Population Variation and Genetic Control of Modular Chromatin Architecture in Humans.

Chromatin state variation at gene regulatory elements is abundant across individuals, yet we understand little about the genetic basis of this variability. Here, we profiled several histone modifications, the transcription factor (TF) PU.1, RNA polymerase II, and gene expression in lymphoblastoid cell lines from 47 whole-genome sequenced individuals. We observed that distinct cis-regulatory elements exhibit coordinated chromatin variation across individuals in the form of variable chromatin modules (VCMs) at sub-Mb scale. VCMs were associated with thousands of genes and preferentially cluster within chromosomal contact domains. We mapped strong proximal and weak, yet more ubiquitous, distal-acting chromatin quantitative trait loci (cQTL) that frequently explain this variation. cQTLs were associated with molecular activity at clusters of cis-regulatory elements and mapped preferentially within TF-bound regions. We propose that local, sequence-independent chromatin variation emerges as a result of genetic perturbations in cooperative interactions between cis-regulatory elements that are located within the same genomic domain.

A Multi-Omics Approach Reveals Features That Permit Robust and Widespread Regulation of IFN-Inducible Antiviral Effectors.

The antiviral state, an initial line of defense against viral infection, is established by a set of IFN-stimulated genes (ISGs) encoding antiviral effector proteins. The effector ISGs are transcriptionally regulated by type I IFNs mainly via activation of IFN-stimulated gene factor 3 (ISGF3). In this study, the regulatory elements of effector ISGs were characterized to determine the (epi)genetic features that enable their robust induction by type I IFNs in multiple cell types. We determined the location of regulatory elements, the DNA motifs, the occupancy of ISGF3 subunits (IRF9, STAT1, and STAT2) and other transcription factors, and the chromatin accessibility of 37 effector ISGs in murine dendritic cells. The IFN-stimulated response element (ISRE) and its tripartite version occurred most frequently in the regulatory elements of effector ISGs than in any other tested ISG subsets. Chromatin accessibility at their promoter regions was similar to most other ISGs but higher than at the promoters of inflammation-related cytokines, which were used as a reference gene set. Most effector ISGs (81.1%) had at least one ISGF3 binding region proximal to the transcription start site (TSS), and only a subset of effector ISGs (24.3%) was associated with three or more ISGF3 binding regions. The IRF9 signals were typically higher, and ISRE motifs were "stronger" (more similar to the canonical sequence) in TSS-proximal versus TSS-distal regulatory regions. Moreover, most TSS-proximal regulatory regions were accessible before stimulation in multiple cell types. Our results indicate that "strong" ISRE motifs and universally accessible promoter regions that permit robust, widespread induction are characteristic features of effector ISGs.

Spatio-temporal dynamics of intra-host variability in SARS-CoV-2 genomes.

During the course of the COVID-19 pandemic, large-scale genome sequencing of SARS-CoV-2 has been useful in tracking its spread and in identifying variants of concern (VOC). Viral and host factors could contribute to variability within a host that can be captured in next-generation sequencing reads as intra-host single nucleotide variations (iSNVs). Analysing 1347 samples collected till June 2020, we recorded 16 410 iSNV sites throughout the SARS-CoV-2 genome. We found ∼42% of the iSNV sites to be reported as SNVs by 30 September 2020 in consensus sequences submitted to GISAID, which increased to ∼80% by 30th June 2021. Following this, analysis of another set of 1774 samples sequenced in India between November 2020 and May 2021 revealed that majority of the Delta (B.1.617.2) and Kappa (B.1.617.1) lineage-defining variations appeared as iSNVs before getting fixed in the population. Besides, mutations in RdRp as well as RNA-editing by APOBEC and ADAR deaminases seem to contribute to the differential prevalence of iSNVs in hosts. We also observe hyper-variability at functionally critical residues in Spike protein that could alter the antigenicity and may contribute to immune escape. Thus, tracking and functional annotation of iSNVs in ongoing genome surveillance programs could be important for early identification of potential variants of concern and actionable interventions.

CMTM6 attenuates cisplatin-induced cell death in OSCC by regulating AKT/c-Myc-driven ribosome biogenesis.

CMTM6, a type 3 transmembrane protein, is known to stabilize the expression of programmed cell death ligand 1 (PD-L1) and hence facilitates the immune evasion of tumor cells. Recently, we demonstrated that CMTM6 is a major driver of cisplatin resistance in oral squamous cell carcinomas (OSCC). However, the detailed mechanism of how CMTM6 rewires cisplatin resistance in OSCC is yet to be explored. RNA sequencing analysis of cisplatin-resistant OSCC lines stably expressing Nt shRNA and CMTM6 shRNA revealed that CMTM6 might be a potential regulator of the ribosome biogenesis network. Knocking down CMTM6 significantly inhibited transcription of 47S precursor rRNA and hindered the nucleolar structure, indicating reduced ribosome biogenesis. When CMTM6 was ectopically over-expressed in CMTM6KD cells, almost all ribosomal machinery components were rescued. Mechanistically, CMTM6 induced the expression of C-Myc, which promotes RNA polymerase I mediated rDNA transcription. In addition to this, CMTM6 was also found to regulate the AKT-mTORC1-dependent ribosome biogenesis and protein synthesis in cisplatin-resistant lines. The nude mice and zebrafish xenograft experiments indicate that blocking ribosome synthesis either by genetic inhibitor (CMTM6KD) or pharmacological inhibitor (CX-5461) significantly restores cisplatin-mediated cell death in chemoresistant OSCC. Overall, our study suggests that CMTM6 is a major regulator of the ribosome biogenesis network and targeting the ribosome biogenesis network is a viable target to overcome chemoresistance in OSCC. The novel combination of CX-5461 and cisplatin deserves further clinical investigation in advanced OSCC.

TRIM16 controls assembly and degradation of protein aggregates by modulating the p62-NRF2 axis and autophagy.

Sequestration of protein aggregates in inclusion bodies and their subsequent degradation prevents proteostasis imbalance, cytotoxicity, and proteinopathies. The underlying molecular mechanisms controlling the turnover of protein aggregates are mostly uncharacterized. Herein, we show that a TRIM family protein, TRIM16, governs the process of stress-induced biogenesis and degradation of protein aggregates. TRIM16 facilitates protein aggregate formation by positively regulating the p62-NRF2 axis. We show that TRIM16 is an integral part of the p62-KEAP1-NRF2 complex and utilizes multiple mechanisms for stabilizing NRF2. Under oxidative and proteotoxic stress conditions, TRIM16 activates ubiquitin pathway genes and p62 via NRF2, leading to ubiquitination of misfolded proteins and formation of protein aggregates. We further show that TRIM16 acts as a scaffold protein and, by interacting with p62, ULK1, ATG16L1, and LC3B, facilitates autophagic degradation of protein aggregates. Thus, TRIM16 streamlines the process of stress-induced aggregate clearance and protects cells against oxidative/proteotoxic stress-induced toxicity in vitro and in vivo Taken together, this work identifies a new mechanism of protein aggregate turnover, which could be relevant in protein aggregation-associated diseases such as neurodegeneration.

Zbtb10 transcription factor is crucial for murine cDC1 activation and cytokine secretion.

Dendritic cell (DC) activation and cytokine production is tightly regulated. In this study, we found that Zbtb10 expression is activation dependent and it is essential for the immunogenic function of cDC1. Zbtb10 knockdown (KD) significantly reduced the expression of co-stimulatory genes CD80 and CD86 along with cytokines including IL-12, IL-6, and IL-10, in activated cDC1 Mutu-DC line. Consequently, the clonal expansion of CD44+ effector T cells in co-cultured CD4+ T cells was drastically reduced owing to significantly reduced IL-2. At the same time, these CD44+ effector T cells were unable to differentiate toward Tbet+ IFNγ+ Th1 subtype. Instead, an increased frequency of Th2 cells expressing GATA3+ and IL-13+ was observed. Interestingly, in Zbtb10 KD condition the co-cultured T cells depicted increased expression of PD1 and LAG3, the T-cell anergic markers. Moreover, the global transcriptome analysis identified that Zbtb10 is pertinent for DC activation and its depletion in cDC1 completely shuts down their immune responses. Mechanistic analysis revealed that Zbtb10 KD enhanced the expression of NKRF (NF-κB repressing factor) leading to drastic suppression of NF-κB related genes. Zbtb10 KD abrogated p65 and RelB nuclear translocation, thereby controlling the activation and maturation of cDC1 and the ensuing adaptive T cell responses.

Global chromatin state analysis reveals lineage-specific enhancers during the initiation of human T helper 1 and T helper 2 cell polarization.

Naive CD4⁺ T cells can differentiate into specific helper and regulatory T cell lineages in order to combat infection and disease. The correct response to cytokines and a controlled balance of these populations is critical for the immune system and the avoidance of autoimmune disorders. To investigate how early cell-fate commitment is regulated, we generated the first human genome-wide maps of histone modifications that reveal enhancer elements after 72 hr of in vitro polarization toward T helper 1 (Th1) and T helper 2 (Th2) cell lineages. Our analysis indicated that even at this very early time point, cell-specific gene regulation and enhancers were at work directing lineage commitment. Further examination of lineage-specific enhancers identified transcription factors (TFs) with known and unknown T cell roles as putative drivers of lineage-specific gene expression. Lastly, an integrative analysis of immunopathogenic-associated SNPs suggests a role for distal regulatory elements in disease etiology.

Toll-like receptor-7 activation in CD8+ T cells modulates inflammatory mediators in patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disorder of unknown etiology with aberrant immunological responses leading to inflammation, swelling and pain of the joints. CD8+ T cells have been known to be one of the major immune modulators in the progression of RA and the presence of toll-like receptors (TLRs) on these cells further accentuate their role in RA. Herein, we report an increased expression of TLR7 in the endosomes of CD8+ T cells of RA patients correlating with disease severity. The stimulation of TLR7 with Imiquimod (IMQ) in these CD8+ T cells drives the signalling cascade via NFkB and pERK activation and hence an increase in the mRNA transcripts of signature cytokines and cytolytic enzymes. However, a parallel synthesis of Tristetraprolin (TTP), an mRNA destabilizing protein prevents the translation of the mRNA transcripts, leading to a rapid degeneration of the target mRNA. We thus report that a direct TLR7 ligation by its agonist increases cytokine transcript signature but not an equivalent protein surge.

NCoR1 fine-tunes type-I IFN response in cDC1 dendritic cells by directly regulating Myd88-IRF7 axis under TLR9.

Plasmacytoid dendritic cells (DCs) are reported to induce robust type-I interferon (IFN) response, whereas cDC1 DCs develop moderate type-I IFN response upon TLR9 stimulation. It is very interesting to understand how this signaling under TLR9 is tightly regulated for the induction of type-I IFNs. Here, we report co-repressor protein NCoR1 as the major factor fine-tuning the signaling pathways regulating IFN-ß expression under TLR9 in cDC1 DCs. We found that NCoR1 knockdown induced a robust IFN-ß-mediated antiviral response upon TLR9 activation in cDC1 DCs. At the molecular level, we showed that NCoR1 directly repressed MyD88-IRF7 signaling axis in cDC1 cells. Therefore, NCoR1 depletion enhanced pIRF7 levels, IFN-ß secretion, and downstream pSTAT1-pSTAT2 signaling, leading to sustained induction of IFN stimulatory genes. Integrative genomic analysis depicted strong enrichment of an antiviral gene-module in CpG-activated NCoR1 knockdown DCs upon TLR9 activation. Moreover, we confirmed our findings in primary DCs derived from splenocytes of WT and NCoR1 DC-/- animals, which showed protection from Sendai and Vesicular Stomatitis viruses upon CpG activation. Ultimately, we identified that NCoR1-HDAC3 complex is involved in repressing the type-I IFN response in cDC1 DCs.

Integrative genomics identifies the corepressor SMRT as a gatekeeper of adipogenesis through the transcription factors C/EBPß and KAISO.

The molecular role of corepressors is poorly understood. Here, we studied the transcriptional function of the corepressor SMRT during terminal adipogenesis. Genome-wide DNA-binding profiling revealed that this corepressor is predominantly located in active chromatin regions and that most distal SMRT binding events are lost after differentiation induction. Promoter-proximal tethering of SMRT in preadipocytes is primarily mediated by KAISO through the conserved TCTCGCGAGA motif. Further characterization revealed that KAISO, similar to SMRT, accelerates the cell cycle and increases fat accumulation upon knockdown, identifying KAISO as an adipogenic repressor that likely modulates the mitotic clonal expansion phase of this process. SMRT-bound promoter-distal sites tend to overlap with C/EBPß-bound regions, which become occupied by proadipogenic transcription factors after SMRT clearance. This reveals a role for SMRT in masking enhancers from proadipogenic factors in preadipocytes. Finally, we identified SMRT as an adipogenic gatekeeper as it directly fine-tunes transcription of pro- and antiadipogenic genes.

Identification and Characterization of Circular Intronic RNAs Derived from Insulin Gene.

Circular RNAs (circRNAs) are a large family of noncoding RNAs that have emerged as novel regulators of gene expression. However, little is known about the function of circRNAs in pancreatic ß-cells. Here, transcriptomic analysis of mice pancreatic islet RNA-sequencing data identified 77 differentially expressed circRNAs between mice fed with a normal diet and a high-fat diet. Surprisingly, multiple circRNAs were derived from the intron 2 of the preproinsulin 2 (Ins2) gene and are termed as circular intronic (ci)-Ins2. The expression of ci-Ins2 transcripts in mouse pancreatic islets, and ßTC6 cells were confirmed by reverse transcription PCR, DNA sequencing, and RNase R treatment experiments. The level of ci-Ins2 was altered in ßTC6 cells upon exposure to elevated levels of palmitate and glucose. Computational analysis predicted the interaction of several RNA-binding proteins with ci-Ins2 and their flanking region, suggesting their role in the ci-Ins2 function or biogenesis. Additionally, bioinformatics analysis predicted the association of several microRNAs with ci-Ins2. Gene ontology and pathway analysis of genes targeted by miRNAs associated with ci-Ins2 suggested the regulation of several key biological processes. Together, our findings indicate that differential expression of circRNAs, especially ci-Ins2 transcripts, may regulate ß-cell function and may play a critical role in the development of diabetes.

Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics.

The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring-based mass spectrometry assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of key TFs, including PPARγ and RXRα, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from ∼250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNA binding model for PPARγ based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPARγ binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPARγ copy number.

TLR4 activation by lipopolysaccharide confers survival advantage to growth factor deprived prostate cancer cells.

BACKGROUND: Prostate cancer (PCa) cells express Toll-like receptor-4 (TLR4), a known pro-tumorigenic molecule for different cancer cells. The cancer cells residing in the avascular region of the tumor confront various metabolic stresses and continuously adapt mechanisms to overcome them. We hypothesized that TLR4 activation might provide direct survival advantage to metabolically stressed PCa cells. METHODS: We first investigated the effect of LPS on survival of serum deprived PCa cells. To understand the molecular mechanisms involved in TLR4 mediated PCa survival, we next investigated change in expression of markers for apoptosis, senescence and autophagy. Ultimately, the effect of LPS on established prostate tumors was confirmed in vivo using a syngeneic rat model for PCa. RESULTS: Lipopolysaccharide (LPS)-mediated TLR4 activation significantly enhanced survival of serum deprived (SD) PC3, DU145 and MAT-LyLu PCa cells. TLR4 inhibition by a specific inhibitor resulted in rapid death of SD-PC3 cells, which was significantly suppressed by LPS. Interestingly, LPS treatment suppressed macroautophagy in SD-PC3 cells and increased expression of CCL2 (C-C motif ligand-2), a known autophagy inhibitor and pro-survival factor. Intra-tumor LPS injection resulted in increased tumor mass, induced TLR4 activation, suppressed autophagy, and increased the macrophage population in MAT-LyLu-tumors. CONCLUSIONS: Our study reveals that bacterial LPS enhance survival of PCa cells under conditions of nutrient stress through TLR4 activation. Moreover, LPS induces overexpression of CCL2 involved in the suppression of starvation-induced macroautophagy in PCa cells, and enhanced macrophage population in prostate tumors in vivo. Taken together, the current study suggests the importance of bacterial infection or TLR4-activation in prostate cancer pathogenesis.

Identification and removal of low-complexity sites in allele-specific analysis of ChIP-seq data.

MOTIVATION: High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. RESULTS: We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays. AVAILABILITY: The R package abs filter for library clonality simulations and detection of amplification-biased sites is available from http://updepla1srv1.epfl.ch/waszaks/absfilter

A yeast one-hybrid and microfluidics-based pipeline to map mammalian gene regulatory networks.

The comprehensive mapping of gene promoters and enhancers has significantly improved our understanding of how the mammalian regulatory genome is organized. An important challenge is to elucidate how these regulatory elements contribute to gene expression by identifying their trans-regulatory inputs. Here, we present the generation of a mouse-specific transcription factor (TF) open-reading frame clone library and its implementation in yeast one-hybrid assays to enable large-scale protein-DNA interaction detection with mouse regulatory elements. Once specific interactions are identified, we then use a microfluidics-based method to validate and precisely map them within the respective DNA sequences. Using well-described regulatory elements as well as orphan enhancers, we show that this cross-platform pipeline characterizes known and uncovers many novel TF-DNA interactions. In addition, we provide evidence that several of these novel interactions are relevant in vivo and aid in elucidating the regulatory architecture of enhancers.

Piperlongumine based nanomedicine impairs glycolytic metabolism in triple negative breast cancer stem cells through modulation of GAPDH & FBP1.

BACKGROUND: Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer and exhibits high rate of chemoresistance, metastasis, and relapse. This can be attributed to the failure of conventional therapeutics to target a sub-population of slow cycling or quiescent cells called as cancer stem cells (CSCs). Therefore, elimination of CSCs is essential for effective TNBC treatment. PURPOSE: Research suggests that breast CSCs exhibit elevated glycolytic metabolism which directly contributes in maintenance of stemness, self-renewability and chemoresistance as well as in tumor progression. Therefore, this study aimed to target rewired metabolism which can serve as Achilles heel for CSCs population and have far reaching effect in TNBC treatment. METHODS: We used two preclinical models, zebrafish and nude mice to evaluate the fate of nanoparticles as well as the therapeutic efficacy of both piperlongumine (PL) and its nanomedicine (PL-NPs). RESULTS: In this context, we explored a phytochemical piperlongumine (PL) which has potent anti-cancer properties but poor pharmacokinetics impedes its clinical translation. So, we developed PLGA based nanomedicine for PL (PL-NPs), and demonstrated that it overcomes the pharmacokinetic limitations of PL, along with imparting advantages of selective tumor targeting through Enhanced Permeability and Retention (EPR) effect in zebrafish xenograft model. Further, we demonstrated that PL-NPs efficiently inhibit glycolysis in CSCs through inhibition of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by modulating glutathione S-transferase pi 1 (GSTP1) and upregulation of fructose-1,6-bisphosphatase 1 (FBP1), a rate-limiting enzyme in gluconeogenesis. We also illustrated that inhibition of glycolysis results in overall tumor regression in two preclinical models. CONCLUSION: This study discusses novel mechanism of action by which PL acts on CSCSs. Taken together our study provides insight into development of PL based nanomedicine which could be exploited in clinics to achieve complete eradication of TNBC by targeting CSCs.

Lactobacillus rhamnosus reduces CD8+T cell mediated inflammation in patients with rheumatoid arthritis.

BACKGROUND: The T cells, components of adaptive immunity participate in immune pathology of the autoimmune inflammatory disorder called rheumatoid arthritis (RA). The presence of TLRs on the surface of the CD8+ T cells and their ability to recognize bacterial moieties adds to the inflammatory burden in case of RA. It has been reported that the gut microbiome is necessary for the crucial shift in the balance between proinflammatory and anti-inflammatory cytokines. The altered gut microbiome and the presence of TLRs emphasizes on the microbiome driven inflammatory responses in case of RA. METHODS: Eighty-nine RA patients participated in this study. Clinical variations like disease duration, number of actively inflamed joints, number and type of bone deformities, CRP, RF, Anti-CCP, ESR, DAS 28 score were recorded for each patient. Co-culture of CD8+T cells and bacteria has been performed with proper culture condition. TLRs and inflammatory mediators' expression level were checked by both qPCR and flow cytometry analysis. RESULTS: We observed in the suppression of pro-inflammatory molecules like Granzyme B and IFNƳ and expression of TLR2 in CD8 + T cells upon treatment with Lactobacillus rhamnosus (L. rhamnosus). Moreover, L. rhamnosus activated CD8+T cells such that they could induce FOXP3 expression in CD4+T cells thereby skewing T cell population towards a regulatory phenotype. On the contrary, TLR4 engagement on CD8+T cell by Escherichia coli (E.coli) increased in inflammatory responses following ERK activation. CONCLUSIONS: Thus, we conclude that L. rhamnosus can effectively suppress CD8+T cell mediated inflammation by a simultaneous decrease of Th1 cells that may potentiate better treatment modalities for RA.

NCoR1 controls immune tolerance in conventional dendritic cells by fine-tuning glycolysis and fatty acid oxidation.

Dendritic cells (DCs) undergo rapid metabolic reprogramming to generate signal-specific immune responses. The fine control of cellular metabolism underlying DC immune tolerance remains elusive. We have recently reported that NCoR1 ablation generates immune-tolerant DCs through enhanced IL-10, IL-27 and SOCS3 expression. In this study, we did comprehensive metabolic profiling of these tolerogenic DCs and identified that they meet their energy requirements through enhanced glycolysis and oxidative phosphorylation (OXPHOS), supported by fatty acid oxidation-driven oxygen consumption. In addition, the reduced pyruvate and glutamine oxidation with a broken TCA cycle maintains the tolerogenic state of the cells. Mechanistically, the AKT-mTOR-HIF-1α-axis mediated glycolysis and CPT1a-driven ß-oxidation were enhanced in these tolerogenic DCs. To confirm these observations, we used synthetic metabolic inhibitors and found that the combined inhibition of HIF-1α and CPT1a using KC7F2 and etomoxir, respectively, compromised the overall transcriptional signature of immunological tolerance including the regulatory cytokines IL-10 and IL-27. Functionally, treatment of tolerogenic DCs with dual KC7F2 and etomoxir treatment perturbed the polarization of co-cultured naïve CD4+ T helper (Th) cells towards Th1 than Tregs, ex vivo and in vivo. Physiologically, the Mycobacterium tuberculosis (Mtb) infection model depicted significantly reduced bacterial burden in BMcDC1 ex vivo and in CD103+ lung DCs in Mtb infected NCoR1DC-/-mice. The spleen of these infected animals also showed increased Th1-mediated responses in the inhibitor-treated group. These findings suggested strong involvement of NCoR1 in immune tolerance. Our validation in primary human monocyte-derived DCs (moDCs) showed diminished NCOR1 expression in dexamethasone-derived tolerogenic moDCs along with suppression of CD4+T cell proliferation and Th1 polarization. Furthermore, the combined KC7F2 and etomoxir treatment rescued the decreased T cell proliferative capacity and the Th1 phenotype. Overall, for the first time, we demonstrated here that NCoR1 mediated control of glycolysis and fatty acid oxidation fine-tunes immune tolerance versus inflammation balance in murine and human DCs.

Long-read 16S-seq reveals nasopharynx microbial dysbiosis and enrichment of Mycobacterium and Mycoplasma in COVID-19 patients: a potential source of co-infection.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major global health concern. This virus infects the upper respiratory tract and causes pneumonia-like symptoms. So far, few studies have shown alterations in nasopharyngeal (NP) microbial diversity, enrichment of opportunistic pathogens and their role in co-infections during respiratory infections. Therefore, we hypothesized that microbial diversity changes, with increase in the population of opportunistic pathogens, during SARS-CoV2 infection in the nasopharynx, which may be involved in co-infection in COVID-19 patients. The 16S rRNA variable regions, V1-V9, of NP samples of control and COVID-19 (symptomatic and asymptomatic) patients were sequenced using the Oxford Nanopore™ technology. Comprehensive bioinformatics analysis for determining alpha/beta diversities, non-metric multidimensional scaling, correlation studies, canonical correspondence analysis, linear discriminate analysis, and dysbiosis index were used to analyze the control and COVID-19-specific NP microbiomes. We observed significant dysbiosis in the COVID-19 NP microbiome with an increase in the abundance of opportunistic pathogens at genus and species levels in asymptomatic/symptomatic patients. The significant abundance of Mycobacteria spp. and Mycoplasma spp. in symptomatic patients suggests their association and role in co-infections in COVID-19 patients. Furthermore, we found strong correlation of enrichment of Mycobacteria and Mycoplasma with the occurrences of chest pain and fever in symptomatic COVID-19 patients. This is the first study from India to show the abundance of Mycobacteria and Mycoplasma opportunistic pathogens in non-hospitalized COVID-19 patients and their relationship with symptoms, indicating the possibility of co-infections.

Increased Extracellular ATP in Plasma of Rheumatoid Arthritis Patients Activates CD8+T Cells.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disorder with genetic and environmental causes often linked with the disease etiology. A disrupted metabolism has often been a characteristic of RA and an altered metabolic state of immune cells has been associated with their phenotypic and functional changes. The energy in the form of ATP produced by the metabolically active cells may thus initiate a cascade of immune responses there by influencing the disease pathogenesis or progression. AIM OF THE STUDY: Through this study we have focused on determining the role of ATP in etiology of RA and aberrant cellular functions. METHODS: Blood samples of 80 healthy controls (HC) and 95 RA patients were screened for extracellular ATP concentration, transcriptome analyses, an inflammatory mediator and the results were statistically analysed. RESULTS: In this study, ATP is shown to be excessive in the plasma of RA patients (453.5 ± 16.09% in RA vs. 233.9 ± 10.07% in HC, p <0.0001) and significantly increases with the disease severity. The abundant extracellular ATP could activate circulating cytotoxic CD8+T cells in RA patients to produce Granzyme B. CONCLUSION: Plasma ATP is thus identified to have a significant potential in progression and prognosis of RA and may thus be studied further to design better therapeutic approaches for the disease.