Objectives Reactive arthritis (ReA) provides a unique opportunity to comprehend how a mucosal infection leads to inflammatory arthritis at a distant site without the apparent invasion of the pathogen. Unfortunately, conventional stool cultures after ReA provide limited information, and there is a dearth of metagenomic studies in ReA. The objective of this study was to identify gut microbiota associated with the development of ReA.Methods Patients with ReA or undifferentiated peripheral spondyloarthritis (UpSpA) were included if they presented within 4 weeks of the onset of the current episode of arthritis. Metagenomic DNA was extracted from the stools of these patients and of 36 age- and sex-similar controls. Sequencing and analysis were done using a standard 16S ribosomal pipeline.Results Of 55 patients, there was no difference between the gut microbiota of postdiarrheal ReA (n = 20) and of upSpA (n = 35). Comparing the gut microbiota of patients vs healthy controls, the patients had significantly higher alpha and beta diversity measures. After stringency filters, Proteobacteria had high abundance while Firmicutes had lesser as compared with the controls. Six families were overexpressed in patients, while another five were overexpressed in controls. Sixteen genera and 18 species were significantly different between patients and controls. At the species level there was strong association of Staphylococcus aureus, Clostridium septicum Klebsiella pneumoniae, Escherichia coli, Empedobacter brevis, Roseburia hominis, Bacillus velezensis and Crassaminicella with ReA.Conclusion The microbiota of classical gut-associated ReA and upSpA is similar. Patients have higher diversities in their gut microbiota compared with healthy controls. Both known and previously unreported species associated with ReA/upSpA were identified.

In vitro cell line model systems are essential in supporting the research community due to their low cost, uniform culturing conditions, homogeneous biological resources, and easy experimental design to study the cause and effect of a gene or a molecule. Human leukemia 60 (HL60) is an in-vitro hematopoietic model system that has been used for decades to study normal myeloid differentiation and leukemia biology. Here, we show that IMDM supplemented with 20% FBS is an optimal culturing condition and induces effective myeloid differentiation compared with RPMI supplemented with 10% FBS when HL60 is induced with 1α,25-dihydroxyvitamin D3 (Vit D3) and all-trans retinoic acid (ATRA). The chromatin organization is compacted, and the repressive epigenetic mark H3K27me3 is enhanced upon HL60-mediated terminal differentiation. Differential gene expression analysis obtained from RNA sequencing in HL60 cells during myeloid differentiation showed the induction of pathways involved in epigenetic regulation, myeloid differentiation, and immune regulation. Using high-throughput transcriptomic data (GSE74246), we show the similarities (genes that did not satisfy |log2FC|>1 and FDR<0.05) and differences (FDR <0.05 and |log2FC|>1) between granulocyte-monocyte progenitor vs HL60 cells, Vit D3 induced monocytes (vMono) in HL60 cells vs primary monocytes (pMono), and HL60 cells vs leukemic blasts at the transcriptomic level. We found striking similarities in biological pathways between these comparisons, suggesting that the HL60 model system can be effectively used for studying myeloid differentiation and leukemic aberrations. The differences obtained could be attributed to the fact that the cellular programs of the leukemic cell line and primary cells are different. We validated several gene expression patterns for different comparisons with CD34+ cells derived from cord blood for myeloid differentiation and AML patients. In addition to the current knowledge, our study further reveals the significance of using HL60 cells as in vitro model system under optimal conditions to understand its potential as normal myeloid differentiation model as well as leukemic model at the molecular level.

MIS-C is a rare, highly inflammatory state resembling incomplete Kawasaki disease, temporarily associated with COVID-19. The pathogenesis is not completely known. RNAseq was carried out on whole blood of six treatment-naïve MIS-C patients. This was compared against RNAseq transcriptomics data of five healthy controls (HC), four Kawasaki Disease (KD) and seven systemic Juvenile Idiopathic Arthritis (sJIA). Using PCA, MIS-C clustered separately from HC, KD and sJIA. Amongst the top 50 significant genes in the three comparisons with HC, KD, and sJIA, common genes were: TMCC2, ITGA2B, DMTN, GFI1B, PF4, QSER1, GRAP2, TUBB1. DSEA revealed that maximum number of hits for overexpressed pathways was for NABA matrisome activation when MIS-C was compared against HC. Cytokine stimulated cellular activation pathways, specifically IL-10 were downregulated. MIS-C had more activated pathways of neutrophil degranulation and acquired immune activation but less of coagulation system or heat-shock system involvement as compared to KD. As compared to sJIA, humoral immune response and complements were activated. Matrisome activation was higher, with increased cell–cell interaction and ECM signalling. This analysis revealed novel insights into the pathogenesis of MIS-C, including the potential role of matrisomes, humoral immune system and down-regulated interleukin-10 pathways.

Type I interferon (IFN-I) response is the first line of host defense against invading viruses. In the absence of definite mouse models, the role of IFN-I in SARS-CoV-2 infection remains perplexing. Here, we develop two mouse models, one with constitutively high IFN-I response (hACE2; Irgm1-/-) and the other with dampened IFN-I response (hACE2; Ifnar1-/-), to comprehend the role of IFN-I response. We report that hACE2; Irgm1-/- mice are resistant to lethal SARS-CoV-2 infection. In contrast, a severe SARS-CoV-2 infection along with immune cell infiltration, cytokine storm, and enhanced pathology is observed in the lungs and brain of hACE2; Ifnar1-/- mice. The hACE2; Irgm1-/-Ifnar1-/- double-knockout mice display loss of the protective phenotype observed in hACE2; Irgm1-/- mice, suggesting that heightened IFN-I response accounts for the observed immunity. Taking the results together, we demonstrate that IFN-I protects from lethal SARS-CoV-2 infection, and Irgm1 (IRGM) could be an excellent therapeutic target against SARS-CoV-2.

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.

Aberrations in transcription and epigenetic factors lead to neoplastic transformation such as acute myeloid leukemia (AML), which is characterized by accumulation of hyperproliferative blasts originating from leukemic stem cells. The use of therapeutic agents designed to lift the differentiation block and reinforce terminal cellular differentiation and growth arrest is 1 way to manage AML pathophysiology. Therefore, understanding these critical regulatory switches is essential for designing selective and effective drug targets for AML. The ATP-dependent SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complex has been implicated in 20% cancers, including AML.1 The catalytic subunit, SMARCA4, drives leukemogenesis by facilitating constitutive Myc expression via enhancer remodeling.2,3 The loss of auxiliary subunits such as ACTL6A leads to proliferation defects in stem cells and bone marrow failure, whereas defects in SMARCD2 affect neutrophil development.4-6 This prompted us to investigate the differential expression of SWI/SNF complex subunits across different stages of blood cell development. We mined several sequencing datasets for the expression of 21 subunits of SWI/SNF complex in various hematopoietic cells and found several of them to be differentially expressed in a cell type-specific manner (supplemental Figure 1). Interestingly, we found that the SMARCD isoforms showed distinct and opposing cell type-specific enrichment. SMARCD1 and SMARCD2 expression was high in CD34+ hematopoietic stem/progenitors (HSPCs), whereas SMARCD3 was specifically enriched in monocytes (Figure 1A; supplemental Figures 1 and 2). The role of SMARCD1 in myeloid differentiation and leukemia has not been investigated thus far, and hence, we validated the above findings using both ex vivo and in vitro hematopoietic models. SMARCD1 expression was high in cord blood-derived CD34+ HSPCs and was significantly reduced in macrophage colony stimulating factor-differentiated HSPCs (Figure 1B). Similarly, SMARCD1 expression was reduced in vitamin D3-differentiated HL-60 cells (Figure 1C). Interestingly, we observed concomitant increase in SMARCD3 expression in differentiated cord blood and promyelocytic human leukemia-60 (HL60) cells, indicating interplay of SMARCD isoforms in hematopoietic differentiation (Figure 1B-C). Next, we investigated the expression of SMARCD1 in patients with AML. Compared with the CD342 compartment, similar enrichment profiles of SMARCD1, SMARD2, and SMARCD3 were observed in the CD34+ AML HSPCs (Figure 1D). The French-American-British (FAB) system classifies AML based on their maturity, and as SMARCD1 expression is enriched in the HSPCs, we assessed its expression across subtypes. Interestingly, we found higher expression of SMARCD1 in undifferentiated AML (M0, M1, M2 subtypes) than in the more differentiated AML FAB subtypes (M3, M4, M5; Figure 1E). These observations indicate a strong correlation between SMARCD1 expression and undifferentiated cell state (both normal and leukemic). Furthermore, the leukemic cell lines show significant dependency on SMARCD1, highlighting its potential role in leukemic cells (supplemental Figure 2G-I). The preferential expression of SMARCD1 in normal and leukemic stem/progenitor cells is attributed to a high promoter accessibility of SMARCD1 in those cell types (supplemental Figure 2E-F).

Treatment of Sprague Dawley rats with 3-methoxy-4-aminoazobenzene (3-MeO-AAB) resulted in striking increase of the activity of hepatic microsomal cytochrome P-450s which could efficiently catalyze the mutagenic activation of hepatocarcinogenic aromatic amines such as a tryptophan-pyrolysate component, Trp P-2, and a glutamic acid-pyrolysate component, Glu P-1. The 3-MeO-AAB-induced cytochrome P-450 (3-MeO-AAB-P-450) was examined for the molecular character by immuno-Western blotting using monoclonal antibody to 3-methylcholanthrene-induced cytochrome P-448 (P-448H; m.w. 54,000).

The presence of activated pancreatic stellate cells (PSCs) in the pancreatic ductal adenocarcinoma (PDAC) microenvironment plays a significant role in cancer progression. Macrophage migration inhibitory factor (MIF) is overexpressed in PDAC tissues and expressed by both cancer and stromal cells. The pathophysiological role of MIF in PDAC‐associated fibroblasts or PSCs is yet to be elucidated. Here we report that the PSCs of mouse or cancer‐associated fibroblast cells (CAFs) of human expresses MIF and its receptors, whose expression gets upregulated upon LPS or TNF‐α stimulation. In vitro functional experiments showed that MIF significantly conferred a survival advantage to CAFs/PSCs upon growth factor deprivation. Genetic or pharmacological inhibition of MIF also corroborated these findings. Further, co‐injection of mouse pancreatic cancer cells with PSCs isolated from Mif−/− or Mif+/+ mice confirmed the pro‐survival effect of MIF in PSCs and also demonstrated the pro‐tumorigenic role of MIF expressed by CAFs in vivo. Differential gene expression analysis and in vitro mechanistic studies indicated that MIF expressed by activated CAFs/PSCs confers a survival advantage to these cells by suppression of interferon pathway induced p53 dependent apoptosis.

Dynamic regulation of phosphorylation and dephosphorylation of histones is essential for eukaryotic transcription, but the enzymes engaged in histone dephosphorylation are not fully explored. Here, we show that the tyrosine phosphatase SHP‐1 dephosphorylates histone H2B and plays a critical role during transition from the initiation to the elongation stage of transcription. Nuclear‐localized SHP‐1 is associated with the Paf1 complex at chromatin and dephosphorylates H2B at tyrosine 121. Moreover, knockout of SHP‐1, or expression of a mutant mimicking constitutive phosphorylation of H2B Y121, leads to a reduction in genome‐wide H2B ubiquitination, which subsequently causes defects in RNA polymerase II‐dependent transcription. Mechanistically, we demonstrate that Y121 phosphorylation precludes H2B's interaction with the E2 enzyme, indicating that SHP‐1‐mediated dephosphorylation of this residue may be a prerequisite for efficient H2B ubiquitination. Functionally, we find that SHP‐1‐mediated H2B dephosphorylation contributes to maintaining basal autophagic flux in cells through the efficient transcription of autophagy and lysosomal genes. Collectively, our study reveals an important modification of histone H2B regulated by SHP‐1 that has a role during eukaryotic transcription.

Type 1 interferon (IFN-I) response is the first line of host defense against invading viruses. In the absence of definite mouse models, the role of IFN-I in SARS-CoV-2 infections remained to be perplexing. Here, we developed two mouse models, one with constitutively high IFN-I response (hACE2; Irgm1 −/− ) and the other with dampened IFN-I response (hACE2; Ifnar1 −/− ) to comprehend the role of IFN-I response during SARS-CoV-2 invasion. We found that hACE2; Irgm1 −/− mice were resistant to lethal SARS-CoV-2 infection with substantially reduced cytokine storm and immunopathology. In striking contrast, a severe SARS-CoV-2 infection along with immune cells infiltration, inflammatory response, and enhanced pathology was observed in the lungs of hACE2; Ifnar1 −/− mice. Additionally, hACE2; Ifnar1 −/− mice were highly susceptible to SARS-CoV-2 neuroinvasion in the brain accompanied by immune cell infiltration, microglia/astrocytes activation, cytokine response, and demyelination of neurons. The hACE2; Irgm1 −/− Ifnar1 −/− double knockout mice or hACE2; Irgm1 −/− mice treated with STING or RIPK2 pharmacological inhibitors displayed loss of the protective phenotypes observed in hACE2; Irgm1 −/− mice suggesting that heightened IFN-I response accounts for the observed immunity. Taken together, we explicitly demonstrate that IFN-I protects from lethal SARS-CoV-2 infection, and Irgm1 (IRGM) could be an excellent therapeutic target. GRAPHICAL ABSTRACT

BACKGROUND:Chemoresistance is one of the major factors for treatment failure in OSCC. Identifying key resistance triggering molecules will be useful strategy for developing novel treatment methods.METHODS:To identify the causative factors of chemoresistance, we performed RNA sequencing and global proteomic profiling of human OSCC lines presenting with sensitive, early and late cisplatin-resistance patterns.RESULTS:From the common set of dysregulated genes from both the analysis, RRBP1 was identified to be upregulated in both early and late cisplatin-resistant cells with respect to the sensitive counterpart. Analysis of OSCC patient sample indicates that RRBP1 expression is upregulated in chemotherapy-non-responder tumours as compared to chemotherapy-responder tumours. Genetic (knockout) or pharmacological (Radezolid, represses expression of RRBP1) inhibition of RRBP1 restores cisplatin-mediated cell death in chemo-resistant OSCC. Mechanistically, RRBP1 regulates Yes-associated protein1 (YAP1), a key protein in the Hippo pathway to induce chemoresistance. The PDC xenograft data suggests that knockout of RRBP1 induces cisplatin-mediated cell death and facilitates a significant reduction of tumour burden.CONCLUSION:Overall, our data suggests that (I) RRBP1 is a major driver of cisplatin-resistance in OSCC, (II) RRBP1 regulates YAP1 expression to mediate cisplatin-resistance, (III) Radezolid represses RRBP1 expression and (IV) targeting RRBP1 reverses cisplatin-induced chemoresistance in advanced OSCC.

AbstractThe coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome corona virus 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 that respiratory infections alter nasopharyngeal (NP) microbiome diversity and enrich opportunistic pathogens. In this study, we have sequenced the 16S rRNA variable regions, V1 through V9, extracted from NP samples of control and COVID-19 (symptomatic and asymptomatic) participants using the Oxford Nanopore™ technology. Comprehensive bioinformatics analysis investigating the alpha/beta diversities, non-metric multidimensional scaling, correlation studies, canonical correspondence analysis, linear discriminate analysis, and dysbiosis index analysis revealed control and COVID-19-specific NP microbiomes. We observed significant dysbiosis in COVID-19 NP microbiome with abundance of opportunistic pathogens such asCutibacterium, Corynebacterium, Oerskovia, andCellulomonasin asymptomatic patients, and ofStreptomycesandMycobacteriaceaefamily in symptomatic patients. Furthermore, we observed sharp rise in enrichment of opportunistic pathogens in symptomatic patients, with abundance ofMycobacteriaandMycoplasma, which strongly correlated with the occurrences of chest pain and fever. Our findings contribute novel insights regarding emergence of opportunistic pathogens in COVID-19 patients and their relationship with symptoms, suggesting their potential role in coinfections.

The type I interferon (IFN) response is the major host arsenal against invading viruses. IRGM is a negative regulator of IFN responses under basal conditions. However, the role of human IRGM during viral infection has remained unclear. In this study, we show that IRGM expression is increased upon viral infection. IFN responses induced by viral PAMPs are negatively regulated by IRGM. Conversely, IRGM depletion results in a robust induction of key viral restriction factors including IFITMs, APOBECs, SAMHD1, tetherin, viperin, and HERC5/6. Additionally, antiviral processes such as MHC-I antigen presentation and stress granule signaling are enhanced in IRGM-deficient cells, indicating a robust cell-intrinsic antiviral immune state. Consistently, IRGM-depleted cells are resistant to the infection with seven viruses from five different families, including Togaviridae, Herpesviridae, Flaviviverdae, Rhabdoviridae, and Coronaviridae. Moreover, we show that Irgm1 knockout mice are highly resistant to chikungunya virus (CHIKV) infection. Altogether, our work highlights IRGM as a broad therapeutic target to promote defense against a large number of human viruses, including SARS-CoV-2, CHIKV, and Zika virus.

Activation of the type 1 interferon response is extensively connected to the pathogenesis of autoimmune diseases. Loss of function of Immunity Related GTPase M (IRGM) has also been associated to several autoimmune diseases, but its mechanism of action is unknown. Here, we found that IRGM is a master negative regulator of the interferon response. Several nucleic acid-sensing pathways leading to interferon-stimulated gene expression are highly activated in IRGM knockout mice and human cells. Mechanistically, we show that IRGM interacts with nucleic acid sensor proteins, including cGAS and RIG-I, and mediates their p62-dependent autophagic degradation to restrain interferon signaling. Further, IRGM deficiency results in defective mitophagy leading to the accumulation of defunct leaky mitochondria that release cytosolic DAMPs and mtROS. Hence, IRGM deficiency increases not only the levels of the sensors, but also those of the stimuli that trigger the activation of the cGAS-STING and RIG-I-MAVS signaling axes, leading to robust induction of IFN responses. Taken together, this study defines the molecular mechanisms by which IRGM maintains interferon homeostasis and protects from autoimmune diseases.

A novel disease, COVID-19, is sweeping the world since end of 2019. While in many countries, the first wave is over, but the pandemic is going through its next phase with a significantly higher infectability. COVID-19 is caused by the novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that seems to be more infectious than any other previous human coronaviruses. To understand any unique traits of the virus that facilitate its entry into the host, we compared the published structures of the viral spike protein of SARS-CoV-2 with other known coronaviruses to determine the possible evolutionary pathway leading to the higher infectivity. The current report presents unique information regarding the amino acid residues that were a) conserved to maintain the binding with ACE2 (Angiotensin-converting enzyme 2), and b) substituted to confer an enhanced binding affinity and conformational flexibility to the SARS-CoV-2 spike protein. The present study provides novel insights into the evolutionary nature and molecular basis of higher infectability and perhaps the virulence of SARS-CoV-2.

Acute myeloid leukemia (AML) is a common and aggressive hematological malignancy. Acquisition of heterogeneous genetic aberrations and epigenetic dysregulation lead to the transformation of hematopoietic stem cells (HSC) into leukemic stem cells (LSC), which subsequently gives rise to immature blast cells and a leukemic phenotype. LSCs are responsible for disease relapse as current chemotherapeutic regimens are not able to completely eradicate these cellular sub-populations. Therefore, it is critical to improve upon the existing knowledge of LSC specific markers, which would allow for specific targeting of these cells more effectively allowing for their sustained eradication from the cellular milieu. Although significant milestones in decoding the aberrant transcriptional network of various cancers, including leukemia, have been achieved, studies on the involvement of post-transcriptional gene regulation (PTGR) in disease progression are beginning to unfold. RNA binding proteins (RBPs) are key players in mediating PTGR and they regulate the intracellular fate of individual transcripts, from their biogenesis to RNA metabolism, via interactions with RNA binding domains (RBDs). in this study, we have used an integrative approach to systematically profile RBP expression and identify key regulatory RBPs involved in normal myeloid development and AML. We have analyzed RNA-seq datasets (GSE74246) of HSCs, common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), monocytes, LSCs, and blasts. We observed that normal and leukemic cells can be distinguished on the basis of RBP expression, which is indicative of their ability to define cellular identity, similar to transcription factors. We identified that distinctly co-expressing modules of RBPs and their subclasses were enriched in hematopoietic stem/progenitor (HSPCs) and differentiated monocytes. We detected expression of DZIP3, an E3 ubiquitin ligase, in HSPCs, knockdown of which promotes monocytic differentiation in cell line model. We identified co-expression modules of RBP genes in LSCs and among these, distinct modules of RBP genes with high and low expression. The expression of several AML-specific RBPs were also validated by quantitative polymerase chain reaction. Network analysis identified densely connected hubs of ribosomal RBP genes (rRBPs) with low expression in LSCs, suggesting the dependency of LSCs on altered ribosome dynamics. In conclusion, our systematic analysis elucidates the RBP transcriptomic landscape in normal and malignant myelopoiesis, and highlights the functional consequences that may result from perturbation of RBP gene expression in these cellular landscapes.

Activation of type 1 interferon response is extensively connected with the antiviral immunity and pathogenesis of autoimmune diseases. Here, we found that IRGM, whose deficiency is linked with the genesis of several autoimmune disorders, is a master negative regulator of the interferon response. Mechanistically, we show that IRGM interacts with nucleic acid sensor proteins, including cGAS and RIG-I, and mediates their autophagic degradation to restrain activation of interferon signaling. Further, IRGM maintains mitophagy flux, and its deficiency results in the accumulation of defunct leaky mitochondria that releases cytosolic DAMPs triggering activation of interferon responses via cGAS-STING and RIG-I-MAVS signaling axis. Due to an enduring type 1 IFN response in IRGM-deficient cells and mice, they were intrinsically resistant to infection of the Japanese Encephalitis virus, Herpes Simplex virus, and Chikungunya virus. Altogether, this study defines the molecular mechanisms by which IRGM maintains interferon homeostasis and protects from autoimmune diseases. Further, it identifies IRGM as a broad therapeutic target for defense against viruses.

Antimicrobial peptides (AMPs) have the potential to serve as an alternative to antibiotic. AMPs usually exert bactericidal activity via direct killing of microbial pathogens. Reports have proposed that by harnessing innate immune activation, AMPs can regulate pathogen invasion and may control infection. It has been reported that AMPs could be utilized to activate the innate mucosal immune response in order to eliminate pathogenic infections. This way of controlling pathogen infection, by activating host immunity, confers the potential to the select AMPs to alleviate the problem of antibiotic resistance. Among various AMPs tested LL-37 and indolicidin, showed promise to be potential candidates for eliciting enhanced host innate immune responses. LL-37 and indolicidin had exhibited substantial innate immune activation in both human and murine macrophages. Dosage for each of the AMPs, however, was high with adverse side effects.