Identification of the proteolytic signature in CVB3-infected cells.

Coxsackievirus B3 (CVB3) encodes proteinases that are essential for processing of the translated viral polyprotein. Viral proteinases also target host proteins to manipulate cellular processes and evade innate antiviral responses to promote replication and infection. While some host protein substrates of the CVB3 3C and 2A cysteine proteinases have been identified, the full repertoire of targets is not known. Here, we utilize an unbiased quantitative proteomics-based approach termed terminal amine isotopic labeling of substrates (TAILS) to conduct a global analysis of CVB3 protease-generated N-terminal peptides in both human HeLa and mouse cardiomyocyte (HL-1) cell lines infected with CVB3. We identified >800 proteins that are cleaved in CVB3-infected HeLa and HL-1 cells including the viral polyprotein, known substrates of viral 3C proteinase such as PABP, DDX58, and HNRNPs M, K, and D and novel cellular proteins. Network and GO-term analysis showed an enrichment in biological processes including immune response and activation, RNA processing, and lipid metabolism. We validated a subset of candidate substrates that are cleaved under CVB3 infection and some are direct targets of 3C proteinase in vitro. Moreover, depletion of a subset of TAILS-identified target proteins decreased viral yield. Characterization of two target proteins showed that expression of 3Cpro-targeted cleaved fragments of emerin and aminoacyl-tRNA synthetase complex-interacting multifunctional protein 2 modulated autophagy and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway, respectively. The comprehensive identification of host proteins targeted during virus infection provides insights into the cellular pathways manipulated to facilitate infection. IMPORTANCE: RNA viruses encode proteases that are responsible for processing viral proteins into their mature form. Viral proteases also target and cleave host cellular proteins; however, the full catalog of these target proteins is incomplete. We use a technique called terminal amine isotopic labeling of substrates (TAILS), an N-terminomics to identify host proteins that are cleaved under virus infection. We identify hundreds of cellular proteins that are cleaved under infection, some of which are targeted directly by viral protease. Revealing these target proteins provides insights into the host cellular pathways and antiviral signaling factors that are modulated to promote virus infection and potentially leading to virus-induced pathogenesis.

Crosstalk Between Innate Immunity and Autophagy in Viral Myocarditis Leading to Dilated Cardiomyopathy.

Viral myocarditis, characterised by inflammation of the heart muscle, presents a significant challenge to global public health, particularly affecting younger individuals and often progressing to dilated cardiomyopathy (DCM), a leading cause of heart failure. Despite ongoing research efforts, viable treatments for this condition remain elusive. Recent studies have shed light on the complex interplay between the innate immune response and autophagy mechanisms, revealing their pivotal roles in the pathogenesis of viral myocarditis and subsequent DCM development. This review aims to delve into the recent advancements in understanding the molecular mechanisms and pathways that intersect innate immunity and autophagy in the context of viral myocarditis. Furthermore, it explores the potential therapeutic implications of these findings, offering insights into promising avenues for the management and treatment of this debilitating condition.

Combining bulk and scRNA-seq to explore the molecular mechanisms governing the distinct efferocytosis activities of a macrophage subpopulation in PDAC.

Pancreatic ductal adenocarcinoma (PDAC), a very aggressive tumour, is currently the third leading cause of cancer-related deaths. Unfortunately, many patients face the issue of inoperability at the diagnostic phase leading to a quite dismal prognosis. The onset of metastatic processes has a crucial role in the elevated mortality rates linked to PDAC. Individuals with metastatic advances receive only palliative therapy and have a grim prognosis. It is essential to carefully analyse the intricacies of the metastatic process to enhance the prognosis for individuals with PDAC. Malignancy development is greatly impacted by the process of macrophage efferocytosis. Our current knowledge about the complete range of macrophage efferocytosis activities in PDAC and their intricate interactions with tumour cells is still restricted. This work aims to resolve communication gaps and pinpoint the essential transcription factor that is vital in the immunological response of macrophage populations. We analysed eight PDAC tissue samples sourced from the gene expression omnibus. We utilized several software packages such as Seurat, DoubletFinder, Harmony, Pi, GSVA, CellChat and Monocle from R software together with pySCENIC from Python, to analyse the single-cell RNA sequencing (scRNA-seq) data collected from the PDAC samples. This study involved the analysis of a comprehensive sample of 22,124 cells, which were classified into distinct cell types. These cell types encompassed endothelial and epithelial cells, PDAC cells, as well as various immune cells, including CD4+ T cells, CD8+ T cells, NK cells, B cells, plasma cells, mast cells, monocytes, DC cells and different subtypes of macrophages, namely C0 macrophage TGM2+, C1 macrophage PFN1+, C2 macrophage GAS6+ and C3 macrophage APOC3+. The differentiation between tumour cells and epithelial cells was achieved by the implementation of CopyKat analysis, resulting in the detection and categorization of 1941 PDAC cells. The amplification/deletion patterns observed in PDAC cells on many chromosomes differ significantly from those observed in epithelial cells. The study of Pseudotime Trajectories demonstrated that the C0 macrophage subtype expressing TGM2+ had the lowest level of differentiation. Additionally, the examination of gene set scores related to efferocytosis suggested that this subtype displayed higher activity during the efferocytosis process compared to other subtypes. The most active transcription factors for each macrophage subtype were identified as BACH1, NFE2, TEAD4 and ARID3A. In conclusion, the examination of human PDAC tissue samples using immunofluorescence analysis demonstrated the co-localization of CD68 and CD11b within regions exhibiting the presence of keratin (KRT) and alpha-smooth muscle actin (α-SMA). This observation implies a spatial association between macrophages, fibroblasts, and epithelial cells. There is variation in the expression of efferocytosis-associated genes between C0 macrophage TGM2+ and other macrophage cell types. This observation implies that the diversity of macrophage cells might potentially influence the metastatic advancement of PDAC. Moreover, the central transcription factor of different macrophage subtypes offers a promising opportunity for targeted immunotherapy in the treatment of PDAC.

Cleavage of 14-3-3ε by the enteroviral 3C protease dampens RIG-I-mediated antiviral signaling.

Viruses have evolved diverse strategies to evade the host innate immune response and promote infection. The retinoic acid-inducible gene I (RIG-I)-like receptors RIG-I and MDA5 are antiviral factors that sense viral RNA and trigger downstream signal via mitochondrial antiviral-signaling protein (MAVS) to activate type I interferon expression. 14-3-3ε is a key component of the RIG-I translocon complex that interacts with MAVS at the mitochondrial membrane; however, the exact role of 14-3-3ε in this pathway is not well understood. In this study, we demonstrate that 14-3-3ε is a direct substrate of both the poliovirus and coxsackievirus B3 (CVB3) 3C proteases (3Cpro) and that it is cleaved at Q236↓G237, resulting in the generation of N- and C-terminal fragments of 27.0 and 2.1 kDa, respectively. While the exogenous expression of wild-type 14-3-3ε enhances IFNB mRNA production during poly(I:C) stimulation, expression of the truncated N-terminal fragment does not. The N-terminal 14-3-3ε fragment does not interact with RIG-I in co-immunoprecipitation assays, nor can it facilitate RIG-I translocation to the mitochondria. Probing the intrinsically disordered C-terminal region identifies key residues responsible for the interaction between 14-3-3ε and RIG-I. Finally, overexpression of the N-terminal fragment promotes CVB3 infection in mammalian cells. The strategic enterovirus 3Cpro-mediated cleavage of 14-3-3ε antagonizes RIG-I signaling by disrupting critical interactions within the RIG-I translocon complex, thus contributing to evasion of the host antiviral response. IMPORTANCE Host antiviral factors work to sense virus infection through various mechanisms, including a complex signaling pathway known as the retinoic acid-inducible gene I (RIG-I)-like receptor pathway. This pathway drives the production of antiviral molecules known as interferons, which are necessary to establish an antiviral state in the cellular environment. Key to this antiviral signaling pathway is the small chaperone protein 14-3-3ε, which facilitates the delivery of a viral sensor protein, RIG-I, to the mitochondria. In this study, we show that the enteroviral 3C protease cleaves 14-3-3ε during infection, rendering it incapable of facilitating this antiviral response. We also find that the resulting N-terminal cleavage fragment dampens RIG-I signaling and promotes virus infection. Our findings reveal a novel viral strategy that restricts the antiviral host response and provides insights into the mechanisms underlying 14-3-3ε function in RIG-I antiviral signaling.

Heart-specific NFAT5 knockout suppresses type I interferon signaling and aggravates coxsackievirus-induced myocarditis.

Nuclear factor of activated T cells 5 (NFAT5) is an osmosensitive transcription factor that is well-studied in renal but rarely explored in cardiac diseases. Although the association of Coxsackievirus B3 (CVB3) with viral myocarditis is well-established, the role of NFAT5 in this disease remains largely unexplored. Previous research has demonstrated that NFAT5 restricts CVB3 replication yet is susceptible to cleavage by CVB3 proteases. Using an inducible cardiac-specific Nfat5-knockout mouse model, we uncovered that NFAT5-deficiency exacerbates cardiac pathology, worsens cardiac function, elevates viral load, and reduces survival rates. RNA-seq analysis of CVB3-infected mouse hearts revealed the significant impact of NFAT5-deficiency on gene pathways associated with cytokine signaling and inflammation. Subsequent in vitro and in vivo investigation validated the disruption of the cytokine signaling pathway in response to CVB3 infection, evidenced by reduced expression of key cytokines such as interferon ß1 (IFNß1), C-X-C motif chemokine ligand 10 (CXCL10), interleukin 6 (IL6), among others. Furthermore, NFAT5-deficiency hindered the formation of stress granules, leading to a reduction of important stress granule components, including plakophilin-2, a pivotal protein within the intercalated disc, thereby impacting cardiomyocyte structure and function. These findings unveil a novel mechanism by which NFAT5 inhibits CVB3 replication and pathogenesis through the promotion of antiviral type I interferon signaling and the formation of cytoplasmic stress granules, collectively identifying NFAT5 as a new cardio protective protein.

The relationship and clinical significance of lactylation modification in digestive system tumors.

Lactylation, an emerging post-translational modification, plays a pivotal role in the initiation and progression of digestive system tumors. This study presents a comprehensive review of lactylation in digestive system tumors, underscoring its critical involvement in tumor development and progression. By focusing on metabolic reprogramming, modulation of the tumor microenvironment, and the molecular mechanisms regulating tumor progression, the potential of targeting lactylation as a therapeutic strategy is highlighted. The research reveals that lactylation participates in gene expression regulation and cell signaling by affecting the post-translational states of histones and non-histone proteins, thereby influencing metabolic pathways and immune evasion mechanisms in tumor cells. Furthermore, this study assesses the feasibility of lactylation as a therapeutic target, providing insights for clinical treatment of gastrointestinal cancers. Future research should concentrate on elucidating the mechanisms of lactylation, developing efficient lactylation inhibitors, and validating their therapeutic efficacy in clinical trials, which could transform current cancer treatment and immunotherapy approaches. In summary, this review emphasizes the crucial role of lactylation in tumorigenesis and progression through a detailed analysis of its molecular mechanisms and clinical significance.

Leptin receptor Gln223Arg and Lys109Arg polymorphisms may be associated with HBV-related hepatocellular carcinoma risk: A system review and meta-analysis.

Increasing evidence has suggested a strong association of hepatocellular carcinoma (HCC) susceptibility and Gln223Arg (rs1137101) and Lys109Arg (rs1137100) polymorphisms in leptin receptor (LEPR) genes. To provide a quantitative assessment for such correlation, we reviewed all related systems and conducted meta-analysis for case and control researches. A literature search of Web of Science, EMBASE, PubMed, Scopus as well as China National Knowledge Infrastructure databases was collected. 95% confidence intervals (95% CIs) together with odds ratios (ORs) were calculated. Five case-control researches consisting of 1323 cases and 1919 control cases were incorporated into meta-analysis. Researches indicated A-allelic and AA genotype of rs1137101 were substantially related to boosted susceptibility of hepatitis B virus (HBV)-related HCC (mutant model, OR = 1.81, 95% CI = 1.36-2.41, p < .001; allelic model, OR = 1.55, 95% CI = 1.32-1.83, p < .001). On the contrary, we observed GG genotype of rs1137101 substantially related to reduced risk of HBV-related HCC (wild model, OR 0.59, 95%CI = 0.46-0.75, p < .001). We observed AA genotype of rs1137100 relevant to boosted HCC risk (mutant model, OR = 1.51, 95%CI = 1.14-2.01, p = .005) as well as in those with HBV-related HCCs (homozygous model, OR = 2.12, 95%CI = 1.49-3.02, p < .001; mutant model, OR = 1.67, 95%CI = 1.23-2.26, p = .001). G-allele and AA genotype of rs1137101 might be in connection with boosted HBV-related HCC susceptibility, and wild-type GG genotype might prevent diseases. AA genotype of rs1137100 might also improve HBV-related HCC susceptibility. Such conclusions ought to be validated by larger and better-designed researches.

A combination of genetically engineered oncolytic virus and melittin-CpG for cancer viro-chemo-immunotherapy.

BACKGROUND: Immunotherapy has emerged as an efficient therapeutic approach for cancer management. However, stimulation of host immune system against cancer cells often fails to achieve promising clinical outcomes mainly owing to the immunosuppressive characteristics of the tumor microenvironment (TME). Combination therapeutics that can trigger sustained immunogenic cell death (ICD) have provided new opportunities for cancer treatment. METHODS: In this study, we designed and applied an ICD inducer regimen, including a genetically engineered oncolytic virus (miRNA-modified coxsackieviruses B3, miR-CVB3), a pore-forming lytic peptide (melittin, found in bee venom), and a synthetic toll-like receptor 9 ligand (CpG oligodeoxynucleotides), for breast cancer and melanoma treatment. We compared the anti-tumor efficacy of miR-CVB3 and CpG-melittin (CpGMel) alone and in combination (miR-CVB3 + CpGMel) and investigated possible mechanisms involved. RESULTS: We demonstrated that miR-CVB3 + CpGMel had no major impact on viral growth, while enhancing the cellular uptake of CpGMel in vitro. We further showed that combination therapy led to significant increases in tumor cell death and release of damage-associated molecular patterns compared with individual treatment. In vivo studies in 4T1 tumor-bearing Balb/c mice revealed that both primary and distant tumors were significantly suppressed, and the survival rate was significantly prolonged after administration of miR-CVB3 + CpGMel compared with single treatment. This anti-tumor effect was accompanied by increased ICD and immune cell infiltration into the TME. Safety analysis showed no significant pathological abnormalities in Balb/c mice. Furthermore, the developed therapeutic regimen also demonstrated a great anti-tumor activity in B16F10 melanoma tumor-bearing C57BL/6 J mice. CONCLUSIONS: Overall, our findings indicate that although single treatment using miR-CVB3 or CpGMel can efficiently delay tumor growth, combining oncolytic virus-based therapy can generate even stronger anti-tumor immunity, leading to a greater reduction in tumor size.

Crosstalk between RNA viruses and DNA sensors: Role of the cGAS-STING signalling pathway.

Despite only comprising half of all known viral species, RNA viruses are disproportionately responsible for many of the worst epidemics in human history, including outbreaks of influenza, poliomyelitis, Ebola, and most recently, the coronavirus disease-2019 (COVID-19) pandemic. The propensity for RNA viruses to replicate in cytosolic compartments has led to an evolutionary arms race and the emergence of cytosolic sensors to recognise and initiate the host innate immune response. Although significant progress has been made in identifying and characterising cytosolic RNA sensors as anti-viral innate immune factors, the potential role for cytosolic DNA sensors in RNA viral infection is only recently being appreciated. Among these, the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway has attracted increasing attention. The cGAS-STING signalling pathway has emerged as a key innate immune signalling axis that is implicated in diverse human diseases from infectious diseases to neurodegeneration and cancer. Here we review the existing literature on RNA viruses and their reciprocal interactions with the cGAS-STING pathway and share insights into RNA virus diversity by touching on the similarities and differences of RNA viral strategies.

A situational analysis of human resource and non-communicable diseases management for community health workers in Chengdu, China: a cross-sectional study.

BACKGROUND: Non-communicable diseases (NCDs) pose a major challenge to health economic cost and residents' health status. Community health workers (CHWs) are the gatekeeper of primary health care. OBJECTIVE: This study aimed to conduct a situational analysis of current human resource and requirements of NCDs-related training among CHWs in Chengdu with regard to address to understand the suggestions for improvement of challenges and barriers. METHODS: A descriptive online cross-sectional survey was conducted among CHWs (doctors and nurses) from 23 districts and counties in Chengdu. Sociodemographic and NCDs-related variables were collected. Univariate analysis and multiple response analysis were used to describe the characteristics of these variables. RESULTS: 711 doctors and 637 nurses completely responded. There were significant differences among gender, age, educational levels, professional title, working year, type of institution, urban circle and registration in general practice between doctors and nurses (P < 0.001). 60.6% of doctors were female, compared to 98.0% for nurses. 58.2% of doctors held a bachelor's degree compared with 45.4% of nurses, while 48.3% of nurses held a junior college degree compared with 25.7% of doctors. Higher levels of professional title and registration in general practice were found in doctors compared with nurses. The proportions of NCDs' category, NCDs-related roles and tasks, NCDs-related training contents and forms that CHWs have attend and hoped to gain more were significantly different between doctors and nurses (P < 0.001). In general, the proportions in nurses were much lower than those of doctors (P < 0.05). The top five diseases managed by CHWs were hypertension, diabetes, cerebrovascular disease, chronic respiratory diseases and mental diseases. The five most reported roles performed among doctors included the distribution of health education (91.4%), following up (85.9%), establishing archives (71.3%), medicine adjustment (64.7%) and treatment implementation (52.0%). The top three diseases managed by nurses were same with doctors. The top four and five tasks were contact with patients or health services (39.6%) and referral (16.6%) in nurses. Most CHWs had received primary and common diseases-related trainings, but they had few opportunities to study in a tertiary hospital (40.4% in doctors and 20.9% in nurses, respectively), attend domestic academic conferences (26.9% in doctors vs. 9.7% in nurses), and take part in training courses (44.9% in nurses). CHWs hoped that the above-discussed training contents and forms could be provided more in the future. Besides basic skills related trainings, some specific skills related trainings should be strengthened. CONCLUSION: The qualifications in doctors were much better than those of nurses. The roles performed by CHWs in NCDs management are varied form common and frequent disease management to subsequent follow up and supervision. CHWs hope to receive more desired and oriented trainings. There is a need for building capacity of CHWs, optimizing and defining CHWs' role, facilitating postgraduate medical education support and strengthening multidisciplinary collaboration would be effective in NCDs management.

Sublethal enteroviral infection exacerbates disease progression in an ALS mouse model.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of the motor neuron system associated with both genetic and environmental risk factors. Infection with enteroviruses, including poliovirus and coxsackievirus, such as coxsackievirus B3 (CVB3), has been proposed as a possible causal/risk factor for ALS due to the evidence that enteroviruses can target motor neurons and establish a persistent infection in the central nervous system (CNS), and recent findings that enteroviral infection-induced molecular and pathological phenotypes closely resemble ALS. However, a causal relationship has not yet been affirmed. METHODS: Wild-type C57BL/6J and G85R mutant superoxide dismutase 1 (SOD1G85R) ALS mice were intracerebroventricularly infected with a sublethal dose of CVB3 or sham-infected. For a subset of mice, ribavirin (a broad-spectrum anti-RNA viral drug) was given subcutaneously during the acute or chronic stage of infection. Following viral infection, general activity and survival were monitored daily for up to week 60. Starting at week 20 post-infection (PI), motor functions were measured weekly. Mouse brains and/or spinal cords were harvested at day 10, week 20 and week 60 PI for histopathological evaluation of neurotoxicity, immunohistochemical staining of viral protein, neuroinflammatory/immune and ALS pathology markers, and NanoString and RT-qPCR analysis of inflammatory gene expression. RESULTS: We found that sublethal infection (mimicking chronic infection) of SOD1G85R ALS mice with CVB3 resulted in early onset and progressive motor dysfunction, and shortened lifespan, while similar viral infection in C57BL/6J, the background strain of SOD1G85R mice, did not significantly affect motor function and mortality as compared to mock infection within the timeframe of the current study (60 weeks PI). Furthermore, we showed that CVB3 infection led to a significant increase in proinflammatory gene expression and immune cell infiltration and induced ALS-related pathologies (i.e., TAR DNA-binding protein 43 (TDP-43) pathology and neuronal damage) in the CNS of both SOD1G85R and C57BL/6J mice. Finally, we discovered that early (day 1) but not late (day 15) administration of ribavirin could rescue ALS-like neuropathology and symptoms induced by CVB3 infection. CONCLUSIONS: Our study identifies a new risk factor that contributes to early onset and accelerated progression of ALS and offers opportunities for the development of novel targeted therapies.

FUS/TLS Suppresses Enterovirus Replication and Promotes Antiviral Innate Immune Responses.

During viral infection, the dynamic virus-host relationship is constantly in play. Many cellular proteins, such as RNA-binding proteins (RBPs), have been shown to mediate antiviral responses during viral infection. Here, we report that the RBP FUS/TLS (fused in sarcoma/translocated in liposarcoma) acts as a host-restricting factor against infection with coxsackievirus B3 (CVB3). Mechanistically, we found that deletion of FUS leads to increased viral RNA transcription and enhanced internal ribosome entry site (IRES)-driven translation, with no apparent impact on viral RNA stability. We further demonstrated that FUS physically interacts with the viral genome, which may contribute to direct inhibition of viral RNA transcription/translation. Moreover, we identified a novel function for FUS in regulating host innate immune response. We show that in the absence of FUS, gene expression of type I interferons and proinflammatory cytokines elicited by viral or bacterial infection is significantly impaired. Emerging evidence suggests a role for stress granules (SGs) in antiviral innate immunity. We further reveal that knockout of FUS abolishes the ability to form SGs upon CVB3 infection or poly(I·C) treatment. Finally, we show that, to avoid FUS-mediated antiviral response and innate immunity, CVB3 infection results in cytoplasmic mislocalization and cleavage of FUS through the enzymatic activity of viral proteases. Together, our findings in this study identify FUS as a novel host antiviral factor which restricts CVB3 replication through direct inhibition of viral RNA transcription and protein translation and through regulation of host antiviral innate immunity.IMPORTANCE Enteroviruses are common human pathogens, including those that cause myocarditis (coxsackievirus B3 [CVB3]), poliomyelitis (poliovirus), and hand, foot, and mouth disease (enterovirus 71). Understanding the virus-host interaction is crucial for developing means of treating and preventing diseases caused by these pathogens. In this study, we explored the interplay between the host RNA-binding protein FUS/TLS and CVB3 and found that FUS/TLS restricts CVB3 replication through direct inhibition of viral RNA transcription/translation and through regulation of cellular antiviral innate immunity. To impede the antiviral role of FUS, CVB3 targets FUS for mislocalization and cleavage. Findings from this study provide novel insights into interactions between CVB3 and FUS, which may lead to novel therapeutic interventions against enterovirus-induced diseases.

Innate immune evasion mediated by picornaviral 3C protease: Possible lessons for coronaviral 3C-like protease?

Severe acute respiratory syndrome coronavirus-2 is the etiological agent of the ongoing pandemic of coronavirus disease-2019, a multi-organ disease that has triggered an unprecedented global health and economic crisis. The virally encoded 3C-like protease (3CLpro ), which is named after picornaviral 3C protease (3Cpro ) due to their similarities in substrate recognition and enzymatic activity, is essential for viral replication and has been considered as the primary drug target. However, information regarding the cellular substrates of 3CLpro and its interaction with the host remains scarce, though recent work has begun to shape our understanding more clearly. Here we summarized and compared the mechanisms by which picornaviruses and coronaviruses have evolved to evade innate immune surveillance, with a focus on the established role of 3Cpro in this process. Through this comparison, we hope to highlight the potential action and mechanisms that are conserved and shared between 3Cpro and 3CLpro . In this review, we also briefly discussed current advances in the development of broad-spectrum antivirals targeting both 3Cpro and 3CLpro .

The papain-like protease of coronaviruses cleaves ULK1 to disrupt host autophagy.

The ongoing pandemic of COVID-19 alongside the outbreaks of SARS in 2003 and MERS in 2012 underscore the significance to understand betacoronaviruses as a global health challenge. SARS-CoV-2, the etiological agent for COVID-19, has infected over 50 million individuals' worldwide with more than ∼1 million fatalities. Autophagy modulators have emerged as potential therapeutic candidates against SARS-CoV-2 but recent clinical setbacks urge for better understanding of viral subversion of autophagy. Using MHV-A59 as a model betacoronavirus, time-course infections revealed significant loss in the protein level of ULK1, a canonical autophagy-regulating kinase, and the concomitant appearance of a possible cleavage fragment. To investigate whether virus-encoded proteases target ULK1, we conducted in-vitro and cellular cleavage assays and identified ULK1 as a novel bona fide substrate of SARS-CoV-2 papain-like protease (PLpro). Mutagenesis studies discovered that ULK1 is cleaved at a conserved PLpro recognition sequence (LGGG) after G499, separating its N-terminal kinase domain from a C-terminal substrate recognition region. Over-expression of SARS-CoV-2 PLpro is sufficient to impair starvation-induced autophagy and disrupt formation of ULK1-ATG13 complex. Finally, we demonstrated a dual role for ULK1 in MHV-A59 replication, serving a pro-viral functions during early replication that is inactivated at late stages of infection. In conclusion, our study identified a new mechanism by which PLpro of betacoronaviruses induces viral pathogenesis by targeting cellular autophagy.

Macrophage polarization-associated lnc-Ma301 interacts with caprin-1 to inhibit hepatocellular carcinoma metastasis through the Akt/Erk1 pathway.

BACKGROUND: Epithelial-mesenchymal transition (EMT) promotes migration, invasion, and metastasis of hepatocellular carcinoma (HCC) cells. The molecular mechanisms behind EMT and metastasis in HCC remain unclear. METHODS: Microarray analysis was used to identify lncRNAs expression during polarization of U937 macrophages from M2 to M1 phenotype. The expression of the identified lncRNA was compared between clinical samples of HCC tissues or adjacent normal tissues, as well as between HCC and normal liver cell lines. lnc-Ma301 was overexpressed or knocked-down in HCC cell lines, and the effects were assessed in vitro and in vivo. Interactions among lnc-Ma301 and its potential downstream targets caprin-1 were investigated in HCC cell lines. Effects of lnc-Ma301 over- and underexpression on the Akt/Erk1 signaling pathways were examined. RESULTS: Microarray analyses identified lnc-Ma301 as one of the most overexpressed long non-coding RNAs during polarization of U937 macrophages from M2 to M1 phenotype. Lnc-Ma301 showed lower expression in HCC tissues than in adjacent normal tissues, and lower expression was associated with worse prognosis. Activation of lnc-Ma301 inhibited cell proliferation, migration and EMT in HCC cell cultures, and it inhibited lung metastasis of HCC tumors in mice. Mechanistic studies suggested that lnc-Ma301 interacts with caprin-1 to inhibit HCC metastasis and EMT through Akt/Erk1 pathway. CONCLUSIONS: Lnc-Ma301 may help regulate onset and metastasis of HCC.

NLRP3 deficiency exacerbates enterovirus infection in mice.

The role for the NOD-like receptor (NLR) P3 inflammasome in enterovirus infection remains controversial. Available data suggest that the NLRP3 inflammasome is protective against enterovirus A71 but detrimental to the host during coxsackievirus B3 (CVB3) infection. CVB3 is a common etiologic agent associated with myocarditis and pancreatitis. Previous findings on the role of NLRP3 in CVB3 were based primarily on indirect evidence. Here, we utilized NLRP3 knockout mice as well as immune and cardiac cells to investigate the direct interplay between CVB3 infection and NLRP3 activation. We demonstrated that NLRP3 knockout mice exhibited more severe disease phenotype after CVB3 infection (significantly higher virus titers), increased myocardial, and pancreatic damage, as well as markedly impaired cardiac function compared to nontransgenic control mice. We further showed that NLRP3 activity was enhanced during early stage of CVB3 infection, as evidenced by increased gene expression and/or secretion of IL-1ß and caspase-1. Finally, we demonstrated that CVB3 inactivates the NLRP3 inflammasome by degrading NLRP3 and its upstream serine/threonine-protein kinase receptor-interacting protein 1/3 via the proteolytic activity of virus-encoded proteinases. Taken together, our results reveal the functional significance of NLRP3 in host antiviral immunity against CVB3 infection and the mechanisms by which CVB3 has evolved to counteract the host defense response.-Wang, C., Fung, G., Deng, H., Jagdeo, J., Mohamud, Y., Xue, Y. C., Jan, E., Hirota, J. A., Luo, H. NLRP3 deficiency exacerbates enterovirus infection in mice.

Single molecule real-time sequencing and RNA-seq unravel the role of long non-coding and circular RNA in the regulatory network during Nile tilapia (Oreochromis niloticus) infection with Streptococcus agalactiae.

BACKGROUND: The tilapia aquaculture industry is facing heavy economic losses due to Streptococcus agalactiae (S. agalactiae) infections. While progress has been made in past years, the lack of a high-quality tilapia genome and transcript annotations makes systematic and comprehensive exploration for a non-coding RNA regulatory network associated with the infection process unfeasible, and it stunts further research focused on disease defense and treatment. Herein, single molecular real time sequencing (SMRT-Seq) and RNA-seq data were utilized to generate a high-quality transcript annotation. In addition, Changes in mRNA and non-coding RNA expression were also analyzed during a S. agalactiae infection in tilapia. FINDINGS: In total, 16.79 Gb of clean data were obtained by sequencing on six SMRT cells, with 712,294 inserts (326,645 full-length non-chimeric reads and 354,188 non-full-length reads). A total of 197,952 consensus transcripts were obtained. Additionally, 55,857 transcript sequences were acquired, with 12,297 previously annotated and 43,560 newly identified transcripts. To further examine the immune response in Oreochromis niloticus following a S. agalactiae infection, a total of 470.62 Gb of clean data was generated by sequencing a library containing 18 S. agalactiae infected tilapia samples. Of the identified genes, 9911 were newly exploited, of which 7102 were functional annotated. Furthermore, 7874 mRNAs, 1281 long non-coding RNAs (out of 21,860 long non-coding RNAs), and 61 circular RNAs (out of 1026 circular RNAs) were found to be differentially expressed during infection, with the 1026 circRNAs not previously identified in tilapia. Moreover, k-means clustering and WGCNA analyses revealed that the immune response of tilapia to a S. agalactiae infection can be divided into three stages: cytokines driven rapid immune response, energy metabolism promotion, and the production of lysosomes and phagosomes. During this response, the head kidney and spleen have synergistic effects, while maintaining independent characteristics. Finally, lncRNA-mRNA (trans and cis), lncRNA-miRNA-mRNA, and circRNA-miRNA-mRNA regulatory networks were constructed and revealed that non-coding RNA is involved in the regulation of immune-related genes. CONCLUSIONS: This study generated a greatly-improved transcript annotation for tilapia using long-read PacBio sequencing technology, and revealed the presence of a regulatory network comprised of non-coding RNAs in Nile tilapia infected with S. agalactiae.

Emerging nanomedicines for effective breast cancer immunotherapy.

Breast cancer continues to be the most frequently diagnosed malignancy among women, putting their life in jeopardy. Cancer immunotherapy is a novel approach with the ability to boost the host immune system to recognize and eradicate cancer cells with high selectivity. As a promising treatment, immunotherapy can not only eliminate the primary tumors, but also be proven to be effective in impeding metastasis and recurrence. However, the clinical application of cancer immunotherapy has faced some limitations including generating weak immune responses due to inadequate delivery of immunostimulants to the immune cells as well as uncontrolled modulation of immune system, which can give rise to autoimmunity and nonspecific inflammation. Growing evidence has suggested that nanotechnology may meet the needs of current cancer immunotherapy. Advanced biomaterials such as nanoparticles afford a unique opportunity to maximize the efficiency of immunotherapy and significantly diminish their toxic side-effects. Here we discuss recent advancements that have been made in nanoparticle-involving breast cancer immunotherapy, varying from direct activation of immune systems through the delivery of tumor antigens and adjuvants to immune cells to altering immunosuppression of tumor environment and combination with other conventional therapies.

Fault Diagnosis for High-Speed Train Axle-Box Bearing Using Simplified Shallow Information Fusion Convolutional Neural Network.

Axle-box bearings are one of the most critical mechanical components of the high-speed train. Vibration signals collected from axle-box bearings are usually nonlinear and nonstationary, caused by the complicated operating conditions. Due to the high reliability and real-time requirement of axle-box bearing fault diagnosis for high-speed trains, the accuracy and efficiency of the bearing fault diagnosis method based on deep learning needs to be enhanced. To identify the axle-box bearing fault accurately and quickly, a novel approach is proposed in this paper using a simplified shallow information fusion-convolutional neural network (SSIF-CNN). Firstly, the time domain and frequency domain features were extracted from the training samples and testing samples before been inputted into the SSIF-CNN model. Secondly, the feature maps obtained from each hidden layer were transformed into a corresponding feature sequence by the global convolution operation. Finally, those feature sequences obtained from different layers were concatenated into one-dimensional as the fully connected layer to achieve the fault identification task. The experimental results showed that the SSIF-CNN effectively compressed the training time and improved the fault diagnosis accuracy compared with a general CNN.

Genomic analysis provides insights into the transmission and pathogenicity of Talaromyces marneffei.

Talaromyces marneffei (T. marneffei) is a medically important opportunistic dimorphic fungus that infects both humans and bamboo rats. However, the mechanisms of transmission and pathogenicity of T. marneffei are poorly understood. In our study, we combined Illumina and PacBio sequencing technologies to sequence and assemble a complete genome of T. marneffei. To elucidate the transmission route and source, we sequenced three additional T. marneffei isolates using Illumina sequencing technology. Variations among isolates were used to develop a multilocus sequence typing (MLST) system comprising five housekeeping genes that can be used to discriminate between isolates derived from different sources. Our analysis revealed that human and bamboo rat share identical genotypes in these five loci. Thus, we hypothesized that T. marneffei is transmitted to humans through inhalation of spores in the surrounding environment into the lungs and that the bamboo rat can serve as an important natural reservoir for pathogens. Furthermore, we also identified temperature-dependent polyketide synthases, non-ribosomal peptide synthetases and secreted proteins as putative pathogenicity-related factors. In addition, we identified antifungal drug targets that can be investigated in future studies to elucidate the mechanisms underlying drug resistance. In summary, our study presents the basic features of the T. marneffei genome and provides insights into the transmission and pathogenicity of T. marneffei, which warrant fundamental experimental research.