Cytosolic DNA-sensing Pathway Research Articles

Oxct1 regulates cardiomyocyte PANoptosis in acute myocardial infarction via succinylating Tfam

Abstract Background Myocardial ischemia leads to various forms of programmed cell death in cardiomyocytes, resulting in acute myocardial infarction (AMI) and the development of heart failure in the long term. PANoptosis is a unique cell death regulatory mechanism orchestrated by the PANoptosome complex, which regulates and intersects multiple forms of programmed cell death. Oxct1 has been demonstrated to play crucial roles in tumors, pressure-overloaded hearts, and cardiac endothelial cells. However, whether Oxct1 is involved in the regulation of PANoptosis and correlated mechanisms during AMI remains unclear. Purpose To elucidate the role of Oxct1 in cardiomyocytes during AMI and uncover the potential mechanisms. Methods The myocardial infarction model induced by anterior descending branch ligation and the primary cardiomyocytes hypoxia model were employed in our experiments. Small interfering RNA (siRNA) and adenovirus were constructed to knock down and overexpress Oxct1 for altering its expression in cardiomyocytes in vitro. Cardiomyocyte-specific knockout mice were constructed to alter Oxct1 expression in vivo. PANoptosis characteristic measurement, including Z-DNA binding protein 1(Zbp1), pyroptosis markers, apoptosis markers, necroptosis markers, ROS, and mitochondrial morphology, were performed to verify the ability of Oxct1 to regulate the PANoptosis of cardiomyocytes. Cardiac morphological, structural, and functional changes were measured to assess the effect of Oxct1 on AMI. RNA sequencing was performed to identify downstream pathways. Immunoprecipitation +Mass spectrometry and co-immunoprecipitation were performed to identify the specific binding factors of Oxct1. Results We found that the expression of Oxct1 was significantly elevated after AMI. Besides, gain- and loss-of-function experiments were constructed both in vitro and in vivo. Functionally, Oxct1 knockdown significantly enhanced cardiomyocyte vitality, reduced ROS production and mtDNA cytoplasmic release in hypoxic cardiomyocytes. In vivo, Oxct1cko mice manifested reduced infarct size, improved cardiac function and alleviated cardiac fibrosis by mitigating PANoptosis. However, Oxct1 overexpression resulted in the opposite effects. Afterwards, RNA-seq analysis results showed that cytosolic DNA-sensing pathway was significantly enriched in Oxct1 knockdown cardiomyocytes. Mechanistically, Oxct1 could bind to mitochondrial transcription factor A(Tfam) and increased its succinylation, promoting Tfam degradation. Downregulation of Tfam resulted in ROS production and mtDNA damage, while mtDNA released into cytoplasm could activate Zbp1 and induce cascade reaction of PANoptosis. Conclusion This study demonstrates that Oxct1 promotes Tfam degradation through succinylation, exacerbating ROS production, mtDNA damage and cytoplasmic release, ultimately leading to cardiomyocyte PANoptosis in AMI.

Unexpected macrophage-mediated myocardial cGas-STING activation in a novel mouse model of stress-related sleep disturbance

Abstract Background Delayed bedtime following stress disorder is prevalent in waves of pandemics and modern life. Considered to be a specific and important contributor to cardiovascular health, stress-related sleep disturbance has an unmet need in steady preclinical models. We previously found exciting corticotropin-releasing hormone (CRH) neurons in the paraventricular nucleus of the hypothalamus (PVH) area of mice could induce 3-hour-long wakefulness. Purpose This study aimed to induce reproducible phenotypes of stress-related sleep disturbance in mice and explore the potential impact on cardiac function. Methods The chemogenetic method of designer receptors exclusively activated by designer drugs (DREADD) system was adopted to mimic stress-related sleep disturbance. We transfected PVH CRH neurons with rAAV-hSyn-DIO-hM3Dq-mCherry and rAAV-Crh-CRE. Prolonged CRH neuron activation was induced by daily intraperitoneal injection of clozapine N-oxide (CNO, 3mg/kg) at 9 am. Bulk RNA-sequencing and bioinformatics analysis were conducted for mechanistic exploration. Results 2-week repeated chemogenetic activation of PVH CRH neurons induced a 5-fold corticosterone release, consistent with increased daily 3-hour wakefulness and corresponding decreases in both rapid eye movement (REM) as well as non-REM sleep. Over 30% of chronic CRH activation mice displayed difficulties in maintaining balance and experienced premature mortality. Mice subjected to prolonged CRH activation showed impaired left ventricular ejection fraction (67.9% versus 48.2%, p=0.0011), and a dilated appearance demonstrated by histological staining. Intriguingly, the number of circulating monocytes increased. Then, we performed bulk RNA-sequencing of heart and bone marrow from CRH-activated and control mice. Differential gene expression and gene set enrichment analysis (GSEA) indicated marked activation of interferon-beta-related pathways in both tissues. Cytosolic DNA-sensing pathway and related key effector genes (cGAS, Cxcl10, CCL5) were found up-regulated in the heart, while mitochondrial oxidative phosphorylation was suppressed. We adopted the CIBERSORT tool to estimate immune infiltration in heart tissues and characterized M1 macrophage as the main pro-inflammatory cell. Further, we screened a set of secreted factors and mediators, which might play active roles in inflamed hearts. Conclusion Taken together, we report a failing heart in a mouse model of stress-related sleep disturbance. The neuro-immune axis involvement and molecular mechanisms merit in-depth explorations.

Nanopore-based full-length transcriptome sequencing of the skin in Pseudopleuronectes yokohamae identifies novel antimicrobial peptide genes

The marbled flounder (Pseudopleuronectes yokohamae) is highly esteemed for its exceptional nutritional value and delicious taste. However, this species has extremely limited transcriptome data, which can offer priceless information for disease protection. In the study, the skin transcriptomic sequencing of P. yokohamae revealed 7.72 GB of clean data using the Nanopore sequencing platform. The results revealed 30,498 transcripts of functional annotations in the P. yokohamae transcriptome. All transcripts were searched in eight functional databases. A total of 10,337 ORFs were obtained, of which 6081 complete ORFs accounted for 58.83% of all predicted CDS. Moreover, 10,195 SSRs were detected. Meanwhile, the non-pecific immunity pathways were investigated for better understanding of the immunological reaction in P. yokohamae, and seven innate immune pathways were identified. The innate-immune related genes were highly expressed in the NOD-like receptor signaling pathway, followed by the C-type lectin receptor signaling pathway, Toll-like receptor signaling pathway, RIG-I-like receptor signaling pathway and Cytosolic DNA-sensing pathway. In this study, four families of antimicrobial peptides (AMPs) in P. yokohamae were analysed for the first time, including piscidins, hepcidins, liver-expressed antimicrobial peptide and defensins. Seven AMPs, including Pypleurocidin-like WF3, Pypleurocidin-like WFX, Pyhepcidin 1, Pyhepcidin-like 1, PyLEAP-2, Pybeta-defensin and Pybeta-defensin-like 1, were further identified. The seven AMPs showed a highly identity in their cDNA and genomic structures and an inducible expression pattern preferable to skin in response to pathogens. The transcriptomic data and investigation of AMPs from P. yokohamae promote a deeper awareness of fish mucosal immunity and provide information in the prevention of fish diseases.

Causal relationship between Interleukin-27 expression levels and osteoporosis: a bidirectional mendelian randomization study

BackgroundThis study aimed to evaluate the causal relationship between Interleukin-27 (IL-27) and osteoporosis by bidirectional Mendelian randomization (MR) analysis.MethodsFirstly, the genome-wide association study summary data of osteoporosis (finn-b-M13_OSTEOPOROSIS) and IL-27 levels (ebi-a-GCST90012017) were picked out from the Integrative Epidemiology Unit (IEU) OpenGWAS database. After filtrating instrumental variables (IVs), the bidirectional MR analysis between IL-27 levels and osteoporosis was performed by MR-Egger, Weighted median, Simple mode, Weighted mode, and Inverse variance weighted (IVW). Subsequently, the sensitivity analysis was adopted to evaluate the reliability of the MR results via the Heterogeneity, Horizontal pleiotropy test and Leave-One-Out (LOO) analysis. Finally, the enrichment analysis of genes corresponding to SNPs related to IL-27 levels derived from eQTLGen database was executed to explore in depth the biological function and regulatory mechanism of these genes on osteoporosis occurrence.ResultsThe bidirectional MR results based on IVW method revealed that IL-27 level as a risk factor was causally related to osteoporosis (P = 0.004, odds ratio (OR) = 1.123, 95% confidence interval (CI) = 1.037–1.217), whereas osteoporosis was not in significant connection with IL-27 levels (P > 0.05). In regard to the sensitivity analysis for forward MR results, there was no heterogeneity and horizontal pleiotropy, and no SNPs relevant to IL-27 levels existed severe bias, suggesting the reliability of forward MR analysis. Furthermore, a total of 74 genes corresponding to 26 SNPs of IL-27 levels were obtained and were mainly involved in immune and inflammatory pathways including MyD88-dependent toll-like receptor signaling pathway, Toll-like receptor signaling pathway, cytosolic DNA-sensing pathway and so forth.ConclusionsThis study supported that IL-27 level as a risk factor was causally connected with osteoporosis and might regulate the disease occurrence and progression by means of immune and inflammatory mechanisms, which could provide important reference and evidence for further exploring the role of IL-27 in the development of osteoporosis.

Circulating Cell-Free DNA Promotes Inflammation in Patients with Dermatomyositis with Anti-NXP2 Antibodies via the cGAS/STING Pathway.

Dermatomyositis (DM) is a rare type I interferon (IFN-I)-driven autoimmune disease, and anti-nuclear matrix protein 2 (NXP2) antibody is related to severe muscle disease and poor prognosis. Circulating cell-free DNA (ccf-DNA), including ccf-mitochondrial DNA and ccf-nuclear DNA, activates cGAS/STING pathway to induce IFN-I production in autoimmune diseases. We investigated whether serum-derived ccf-DNA played a pathogenic role on skeletal muscle in anti-NXP2 antibody-positive DM. Serum ccf-DNA levels were measured, and correlations between ccf-DNA and clinicopathological indicators were performed. RNA sequencing, immunofluorescence, western blotting and RT-qPCR were performed on skeletal muscle samples. The serum-induced expression of p-STING in C2C12 cells was assessed in vitro. We found that increased ccf-DNA levels were positively correlated with MYOACT scores in anti-NXP2 antibody-positive DM. RNA sequencing and immunofluorescence results revealed that the cytosolic DNA-sensing pathway was upregulated and that increased cytosolic dsDNA was colocalised with cGAS in skeletal muscle in anti-NXP2 antibody-positive DM. Western blot analysis revealed activation of the cGAS/STING pathway in patients with perifascicular atrophy (PFA) but not in patients without PFA. RT-qPCR showed increased IFN-I scores in both patients with PFA and patients without PFA. Sera from patients with PFA increased p-STING expression in C2C12 cells, and DNase I treatment and STING inhibitor efficiently inhibited p-STING expression, respectively. Increased ccf-DNA levels may be potential biomarkers for monitoring disease activity in anti-NXP2 antibody-positive DM. Activation of the cGAS/STING pathway is associated with PFA. Our findings identify the pathogenic role of ccf-DNA on skeletal muscle via the cGAS/STING pathway.

TTK inhibitor OSU13 promotes immunotherapy responses by activating tumor STING.

TTK spindle assembly checkpoint kinase is an emerging cancer target. This preclinical study explored the antitumor mechanism of TTK inhibitor OSU13 to define a strategy for clinical development. We observed prominent antitumor activity of OSU13 in melanoma, colon and breast cancer cells, organoids derived from patients with melanoma, and mice bearing colon tumors associated with G2 cell cycle arrest, senescence, and apoptosis. OSU13-treated cells displayed DNA damage and micronuclei that triggered the cytosolic DNA-sensing cGAS/STING pathway. STING was required for the induction of several proteins involved in T cell recruitment and activity. Tumors from OSU13-treated mice showed an increased proportion of T and NK cells and evidence of PD-1/PD-L1 immune checkpoint activation. Combining a low-toxicity dose of OSU13 with anti-PD-1 checkpoint blockade resulted in prominent STING- and CD8+ T cell-dependent tumor inhibition and improved survival. These findings provide a rationale for utilizing TTK inhibitors in combination with immunotherapy in STING-proficient tumors.

Micro-and nano-plastics induce kidney damage and suppression of innate immune function in zebrafish (Danio rerio) larvae

Aquatic environments serve as critical repositories for pollutants and have significantly accumulated micro- and nanoplastics (MNPs) due to the extensive production and application of plastic products. While the disease resistance and immunity of fish are closely linked to the condition of their aquatic habitats, the specific effects of nanoplastics (NPs) and microplastics (MPs) within these environments on fish immune functions are still not fully understood. The present study utilized zebrafish (Danio rerio) embryos and larvae as model organisms to examine the impacts of polystyrene NPs (100 nm) and MPs (5 μm) on fish immune responses. Our findings reveal that NPs and MPs tend to accumulate on the surfaces of embryos and within the intestines of larvae, triggering oxidative stress and significantly increasing susceptibility to Edwardsiella piscicida infection in zebrafish larvae. Transmission electron microscopy examined that both NPs and MPs inflicted damage to the kidney, an essential immune organ, with NPs predominantly inducing endoplasmic reticulum stress and MPs causing lipid accumulation. Transcriptomic analysis further demonstrated that both NPs and MPs significantly suppress the expression of key innate immune pathways, notably the C-type lectin receptor signaling pathway and the cytosolic DNA-sensing pathway. Within these pathways, the immune factor interleukin-1 beta (il1b) was consistently downregulated in both exposure groups. Furthermore, exposure to E. piscicida resulted in restricted upregulation of il1b mRNA and protein levels, likely contributing to diminished disease resistance in zebrafish larvae exposed to MNPs. Our findings suggest that NPs and MPs similarly impair the innate immune function of zebrafish larvae and weaken their disease resistance, highlighting the significant environmental threat posed by these pollutants.

Transcriptomic analysis reveals molecular characterization and immune landscape of PANoptosis-related genes in atherosclerosis.

Atherosclerosis is a chronic inflammatory disease characterized by abnormal lipid deposition in the arteries. Programmed cell death is involved in the inflammatory response of atherosclerosis, but PANoptosis, as a new form of programmed cell death, is still unclear in atherosclerosis. This study explored the key PANoptosis-related genes involved in atherosclerosis and their potential mechanisms through bioinformatics analysis. We evaluated differentially expressed genes (DEGs) and immune infiltration landscape in atherosclerosis using microarray datasets and bioinformatics analysis. By intersecting PANoptosis-related genes from the GeneCards database with DEGs, we obtained a set of PANoptosis-related genes in atherosclerosis (PANoDEGs). Functional enrichment analysis of PANoDEGs was performed and protein-protein interaction (PPI) network of PANoDEGs was established. The machine learning algorithms were used to identify the key PANoDEGs closely linked to atherosclerosis. Receiver operating characteristic (ROC) analysis was used to assess the diagnostic potency of key PANoDEGs. CIBERSORT was used to analyze the immune infiltration patterns in atherosclerosis, and the Spearman method was used to study the relationship between key PANoDEGs and immune infiltration abundance. The single gene enrichment analysis of key PANoDEGs was investigated by GSEA. The transcription factors and target miRNAs of key PANoDEGs were predicted by Cytoscape and online database, respectively. The expression of key PANoDEGs was validated through animal and cell experiments. PANoDEGs in atherosclerosis were significantly enriched in apoptotic process, pyroptosis, necroptosis, cytosolic DNA-sensing pathway, NOD-like receptor signaling pathway, lipid and atherosclerosis. Four key PANoDEGs (ZBP1, SNHG6, DNM1L, and AIM2) were found to be closely related to atherosclerosis. The ROC curve analysis demonstrated that the key PANoDEGs had a strong diagnostic potential in distinguishing atherosclerotic samples from control samples. Immune cell infiltration analysis revealed that the proportion of initial B cells, plasma cells, CD4 memory resting T cells, and M1 macrophages was significantly higher in atherosclerotic tissues compared to normal tissues. Spearman analysis showed that key PANoDEGs showed strong correlations with immune cells such as T cells, macrophages, plasma cells, and mast cells. The regulatory networks of the four key PANoDEGs were established. The expression of key PANoDEGs was verified in further cell and animal experiments. This study evaluated the expression changes of PANoptosis-related genes in atherosclerosis, providing a reference direction for the study of PANoptosis in atherosclerosis and offering potential new avenues for further understanding the pathogenesis and treatment strategies of atherosclerosis.

Discovery of a triphenylamine-based ligand that targets mitochondrial DNA G-quadruplexes and activates the cGAS-STING immunomodulatory pathway

Stabilization of G-quadruplex (G4) structures in mitochondria leads to the damage of mitochondrial DNA (mtDNA), making mtDNA G4s a promising target in the field of cancer therapy in recent years. Damaged mtDNA released into the cytosol can stimulate cytosolic DNA-sensing pathways, and cGAS-STING pathway is a typical one with potent immunostimulatory effects. A few small molecule ligands of mtDNA G4s are identified with antitumor efficacy, but little is known about their results and mechanisms on immunomodulation. In this study, we engineered a series of triphenylamine-based analogues targeting mtDNA G4s, and A6 was determined as the most promising compound. Cellular studies indicated that A6 caused severe mtDNA damage. Then, damaged mtDNA stimulated cGAS-STING pathway, resulting in the following cytokine production of tumor cells and the maturation of DCs. In vivo experiments certified that A6 exerted suppressive influences on tumor growth and metastasis in 4T1 cell-bearing mice by the regulation of TME, including the remodeling of macrophages and the activation of T cells. To our knowledge, it is the first time to report a ligand targeting mtDNA G4s to activate the cGAS-STING immunomodulatory pathway, providing a novel strategy for the future development of mtDNA G4-based antitumor agents.

Abstract 5669: Rare germline variants on SI gene and the possibility of colorectal cancer development

Abstract Research on cancer and germline variants has been ongoing. The germline variant in CPGs has been linked to cancer development. BRCA 1/2 is a typical case and oncometabolite production due to germline variants of the FH gene can affect the cancer progression. Among the genes related to IEMs, SI (Sucrase-isomaltase) is an enzyme that converts sucrose into glucose and fructose to allow normal metabolism in the small intestine. In addition, SI is highly specifically expressed in the gastrointestinal tract (nTPM = 435.9). So, we focused on and hypothesized the biological traits of the SI gene and alteration caused by germline variants could affect colorectal cancer progression. We used the public cancer databases PCAWG and TCGA and the healthy cohorts 1000G. We confirmed carriers with SI germline variants are the top gene with a high prevalence of IEM-related genes. We also established the hypothesis the SI gene may match “Knudson’s Two-hit hypothesis”. Depending on whether germline variants in IEM-related genes or not, we performed regression and utilized LOH (Loss of heterozygosity) information for each cancer sample from the public database. In COAD and STAD which are in the gastrointestinal tract, SI was confirmed to have a high tendency for LOH according to germline prevalence. Next, we performed DEG analysis and found SI germline carriers had upregulated genes related to the IFIT (Interferon-induced proteins with tetratricopeptide repeats) family. STRING network analysis also showed significance in pathways associated with “Interferon alpha/beta signaling (FDR = 0.00058)”, “Antiviral defense (FDR = 0.0016)”, “Innate immunity (FDR = 0.0317)”, and “Tetratricopeptide repeats (FDR = 0.0413)”. The following GSEA revealed that “KEGG_CYTOSOLIC DNA-SENSING PATHWAY” and “HALLMARK_INTERFERON_ALPHA_RESPONSE” were enriched in the SI germline carriers. In summary, it can be suggested immune regulation caused by SI germline variants may lead to cancer progression. We executed a follow-up analysis using the genes with low expression levels to the SI germline carriers. Among the down-regulated genes in the DEG, REG4 (Regenerating Family Member 4) and RELMβ (Resistin-Like Molecule Beta) genes were confirmed to express and function specifically only in the gastrointestinal tract. RELMβ is essential for preserving energy balance and glucose metabolism. As a validation at the protein level, we conducted IHC (Immunohistochemistry) on tissues from Korean colorectal cancer patients and confirmed lower REG4 and RELMβ expression were associated with poor prognosis. Our results suggest that SI germline carriers may cause colorectal cancer by affecting the immune system due to decreased SI enzyme function. Additionally, the decreased REG4 and RELMβ expression levels are associated with the SI germline variant and may affect cancer progression. Further analyses and experimental validations are still required. Citation Format: Seokhyeon Kim, Jeong Mo Bae, Youngil Koh, Sung-Soo Yoon. Rare germline variants on SI gene and the possibility of colorectal cancer development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5669.

The activation of the adaptor protein STING depends on its interactions with the phospholipid PI4P.

Activation of the endoplasmic reticulum (ER)-resident adaptor protein STING, a component of a cytosolic DNA-sensing pathway, induces the transcription of genes encoding type I interferons (IFNs) and other proinflammatory factors. Because STING is activated at the Golgi apparatus, control of the localization and activation of STING is important in stimulating antiviral and antitumor immune responses. Through a genome-wide CRISPR interference screen, we found that STING activation required the Golgi-resident protein ACBD3, which promotes the generation of phosphatidylinositol 4-phosphate (PI4P) at the trans-Golgi network, as well as other PI4P-associated proteins. Appropriate localization and activation of STING at the Golgi apparatus required ACBD3 and the PI4P-generating kinase PI4KB. In contrast, STING activation was enhanced when the lipid-shuttling protein OSBP, which removes PI4P from the Golgi apparatus, was inhibited by the US Food and Drug Administration-approved antifungal itraconazole. The increase in the abundance of STING-activating phospholipids at the trans-Golgi network resulted in the increased production of IFN-β and other cytokines in THP-1 cells. Furthermore, a mutant STING that could not bind to PI4P failed to traffic from the ER to the Golgi apparatus in response to a STING agonist, whereas forced relocalization of STING to PI4P-enriched areas elicited STING activation in the absence of stimulation with a STING agonist. Thus, PI4P is critical for STING activation, and manipulating PI4P abundance may therapeutically modulate STING-dependent immune responses.

Multi-Omics Analysis Reveals the IFI6 Gene as a Prognostic Indicator and Therapeutic Target in Esophageal Cancer.

The role of the IFI6 gene has been described in several cancers, but its involvement in esophageal cancer (ESCA) remains unclear. This study aimed to identify novel prognostic indicators for ESCA-targeted therapy by investigating IFI6's expression, epigenetic mechanisms, and signaling activities. We utilized public data from the Gene Expression Omnibus (GEO) and the Cancer Genome Atlas (TCGA) to analyze IFI6's expression, clinical characteristics, gene function, pathways, and correlation with different immune cells in ESCA. The TIMER2.0 database was employed to assess the pan-cancer expression of IFI6, while UALCAN was used to examine its expression across tumor stages and histology subtypes. Additionally, the KEGG database helped identify related pathways. Our findings revealed 95 genes positively correlated and 15 genes negatively correlated with IFI6 in ESCA. IFI6 was over-expressed in ESCA and other cancers, impacting patient survival and showing higher expression in tumor tissues than normal tissues. IFI6 was also correlated with CD4+ T cells and B cell receptors (BCRs), both essential in immune response. GO Biological Process (GO BP) enrichment analysis indicated that IFI6 was primarily associated with the Type I interferon signaling pathway and the defense response to viruses. Intriguingly, KEGG pathway analysis demonstrated that IFI6 and its positively correlated genes in ESCA were mostly linked to the Cytosolic DNA-sensing pathway, which plays a crucial role in innate immunity and viral defense, and the RIG-I-like receptor (RLR) signaling pathway, which detects viral infections and activates immune responses. Pathways related to various viral infections were also identified. It is important to note that our study relied on online databases. Given that ESCA consists of two distinct subgroups (ESCC and EAC), most databases combine them into a single category. Future research should focus on evaluating IFI6 expression and its impact on each subgroup to gain more specific insights. In conclusion, inhibiting IFI6 using targeted therapy could be an effective strategy for treating ESCA considering its potential as a biomarker and correlation with immune cell factors.

Activation of cGAS-STING suppresses coxsackievirus replication via interferon-dependent signaling

Coxsackievirus B3 (CVB3) is a non-enveloped, single-stranded, positive RNA virus known for its role in provoking inflammatory diseases that affect the heart, pancreas, and brain, leading to conditions such as myocarditis, pancreatitis, and meningitis. Currently, there are no FDA-approved drugs treating CVB3 infection; therefore, identifying potential molecular targets for antiviral drug development is imperative. In this study, we examined the possibility of activating the cyclic GMP–AMP (cGAMP) synthase (cGAS)–stimulator of interferon genes (STING) pathway, a cytosolic DNA-sensing pathway that triggers a type-I interferon (IFN) response, in inhibiting CVB3 infection. We found that activation of the cGAS-STING pathway through the application of cGAS (poly dA:dT and herring testes DNA) or STING agonists (2′3′-cGAMP and diamidobenzimidazole), or the overexpression of STING, significantly suppresses CVB3 replication. Conversely, gene-silencing of STING enhances viral replication. Mechanistically, we demonstrated that cGAS-STING activation combats CVB3 infection by inducing IFN response. Notably, we discovered that knockdown of IFN-α/β receptor, a key membrane receptor in type-I IFN signaling, or inhibition of the downstream JAK1/2 signaling with ruxolitinib, mitigates the effects of STING activation, resulting in increased viral protein production. Furthermore, we investigated the interplay between CVB3 and the cGAS-STING pathway. We showed that CVB3 does not trigger cGAS-STING activation; instead, it antagonizes STING and the downstream TBK1 activation induced by cGAMP. In summary, our results provide insights into the interaction of an RNA virus and the DNA-sensing pathway, highlighting the potential for agonist activation of the cGAS-STING pathway in the development of anti-CVB3 drugs.

Microbiome-host interactions in the pathogenesis of acute exacerbation of chronic obstructive pulmonary disease.

To explore the underlying mechanisms the airway microbiome contributes to Acute Exacerbation of Chronic Obstructive Pulmonary Disease(AECOPD). We enrolled 31 AECOPD patients and 26 stable COPD patients, their sputum samples were collected for metagenomic and RNA sequencing, and then subjected to bioinformatic analyses. The expression of host genes was validated by Quantitative Real-time PCR(qPCR) using the same batch of specimens. Our results indicated a higher expression of Rothia mucilaginosa(p=0.015) in the AECOPD group and Haemophilus influenzae(p=0.005) in the COPD group. The Different expressed genes(DEGs) detected were significantly enriched in "type I interferon signaling pathway"(p<0.001, q=0.001) in gene function annotation, and "Cytosolic DNA-sensing pathway"(p=0.002, q=0.024), "Toll-like receptor signaling pathway"(p=0.006, q=0.045), and "TNF signaling pathway"(p=0.006, q=0.045) in KEGG enrichment analysis. qPCR amplification experiment verified that the expression of OASL and IL6 increased significantly in the AECOPD group. Pulmonary bacteria dysbiosis may regulate the pathogenesis of AECOPD through innate immune system pathways like type I interferon signaling pathway and Toll-like receptor signaling pathway.

In silico characterization and identification of compound heterozygous variants in H/ACA Ribonucleoprotein Assembly Factor (SHQ1) from Indian population.

H/ACA small nucleolar ribonucleoproteins (snoRNP) form a complex with multiple proteins to accomplish the pseudouridylation of rRNA. The assembly of H/ACA small nucleolar ribonucleoproteins (snoRNP) is initiated by H/ACA ribonucleoprotein Assembly factor, that is, SHQ1. Mutations in SHQ1 have been reported to cause two disorders namely, dystonia-35 childhood onset (OMIM*619921) and neurodevelopmental disorder with seizures and dystonia (OMIM*619922), both of which are inherited in an autosomal recessive manner. Considering the high genetic and clinical diversity of SHQ1-related clinical features and the importance of SHQ1 in the assembly of the H/ACA snoRNP complex, it is important to take a systematic approach to delineate the genetic diagnosis and impact of mutations on protein structure and stability. Whole exome sequencing followed by Sanger validation was performed in an individual with the clinical features of neurodevelopmental disorder with seizures and dystonia (OMIM*619922). Protein modeling studies of all the reported SHQ1 variants to date were performed using freely available web servers Interactive Tree of Life, String, BioGrid, ShinyGO, DAVID, and Pathvix. Protein structures were visualized using Pymol. We identified compound heterozygous variants, one known frameshift deletion c. 828_831del, p.(Asp277Serfs*27) and the other novel missense variant c. 1157A>C, p.(Tyr386Ser) found in an individual with neurodevelopmental disorder, seizures, movement disorder, and hypomyelination leukodystrophy on neuroimaging. Protein-interactome studies identified potential genetic interactors that include GAR1, NAF1, TRUB1, UTP15, DKC1, NOP10, NPHOSPH 10, KRR1, NOP58, NOP56, FBL, RRP9, NHP2, RUVBL1, and RUVBL2. Ribosome biogenesis in eukaryotes, RNA polymerase, RNA transport, spliceosome, ribosome, cytosolic DNA-sensing pathway, DNA replication, mismatch repair, base excision repair, nucleotide excision repair, and basal transcription factors process were identified as the linked pathways with the prioritized genes. In conclusion, a sophisticated genotype and phenotype correlation followed by linking the genes to the key biological pathways opens new avenues to understand disease pathology and plan for therapeutic interventions.

A Novel Pyroptosis-Based Prognostic Model Correlated with the Parainflammatory Immune Microenvironment of Pancreatic Cancer

Background. Pyroptosis has a dual function in malignant tumor progression and management. The action of pyroptosis-related genes (PRGs) in pancreatic cancer (PC), however, remains uncertain. Methods. Differential expression analyses of 57 PRGs were conducted in the TCGA TARGET GTEx dataset. The candidate genes were determined using LASSO Cox regression and random forest analyses. A risk model was developed with the TCGA dataset and validated with the ICGC dataset. Results. Three prognosis-related PRGs (BAK1, GSDMC, and IL18) were chosen to create a risk model. High-risk patients from the TCGA and ICGC cohorts had an unfavorable overall survival (all p&lt;0.05). The risk modelʼs accuracy and independent predictability were assessed by receiver operating characteristic curves and multivariate Cox regression analysis, respectively. High-risk patients possessed different molecular pathways, higher KRAS and TP53 mutations, increased expression of PD-L1, C1 immune subtype, and immunosuppressive microenvironment characterized by parainflammation compared to low-risk patients. KRAS and TP53 mutations participated in different inflammatory pathways and played different prognostic roles between the two risk groups. KRAS mutations in high-risk patients caused a more unfavorable prognosis than wild-type KRAS (p=0.016), whereas TP53 mutations in low-risk patients exhibited a poorer outcome than wild-type TP53 (p=0.009). Spearman correlation analyses revealed that the parainflammatory response in PC might be implicated in GSDMC-mediated pyroptosis via cytosolic DNA-sensing pathways under hypoxic conditions. Furthermore, the risk scores were significantly and positively related to the expression of HNRNPC, RBM15, YTHDF1, and YTHDF2, as well as sensitivity to gemcitabine, cisplatin, and erlotinib. Conclusions. This study created a novel pyroptosis-based risk model related to the parainflammatory immune microenvironment, which might help identify novel biomarkers, evaluate the tumor immune microenvironment, and develop management strategies for PC patients.

Identification of novel biomarkers in obstructive sleep apnea via integrated bioinformatics analysis and experimental validation

Background Obstructive sleep apnea (OSA) is a complex and multi-gene inherited disease caused by both genetic and environmental factors. However, due to the high cost of diagnosis and complex operation, its clinical application is limited. This study aims to explore potential target genes associated with OSA and establish a corresponding diagnostic model. Methods This study used microarray datasets from the Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs) related to OSA and perform functional annotation and pathway analysis. The study employed multi-scale embedded gene co-expression network analysis (MEGENA) combined with least absolute shrinkage and selection operator (LASSO) regression analysis to select hub genes and construct a diagnostic model for OSA. In addition, the study conducted correlation analysis between hub genes and OSA-related genes, immunoinfiltration, gene set enrichment analysis (GSEA), miRNA network analysis, and identified potential transcription factors (TFs) and targeted drugs for hub genes. Finally, the study used chronic intermittent hypoxia (CIH) mouse model to simulate OSA hypoxic conditions and verify the expression of hub genes in CIH mice. Results In this study, a total of 401 upregulated genes and 275 downregulated genes were identified, and enrichment analysis revealed that these differentially expressed genes may be associated with pathways such as vasculature development, cellular response to cytokine stimulus, and negative regulation of cell population proliferation. Through MEGENA combined with LASSO regression, seven OSA hub genes were identified, including C12orf54, FOS, GPR1, OR9A4, MYO5B, RAB39B, and KLHL4. The diagnostic model constructed based on these genes showed strong stability. The expression levels of hub genes were significantly correlated with the expression levels of OSA-related genes and mainly acted on pathways such as the JAK/STAT signaling pathway and the cytosolic DNA-sensing pathway. Drug-target predictions for hub genes were made using the Connectivity Map (CMap) database and the Drug-Gene Interaction database (Dgidb), which identified targeted therapeutic drugs for the hub genes. In vivo experiments showed that the hub genes were all decreasing in the OSA mouse model. Conclusions This study identified novel biomarkers for OSA and established a reliable diagnostic model. The transcriptional changes identified may help to reveal the pathogenesis, mechanisms, and sequelae of OSA.

Identification of a novel ferroptosis-related gene signature associated with retinal degeneration induced by light damage in mice

BackgroundNeurodegenerative retinal diseases such as retinitis pigmentosa are serious disorders that may cause irreversible visual impairment. Ferroptosis is a novel type of programmed cell death, and the involvement of ferroptosis in retinal degeneration is still unclear. This study aimed to investigate the related ferroptosis genes in a mice model of retinal degeneration induced by light damage. MethodsA public dataset of GSE10528 deriving from the Gene Expression Omnibus database was analyzed to identify the differentially expressed genes (DEGs). Gene set enrichment analysis between light damage and control group was conducted. The differentially expressed ferroptosis-related genes (DE-FRGs) were subsequently identified by intersecting the DEGs with a ferroptosis genes dataset retrieved from the FerrDb database. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were further performed using the DE-FRGs. A protein-protein interaction (PPI) network was constructed to identify hub ferroptosis-related genes (HFRGs). The microRNAs (miRNAs)-HFRGs, transcription factors (TFs)-HFRGs networks as well as target drugs potentially interacting with HFRGs were analyzed utilizing bioinformatics algorithms. ResultsA total of 932 DEGs were identified between the light damage and control group. Among these, 25 genes were associated with ferroptosis. GO and KEGG analyses revealed that these DE-FRGs were mainly enriched in apoptotic signaling pathway, response to oxidative stress and autophagy, ferroptosis, necroptosis and cytosolic DNA-sensing pathway. Through PPI network analysis, six hub ferroptosis-related genes (Jun, Stat3, Hmox1, Atf3, Hspa5 and Ripk1) were ultimately identified. All of them were upregulated in light damage retinas, as verified by the GSE146176 dataset. Bioinformatics analyses predicated that 116 miRNAs, 23 TFs and several potential therapeutic compounds might interact with the identified HFRGs. ConclusionOur study may provide novel potential biomarkers, therapeutic targets and new insights into the ferroptosis landscape in retinal neurodegenerative diseases.

CSIG-09. RADIOTHERAPY WITH CONCURRENT INHIBITION OF CHEK2 ACTIVATES STING IN GLIOMAS

Abstract Using an in vivo CRISPR screen approach, we demonstrated the contribution of glioma cell intrinsic checkpoint kinase 2 (Chek2) in the evasion of tumor cells from CD8+ T cell recognition. Genetic depletion or pharmacological inhibition of Chek2 with blood-brain-barrier permeable drugs that are being evaluated in clinical trials, in combination with standard of care radiotherapy and PD-1/PD-L1 blockade, led to survival benefit in multiple preclinical glioma models. Moreover, we observed that Chek2 inhibition or depletion is associated with activation of cytosolic DNA-sensing STING pathway in the glioma cells. A positive correlation was identified between STING pathway activity and T cell infiltration in the TCGA glioblastoma dataset. STING activation has been shown to promote robust immune response in glioblastoma. Building upon these results, we evaluated the impact of radiotherapy on Chek2 and STING signaling. After applying 2 Gy radiotherapy, a 20 fold increase of Chek2 activation was observed within the first hour in the in vitro grown glioblastoma patient derived cells, specifically GBM6 and GBM43 cells. Chek2 activation levels returned to the baseline within 6 hours in these cells. However, STING activation was moderate to absent at different time intervals within the first 48 hours post 2 Gy radiotherapy. Interestingly, when the in vitro-grown GBM6 cells were pretreated with Chek1/2 inhibitor prexasertib for 48 hours prior to the 2 Gy radiotherapy exposure, there was 10 fold reduction in the activation of Chek2 as compared to the radiotherapy alone group. Concomitantly, a 2-fold increase in the activation of STING was noted for up to 48 h in the pretreated group, as compared to the radiotherapy alone group. Our in vitro findings suggest an inverse relationship between the activation of Chek2 and STING. In conclusion, radiotherapy with concurrent inhibition of Chek2 offers a novel therapeutic combination to modulate STING activation in gliomas.

In silico based analysis to explore genetic linkage between atherosclerosis and its potential risk factors

Atherosclerosis (ATH) is a chronic cardiovascular disease characterized by plaque formation in arteries, and it is a major cause of illness and death. Although therapeutic advances have significantly improved the prognosis of ATH, missing therapeutic targets pose a significant residual threat. This research used a systems biology approach to identify the molecular biomarkers involved in the onset and progression of ATH, analysing microarray gene expression datasets from ATH and tissues impacted by risk factors such as high cholesterol, adipose tissue, smoking, obesity, sedentary lifestyle, stress, alcohol consumption, hypertension, hyperlipidaemia, high fat, diabetes to find the differentially expressed genes (DEGs). Bioinformatic analyses of Protein-Protein Interaction (PPI), Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) were conducted on differentially expressed genes, revealing metabolic and signaling pathways (the chemokine signaling pathway, cytokine-cytokine receptor interaction, the cytosolic DNA-sensing pathway, the peroxisome proliferator-activated receptors signaling pathway, and the nuclear factor-kappa B signaling pathway), ten hubs proteins (CCL5, CCR1, TLR1, CCR2, FCGR2A, IL1B, CD163, AIF1, CXCL-1 and TNF), five transcription factors (YY1, FOXL1, FOXC1, SRF, and GATA2), and five miRNAs (mir-27a-3p, mir-124–3p, mir-16–5p, mir-129-2-3p, mir-1-3p). These findings identify potential biomarkers that may increase knowledge of the mechanisms underlying ATH and their connection to risk factors, aiding in the development of new therapies.