Correlation of severity & clinical outcomes of COVID-19 with virus variants: A prospective, multicentre hospital network study.

BACKGROUND OBJECTIVES: The clinical course of COVID-19 and its prognosis are influenced by both viral and host factors. The objectives of this study were to develop a nationwide platform to investigate the molecular epidemiology of SARS-CoV-2 (Severe acute respiratory syndrome Corona virus 2) and correlate the severity and clinical outcomes of COVID-19 with virus variants. METHODS: A nationwide, longitudinal, prospective cohort study was conducted from September 2021 to December 2022 at 14 hospitals across the country that were linked to a viral sequencing laboratory under the Indian SARS-CoV-2 Genomics Consortium. All participants (18 yr and above) who attended the hospital with a suspicion of SARS-CoV-2 infection and tested positive by the reverse transcription-PCR method were included. The participant population consisted of both hospitalized as well as outpatients. Their clinical course and outcomes were studied prospectively. Nasopharyngeal samples collected were subjected to whole genome sequencing to detect SARS-CoV-2 variants. RESULTS: Of the 4972 participants enrolled, 3397 provided samples for viral sequencing and 2723 samples were successfully sequenced. From this, the evolution of virus variants of concern including Omicron subvariants which emerged over time was observed and the same reported here. The mean age of the study participants was 41 yr and overall 49.3 per cent were female. The common symptoms were fever and cough and 32.5 per cent had comorbidities. Infection with the Delta variant evidently increased the risk of severe COVID-19 (adjusted odds ratio: 2.53, 95% confidence interval: 1.52, 4.2), while Omicron was milder independent of vaccination status. The independent risk factors for mortality were age >65 yr, presence of comorbidities and no vaccination. INTERPRETATION CONCLUSIONS: The authors believe that this is a first-of-its-kind study in the country that provides real-time data of virus evolution from a pan-India network of hospitals closely linked to the genome sequencing laboratories. The severity of COVID-19 could be correlated with virus variants with Omicron being the milder variant.

A role for the cystathionine-ß-synthase /H2S axis in astrocyte dysfunction in the aging brain.

Astrocytic dysfunction is central to age-related neurodegenerative diseases. However, the mechanisms leading to astrocytic dysfunction are not well understood. We identify that among the diverse cellular constituents of the brain, murine and human astrocytes are enriched in the expression of CBS. Depleting CBS in astrocytes causes mitochondrial dysfunction, increases the production of reactive oxygen species (ROS) and decreases cellular bioenergetics that can be partially rescued by exogenous H2S supplementation or by re-expressing CBS. Conversely, the CBS/H2S axis, associated protein persulfidation and proliferation are decreased in astrocytes upon oxidative stress which can be rescued by exogenous H2S supplementation. Here we reveal that in the aging brain, the CBS/H2S axis is downregulated leading to decreased protein persulfidation, together augmenting oxidative stress. Our findings uncover an important protective role of the CBS/H2S axis in astrocytes that may be disrupted in the aged brain.

Pancreatic Ubap2 deletion regulates glucose tolerance, inflammation, and protection from cerulein-induced pancreatitis.

Ubiquitin-binding associated protein 2 (UBAP2) is reported to promote macropinocytosis and pancreatic adenocarcinoma (PDAC) growth, however, its role in normal pancreatic function remains unknown. We addressed this knowledge gap by generating UBAP2 knockout (U2KO) mice under a pancreas-specific Cre recombinase (Pdx1-Cre). Pancreatic architecture remained intact in U2KO animals, but they demonstrated slight glucose intolerance compared to controls. Upon cerulein challenge to induce pancreatitis, U2KO animals had reduced levels of several pancreatitis-relevant cytokines, amylase and lipase in the serum, reduced tissue damage, and lessened neutrophil infiltration into the pancreatic tissue. Mechanistically, cerulein-challenged U2KO animals revealed reduced NF-κB activation compared to controls. In vitro promoter binding studies confirmed the reduction of NF-κB binding to its target molecules supporting UBAP2 as a new regulator of inflammation in pancreatitis and may be exploited as a therapeutic target in future to inhibit pancreatitis.

High Persistence of Novel Polyfluoroalkyl Betaines in Aerobic Soils.

Some contemporary aqueous film-forming foams (AFFFs) contain n:3 and n:1:2 fluorotelomer betaines (FTBs), which are often detected at sites impacted by AFFFs. As new chemical replacements, little is known about their environmental fate. For the first time, we investigated the biotransformation potential of 5:3 and 5:1:2 FTBs and a commercial AFFF that mainly contains n:3 and n:1:2 FTBs (n = 5, 7, 9, 11, and 13). Although some polyfluoroalkyl compounds are precursors to perfluoroalkyl acids, 5:3 and 5:1:2 FTBs exhibited high persistence, with no significant changes even after 120 days of incubation. While the degradation of 5:3 FTB into suspected products such as fluorotelomer acids or perfluoroalkyl carboxylic acids (PFCAs) could not be conclusively confirmed, we did identify a potential biotransformation product, 5:3 fluorotelomer methylamine. Similarly, 5:1:2 FTB did not break down or produce short-chain hydrogen-substituted polyfluoroalkyl acids (n:2 H-FTCA), hydrogen-substituted PFCA (2H-PFCA), or any other products. Incubating the AFFF in four soils with differing properties and microbial communities resulted in 0.023-0.25 mol % PFCAs by day 120. Most of the products are believed to be derived from n:2 fluorotelomers, minor components of the AFFF. Therefore, the findings of the study cannot be fully explained by the current understanding of structure-biodegradability relationships.

The deubiquitinase USP10 protects pancreatic cancer cells from endoplasmic reticulum stress.

The ubiquitin-specific peptidase 10 (USP10) plays a context-specific, pro or anti-tumorigenic role in different malignancies. However, the role of USP10 in pancreatic cancer remains unclear. Our protein and RNA level analysis from archived specimens and public databases show that USP10 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and expression correlates with poor overall patient survival. Phenotypically, silencing USP10 decreased viability, clonal growth and invasive properties of pancreatic cancer cells. Mechanistically, silencing USP10 upregulated BiP and induced endoplasmic reticulum (ER) stress that led to an unfolded protein response (UPR) and upregulation of PERK, IRE1α. Decreased cell viability of USP10 silenced cells could be rescued by a chemical chaperone that promotes protein folding. Our studies suggest that USP10 by protecting pancreatic cancer cells from ER stress may support tumor progression.

KRCC1, a modulator of the DNA damage response.

The lysine-rich coiled-coil 1 (KRCC1) protein is overexpressed in multiple malignancies, including ovarian cancer, and overexpression correlates with poor overall survival. Despite a potential role in cancer progression, the biology of KRCC1 remains elusive. Here, we characterize the biology of KRCC1 and define its role in the DNA damage response and in cell cycle progression. We demonstrate that KRCC1 associates with the checkpoint kinase 1 (CHK1) upon DNA damage and regulates the CHK1-mediated checkpoint. KRCC1 facilitates RAD51 recombinase foci formation and augments homologous recombination repair. Furthermore, KRCC1 is required for proper S-phase progression and subsequent mitotic entry. Our findings uncover a novel component of the DNA damage response and a potential link between cell cycle, associated damage response and DNA repair.

Disabling partners in crime: Gold nanoparticles disrupt multicellular communications within the tumor microenvironment to inhibit ovarian tumor aggressiveness.

The tumor microenvironment (TME) plays a key role in the poor prognosis of many cancers. However, there is a knowledge gap concerning how multicellular communication among the critical players within the TME contributes to such poor outcomes. Using epithelial ovarian cancer (EOC) as a model, we show how crosstalk among cancer cells (CC), cancer associated fibroblasts (CAF), and endothelial cells (EC) promotes EOC growth. We demonstrate here that co-culturing CC with CAF and EC promotes CC proliferation, migration, and invasion in vitro and that co-implantation of the three cell types facilitates tumor growth in vivo. We further demonstrate that disruption of this multicellular crosstalk using a gold nanoparticle (GNP) inhibits these pro-tumorigenic phenotypes in vitro as well as tumor growth in vivo. Mechanistically, GNP treatment reduces expression of several tumor-promoting cytokines and growth factors, resulting in inhibition of MAPK and PI3K-AKT activation and epithelial-mesenchymal transition - three key oncogenic signaling pathways responsible for the aggressiveness of EOC. The current work highlights the importance of multicellular crosstalk within the TME and its role for the aggressive nature of EOC, and demonstrates the disruption of these multicellular communications by self-therapeutic GNP, thus providing new avenues to interrogate the crosstalk and identify key perpetrators responsible for poor prognosis of this intractable malignancy.

Gold Nanoparticles Disrupt the IGFBP2/mTOR/PTEN Axis to Inhibit Ovarian Cancer Growth.

By exploiting the self-therapeutic properties of gold nanoparticles (GNPs) a molecular axis that promotes the growth of high-grade serous ovarian cancer (HGSOC), one of the deadliest gynecologic malignancies with poorly understood underlying molecular mechanisms, has been identified. The biodistribution and toxicity of GNPs administered by intravenous or intraperitoneal injection, both as a single dose or by repeated dosing over two weeks are first assessed; no biochemical or histological toxicity to vital organs is found. Using an orthotopic patient-derived xenograft (PDX) model of HGSOC, the authors then show that GNP treatment robustly inhibits tumor growth. Investigating the molecular mechanisms underlying the GNP efficacy reveals that GNPs downregulate insulin growth factor binding protein 2 (IGFBP2) by disrupting its autoregulation via the IGFBP2/mTOR/PTEN axis. This mechanism is validated by treating a cell line-based human xenograft tumor with GNPs and an mTOR dual-kinase inhibitor (PI-103), either individually or in combination with GNPs; GNP and PI-103 combination therapy inhibit ovarian tumor growth similarly to GNPs alone. This report illustrates how the self-therapeutic properties of GNPs can be exploited as a discovery tool to identify a critical signaling axis responsible for poor prognosis in ovarian cancer and provides an opportunity to interrogate the axis to improve patient outcomes.

Characterization of the rare microbiome of rice paddy soil from arsenic contaminated hotspot of West Bengal and their interrelation with arsenic and other geochemical parameters.

Rare microbial taxa [bacterial and archaeal operational taxonomic units (OTUs) with mean relative abundance ≤ 0.001%] were critical for ecosystem function, yet, their identity and function remained incompletely understood, particularly in arsenic (As) contaminated rice soils. In the present study we have characterized the rare populations of the As-contaminated rice soil microbiomes from West Bengal (India) in terms of their identity, interaction and potential function. Major proportion of the OTUs (73% of total 38,289 OTUs) was represented by rare microbial taxa (henceforth mentioned as rare taxa), which covered 4.5-15.7% of the different communities. Taxonomic assignment of the rare taxa showed their affiliation to members of Gamma-, Alpha-, Delta- Proteobacteria, Actinobacteria, and Acidobacteria. SO42-, NO3-, NH4+and pH significantly impacted the distribution of rare taxa. Rare taxa positively correlated with As were found to be more frequent in relatively high As soil while the rare taxa negatively correlated with As were found to be more frequent in relatively low As soil. Co-occurrence-network analysis indicated that rare taxa whose abundance were correlated strongly (R > 0.8) with As also had strong association (R > 0.8) with PO42-, NO3-, and NH4+. Correlation analysis indicated that the rare taxa were likely to involved in two major guilds one, involved in N-metabolism and the second involved in As/Fe as well as other metabolisms. Role of the rare taxa in denitrification and dissimilatory NO3- reduction (DNRA), As biotransformation, S-, H-, C- and Fe-, metabolism was highlighted from 16S rRNA gene-based predictive analysis. Phylogenetic analysis of rare taxa indicated signatures of inhabitant rice soil microorganisms having significant roles in nitrogen (N) cycle and As-Fe metabolism. This study provided critical insights into the taxonomic identity, metabolic potentials and importance of the rare taxa in As biotransformation and biogeochemical cycling of essential nutrients in As-impacted rice soils.

Small Non-Coding-RNA in Gynecological Malignancies.

Gynecologic malignancies, which include cancers of the cervix, ovary, uterus, vulva, vagina, and fallopian tube, are among the leading causes of female mortality worldwide, with the most prevalent being endometrial, ovarian, and cervical cancer. Gynecologic malignancies are complex, heterogeneous diseases, and despite extensive research efforts, the molecular mechanisms underlying their development and pathology remain largely unclear. Currently, mechanistic and therapeutic research in cancer is largely focused on protein targets that are encoded by about 1% of the human genome. Our current understanding of 99% of the genome, which includes noncoding RNA, is limited. The discovery of tens of thousands of noncoding RNAs (ncRNAs), possessing either structural or regulatory functions, has fundamentally altered our understanding of genetics, physiology, pathophysiology, and disease treatment as they relate to gynecologic malignancies. In recent years, it has become clear that ncRNAs are relatively stable, and can serve as biomarkers for cancer diagnosis and prognosis, as well as guide therapy choices. Here we discuss the role of small non-coding RNAs, i.e., microRNAs (miRs), P-Element induced wimpy testis interacting (PIWI) RNAs (piRNAs), and tRNA-derived small RNAs in gynecological malignancies, specifically focusing on ovarian, endometrial, and cervical cancer.

Gold nanoparticles inhibit activation of cancer-associated fibroblasts by disrupting communication from tumor and microenvironmental cells.

Cancer-associated fibroblasts (CAFs) are a major constituent of the tumor microenvironment (TME) and an important contributor to cancer progression and therapeutic resistance. Regulation of CAF activation is a promising strategy to influence cancer outcomes. Here, we report that ovarian cancer cells (OCs) and TME cells promote the activation of ovarian CAFs, whereas gold nanoparticles (GNPs) of 20 nm in diameter inhibit the activation, as demonstrated by the changes in cell morphology, migration, and molecular markers. GNPs exert the effect by altering the levels of multiple fibroblast activation or inactivation proteins, such as TGF-ß1, PDGF, uPA and TSP1, secreted by OCs and TME cells. Thus, GNPs represent a potential tool to help understand multicellular communications existing in the TME as well as devise strategies to disrupt the communication.

Targeting the TGFß pathway in uterine carcinosarcoma.

Uterine carcinosarcoma (UCS) is a relatively infrequent, but extremely aggressive endometrial malignancy. Although surgery and chemotherapy have improved outcomes, overall survival (OS) remains dismal due to the lack of targeted therapy and biphasic (epithelial and mesenchymal) nature that renders the tumor aggressive and difficult to manage. Here we report a role of transforming growth factor-ß (TGFß) in maintaining epithelial to mesenchymal transition (EMT) phenotype and aggressiveness in UCS. Using a 3D-culture system, we evaluated the efficacy of the transforming growth factor-ß receptor-I (TGFßR1) kinase inhibitor Galunisertib (GLT), alone and in combination with standard chemotherapeutic drugs used for the management of UCS. We demonstrate that GLT by inhibiting canonical and non-canonical signaling emanating from transforming growth factor-ß1 (TGFß1) reduces cellular viability, invasion, clonal growth and differentiation. Interestingly, GLT sensitizes UCS cells to chemotherapy both in vitro and in in vivo preclinical tumor model. Hence, targeting TGFß signaling, in combination with standard chemotherapy, may be exploited as an important strategy to manage the clinically challenging UCS.

Cystathione ß-synthase regulates HIF-1α stability through persulfidation of PHD2.

The stringent expression of the hypoxia inducible factor-1α (HIF-1α) is critical to a variety of pathophysiological conditions. We reveal that, in normoxia, enzymatic action of cystathionine ß-synthase (CBS) produces H2S, which persulfidates prolyl hydroxylase 2 (PHD2) at residues Cys21 and Cys33 (zinc finger motif), augmenting prolyl hydroxylase activity. Depleting endogenous H2S either by hypoxia or by inhibiting CBS via chemical or genetic means reduces persulfidation of PHD2 and inhibits activity, preventing hydroxylation of HIF-1α, resulting in stabilization. Our in vitro findings are further supported by the depletion of CBS in the zebrafish model that exhibits axis defects and abnormal intersegmental vessels. Exogenous H2S supplementation rescues both in vitro and in vivo phenotypes. We have identified the persulfidated residues and defined their functional significance in regulating the activity of PHD2 via point mutations. Thus, the CBS/H2S/PHD2 axis may provide therapeutic opportunities for pathologies associated with HIF-1α dysregulation in chronic diseases.

MicroRNA-195 controls MICU1 expression and tumor growth in ovarian cancer.

MICU1 is a mitochondrial inner membrane protein that inhibits mitochondrial calcium entry; elevated MICU1 expression is characteristic of many cancers, including ovarian cancer. MICU1 induces both glycolysis and chemoresistance and is associated with poor clinical outcomes. However, there are currently no available interventions to normalize aberrant MICU1 expression. Here, we demonstrate that microRNA-195-5p (miR-195) directly targets the 3' UTR of the MICU1 mRNA and represses MICU1 expression. Additionally, miR-195 is under-expressed in ovarian cancer cell lines, and restoring miR-195 expression reestablishes native MICU1 levels and the associated phenotypes. Stable expression of miR-195 in a human xenograft model of ovarian cancer significantly reduces tumor growth, increases tumor doubling times, and enhances overall survival. In conclusion, miR-195 controls MICU1 levels in ovarian cancer and could be exploited to normalize aberrant MICU1 expression, thus reversing both glycolysis and chemoresistance and consequently improving patient outcomes.

Ubiquitin-binding associated protein 2 regulates KRAS activation and macropinocytosis in pancreatic cancer.

Macropinocytosis supports the metabolic requirement of RAS-transformed pancreatic ductal adenocarcinoma cells (PDACs). However, regulators of RAS-transformation (activation) that lead to macropinocytosis have not been identified. Herein, we report that UBAP2 (ubiquitin-binding associated protein 2), regulates the activation of KRAS and macropinocytosis in pancreatic cancer. We demonstrate that UBAP2 is highly expressed in both pancreatic cancer cell lines and tumor tissues of PDAC patients. The expression of UBAP2 is associated with poor overall survival in several cancers, including PDAC. Silencing UBAP2 decreases the levels of activated KRAS, and inhibits macropinocytosis, and tumor growth in vivo. Using a UBAP2-deletion construct, we demonstrate that the UBA-domain of UBAP2 is critical for the regulation of macropinocytosis and maintaining the levels of activated KRAS. In addition, UBAP2 regulates RAS downstream signaling and helps maintain RAS in the GTP-bound form. However, the exact mechanism by which UBAP2 regulates KRAS activation is unknown and needs further investigation. Thus, UBAP2 may be exploited as a potential therapeutic target to inhibit macropinocytosis and tumor growth in activated KRAS-driven cancers.

Cystathionine beta synthase regulates mitochondrial dynamics and function in endothelial cells.

Mutations in the human cystathionine beta synthase (CBS) gene are known to cause endothelial dysfunction responsible for cardiovascular and neurovascular diseases. CBS is the predominant hydrogen sulfide (H2 S)-producing enzyme in endothelial cells (ECs). Recently, H2 S was shown to attenuate ROS and improve mitochondrial function. Mitochondria are metabolic organelles that actively transform their ultrastructure to mediate their function. Therefore, we questioned whether perturbation of CBS/H2 S activity could drive mitochondrial dysfunction via mitochondrial dynamics in ECs. Here we demonstrate that silencing CBS induces mitochondria fragmentation, attenuates efficient oxidative phosphorylation, and decreases EC function. Mechanistically, CBS silencing significantly elevates ROS production, thereby leading to reduced mitofusin 2 (MFN2) expression, decouple endoplasmic reticulum-mitochondria contacts, increased mitochondria fission, enhanced receptor-mediated mitophagy, and increased EC death. These defects were significantly rescued by the treatment of H2 S donors. Taken together our data highlights a novel signaling axis that mechanistically links CBS with mitochondrial function and ER-mitochondrial tethering and could be considered as a new therapeutic approach for the intervention of EC dysfunction-related pathologies.

KRCC1: A potential therapeutic target in ovarian cancer.

Using a systems biology approach to prioritize potential points of intervention in ovarian cancer, we identified the lysine rich coiled-coil 1 (KRCC1), as a potential target. High-grade serous ovarian cancer patient tumors and cells express significantly higher levels of KRCC1 which correlates with poor overall survival and chemoresistance. We demonstrate that KRCC1 is predominantly present in the chromatin-bound nuclear fraction, interacts with HDAC1, HDAC2, and with the serine-threonine phosphatase PP1CC. Silencing KRCC1 inhibits cellular plasticity, invasive properties, and potentiates apoptosis resulting in reduced tumor growth. These phenotypes are associated with increased acetylation of histones and with increased phosphorylation of H2AX and CHK1, suggesting the modulation of transcription and DNA damage that may be mediated by the action of HDAC and PP1CC, respectively. Hence, we address an urgent need to develop new targets in cancer.

Biodegradation of Unpretreated Low-Density Polyethylene (LDPE) by Stenotrophomonas sp. and Achromobacter sp., Isolated From Waste Dumpsite and Drilling Fluid.

Exploring the catabolic repertoire of natural bacteria for biodegradation of plastics is one of the priority areas of biotechnology research. Low Density Polyethylene (LDPE) is recalcitrant and poses serious threats to our environment. The present study explored the LDPE biodegradation potential of aerobic bacteria enriched from municipal waste dumpsite and bentonite based drilling fluids from a deep subsurface drilling operation. Considerable bacterial growth coupled with significant weight loss of the LDPE beads (∼8%), change in pH to acidic condition and biofilm cell growth around the beads (CFU count 105-106/cm2) were noted for two samples (P and DF2). The enriched microbial consortia thus obtained displayed high (65-90%) cell surface hydrophobicity, confirming their potential toward LDPE adhesion as well as biofilm formation. Two LDPE degrading bacterial strains affiliated to Stenotrophomonas sp. and Achromobacter sp. were isolated as pure culture from P and DF2 enrichments. 16S rRNA gene sequences of these isolates indicated their taxonomic novelty. Further biodegradation studies provided strong evidence toward the LDPE metabolizing ability of these two organisms. Atomic Fore Microscopy (AFM) and Scanning Electron Microscopy (SEM) revealed considerable damage (in terms of formation of cracks, grooves, etc.) on the micrometric surface of the LDPE film. Analysis of the average roughness (Ra), root mean square roughness (Rq), average height (Rz), maximum peak height (Rp), and maximum valley depth (Rv) (nano-roughness parameters) through AFM indicated 2-3 fold increase in nano-roughness of the LDPE film. FTIR analysis suggested incorporation of alkoxy (1000-1090 cm-1), acyl (1220 cm-1), nitro (1500-1600 cm-1), carbonyl (1720 cm-1) groups into the carbon backbone, formation of N-O stretching (1360 cm-1) and chain scission (905 cm-1) in the microbially treated LDPEs. Increase in carbonyl index (15-20 fold), double bond index (1.5-2 fold) and terminal double bond index (30-40 fold) confirmed that biodegraded LDPEs had undergone oxidation, vinylene formation and chain scission. The data suggested that oxidation and dehydrogenation could be the key steps allowing formation of low molecular weight products suitable for their further mineralization by the test bacteria. The study highlighted LDPE degrading ability of natural bacteria and provided the opportunity for their development in plastic remediation process.

Gold Nanoparticle Transforms Activated Cancer-Associated Fibroblasts to Quiescence.

Activated cancer-associated fibroblasts (CAFs) play a major role in the poor outcome in many diseases including pancreatic cancer. Normally quiescent with high lipid content and low proliferative capacity, CAFs receiving cues from cancer cells in the tumor microenvironment become activated and transformed into a lipid-deprived and highly proliferative myofibroblast type phenotype. Therefore, reversal of activated fibroblasts to the quiescence state is an important area of investigation that may help the therapeutic management of a number of diseases including pancreatic cancer. Here, we describe a unique biological function of gold nanoparticles (GNPs) and demonstrate that GNPs may be used to transform activated CAFs to quiescence and provide insights into the underlying molecular mechanisms. Using immortalized and primary patient derived CAFs, we demonstrate that GNPs enhanced lipid content in the cells by inducing expression of lipogenesis genes such as FASN, SREBP2, and FABP3. Using pharmacological inhibitors of lipolysis, lipophagy, and fatty acid oxidation, we further demonstrate that CAFs utilized a GNP-induced endogenously synthesized lipid to maintain the quiescent phenotype. Consequently, treatment with GNP sensitizes CAF to FASN inhibitor or FASN siRNA. Hence, GNPs may be used as a tool to probe mechanisms of quiescence in CAFs and help device strategies to target the stromal compartment exploiting the mechanisms of lipid utilization.