BRCA1-regulated nuclear innate sensing of Herpesviral genome by IFI16 and IFI16’s acetylation is critical for its cytoplasmic trafficking and induction of innate responses: DOI: 10.14800/ics.1076

Sensing of invading DNA virus genomes appear to be triggered by a number of host cell DNA sensors depending on their subcellular localization which stimulate innate anti-viral responses such as the activation of type-I interferons (IFNs) and/or inflammasomes resulting in the  production of inflammatory IL-1? and IL-18 cytokines. With growing understanding of diverse identities whether these proteins function alone or  with other host cell molecules and the post-translational modifications affecting their functions are under intense investigations. Nuclear resident IFI16 have been shown to sense the episomal DNA genomes of herpes viruses resulting in the induction of IFI16-inflammasome and/or interferon responses. Here, we highlight our recent finding regarding the role of cellular  BRCA1, a transcription factor and DNA damage response protein, forming a distinct complex with IFI16 to regulate the nuclear innate sensing of herpes viral DNA and subsequent IFI16-ASC-procaspase-1 inflammasome complex formation and distribution to the cytoplasm leading into caspase-1 and IL-1? production. BRCA1 is also responsible for the cytoplasmic IFI16-STING signalosome activation and induction of IFN-? during de novo KSHV and HSV-1 infection. Our concurrent studies have also revealed that the histone acetyl transferase p300 mediated acetylation of nuclear IFI16 is a dynamic post-genome recognition event responsible for Ran dependent nuclear to cytoplasmic trafficking of IFI16 during herpesvirus infection. This post-translational modification is essential for IFI16-ASC interaction and inflammasome activation as well as for the association with STING in the cytoplasm resulting in IRF-3 phosphorylation, nuclear pIRF-3 localization and interferon-? production. Collectively, these comprehensive studies highlight that BRCA1 plays a hitherto unidentified immunomodulatory role to facilitate the anti-viral functions of IFI16 and acetylation of nuclear IFI16 is a necessary post-translational modification for innate responses during herpesvirus infection. These studies open up a new understanding of virus-host interplay, viral genome sensing and host innate anti-viral defense mechanisms

BRCA1-regulated nuclear innate sensing of Herpesviral genome by IFI16 and IFI16’s acetylation is critical for its cytoplasmic trafficking and induction of innate responses

Sensing of invading DNA virus genomes appear to be triggered by a number of host cell DNA sensors depending on their subcellular localization which stimulate innate anti-viral responses such as the activation of type-I interferons (IFNs) and/or inflammasomes resulting in the  production of inflammatory IL-1β and IL-18 cytokines. With growing understanding of diverse identities whether these proteins function alone or  with other host cell molecules and the post-translational modifications affecting their functions are under intense investigations. Nuclear resident IFI16 have been shown to sense the episomal DNA genomes of herpes viruses resulting in the induction of IFI16-inflammasome and/or interferon responses. Here, we highlight our recent finding regarding the role of cellular  BRCA1, a transcription factor and DNA damage response protein, forming a distinct complex with IFI16 to regulate the nuclear innate sensing of herpes viral DNA and subsequent IFI16-ASC-procaspase-1 inflammasome complex formation and distribution to the cytoplasm leading into caspase-1 and IL-1β production. BRCA1 is also responsible for the cytoplasmic IFI16-STING signalosome activation and induction of IFN-β during de novo KSHV and HSV-1 infection. Our concurrent studies have also revealed that the histone acetyl transferase p300 mediated acetylation of nuclear IFI16 is a dynamic post-genome recognition event responsible for Ran dependent nuclear to cytoplasmic trafficking of IFI16 during herpesvirus infection. This post-translational modification is essential for IFI16-ASC interaction and inflammasome activation as well as for the association with STING in the cytoplasm resulting in IRF-3 phosphorylation, nuclear pIRF-3 localization and interferon-β production. Collectively, these comprehensive studies highlight that BRCA1 plays a hitherto unidentified immunomodulatory role to facilitate the anti-viral functions of IFI16 and acetylation of nuclear IFI16 is a necessary post-translational modification for innate responses during herpesvirus infection. These studies open up a new understanding of virus-host interplay, viral genome sensing and host innate anti-viral defense mechanisms

Intravesical Mycobacterium brumae triggers both local and systemic immunotherapeutic responses against bladder cancer in mice

The standard treatment for high-risk non-muscle invasive bladder cancer (BC) is the intravesical administration of live Mycobacterium bovis BCG. Previous studies suggest improving this therapy by implementing non-pathogenic mycobacteria, such as Mycobacterium brumae, and/or different vehicles for mycobacteria delivery, such as an olive oil (OO)-in-water emulsion. While it has been established that BCG treatment activates the immune system, the immune effects of altering the mycobacterium and/or the preparation remain unknown. In an orthotopic murine BC model, local immune responses were assessed by measuring immune cells into the bladder and macromolecules in the urine by flow cytometry and multiplexing, respectively. Systemic immune responses were analyzed by quantifying sera anti-mycobacteria antibody levels and recall responses of ex vivo splenocytes cultured with mycobacteria antigens. In both BCG- and M. brumae-treated mice, T, NK, and NKT cell infiltration in the bladder was significantly increased. Notably, T cell infiltration was enhanced in OO-in-water emulsified mycobacteria-treated mice, and urine IL-6 and KC concentrations were elevated. Furthermore, mycobacteria treatment augmented IgG antibody production and splenocyte proliferation, especially in mice receiving OO-in-water emulsified mycobacteria. Our data demonstrate that intravesical mycobacterial treatment triggers local and systemic immune responses, which are most significant when OO-in-water emulsified mycobacteria are used

Interferon-γ-inducible protein 16 (IFI16) is required for the maintenance of Epstein-Barr virus latency

Abstract Background Epstein-Barr virus (EBV) exhibits both lytic and latent (Lat. I, II, and III) phases in an infected individual. It’s during the latent phase of EBV that all EBV-associated cancers, including Burkitt’s lymphoma, nasopharyngeal carcinoma and lymphoproliferative disease arise. Interferon-γ-inducible protein 16 (IFI16) is a well-established innate immune sensor and viral transcriptional regulator involved in response to invading DNA viruses. During latency, IFI16 remains in the nucleus, in part bound to the EBV genome; however, neither its role in EBV lytic cycle or latency has been established. Methods Short interfering RNA against IFI16 and IFI16 overexpression were used to identify the role of IFI16 in the maintenance of EBV latency I. We also studied how induction of the lytic cycle affected IFI16 using the EBV positive, latently infected Akata or MUTU-1 cell lines. Akata cells were induced with TPA and MUTU-1 cells with TGF-β up to 96 h and changes in IFI16 protein were analyzed by Western blotting and immunofluorescence microscopy. To assess the mechanism of IFI16 decrease, EBV DNA replication and late lytic transcripts were blocked using the viral DNA polymerase inhibitor phosphonoacetic acid. Results Knockdown of IFI16 mRNA by siRNA resulted in enhanced levels of EBV lytic gene expression from all temporal gene classes, as well as an increase in the total EBV genome abundance, whereas overexpression of exogenous IFI16 reversed these effects. Furthermore, 96 h after induction of the lytic cycle with either TPA (Akata) or TGF-β (MUTU-1), IFI16 protein levels decreased up to 80% as compared to the EBV-negative cell line BJAB. Reduction in IFI16 was observed in cells expressing EBV lytic envelope glycoprotein. The decreased levels of IFI16 protein do not appear to be dependent on late lytic transcripts of EBV but suggest involvement of the immediate early, early, or a combination of both gene classes. Conclusions Reduction of IFI16 protein levels following lytic cycle induction, as well as reactivation from latency after IFI16 mRNA knockdown suggests that IFI16 is crucial for the maintenance of EBV latency. More importantly, these results identify IFI16 as a unique host factor protein involved in the EBV lifecycle, making it a potential therapeutic target to combat EBV-related malignancies

Effect of an Integrated Physiotherapy Protocol on Knee Osteoarthritis Patients: A Preliminary Study

Background: Exercise therapy can potentially relieve symptoms and improve functional status of the knee osteoarthritis population. Despite the proved practical benefits, there is no standard, comprehensive physiotherapeutic protocol available targeting the physical and physiological impairment cluster associated with disease. Osteoarthritis is a whole joint disease, affecting joint cartilage, ligaments, menisci and joint associated muscles, from variable pathophysiological processes. Hence, there is a need to develop a physiotherapy protocol to address the multi-structural physical, physiological and functional impairments associated with the disease. Objective: The objective of the present study is to evaluate the efficacy of designed, therapist supervised, patient education, progressive resistance exercises, passive stretching exercises, soft tissue manipulation, muscle energy technique, Maitland mobilization, aerobic exercise, and neuromuscular training physiotherapy protocol on pain, disability, balance, and physical functional performance in knee osteoarthritis patients. Methodology: The preliminary study was conducted on a (n = 60) sample of convenience. The samples were randomly allocated into two study groups, intervention, and control group. The control group was advised on a basic home program. On the other hand, the treatment of the intervention group was designed with a therapist supervised Physiotherapy Protocol. The outcome variables studied were the Visual Analogue Scale, Modified WOMAC Scale, Timed Up and Go Test, Functional Reach Test, 40 m Fast Paced Walk Test, Stair Climb Test, 30 s Chair Stand Test. Results: The results of the study revealed a significant improvement among most of the studied outcome measures in the intervention group, hence the designed supervised physiotherapy protocol was found effective in relieving multiple physiological impairments associated with this whole joint disease

Apigenin enhances sorafenib anti-tumour efficacy in hepatocellular carcinoma

Background: The “one drug-one target” paradigm has various limitations affecting drug efficacy, such as resistance profiles and adverse effects. Combinational therapies help reduce unexpected off-target effects and accelerate therapeutic efficacy. Sorafenib- an FDA-approved drug for liver cancer, has multiple limitations. Therefore, it is recommended to identify an agent that increases its effectiveness and reduces toxicity. In this regard, Apigenin, a plant flavone, would be an excellent option to explore. Methods: We used in silico, in vitro, and animal models to explore our hypothesis. For the in vitro study, HepG2 and Huh7 cells were exposed to Apigenin (12-96 μM) and Sorafenib (1-10 μM). For the in vivo study, Diethylnitrosamine (DEN) (25 mg/kg) induced tumor-bearing animals were given Apigenin (50 mg/kg) or Sorafenib (10 mg/kg) alone and combined. Apigenin's bioavailability was checked by UPLC. Tumor nodules were studied macroscopically and by Scanning Electron Microscopy (SEM). Biochemical analysis, histopathology, immunohistochemistry, and qRT-PCR were done. Results: The results revealed Apigenin's good bioavailability. In silico study showed binding affinity of both chemicals with p53, NANOG, ß-Catenin, c-MYC, and TLR4. We consistently observed a better therapeutic efficacy in combination than alone treatment. Combination treatment showed i) better cytotoxicity, apoptosis induction, and cell cycle arrest of tumor cells, ii) tumor growth reduction, iii) increased expression of p53 and decreased Cd10, Nanog, ß-Catenin, c-Myc, Afp, and Tlr4. Conclusions: In conclusion, Apigenin could enhance the therapeutic efficacy of Sorafenib against liver cancer and may be a promising therapeutic approach for treating HCC. However, further research is imperative to gain more in-depth mechanistic insights

EphrinA2 regulates clathrin mediated KSHV endocytosis in fibroblast cells by coordinating integrin-associated signaling and c-Cbl directed polyubiquitination.

Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with human dermal endothelial cell surface tyrosine kinase EphrinA2 (EphA2) and integrins (α3β1 and αVβ3) in the lipid raft (LR) region, and EphA2 regulates macropinocytic virus entry by coordinating integrin-c-Cbl associated signaling. In contrast, KSHV enters human foreskin fibroblast (HFF) cells by LR-independent clathrin mediated endocytosis. The present studies conducted to identify the key molecules regulating KSHV entry in HFF cells showed that KSHV induces association with integrins (αVβ5, αVβ3 and α3β1) and EphA2 in non-LR regions early during infection and activates EphA2, which in turn associates with phosphorylated c-Cbl, myosin IIA, FAK, Src, and PI3-K, as well as clathrin and its adaptor AP2 and effector Epsin-15 proteins. EphA2 knockdown significantly reduced these signal inductions, virus internalization and gene expression. c-Cbl knockdown ablated the c-Cbl mediated K63 type polyubiquitination of EphA2 and clathrin association with EphA2 and KSHV. Mutations in EphA2's tyrosine kinase domain (TKD) or sterile alpha motif (SAM) abolished its interaction with c-Cbl. Mutations in tyrosine kinase binding (TKB) or RING finger (RF) domains of c-Cbl resulted in very poor association of c-Cbl with EphA2 and decreased EphA2 polyubiquitination. These studies demonstrated the contributions of these domains in EphA2 and c-Cbl association, EphA2 polyubiquitination and virus-EphA2 internalization. Collectively, these results revealed for the first time that EphA2 influences the tyrosine phosphorylation of clathrin, the role of EphA2 in clathrin mediated endocytosis of a virus, and c-Cbl mediated EphA2 polyubiquitination directing KSHV entry in HFF cells via coordinated signal induction and progression of endocytic events, all of which suggest that targeting EphA2 and c-Cbl could block KSHV entry and infection

Systems-wide view of host-pathogen interactions across COVID-19 severities using integrated omics analysis

Summary: The mechanisms explaining the variability in COVID-19 clinical manifestations (mild, moderate, and severe) are not fully understood. To identify key gene expression markers linked to disease severity, we employed an integrated approach, combining host-pathogen protein-protein interaction data and viral-induced host gene expression data. We analyzed an RNA-seq dataset from peripheral blood mononuclear cells across 12 projects representing the spectrum of disease severity. We identified genes showing differential expression across mild, moderate, and severe conditions. Enrichment analysis of the pathways in host proteins targeted by each of the SARS-CoV-2 proteins revealed a strong association with processes related to ribosomal biogenesis, translation, and translocation. Interestingly, most of these pathways and associated cellular machinery, including ribosomal biogenesis, ribosomal proteins, and translation, were upregulated in mild conditions but downregulated in severe cases. This suggests that COVID-19 exhibits a paradoxical host response, boosting host/viral translation in mild cases but slowing it in severe cases