptors in CBASS and CRISPR immune defense and extend the importance of linkage specificity beyond mammalian cGAS-STING signaling.

BACKGROUND: Viridans group streptococci of the Streptococcus mitis-oralis subgroup are important endovascular pathogens. They can rapidly develop high-level and durable non-susceptibility to daptomycin both in vitro and in vivo upon exposure to daptomycin. Two consistent genetic adaptations associated with this phenotype (i.e., mutations in cdsA and pgsA) lead to the depletion of the phospholipids, phosphatidylglycerol and cardiolipin, from the bacterial membrane. Such alterations in phospholipid biosynthesis will modify carbon flow and change the bacterial metabolic status. To determine the metabolic differences between daptomycin-susceptible and non-susceptible bacteria, the physiology and metabolomes of S. mitis-oralis strains 351 (daptomycin-susceptible) and 351-D10 (daptomycin non-susceptible) were analyzed. S. mitis-oralis strain 351-D10 was made daptomycin non-susceptible through serial passage in the presence of daptomycin. RESULTS: Daptomycin non-susceptible S. mitis-oralis had significant alterations in glucose catabolism and a re-balancing of the redox status through amino acid biosynthesis relative to daptomycin susceptible S. mitis-oralis. These changes were accompanied by a reduced capacity to generate biomass, creating a fitness cost in exchange for daptomycin non-susceptibility. CONCLUSIONS: S. mitis-oralis metabolism is altered in daptomycin non-susceptible bacteria relative to the daptomycin susceptible parent strain. As demonstrated in Staphylococcus aureus, inhibiting the metabolic changes that facilitate the transition from a daptomycin susceptible state to a non-susceptible one, inhibits daptomycin non-susceptibility. By preventing these metabolic adaptations in S. mitis-oralis, it should be possible to deter the formation of daptomycin non-susceptibility.

In many viruses, including rotavirus (RV), the major pathogen of infantile gastroenteritis, capping of viral messenger RNAs is a pivotal step for efficient translation of the viral genome. In RV, VP3 caps the nascent transcripts synthesized from the genomic dsRNA segments by the RV polymerase VP1 within the particle core. Here, from cryo-electron microscopy, x-ray crystallography, and biochemical analyses, we show that VP3 forms a stable tetrameric assembly with each subunit having a modular domain organization, which uniquely integrates five distinct enzymatic steps required for capping the transcripts. In addition to the previously known guanylyl- and methyltransferase activities, we show that VP3 exhibits hitherto unsuspected RNA triphosphatase activity necessary for initiating transcript capping and RNA helicase activity likely required for separating the RNA duplex formed transiently during endogenous transcription. From our studies, we propose a new mechanism for how VP3 inside the virion core caps the nascent transcripts exiting from the polymerase.

Bacteria are continually challenged by foreign invaders, including bacteriophages, and have evolved a variety of defenses against these invaders. Here, we describe the structural and biochemical mechanisms of a bacteriophage immunity pathway found in a broad array of bacteria, including E. coli and Pseudomonas aeruginosa. This pathway uses eukaryotic-like HORMA domain proteins that recognize specific peptides, then bind and activate a cGAS/DncV-like nucleotidyltransferase (CD-NTase) to generate a cyclic triadenylate (cAAA) second messenger; cAAA in turn activates an endonuclease effector, NucC. Signaling is attenuated by a homolog of the AAA+ ATPase Pch2/TRIP13, which binds and disassembles the active HORMA-CD-NTase complex. When expressed in non-pathogenic E. coli, this pathway confers immunity against bacteriophage λ through an abortive infection mechanism. Our findings reveal the molecular mechanisms of a bacterial defense pathway integrating a cGAS-like nucleotidyltransferase with HORMA domain proteins for threat sensing through protein detection and negative regulation by a Trip13 ATPase.

5,7-Di-N-acetyllegionaminic acid (Leg5,7Ac2) is a bacterial nonulosonic acid (NulO) analogue of sialic acids, an important class of monosaccharides in mammals and in some bacteria. To develop efficient one-pot multienzyme (OPME) glycosylation systems for synthesizing Leg5,7Ac2-glycosides, Legionella pneumophila cytidine 5'-monophosphate (CMP)-Leg5,7Ac2 synthetase (LpCLS) was cloned and characterized. It was successfully used in producing Leg5,7Ac2-glycosides from chemoenzymatically synthesized Leg5,7Ac2 using a one-pot two-enzyme system or from its chemically synthesized six-carbon monosaccharide precursor 2,4-diacetamido-2,4,6-trideoxymannose (6deoxyMan2,4diNAc) in a one-pot three-enzyme system. In addition, LpCLS was shown to tolerate Neu5Ac7NAc, a C9-hydroxyl analogue of Leg5,7Ac2 and also a stable analogue of 7-O-acetylneuraminic acid (Neu5,7Ac2), to allow OPME synthesis of the corresponding α2-3-linked sialosides, from chemically synthesized six-carbon monosaccharide precursor 4-N-acetyl-4-deoxy-N-acetylmannosamine (ManNAc7NAc).

Dysfunction of the endoplasmic reticulum (ER) in insulin‐producing beta cells results in cell loss and diabetes mellitus. Here we report on five individuals from three different consanguineous families with infancy‐onset diabetes mellitus and severe neurodevelopmental delay caused by a homozygous p.(Arg371Ser) mutation in FICD. The FICD gene encodes a bifunctional Fic domain‐containing enzyme that regulates the ER Hsp70 chaperone, BiP, via catalysis of two antagonistic reactions: inhibitory AMPylation and stimulatory deAMPylation of BiP. Arg371 is a conserved residue in the Fic domain active site. The FICDR371S mutation partially compromises BiP AMPylation in vitro but eliminates all detectable deAMPylation activity. Overexpression of FICDR371S or knock‐in of the mutation at the FICD locus of stressed CHO cells results in inappropriately elevated levels of AMPylated BiP and compromised secretion. These findings, guided by human genetics, highlight the destructive consequences of de‐regulated BiP AMPylation and raise the prospect of tuning FICD's antagonistic activities towards therapeutic ends. Synopsis: Modification of the endoplasmic reticulum (ER) chaperone BiP by FICD, an enzyme that adds or removes an adenosine monophosphate (AMP), regulates protein folding homeostasis. Secretory cells that produce the hormone insulin or promote neurodevelopment depend on intact ER function. Homozygosity of an Arg371Ser mutation in FICD causes infancy‐onset diabetes mellitus and neurodevelopmental delay.The Arg371Ser corrupts FICD by inactivating its ability to remove AMP from BiP.Inappropriately, elevated levels of AMPylated BiP interfere with secretion, suggesting a molecular mechanism for this genetic disorder. [ABSTRACT FROM AUTHOR]

The accumulation of DNA in the cytosol serves as a key immunostimulatory signal associated with infections, cancer and genomic damage1,2. Cytosolic DNA triggers immune responses by activating the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway3. The binding of DNA to cGAS activates its enzymatic activity, leading to the synthesis of a second messenger, cyclic guanosine monophosphate-adenosine monophosphate (2'3'-cGAMP)4-7. This cyclic dinucleotide (CDN) activates STING8, which in turn activates the transcription factors interferon regulatory factor 3 (IRF3) and nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), promoting the transcription of genes encoding type I interferons and other cytokines and mediators that stimulate a broader immune response. Exogenous 2'3'-cGAMP produced by malignant cells9 and other CDNs, including those produced by bacteria10-12 and synthetic CDNs used in cancer immunotherapy13,14, must traverse the cell membrane to activate STING in target cells. How these charged CDNs pass through the lipid bilayer is unknown. Here we used a genome-wide CRISPR-interference screen to identify the reduced folate carrier SLC19A1, a folate-organic phosphate antiporter, as the major transporter of CDNs. Depleting SLC19A1 in human cells inhibits CDN uptake and functional responses, and overexpressing SLC19A1 increases both uptake and functional responses. In human cell lines and primary cells ex vivo, CDN uptake is inhibited by folates as well as two medications approved for treatment of inflammatory diseases, sulfasalazine and the antifolate methotrexate. The identification of SLC19A1 as the major transporter of CDNs into cells has implications for the immunotherapeutic treatment of cancer13, host responsiveness to CDN-producing pathogenic microorganisms11 and-potentially-for some inflammatory diseases.

Migration through 3D constrictions can cause nuclear rupture and mislocalization of nuclear proteins, but damage to DNA remains uncertain, as does any effect on cell cycle. Here, myosin II inhibition rescues rupture and partially rescues the DNA damage marker γH2AX, but an apparent block in cell cycle appears unaffected. Co-overexpression of multiple DNA repair factors or antioxidant inhibition of break formation also exert partial effects, independently of rupture. Combined treatments completely rescue cell cycle suppression by DNA damage, revealing a sigmoidal dependence of cell cycle on excess DNA damage. Migration through custom-etched pores yields the same damage threshold, with ∼4-µm pores causing intermediate levels of both damage and cell cycle suppression. High curvature imposed rapidly by pores or probes or else by small micronuclei consistently associates nuclear rupture with dilution of stiff lamin-B filaments, loss of repair factors, and entry from cytoplasm of chromatin-binding cGAS (cyclic GMP-AMP synthase). The cell cycle block caused by constricted migration is nonetheless reversible, with a potential for DNA misrepair and genome variation.

We investigated the ability of several recent clinical viridans group streptococci (VGS) bloodstream isolates (Streptococcus mitis/S. oralis subgroup) from daptomycin (DAP)-naive patients to develop DAP resistance in vitro All strains rapidly developed high-level and stable DAP resistance. Substitutions in two enzymes involved in the cardiolipin biosynthesis pathway were identified, i.e., CdsA (phosphatidate cytidylyltransferase) and PgsA (CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyltransferase). These mutations were associated with complete disappearance of phosphatidylglycerol and cardiolipin from cell membranes. DAP interactions with the cell membrane differed in isolates with PgsA versus CdsA substitutions.

Abstract Dysfunction of the endoplasmic reticulum (ER) in insulin‐producing beta cells results in cell loss and diabetes mellitus. Here we report on five individuals from three different consanguineous families with infancy‐onset diabetes mellitus and severe neurodevelopmental delay caused by a homozygous p.(Arg371Ser) mutation in FICD. The FICD gene encodes a bifunctional Fic domain‐containing enzyme that regulates the ER Hsp70 chaperone, BiP, via catalysis of two antagonistic reactions: inhibitory AMPylation and stimulatory deAMPylation of BiP. Arg371 is a conserved residue in the Fic domain active site. The FICDR371S mutation partially compromises BiP AMPylation in vitro but eliminates all detectable deAMPylation activity. Overexpression of FICDR371S or knock‐in of the mutation at the FICD locus of stressed CHO cells results in inappropriately elevated levels of AMPylated BiP and compromised secretion. These findings, guided by human genetics, highlight the destructive consequences of de‐regulated BiP AMPylation and raise the prospect of tuning FICD's antagonistic activities towards therapeutic ends.

Background: Oncolytic viral therapy is a promising method for tumor treatment. Currently, several oncolytic viruses (OVs) have been used as tumor therapy at different phases of research and clinical trials. OVs not only directly lyse tumor cells due to viral replication but also initiate host antitumor immune responses. Previous studies have primarily focused on how OVs activate adaptive immune responses in immune cells. However, the role of innate immune responses in tumors induced by OVs remains unclear. Methods: To determine the innate immune responses induced by vesicular stomatitis virus (VSV), the mutant VSVΔM51 strain was used for the infection and quantitative polymerase chain reaction (qPCR) was employed to measure the transcriptional levels of antiviral genes. The knockdown efficiency of RIG‐I was examined by qPCR. Viral titers were measured by plaque assays. Tumor models were established by intradermally implanting RIG‐I‐knockdown and control LLC cells into the flank of wild type C57BL/6J mice. When the tumors reached approximately 50mm3, they were infected with VSVΔM51 via intratumoral injections to examine its therapeutic effect. Results: Infection with VSVΔM51 triggered remarkable innate immune responses in several tumor cell lines through the cytoplasmic RIG‐I sensing pathway. Moreover, we found that intratumoral injection of VSVΔM51 effectively reduced tumor growth in murine LCC lung cancer model. Importantly, VSVΔM51‐induced antitumor therapy was more effective in murine LLC tumor model established using Rig‐I‐knockdown cells compared with the tumor model established using control cells. Conclusion: RIG‐I‐mediated innate immune signaling in tumor cells plays a negative role in regulating antitumor therapy with VSVΔM51 virus. [ABSTRACT FROM AUTHOR]

Pediatric high-grade gliomas (pHGGs) are the leading cause of cancer-related deaths in children in the USA. Sixteen percent of hemispheric pediatric and young adult HGGs encode Gly34Arg/Val substitutions in the histone H3.3 (H3.3-G34R/V). The mechanisms by which H3.3-G34R/V drive malignancy and therapeutic resistance in pHGGs remain unknown. Using a syngeneic, genetically engineered mouse model (GEMM) and human pHGG cells encoding H3.3-G34R, we demonstrate that this mutation led to the downregulation of DNA repair pathways. This resulted in enhanced susceptibility to DNA damage and inhibition of the DNA damage response (DDR). We demonstrate that genetic instability resulting from improper DNA repair in G34R-mutant pHGG led to the accumulation of extrachromosomal DNA, which activated the cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) pathway, inducing the release of immune-stimulatory cytokines. We treated H3.3-G34R pHGG-bearing mice with a combination of radiotherapy (RT) and DNA damage response inhibitors (DDRi) (i.e., the blood-brain barrier-permeable PARP inhibitor pamiparib and the cell-cycle checkpoint CHK1/2 inhibitor AZD7762), and these combinations resulted in long-term survival for approximately 50% of the mice. Moreover, the addition of a STING agonist (diABZl) enhanced the therapeutic efficacy of these treatments. Long-term survivors developed immunological memory, preventing pHGG growth upon rechallenge. These results demonstrate that DDRi and STING agonists in combination with RT induced immune-mediated therapeutic efficacy in G34-mutant pHGG. [ABSTRACT FROM AUTHOR]

Folate derivatives are important cofactors for enzymes in several metabolic processes. Folate-related inhibition and resistance mechanisms in bacteria are potential targets for antimicrobial therapies and therefore a significant focus of current research. Here, we report that the activity of Escherichia coli poly-γ-glutamyl tetrahydrofolate/dihydrofolate synthase (FolC) is regulated by glutamate/glutamine-sensing uridylyltransferase (GlnD), THF-dependent tRNA modification enzyme (MnmE), and UDP-glucose dehydrogenase (Ugd) as shown by direct in vitro protein-protein interactions. Using kinetics analyses, we observed that GlnD, Ugd, and MnmE activate FolC many-fold by decreasing the K half of FolC for its substrate l-glutamate. Moreover, FolC inhibited the GTPase activity of MnmE at low GTP concentrations. The growth phenotypes associated with these proteins are discussed. These results, obtained using direct in vitro enzyme assays, reveal unanticipated networks of allosteric regulatory interactions in the folate pathway in E. coli and indicate regulation of polyglutamylated tetrahydrofolate biosynthesis by the availability of nitrogen sources, signaled by the glutamine-sensing GlnD protein.

Detection of cytosolic DNA by the enzyme cGAS triggers the production of cGAMP, a second messenger that binds and activates the adaptor protein STING, which leads to interferon (IFN) production. Here, we found that in vivo natural killer (NK) cell killing of tumor cells, but not of normal cells, depends on STING expression in non-tumor cells. Experiments using transplantable tumor models in STING- and cGAS-deficient mice revealed that cGAS expression by tumor cells was critical for tumor rejection by NK cells. In contrast, cGAS expression by host cells was dispensable, suggesting that tumor-derived cGAMP is transferred to non-tumor cells, where it activates STING. cGAMP administration triggered STING activation and IFN-β production in myeloid cells and B cells but not NK cells. Our results reveal that the anti-tumor response of NK cells critically depends on the cytosolic DNA sensing pathway, similar to its role in defense against pathogens, and identify tumor-derived cGAMP as a major determinant of tumor immunogenicity with implications for cancer immunotherapy.

Plasmacytoid dendritic cells (pDC) are essential for immune competence. Here we show that pDC precursor differentiated from human CD34+ hematopoietic stem and progenitor cells (HSPC) has low surface expression of pDC markers, and has limited induction of type I interferon (IFN) and IL-6 upon TLR7 and TLR9 agonists treatment; by contrast, cGAS or RIG-I agonists-mediated activation is not altered. Importantly, after priming with type I and II IFN, these precursor pDCs attain a phenotype and functional activity similar to that of peripheral blood-derived pDCs. Data from CRISPR/Cas9-mediated genome editing of HSPCs further show that HSPC-pDCs with genetic modifications can be obtained, and that expression of the IFN-α receptor is essential for the optimal function, but dispensable for the differentiation, of HSPC-pDC percursor. Our results thus demonstrate the biological effects of IFNs for regulating pDC function, and provide the means of generating of gene-modified human pDCs.

Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs.

Background: Current immunotherapy strategies have contrasting clinical results in human lung cancer patients as small-cell lung cancers (SCLC) often show features of immunological cold tumours. Topoisomerase 1 (TOP1) poisons are effective antitumor drugs with good efficacy against lung cancers.Methods: We used molecular, genetic and bioinformatic approaches to determine the mechanism of micronuclei formation induced by two TOP1 poisons in different human cancer cells, including SCLC cell lines.Results: TOP1 poisons stimulate similar levels of micronuclei in all tested cell lines but downstream effects can vary markedly. TOP1 poisons increase micronuclei levels with a mechanism involving R-loops as overexpression of RNaseH1 markedly reduces or abolishes both H2AX phosphorylation and micronuclei formation. TOP1 poison-induced micronuclei activate the cGAS/STING pathway leading to increased expression of immune genes in HeLa cells, but not in human SCLC cell lines, mainly due to lack of STING and/or cGAS expression. Moreover, the expression of STING and antigen-presenting machinery genes is generally downregulated in patient tumours of human lung cancer datasets.Conclusions: Altogether, our data reveal an immune signalling mechanism activated by TOP1 poisons, which is often impaired in human SCLC tumours. [ABSTRACT FROM AUTHOR]

Background: The cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS) plays critical functions in innate immune responses via the production of the second messenger cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), which stimulates the adaptor stimulator of interferon genes (STING). However, the clinical relevance and prognostic value of the cGAS-STING pathway in human cancers remains largely unexplored.Methods: A gene signature related to the cGAS-STING score was identified. The pan-cancer landscape of cGAS-STING expression was calculated using the RNAseq data acquired from the TCGA cohort. Tumor-infiltrating immune cells (TIICs) were determined by the ssGSEA method. Kaplan-Meier curves, Cox regression analyses, and the area under the curve (AUC) were employed to decipher the predictive value of cGAS-STING risk score and TIICs across several human cancers.Results: Most tumor tissues displayed a higher cGAS-STING score compared with their corresponding nontumor tissues, except for prostate adenocarcinoma (PRAD) and uterine corpus endometrial carcinoma (UCEC). Higher cGAS-STING score was closely associated with poor clinical outcome of kidney renal clear cell carcinoma (KIRC) and kidney renal papillary cell carcinoma (KIRP), whereas the cGAS-STING score predicted a better prognosis in pheochromocytoma and paraganglioma (PCPG). Enrichment analysis showed that cGAS-STING was profoundly implicated in diverse immune-related pathways in KIRC, KIRP, and PCPG. Significant positive correlations were noticed between cGAS-STING score and TIICs, including activated CD8+ T cells, activated CD4+ T cells, monocytes, and mast cells. Finally, the cGAS-STING score was revealed to be an independent prognostic factor for KIRC patients and possessed a strong predictive power for the prognostic evaluation of KIRC and KIRP patients.Conclusions: We constructed a cGAS-STING gene signature to predict survival and tumor immunity across human cancers, which can serve as a novel prognostic indicator and therapeutic target, especially in KIRC and KIRP. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells recognize and kill cancer cells and infected cells by engaging cell surface ligands that are induced preferentially or exclusively on these cells. These ligands are recognized by activating receptors on NK cells, such as NKG2D. In addition to activation by cell surface ligands, the acquisition of optimal effector activity by NK cells is driven in vivo by cytokines and other signals. This review addresses a developing theme in NK cell biology: that NK-activating ligands on cells, and the provision of cytokines and other signals that drive high effector function in NK cells, are driven by abnormalities that arise from transformation or the infected state. The pathways include genomic damage, which causes self DNA to be exposed in the cytosol of affected cells, where it activates the DNA sensor cGAS. The resulting signaling induces NKG2D ligands and also mobilizes NK cell activation. Other key pathways that regulate NKG2D ligands include PI-3 kinase activation, histone acetylation, and the integrated stress response. This review summarizes the roles of these pathways and their relevance in both viral infections and cancer.

The aldehyde dehydrogenase 2 (ALDH2) rs671 polymorphism commonly exists in the East Asian populations and is associated with high risks of cardiovascular disease (CVD). However, the cellular and molecular mechanisms that underlie the ALDH2 rs671 mutant-linked high CVD remain elusive. Here, we show that macrophages derived from human ALDH2 rs671 carriers and ALDH2 knockout mice exhibited an enhanced pro-inflammatory macrophage phenotype and an impaired anti-inflammatory macrophage phenotype. Transplanting bone marrow from ALDH2 -/- ApoE -/- to ApoE -/- mice significantly increased atherosclerotic plaque growth and pro-inflammatory macrophage polarization in vivo. Mechanistically, ALDH2 inhibited activation of the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway in macrophages. Pharmacological inhibition of cGAS by RU.521 completely neutralized ALDH2-deficiency-induced macrophage polarization. In-depth mechanistic investigation showed that ALDH2 accelerated cGAS K48-linked polyubiquitination degradation at lysine 282 in macrophages by reducing the interaction between ubiquitin-specific protease 14 (USP14) and cGAS, mainly through its enzymatic role in mitigating 4-hydroxy-2-nonenal (4-HNE) accumulation. Consistently, USP14 knockdown in bone marrow cells alleviated proinflammatory responses in macrophages and protected against atherosclerosis. Our findings provide new mechanistic insights of ALDH2 deficiency-associated proinflammation and atherosclerosis and new therapeutic and preventive paradigms for treatment of atherosclerosis-associated CVD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Group I and II introns are large catalytic RNAs (ribozymes) that are frequently encountered in fungal mitochondrial genomes. The discovery of respiratory mutants linked to intron splicing defects demonstrated that for the efficient removal of organellar introns there appears to be a requirement of protein splicing factors. These splicing factors can be intron-encoded proteins with maturase activities that usually promote the splicing of the introns that encode them ( cis -acting) and/or nuclear-encoded factors that can promote the splicing of a range of different introns ( trans -acting). Compared to plants organellar introns, fungal mitochondrial intron splicing is still poorly explored, especially in terms of the synergy of nuclear factors with intron-encoded maturases that has direct impact on splicing through their association with intron RNA. In addition, nuclear-encoded accessory factors might drive the splicing impetus through translational activation, mitoribosome assembly, and phosphorylation-mediated RNA turnover. This review explores protein-assisted splicing of introns by nuclear and mitochondrial-encoded maturases as a means of mitonuclear interplay that could respond to environmental and developmental factors promoting phenotypic adaptation and potentially speciation. It also highlights key evolutionary events that have led to changes in structure and ATP-dependence to accommodate the dual functionality of nuclear and organellar splicing factors., Competing Interests: The authors have no competing interests to disclose.

Cytoplasmic mRNA decay is effected by exonucleolytic degradation in either the 5' to 3' or 3' to 5' direction. Pervasive terminal uridylation is implicated in mRNA degradation, however, its functional relevance for bulk mRNA turnover remains poorly understood. In this study, we employ genome-wide 3'-RACE (gw3'-RACE) in the model system fission yeast to elucidate the role of uridylation in mRNA turnover. We observe widespread uridylation of shortened poly(A) tails, promoting efficient 5' to 3' mRNA decay and ensuring timely and controlled mRNA degradation. Inhibition of this uridylation process leads to excessive deadenylation and enhanced 3' to 5' mRNA decay accompanied by oligouridylation. Strikingly we found that uridylation of poly(A) tails and oligouridylation of non-polyadenylated substrates are catalysed by different terminal uridyltransferases Cid1 and Cid16 respectively. Our study sheds new light on the intricate regulatory mechanisms underlying bulk mRNA turnover, demonstrating the role of uridylation in modulating mRNA decay pathways., (© 2024. The Author(s).)

Flavin adenine dinucleotide (FAD) is an essential cofactor for numerous flavoenzymes present in all living organisms. The biosynthesis of FAD from riboflavin involves two sequential reactions catalyzed by riboflavin kinase and flavin adenine dinucleotide synthase (FADS). Entamoeba histolytica , the protozoan parasite responsible for amebiasis, apparently lacks a gene encoding FADS that share similarity with bacterial and eukaryotic canonical FADS, yet it can synthesize FAD. In this study, we have identified the gene responsible for FADS and thoroughly characterized physiological and biochemical properties of FADS from E. histolytica . Phylogenetic analysis revealed that the gene was likely laterally transferred from archaea. The kinetic properties of recombinant EhFADS were consistent with the notion that EhFADS is of archaeal origin, exhibiting K M and k cat values similar to those of the arachaeal enzyme while significantly differing from the human counterpart. Repression of gene expression of EhFADS by epigenetic gene silencing caused substantial reduction in FAD levels and parasite growth, underscoring the importance of EhFADS for the parasite. Furthermore, we demonstrated that EhFADS gene silencing reduced thioredoxin reductase activity, which requires FAD as a cofactor and makes the ameba more susceptible to metronidazole. In summary, this study unveils unique evolutionary and biochemical features of EhFADS and underscores its significance as a promising drug target in combating human amebiasis.IMPORTANCEFAD is important for all forms of life, yet its role and metabolism are still poorly studied in E. histolytica , the protozoan parasite causing human amebiasis. Our study uncovers the evolutionary unique key enzyme, archaeal-type FADS for FAD biosynthesis from E. histolytica for the first time. Additionally, we showed the essentiality of this enzyme for parasite survival, highlighting its potential as target for drug development against E. histolytica infections., Competing Interests: The authors declare no conflict of interest.

Cellular senescence, a stress-induced stable proliferation arrest associated with an inflammatory senescence-associated secretory phenotype (SASP), is a cause of aging. In senescent cells, cytoplasmic chromatin fragments (CCFs) activate SASP via the anti-viral cGAS/STING pathway. Promyelocytic leukemia (PML) protein organizes PML nuclear bodies (NBs), which are also involved in senescence and anti-viral immunity. The HIRA histone H3.3 chaperone localizes to PML NBs in senescent cells. Here, we show that HIRA and PML are essential for SASP expression, tightly linked to HIRA's localization to PML NBs. Inactivation of HIRA does not directly block expression of nuclear factor κB (NF-κB) target genes. Instead, an H3.3-independent HIRA function activates SASP through a CCF-cGAS-STING-TBK1-NF-κB pathway. HIRA physically interacts with p62/SQSTM1, an autophagy regulator and negative SASP regulator. HIRA and p62 co-localize in PML NBs, linked to their antagonistic regulation of SASP, with PML NBs controlling their spatial configuration. These results outline a role for HIRA and PML in the regulation of SASP., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Radiation injury to the intestine is one of the most common complications in patients undergoing abdominal or pelvic cavity radiotherapy. In this study, we investigated the potential protective effect of Lactobacillus rhamnosus GG (LGG) on radiation-induced intestinal injury and its underlying mechanisms. Mice were assigned to a control group, a 10 Gy total abdominal irradiation (TAI) group, or a group pretreated with 10 8 CFU LGG for three days before TAI. Small intestine and gut microbiota were analyzed 3.5 days post-exposure. LGG intervention improved intestinal structure, reduced jejunal DNA damage, and inhibited the inflammatory cGAS/STING pathway. Furthermore, LGG reduced M1 proinflammatory macrophage and CD8+ T cell infiltration, restoring the balance between Th17 and Treg cells in the inflamed jejunum. LGG also partially restored the gut microbiota. These findings suggest the possible therapeutic radioprotective effect of probiotics LGG in alleviating radiation-induced intestinal injury by maintaining immune homeostasis and reshaping gut microbiota., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

MPIase is a glycolipid involved in protein insertion into and preprotein translocation across the cytoplasmic membranes of E. coli. MPIase is upregulated in the cold conditions to overcome the cold-sensitive protein export. CdsA, a CDP-diacylglycerol synthase, catalyzes the first reaction in MPIase biosynthesis. An open reading frame for a peptide of 50 amino acids is encoded immediately after ispU, a neighboring upstream gene of cdsA, and overlaps cdsA to a large extent. Mutational analysis revealed that the expression of this peptide is essential for upregulation of MPIase in the cold. Consistently, expression of this peptide in trans resulted in cold upregulation of MPIase. We therefore named this peptide MucA after its function (MPIase upregulation in the cold). When the partially purified MucA was added to the reaction of the intermediate in MPIase biosynthesis, a significant increase in the product formation was observed, supporting the function of MucA. The possible role of MucA in MPIase biosynthesis is discussed., Competing Interests: Declaration of competing interest The authors declare that they have no conflicts of interest with regard to the content of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Summary: Background. Uterine leiomyosarcoma is a rare aggressive smooth muscle cancer with poor survival rates. RNA Polymerase I (Pol I) activity is elevated in many cancers supporting tumour growth and prior studies in uterine leiomyosarcoma revealed enlarged nucleoli and upregulated Pol I activity-related genes. This study aimed to investigate the anti-tumour potential of CX-5461, a Pol I transcription inhibitor currently being evaluated in clinical trials for several cancers, against the human uterine leiomyosarcoma cell line, SK-UT-1. Methods. SK-UT-1 was characterised using genome profiling and western blotting. The anti-tumour effects of CX-5461 were investigated using cell proliferation assays, expression analysis using qRT-PCR, and BrdU/PI based cell cycle analysis. Results. Genetic analysis of SK-UT-1 revealed mutations in TP53, RB1, PTEN, APC and TSC1 & 2, all potentially associated with increased Pol I activity. Protein expression analysis showed dysregulated p53, RB1 and c-Myc. CX-5461 treatment resulted in an anti-proliferation response, G2 phase cell-cycle arrest and on-target activity demonstrated by reduced ribosomal DNA transcription. Conclusions. SK-UT-1 was confirmed as a representative model of uterine leiomyosarcoma and CX-5461 has significant potential as a novel adjuvant for this rare cancer. [ABSTRACT FROM AUTHOR]

Aim: Although Enterococcus is among bacteria of normal gut flora in animals, these bacteria are also causative agents of nosocomial infections. E. faecium and E. faecalis are the most common enterococci isolated from patients. E. faecium is intrinsically resistant to many antibiotics but susceptible to macrolides and lincosamides. The most common resistance mechanisms to lincosamides are modification of target by methylases and inactivation of lincosamides by nucleotidyltransferase. In present work mechanism of resistance, transferability of resistance and characterisation of genetic support of lincosamide resistance in E. faecium ADU1 which is resistant to macrolides and lincosamides was studied. Materials andAntimicrobial susceptibilities were determined by agar dilution MICs and disk diffusion methods. Lincosamid inactivation of E. faecium ADU1 was tested by Gots' test. Presence of known erm and lnu genes was checked by PCR. E.faecalis JH2-2 was used as recipient strain for conjugation experiment to test transferability of resistance. Chromosomal changes in receptive strains were tested by PFGE. To analyse genetic support of resistance gene invers PCR method was used and amplicon was sequence and homologies were checked with gene bank. Results. A total of 57 environmental Enterococci was tested for resistance to lincosamide by inactivation and 16% were found to be positive. One of these isolates, ADU1 was studied further. The MICs of ADU1 strain for clindamycin and erythromycin were both > 128 µg/ml. Gots' test showed inactivation of lincosamides by ADU1 strain which was shown to carry ermB and lnuB by PCR. Conjugation experiments showed co-transfer of lincosamid resistance with erythromycin resistance. No change in chromosomal restriction profile of transconjugants was observed by PFGE which showed possible transfer of resistance by plasmid. A fragment of 2500bp was obtained by invers PCR. Sequence analysis of this fragment showed that partial homology with pEF418 which was found in Enterococcus faecalis. Conclusion. Resistance to lincosamides is conferred by many different nucleotidyltransferases and disseminated among a large spectrum of bacteria genus and species via mobile conjugative genetic elements. E. faecium ADU1 was resistant to lincosamides by inactivation due to lnuB gene which is carried by conjugative plasmid pEF418. Presence of multiple resistance genes on a transferable plasmid is important for dissemination of antibiotic resistance. Our study is the first study which showed presence of lnuB gene in an Enterococcus isolated in Turkey. Large survey studies should be done to evaluate dissemination of lincosamide resistance by inactivation. [ABSTRACT FROM AUTHOR]