ABSTRACT
Second-generation bioenergy, a carbon neutral or negative renewable resource, is crucial to achieving India's net-zero emission targets. Crop residues are being targeted as a bioenergy resource as they are otherwise burned on-field, leading to significant pollutant emissions. But estimating their bioenergy potential is problematic because of broad assumptions about their surplus fractions. Here, we use comprehensive surveys and multivariate regression models to estimate the bioenergy potential of surplus crop residues in India. These are with high sub-national and crop disaggregation that can facilitate the development of efficient supply chain mechanisms for its widespread usage. The estimated potential for 2019 of 1313 PJ can increase the present bioenergy installed capacity by 82% but is likely insufficient alone to meet India's bioenergy targets. The shortage of crop residue for bioenergy, combined with the sustainability concerns raised by previous studies, imply a need to reassess the strategy for the use of this resource.
Subject(s)
Agriculture , Environmental Pollutants , India , CarbonABSTRACT
The applications of gene therapy-based treatment of cancers were started almost two decades back as a boon over the chemotherapeutic treatment strategies. Gene therapy helps in correcting the genetic sequences for treatment of cancers, thus also acts like a vaccine to induce the cellular and humoral immunity. However, the cancer vaccines typically suffer from a series of biopharmaceutical challenges due to poor solubility, low systemic availability and lack of targeting ability. Owing to these challenges, the physicians and pharmaceutical scientists have explored the applications of nanocarriers as quite promising systems for effective treatment against the tumors. A series of nanotherapeutic systems are available to date for diverse drug therapy applications. Systematic understanding on the preparation, evaluation and application of nanomedicines as a carrier system for delivering the cancer vaccines is highly important. The present review article provides an in-depth understanding on the challenges associated with cancer vaccine delivery and current opportunities with diverse nanomedicinal carriers being available for treatment of cancers.
Subject(s)
Antineoplastic Agents/administration & dosage , Cancer Vaccines/administration & dosage , Drug Delivery Systems , Nanomedicine , Neoplasms/drug therapy , Vaccination/methods , Animals , Humans , Neoplasms/pathologyABSTRACT
Viruses cause a variety of diseases in humans and other organisms. The most important defense mechanism against viral infections is initiated when the viral genome is sensed by host proteins, and this results in interferon production and pro-inflammatory cytokine responses. The sensing of the viral genome or its replication intermediates within host cells is mediated by cytosolic proteins. For example, cGAS and IFI16 recognize non-self DNA, and RIG-I and MDA5 recognize non-self RNA. Once these sensors are activated, they trigger a cascade of reactions activating downstream molecules, which eventually results in the transcriptional activation of type I and III interferons, which play a critical role in suppressing viral propagation, either by directly limiting their replication or by inducing host cells to inhibit viral protein synthesis. The immune response against viruses relies solely upon sensing of viral genomes and their downstream signaling molecules. Although DNA and RNA viruses are sensed by distinct classes of receptor proteins, there is a possibility of overlap between the viral DNA and viral RNA sensing mechanisms. In this review, we focus on various host sensing molecules and discuss the associated signaling pathways that are activated in response to different viral infections. We further highlight the possibility of crosstalk between the cGAS-STING and the RIG-I-MAVS pathways to limit viral infections. This comprehensive review delineates the mechanisms by which different viruses evade host cellular responses to sustain within the host cells.
Subject(s)
Nucleotidyltransferases , RNA Virus Infections , DNA, Viral , Humans , Immunity, Innate , Interferons , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Signal TransductionABSTRACT
Hepatitis C virus (HCV) replication and assembly occur at the specialized site of endoplasmic reticulum (ER) membranes and lipid droplets (LDs), respectively. Recently, several host proteins have been shown to be involved in HCV replication and assembly. In the present study, we demonstrated the important relationship among osteopontin (OPN), the ER, and LDs. OPN is a secreted phosphoprotein, and overexpression of OPN in hepatocellular carcinoma (HCC) tissue can lead to invasion and metastasis. OPN expression is also enhanced in HCV-associated HCC. Our recent studies have demonstrated the induction, proteolytic cleavage, and secretion of OPN in response to HCV infection. We also defined the critical role of secreted OPN in human hepatoma cell migration and invasion through binding to receptors integrin αVß3 and CD44. However, the role of HCV-induced OPN in the HCV life cycle has not been elucidated. In this study, we showed a significant reduction in HCV replication, assembly, and infectivity in HCV-infected cells transfected with small interfering RNA (siRNA) against OPN, αVß3, and CD44. We also observed the association of endogenous OPN with HCV proteins (NS3, NS5A, NS4A/B, NS5B, and core). Confocal microscopy revealed the colocalization of OPN with HCV NS5A and core in the ER and LDs, indicating a possible role for OPN in HCV replication and assembly. Interestingly, the secreted OPN activated HCV replication, infectivity, and assembly through binding to αVß3 and CD44. Collectively, these observations provide evidence that HCV-induced OPN is critical for HCV replication and assembly.IMPORTANCE Recently, our studies uncovered the critical role of HCV-induced endogenous and secreted OPN in migration and invasion of hepatocytes. However, the role of OPN in the HCV life cycle has not been elucidated. In this study, we investigated the importance of OPN in HCV replication and assembly. We demonstrated that endogenous OPN associates with HCV NS3, NS5A, NS5B, and core proteins, which are in close proximity to the ER and LDs. Moreover, we showed that the interactions of secreted OPN with cell surface receptors αVß3 and CD44 are critical for HCV replication and assembly. These observations provide evidence that HCV-induced endogenous and secreted OPN play pivotal roles in HCV replication and assembly in HCV-infected cells. Taken together, our findings clearly demonstrate that targeting OPN may provide opportunities for therapeutic intervention of HCV pathogenesis.
Subject(s)
Hepatitis C/virology , Hyaluronan Receptors/metabolism , Integrin alphaVbeta3/metabolism , Lipid Droplets/metabolism , Osteopontin/metabolism , Viral Nonstructural Proteins/metabolism , Virus Assembly , Virus Replication , Carcinoma, Hepatocellular/metabolism , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/virology , DNA Replication , Hepacivirus/physiology , Hepatitis C/metabolism , Humans , Hyaluronan Receptors/genetics , Integrin alphaVbeta3/genetics , Liver Neoplasms/metabolism , Liver Neoplasms/pathology , Liver Neoplasms/virology , Osteopontin/genetics , RNA, Viral , Tumor Cells, Cultured , Viral Nonstructural Proteins/geneticsABSTRACT
Kaposi's sarcoma-associated herpesvirus (KSHV) binding to the endothelial cell surface heparan sulfate is followed by sequential interactions with α3ß1, αVß3 and αVß5 integrins and Ephrin A2 receptor tyrosine kinase (EphA2R). These interactions activate host cell pre-existing FAK, Src, PI3-K and RhoGTPase signaling cascades, c-Cbl mediated ubiquitination of receptors, recruitment of CIB1, p130Cas and Crk adaptor molecules, and membrane bleb formation leading to lipid raft dependent macropinocytosis of KSHV into human microvascular dermal endothelial (HMVEC-d) cells. The Endosomal Sorting Complexes Required for Transport (ESCRT) proteins, ESCRT-0, -I, -II, and-III, play a central role in clathrin-mediated internalized ubiquitinated receptor endosomal trafficking and sorting. ESCRT proteins have also been shown to play roles in viral egress. We have recently shown that ESCRT-0 component Hrs protein associates with the plasma membrane during macropinocytosis and mediates KSHV entry via ROCK1 mediated phosphorylation of NHE1 and local membrane pH change. Here, we demonstrate that the ESCRT-I complex Tsg101 protein also participates in the macropinocytosis of KSHV and plays a role in KSHV trafficking. Knockdown of Tsg101 did not affect virus entry in HMVEC-d and human umbilical vein endothelial (HUVEC) cells but significantly inhibited the KSHV genome entry into the nucleus and consequently viral gene expression in these cells. Double and triple immunofluorescence, proximity ligation immunofluorescence and co-immuoprecipitation studies revealed the association of Tsg101 with the KSHV containing macropinosomes, and increased levels of Tsg101 association/interactions with EphA2R, c-Cbl, p130Cas and Crk signal molecules, as well as with upstream and downstream ESCRT components such as Hrs (ESCRT-0), EAP45 (ESCRT-II), CHMP6 (ESCRT-III) and CHMP5 (ESCRT-III) in the KSHV infected cells. Tsg101 was also associated with early (Rab5) and late endosomal (Rab7) stages of KSHV intracellular trafficking, and CHMP5 (ESCRT-III) was also associated with Rab 5 and Rab 7. Knockdown of Tsg101 significantly inhibited the transition of virus from early to late endosomes. Collectively, our studies reveal that Tsg101 plays a role in the trafficking of macropinocytosed KSHV in the endothelial cells which is essential for the successful viral genome delivery into the nucleus, viral gene expression and infection. Thus, ESCRT molecules could serve as therapeutic targets to combat KSHV infection.
Subject(s)
DNA-Binding Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Endothelial Cells/virology , Herpesviridae Infections , Host-Parasite Interactions/physiology , Transcription Factors/metabolism , Virus Internalization , Blotting, Western , Fluorescent Antibody Technique , Herpesvirus 8, Human , Humans , Immunoprecipitation , Microscopy, Fluorescence , Pinocytosis , Real-Time Polymerase Chain Reaction , TransfectionABSTRACT
IFI16 (gamma-interferon-inducible protein 16), a predominantly nuclear protein involved in transcriptional regulation, also functions as an innate immune response DNA sensor and induces the IL-1ß and antiviral type-1 interferon-ß (IFN-ß) cytokines. We have shown that IFI16, in association with BRCA1, functions as a sequence independent nuclear sensor of episomal dsDNA genomes of KSHV, EBV and HSV-1. Recognition of these herpesvirus genomes resulted in IFI16 acetylation, BRCA1-IFI16-ASC-procaspase-1 inflammasome formation, cytoplasmic translocation, and IL-1ß generation. Acetylated IFI16 also interacted with cytoplasmic STING and induced IFN-ß. However, the identity of IFI16 associated nuclear proteins involved in STING activation and the mechanism is not known. Mass spectrometry of proteins precipitated by anti-IFI16 antibodies from uninfected endothelial cell nuclear lysate revealed that histone H2B interacts with IFI16. Single and double proximity ligation microscopy, immunoprecipitation, EdU-genome labeled virus infection, and chromatin immunoprecipitation studies demonstrated that H2B is associated with IFI16 and BRCA1 in the nucleus in physiological conditions. De novo KSHV and HSV-1 infection as well as latent KSHV and EBV infection induces the cytoplasmic distribution of H2B-IFI16, H2B-BRCA1 and IFI16-ASC complexes. Vaccinia virus (dsDNA) cytoplasmic replication didn't induce the redistribution of nuclear H2B-IFI16 or H2B into the cytoplasm. H2B is critical in KSHV and HSV-1 genome recognition by IFI16 during de novo infection. Viral genome sensing by IFI16-H2B-BRCA1 leads to BRCA1 dependent recruitment of p300, and acetylation of H2B and IFI16. BRCA1 knockdown or inhibition of p300 abrogated the acetylation of H2B-IFI16 or H2B. Ran-GTP protein mediated the translocation of acetylated H2B and IFI16 to the cytoplasm along with BRCA1 that is independent of IFI16-ASC inflammasome. ASC knockdown didn't affect the acetylation of H2B, its cytoplasmic transportation, and the association of STING with IFI16 and H2B during KSHV infection. Absence of H2B didn't affect IFI16-ASC association and cytoplasmic distribution and thus demonstrating that IFI16-H2B complex is independent of IFI16-ASC-procaspase-1-inflammasome complex formed during infection. The H2B-IFI16-BRCA1 complex interacted with cGAS and STING in the cytoplasm leading to TBK1 and IRF3 phosphorylation, nuclear translocation of pIRF3 and IFN-ß production. Silencing of H2B, cGAS and STING inhibited IFN-ß induction but not IL-1ß secretion, and cGAMP activity is significantly reduced by H2B and IFI16 knockdown during infection. Silencing of ASC inhibited IL-1ß secretion but not IFN-ß secretion during de novo KSHV and HSV-1 infection. These studies identify H2B as an innate nuclear sensor mediating a novel extra chromosomal function, and reveal that two IFI16 complexes mediate KSHV and HSV-1 genome recognition responses, with recognition by the IFI16-BRCA1-H2B complex resulting in IFN-ß responses and recognition by IFI16-BRCA1 resulting in inflammasome responses.
Subject(s)
Genome, Viral , Herpesviridae Infections/immunology , Histones/immunology , Interferon-beta/immunology , Nuclear Proteins/immunology , Phosphoproteins/immunology , Blotting, Western , Cells, Cultured , Chromatin Immunoprecipitation , Cytoplasm/immunology , Enzyme-Linked Immunosorbent Assay , Herpesviridae/immunology , Humans , Immunity, Innate , Immunoprecipitation , Inflammasomes/immunology , Interferon-beta/biosynthesis , Microscopy, FluorescenceABSTRACT
Hepatitis C virus (HCV) relies on host lipids and lipid droplets for replication and morphogenesis. The accumulation of lipid droplets in infected hepatocytes manifests as hepatosteatosis, a common pathology observed in chronic hepatitis C patients. One way by which HCV promotes the accumulation of intracellular lipids is through enhancing de novo lipogenesis by activating the sterol regulatory element-binding proteins (SREBPs). In general, activation of SREBPs occurs during cholesterol depletion. Interestingly, during HCV infection, the activation of SREBPs occurs under normal cholesterol levels, but the underlying mechanisms are still elusive. Our previous study has demonstrated the activation of the inflammasome complex in HCV-infected human hepatoma cells. In this study, we elucidate the potential link between chronic hepatitis C-associated inflammation and alteration of lipid homeostasis in infected cells. Our results reveal that the HCV-activated NLRP3 inflammasome is required for the up-regulation of lipogenic genes such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, fatty acid synthase, and stearoyl-CoA desaturase. Using pharmacological inhibitors and siRNA against the inflammasome components (NLRP3, apoptosis-associated speck-like protein containing a CARD, and caspase-1), we further show that the activation of the NLRP3 inflammasome plays a critical role in lipid droplet formation. NLRP3 inflammasome activation in HCV-infected cells enables caspase-1-mediated degradation of insulin-induced gene proteins. This subsequently leads to the transport of the SREBP cleavage-activating protein·SREBP complex from the endoplasmic reticulum to the Golgi, followed by proteolytic activation of SREBPs by S1P and S2P in the Golgi. Typically, inflammasome activation leads to viral clearance. Paradoxically, here we demonstrate how HCV exploits the NLRP3 inflammasome to activate SREBPs and host lipid metabolism, leading to liver disease pathogenesis associated with chronic HCV.
Subject(s)
Carrier Proteins/metabolism , Hepacivirus/physiology , Hepatocytes/virology , Inflammasomes/metabolism , Lipogenesis , Sterol Regulatory Element Binding Protein 1/agonists , Sterol Regulatory Element Binding Protein 2/agonists , CARD Signaling Adaptor Proteins , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Caspase 1/chemistry , Caspase 1/genetics , Caspase 1/metabolism , Cell Line, Tumor , Cysteine Proteinase Inhibitors/pharmacology , Cytoskeletal Proteins/antagonists & inhibitors , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Endopeptidases/chemistry , Endopeptidases/metabolism , Enzyme Induction/drug effects , Golgi Apparatus/drug effects , Golgi Apparatus/metabolism , Golgi Apparatus/pathology , Golgi Apparatus/virology , Hepacivirus/drug effects , Hepatitis C, Chronic/metabolism , Hepatitis C, Chronic/pathology , Hepatitis C, Chronic/physiopathology , Hepatitis C, Chronic/virology , Hepatocytes/drug effects , Hepatocytes/metabolism , Hepatocytes/pathology , Host-Pathogen Interactions/drug effects , Humans , Inflammasomes/drug effects , Intracellular Signaling Peptides and Proteins/metabolism , Lipogenesis/drug effects , Membrane Proteins/metabolism , NLR Family, Pyrin Domain-Containing 3 Protein , Non-alcoholic Fatty Liver Disease/etiology , Proprotein Convertases/chemistry , Proprotein Convertases/metabolism , Protein Transport/drug effects , Proteolysis/drug effects , RNA Interference , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Sterol Regulatory Element Binding Protein 1/metabolism , Sterol Regulatory Element Binding Protein 2/metabolismABSTRACT
UNLABELLED: Kaposi's sarcoma-associated herpesvirus (KSHV) enters human dermal microvascular endothelial cells (HMVEC-d), its naturalin vivotarget cells, by lipid raft-dependent macropinocytosis. The internalized viral envelope fuses with the macropinocytic membrane, and released capsid is transported to the nuclear vicinity, resulting in the nuclear entry of viral DNA. The endosomal sorting complexes required for transport (ESCRT) proteins, which include ESCRT-0, -I, -II, and -III, play a central role in endosomal trafficking and sorting of internalized and ubiquitinated receptors. Here, we examined the role of ESCRT-0 component Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) in KSHV entry into HMVEC-d by macropinocytosis. Knockdown of Hrs by short hairpin RNA (shRNA) transduction resulted in significant decreases in KSHV entry and viral gene expression. Immunofluorescence analysis (IFA) and plasma membrane isolation and proximity ligation assay (PLA) demonstrated the translocation of Hrs from the cytosol to the plasma membrane of infected cells and association with α-actinin-4. In addition, infection induced the plasma membrane translocation and activation of the serine/threonine kinase ROCK1, a downstream target of the RhoA GTPase. Hrs knockdown reduced these associations, suggesting that the recruitment of ROCK1 is an Hrs-mediated event. Interaction between Hrs and ROCK1 is essential for the ROCK1-induced phosphorylation of NHE1 (Na(+)/H(+)exchanger 1), which is involved in the regulation of intracellular pH. Thus, our studies demonstrate the plasma membrane association of ESCRT protein Hrs during macropinocytosis and suggest that KSHV entry requires both Hrs- and ROCK1-dependent mechanisms and that ROCK1-mediated phosphorylation of NHE1 and pH change is an essential event required for the macropinocytosis of KSHV. IMPORTANCE: Macropinocytosis is the major entry pathway of KSHV in human dermal microvascular endothelial cells, the natural target cells of KSHV. Although the role of ESCRT protein Hrs has been extensively studied with respect to endosomal movement and sorting of ubiquitinated proteins into lysosomes, its function in macropinocytosis is not known. In the present study, we demonstrate for the first time that upon KSHV infection, the endogenous Hrs localizes to the plasma membrane and the membrane-associated Hrs facilitates assembly of signaling molecules, macropinocytosis, and virus entry. Hrs recruits ROCK1 to the membrane, which is required for the activation of NHE1 and an increase in submembranous intracellular pH occurring during macropinocytosis. These studies demonstrate that the localization of Hrs from the cytosol to the plasma membrane is important for coupling membrane dynamics to the cytosolic signaling events during macropinocytosis of KSHV.
Subject(s)
Endosomal Sorting Complexes Required for Transport/physiology , Endothelium, Vascular/virology , Herpesvirus 8, Human/physiology , Phosphoproteins/physiology , Pinocytosis , Virus Internalization , Actinin/metabolism , Cell Line , Cell Membrane/virology , Dermis/blood supply , Dermis/virology , Endosomal Sorting Complexes Required for Transport/genetics , Gene Expression , Gene Knockdown Techniques , Humans , Microvessels/cytology , Microvessels/virology , Phosphoproteins/genetics , rho-Associated Kinases/metabolismABSTRACT
UNLABELLED: IFI16 (interferon gamma-inducible protein 16) recognizes nuclear episomal herpesvirus (Kaposi's sarcoma-associated herpesvirus [KSHV], Epstein-Barr virus [EBV], and herpes simplex virus 1 [HSV-1]) genomes and induces the inflammasome and interferon beta responses. It also acts as a lytic replication restriction factor and inhibits viral DNA replication (human cytomegalovirus [HCMV] and human papillomavirus [HPV]) and transcription (HSV-1, HCMV, and HPV) through epigenetic modifications of the viral genomes. To date, the role of IFI16 in the biology of latent viruses is not known. Here, we demonstrate that knockdown of IFI16 in the latently KSHV-infected B-lymphoma BCBL-1 and BC-3 cell lines results in lytic reactivation and increases in levels of KSHV lytic transcripts, proteins, and viral genome replication. Similar results were also observed during KSHV lytic cycle induction in TREX-BCBL-1 cells with the doxycycline-inducible lytic cycle switch replication and transcription activator (RTA) gene. Overexpression of IFI16 reduced lytic gene induction by the chemical agent 12-O-tetradecoylphorbol-13-acetate (TPA). IFI16 protein levels were significantly reduced or absent in TPA- or doxycycline-induced cells expressing lytic KSHV proteins. IFI16 is polyubiquitinated and degraded via the proteasomal pathway. The degradation of IFI16 was absent in phosphonoacetic acid-treated cells, which blocks KSHV DNA replication and, consequently, late lytic gene expression. Chromatin immunoprecipitation assays of BCBL-1 and BC-3 cells demonstrated that IFI16 binds to KSHV gene promoters. Uninfected epithelial SLK and osteosarcoma U2OS cells transfected with KSHV luciferase promoter constructs confirmed that IFI16 functions as a transcriptional repressor. These results reveal that KSHV utilizes the innate immune nuclear DNA sensor IFI16 to maintain its latency and repression of lytic transcripts, and a late lytic KSHV gene product(s) targets IFI16 for degradation during lytic reactivation. IMPORTANCE: Like all herpesviruses, latency is an integral part of the life cycle of Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent for many human cancers. Herpesviruses utilize viral and host factors to successfully evade the host immune system to maintain latency. Reactivation is a complex event where the latent episomal viral genome springs back to active transcription of lytic cycle genes. Our studies reveal that KSHV has evolved to utilize the innate immune sensor IFI16 to keep lytic cycle transcription in dormancy. We demonstrate that IFI16 binds to the lytic gene promoter, acts as a transcriptional repressor, and thereby helps to maintain latency. We also discovered that during the late stage of lytic replication, KSHV selectively degrades IFI16, thus relieving transcriptional repression. This is the first report to demonstrate the role of IFI16 in latency maintenance of a herpesvirus, and further understanding will lead to the development of strategies to eliminate latent infection.
Subject(s)
Herpesvirus 8, Human/physiology , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Virus Latency , Virus Replication , Cell Line, Tumor , Gene Expression , Gene Knockdown Techniques , Host-Pathogen Interactions , Humans , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , ProteolysisABSTRACT
The IL-1ß and type I interferon-ß (IFN-ß) molecules are important inflammatory cytokines elicited by the eukaryotic host as innate immune responses against invading pathogens and danger signals. Recently, a predominantly nuclear gamma-interferon-inducible protein 16 (IFI16) involved in transcriptional regulation has emerged as an innate DNA sensor which induced IL-1ß and IFN-ß production through inflammasome and STING activation, respectively. Herpesvirus (KSHV, EBV, and HSV-1) episomal dsDNA genome recognition by IFI16 leads to IFI16-ASC-procaspase-1 inflammasome association, cytoplasmic translocation and IL-1ß production. Independent of ASC, HSV-1 genome recognition results in IFI16 interaction with STING in the cytoplasm to induce interferon-ß production. However, the mechanisms of IFI16-inflammasome formation, cytoplasmic redistribution and STING activation are not known. Our studies here demonstrate that recognition of herpesvirus genomes in the nucleus by IFI16 leads into its interaction with histone acetyltransferase p300 and IFI16 acetylation resulting in IFI16-ASC interaction, inflammasome assembly, increased interaction with Ran-GTPase, cytoplasmic redistribution, caspase-1 activation, IL-1ß production, and interaction with STING which results in IRF-3 phosphorylation, nuclear pIRF-3 localization and interferon-ß production. ASC and STING knockdowns did not affect IFI16 acetylation indicating that this modification is upstream of inflammasome-assembly and STING-activation. Vaccinia virus replicating in the cytoplasm did not induce nuclear IFI16 acetylation and cytoplasmic translocation. IFI16 physically associates with KSHV and HSV-1 genomes as revealed by proximity ligation microscopy and chromatin-immunoprecipitation studies which is not hampered by the inhibition of acetylation, thus suggesting that acetylation of IFI16 is not required for its innate sensing of nuclear viral genomes. Collectively, these studies identify the increased nuclear acetylation of IFI16 as a dynamic essential post-genome recognition event in the nucleus that is common to the IFI16-mediated innate responses of inflammasome induction and IFN-ß production during herpesvirus (KSHV, EBV, HSV-1) infections.
Subject(s)
Herpesviridae Infections/metabolism , Immunity, Innate/immunology , Interferon-beta/biosynthesis , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Protein Transport/immunology , Acetylation , Blotting, Western , Cell Line , Cell Nucleus/metabolism , Chromatin Immunoprecipitation , Cytoplasm/immunology , Cytoplasm/metabolism , Herpesviridae Infections/immunology , Herpesvirus 1, Human/immunology , Herpesvirus 1, Human/metabolism , Humans , Immunoprecipitation , Inflammasomes/immunology , Inflammasomes/metabolism , Microscopy, Fluorescence , Polymerase Chain Reaction , RNA, Small Interfering , TransfectionABSTRACT
The innate immune system pattern recognition receptors (PRR) are the first line of host defenses recognizing the various pathogen- or danger-associated molecular patterns and eliciting defenses by regulating the production of pro-inflammatory cytokines such as IL-1ß, IL-18 or interferon ß (IFN-ß). NOD-like receptors (NLRs) and AIM2-like receptors (ALRs) are cytoplasmic inflammasome sensors of foreign molecules, including DNA. IFI16, a sequence-independent nuclear innate sensor ALR, recognizes episomal dsDNA genomes of herpes viruses such as KSHV, EBV, and HSV-1 in the infected cell nuclei, forms an inflammasome complex with ASC and procaspase1, and relocates into the cytoplasm leading into Caspase-1 and IL-1ß generation. IFI16 also induces IFN-ß during HSV-1 infection via the cytoplasmic STING-TBK1-IRF3 pathway. Thus far, whether IFI16 recognizes foreign DNA directly or utilizes other host protein(s) is unknown. Here, we demonstrate that BRCA1, a DNA damage repair sensor and transcription regulator, is in complex with IFI16 in the host cell nucleus, and their association increases in the presence of nuclear viral genomes during de novo KSHV, EBV and HSV-1 infection, and in latent KSHV or EBV infection, but not by DNA damage responses (DDR) induced by bleomycin and vaccinia virus cytoplasmic dsDNA. BRCA1 is a constituent of the triggered IFI16-inflammasome and is translocated into the cytoplasm after genome recognition along with the IFI16-inflammasome. The absence of BRCA1 abrogated IFI16-viral genome association, inflammasome assembly, IFI16 cytoplasmic localization, and Caspase-1 and IL-1ß production. The absence of BRCA1 also abolished the cytoplasmic IFI16-STING interaction, downstream IRF3 phosphorylation, nuclear translocation of pIRF3 and IFN-ß production during de novo KSHV and HSV-1 infection. These findings highlight that BRCA1 plays a hitherto unidentified innate immunomodulatory role by facilitating nuclear foreign DNA sensing by IFI16, subsequent assembly and cytoplasmic distribution of IFI16-inflammasomes leading into IL-1ß formation and the induction of IFN-ß via cytoplasmic signaling through IFI16-STING, TBK1 and IRF3.
Subject(s)
BRCA1 Protein/metabolism , DNA, Viral/genetics , Herpesvirus 1, Human/genetics , Inflammasomes/metabolism , Interferon-beta/metabolism , Nuclear Proteins/metabolism , Phosphoproteins/metabolism , Cell Line , DNA-Binding Proteins/metabolism , Humans , Signal Transduction/geneticsABSTRACT
Osteopontin (OPN) is a secreted phosphoprotein, originally characterized in malignant-transformed epithelial cells. OPN is associated with tumor metastasis of several tumors and is overexpressed in hepatocellular carcinoma (HCC) tissue involving HCC invasion and metastasis. Importantly, OPN is significantly up-regulated in liver injury, inflammation, and hepatitis C virus (HCV)-associated HCC. However, the underlying mechanisms of OPN activation and its role in HCV-mediated liver disease pathogenesis are not known. In this study, we investigated the mechanism of OPN activation in HCV-infected cells. We demonstrate that HCV-mediated Ca(2+) signaling, elevation of reactive oxygen species, and activation of cellular kinases such as p38 MAPK, JNK, PI3K, and MEK1/2 are involved in OPN activation. Incubation of HCV-infected cells with the inhibitors of AP-1 and Sp1 and site-directed mutagenesis of AP-1- and Sp1-binding sites on the OPN promoter suggest the critical role of AP-1 and Sp1 in OPN promoter activation. In addition, we show the in vivo interactions of AP-1 and Sp1 with the OPN promoter using chromatin immunoprecipitation assay. We also show the calpain-mediated processing of precursor OPN (â¼75 kDa) into â¼55-, â¼42-, and â¼36-kDa forms of OPN in HCV-infected cells. Furthermore, we demonstrate the critical role of HCV-induced OPN in increased phosphorylation of Akt and GSK-3ß followed by the activation of ß-catenin, which can lead to EMT of hepatocytes. Taken together, these studies provide an insight into the mechanisms of OPN activation that is relevant to the metastasis of HCV-associated HCC.
Subject(s)
Carcinoma, Hepatocellular/metabolism , Epithelial-Mesenchymal Transition , Hepatitis C/metabolism , Hepatocytes/metabolism , Liver Neoplasms/metabolism , Osteopontin/metabolism , Calcium Signaling/genetics , Calpain/genetics , Calpain/metabolism , Carcinoma, Hepatocellular/genetics , Carcinoma, Hepatocellular/pathology , Carcinoma, Hepatocellular/virology , Cell Line, Tumor , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Hepacivirus , Hepatitis C/genetics , Hepatitis C/pathology , Hepatocytes/pathology , Hepatocytes/virology , Humans , Liver Neoplasms/genetics , Liver Neoplasms/pathology , Liver Neoplasms/virology , Neoplasm Metastasis , Osteopontin/genetics , Protein Processing, Post-Translational/genetics , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolism , Sp1 Transcription Factor/genetics , Sp1 Transcription Factor/metabolism , Transcription Factor AP-1 , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
Chronic hepatitis C is a lethal blood-borne infection often associated with a number of pathologies such as insulin resistance and other metabolic abnormalities. Insulin is a key hormone that regulates the expression of metabolic pathways and favors homeostasis. In this study, we demonstrated the molecular mechanism of hepatitis C virus (HCV) nonstructural protein 5A (NS5A)-induced metabolic dysregulation. We showed that transient expression of HCV NS5A in human hepatoma cells increased lipid droplet formation through enhanced lipogenesis. We also showed increased transcriptional expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1α and diacylglycerol acyltransferase-1 (DGAT-1) in NS5A-expressing cells. On the other hand, there was significantly reduced transcriptional expression of microsomal triglyceride transfer protein (MTP) and peroxisome proliferator-activated receptor γ (PPARγ) in cells expressing HCV NS5A. Furthermore, increased gluconeogenic gene expression was observed in HCV-NS5A-expressing cells. In addition, it was also shown that HCV-NS5A-expressing hepatoma cells show serine phosphorylation of IRS-1, thereby hampering metabolic activity and contributing to insulin resistance. Therefore, this study reveals that HCV NS5A is involved in enhanced gluconeogenic and lipogenic gene expression, which triggers metabolic abnormality and impairs insulin signaling pathway.
Subject(s)
Gluconeogenesis/physiology , Hepacivirus/metabolism , Lipogenesis/physiology , Viral Nonstructural Proteins/physiology , Carcinoma, Hepatocellular/metabolism , Cell Line, Tumor , Gene Expression Regulation , Humans , Insulin Receptor Substrate Proteins/genetics , Insulin Receptor Substrate Proteins/metabolism , Insulin Resistance , Liver Neoplasms/metabolism , Signal TransductionABSTRACT
The study focused on detecting and characterizing microplastics in outdoor and indoor air in Ranchi, Jharkhand, India during post-monsoon (2022) and winter (2023). Stereo microscopic analysis showed that plastic fibres had a dominant presence, fragments were less abundant, whereas fewer films could be detected in indoor and outdoor air. The atmospheric deposition of microplastics outdoors observed 465 ± 27 particles/m2/day in PM10 and 12104 ± 665 and 13833 ± 1152 particles/m2/day in PM2.5 in quartz and PTFE, respectively during the post-monsoon months. During winter, microplastic deposition rates in PM10 samples were found to be 689 ± 52 particles/m2/day and 19789 ± 2957 and 30087 ± 13402 in quartz and PTFE particles/m2/day respectively in PM2.5. The mean deposition rate in indoor environment during post-monsoon was 8.3 × 104 and 1.03 × 105 particles/m2/day in winter. During the post-monsoon period in PM10, there were fibres from 7.7 to 40 µm and fragments from 2.3 µm to 8.6 µm. Indoor atmospheric microplastics, fibres ranged from 1.2 to 47 µm and fragments from 0.9 to 16 µm present respectively during the post-monsoon season. Fibres and fragment sizes witnessed during winter were 3.6-6.9 µm and 2.3-34 µm, respectively. Indoor air films measured in the range of 4.1-9.6 µm. Fourier transform infrared analysis showed that outdoor air contained polyethylene, polypropylene, Polystyrene, whereas indoor air had polyvinyl chloride. Polyethylene mainly was present in outdoor air, with lesser polypropylene and polystyrene than indoors, where polyvinyl chloride and polyethylene were in dominant proportions. Elemental mapping of outdoor and indoor air samples showed the presence of elements on the microplastics. The HYSPLIT models suggest that the particles predominantly were coming from North-West during the post-monsoon season. Principal component analysis indicated wind speed and direction influencing the abundance of microplastics. Microplastics concentration showed strong seasonal influence and potential to act as reservoir of contaminants.
Subject(s)
Air Pollutants , Air Pollution, Indoor , Air Pollution, Indoor/analysis , Air Pollutants/analysis , Microplastics/analysis , Plastics/analysis , Environmental Monitoring , Polypropylenes/analysis , Polystyrenes/analysis , Polyvinyl Chloride/analysis , Quartz , India , Polyethylene/analysis , Particulate Matter/analysis , PolytetrafluoroethyleneABSTRACT
The SARS-CoV-2 main protease Mpro, essential for viral replication is an important drug target. It plays a critical role in processing viral polyproteins necessary for viral replication assembly. One of the predominant SARS-CoV-2 Mpro mutations of Omicron variant is Pro132His. Structurally, this mutation site is located â¼22 Å away from the catalytic site. The solved crystal structure of this mutant in complex with inhibitors as well as its reported catalytic efficiency did not show any difference with respect to the wild type. Thus, the mutation was concluded to be non-allosteric. Based on microsecond long MD simulation of the Pro132His mutant and wild type, we show that Pro132His mutation affects the conformational equilibrium with more population of conformational substates having open catalytic site, modulated by the dynamics of the catalytic site entry loop, implying the allosteric nature of this mutation. The structural analysis indicates that rearrangement of hydrogen bonds between His132 and adjacent residues enhances the dynamics of the linker, which in turn is augmented by the inherent dynamic flexibility of the catalytic pocket entry site due to the presence of charged residues. The altered dynamics leading to loss of secondary structures corroborate well with the reported compromised thermal stability.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/genetics , Catalytic Domain , Molecular Dynamics Simulation , Protease Inhibitors/chemistry , Molecular Docking SimulationABSTRACT
Burning of fossil fuels in the form of coal or gasoline in thermal power plants, industries, and automobiles is a prime source of nitrogen dioxide (NO2), a major air pollutant causing health problems. In this paper, spatio-temporal unevenness of NO2 concentrations via both spaceborne Sentinel-5P and ground-based in situ data have been studied for the period of 2017-2021. Annual and seasonal distribution of TROPOMI-NO2 depict consistency over the Jharkhand region, highlighting six hotspot regions. As compared to 2019, a notable dip of 11% in the spatial annual average TROPOMI-NO2 was achieved in 2020, which were elevated again by 22% in 2021 as the lockdown gradually goes out of the picture. Among eight ground-monitoring stations, Tata and Golmuri stations always displayed a higher level of TROPOMI-NO2 ranges up to 15.2 ×1015molecules.cm-2 and 16.9 ×1015molecules.cm-2 respectively, as being located in the highly industrialised district of Jamshedpur. A big percentage reduction of up to 30% in TROPOMI-NO2 has been reported in Jharia and Bastacola stations in Dhanbad in the lockdown phase of 2020 compared to 2019. Good agreement between TROPOMI-NO2 and surface-NO2 has been achieved with R = 0.8 and R = 0.71 during winter and post-monsoon respectively. Among four meteorological parameters, TROPOMI-NO2 was majorly found to be influenced by precipitation, having R = 0.6-0.8 for almost all stations. More advanced satellite algorithms and ground-based data may be used to estimate NO2 in places where monitoring facilities are limited and thus can help in air pollution control policy.
Subject(s)
Air Pollutants , Air Pollution , COVID-19 , Humans , Air Pollution/analysis , Nitrogen Dioxide/analysis , Environmental Monitoring , Communicable Disease Control , Air Pollutants/analysisABSTRACT
The spatial coverage of PM2.5 monitoring is non-uniform across India due to the limited number of ground monitoring stations. Alternatively, Modern-Era Retrospective Analysis for Research and Applications, Version 2 (MERRA-2), is an atmospheric reanalysis data used for estimating PM2.5. MERRA-2 does not explicitly measure PM2.5 but rather follows an empirical model. MERRA-2 data were spatiotemporally collocated with ground observation for validation across India. Significant underestimation in MERRA-2 prediction of PM2.5 was observed over many monitoring stations ranging from -20 to 60 µg m-3. The utility of Machine Learning (ML) models to overcome this challenge was assessed. MERRA-2 aerosol and meteorological parameters were the input features used to train and test the individual ML models and compare them with the stacking technique. Initially, with 10% of randomly selected data, individual model performance was assessed to identify the best model. XGBoost (XGB) was the best model (r2 = 0.73) compared to Random Forest (RF) and LightGBM (LGBM). Stacking was then applied by keeping XGB as a meta-regressor. Stacked model results (r2 = 0.77) outperformed the best standalone estimate of XGB. Stacking technique was used to predict hourly and daily PM2.5 in different regions across India and each monitoring station. The eastern region exhibited the best hourly prediction (r2 = 0.80) and substantial reduction in Mean Bias (MB = -0.03 µg m-3), followed by the northern region (r2 = 0.63 and MB = -0.10 µg m-3), which showed better output due to the frequent observation of PM2.5 >100 µg m-3. Due to sparse data availability to train the ML models, the lowest performance was for the central region (r2 = 0.46 and MB = -0.60 µg m-3). Overall, India's PM2.5 prediction was good on an hourly basis compared to a daily basis using the ML stacking technique.
Subject(s)
Machine Learning , Meteorology , Retrospective Studies , India , Particulate MatterABSTRACT
AIM: To assess chest ultrasound (US) diagnostic accuracy in pneumothorax diagnosing. MATERIAL AND METHODS: Prospec-tive studies related to the US pneumothorax diagnostic accuracy in trauma patients were extensively searched from 2000 up to November 2020. The studies features and findings were gathered using a standardised form and the methodological quality of the investigations was evaluated using the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2). RESULTS: Twelve articles were finally chosen for quantitative analysis. The overall sensitivity of US scan in pneumothorax diagnosis was 89% (95%CI 86-91%). Specificity was 96% (95%CI 95-97%). The diagnostic odds ratio was 193.94 (59.009-637.40) at 95%CI, thus demonstrating high chest US accuracy in pneumothorax diagnosis. CONCLUSION: Despite the limitations of the included studies, this systematic review and meta-analysis concluded that chest US is a reliable method for diagnosing pneu-mothorax in traumatized patients.