Biology subjects

Sarin, S. K.

Publications and source records attributed to Sarin, S. K..

13 recordsLinked to original sources

bioRxiv2026

Phytometabolite-Enriched Edible Plant-Derived Extracellular Vesicles Exhibit Source-Specific Bioactives with Distinct Pharmacological Potential

Edible plant derived extracellular vesicles (PDEVs) are emerging as biocompatible, orally deliverable nanocarriers with therapeutic potential; however, their phytometabolite cargo, gastrointestinal stability, and source specific biological functions remain poorly characterized. Here, PDEVs were isolated from four phytochemically distinct plant based foods (black carrot, ginger, garlic, and turmeric), selected for their diverse bioactivity, and characterized by transmission electron microscopy, nanoparticle tracking analysis, and zeta potential. Gastrointestinal stability was evaluated in simulated digestion model. Source specific phytometabolites were profiled by untargeted LC MS MS metabolomics. Functionally validated in ammonia stressed epithelial cells and steatotic hepatocytes. PDEVs exhibited characteristic cup shaped morphology with particle sizes ranging from 60 to 214 nm and zeta potentials of -6.0 to -49.0 mV. PDEVs retained colloidal stability, supporting their suitability for oral delivery. We identified 572 phytometabolites with distinct source specific signatures, including lignin and quercetin in carrot EVs, [6] gingerol and silymarin in ginger EVs, diosgenin in garlic EVs, and curcumin in turmeric EVs. These metabolites found associated to antioxidant, anti inflammatory, epithelial barrier, lipid metabolic, and apoptotic pathways. Functional validation demonstrated carrot EVs significantly enhanced epithelial barrier integrity by increasing claudin (>8-fold, p<0.05), occludin (>2-fold, p<0.05). Ginger EVs restored ZO 1 while suppressing cyclin D1 and MMP9(p<0.05). Garlic and turmeric EVs attenuated inflammatory signaling by reducing STAT3, AKT1, and TNF , whereas turmeric EVs additionally decreased caspase 3 and PTGS2(p<0.01). In steatotic hepatocytes, garlic EVs significantly reduced PNPLA3 (p<0.001) and SREBP 1c while increasing PPAR- (p=0.002). Hence, our results indicate that edible PDEVs are gastrointestinally stable, phytometabolite enriched nanocarriers with distinct source specific functional properties, supporting their potential as orally deliverable nutraceuticals for improving gut liver functions.

pharmacology and toxicology↗

bioRxiv2025

Increased versican and fibrosis in mesenteric lymph nodes disrupts immune surveillance and drives systemic bacterial dissemination in cirrhosis

Background and ObjectiveMesenteric lymph nodes (MLN) are immunological barriers against bacterial translocation (BT). Enhanced gut BT through MLN facilitates bacterial spread and higher mortality in cirrhosis. We aimed to elucidate mechanisms underlying MLN failure to effectively contain BT during advanced cirrhosis. DesignBT and immune cells were analyzed in lymphoid organs and circulation of control and CCl4 models, with and without MLN (MLNx). MLN proteomics identified versican (VCAN) as major upregulated protein in cirrhosis, whose immunomodulatory function was examined in vitro and in vivo in CCl and (Bile duct ligation) BDL models. Plasma VCAN were measured in end-stage cirrhosis patients and analyzed as mortality predictor. ResultsIn control rats, bacteria were confined to MLN, whereas cirrhotics showed BT to MLN, lymph, and portal blood. Compared to control, CCl4 rats had increased activated Th-cells in MLN but reduced in circulation. In control-MLNx rats, activated Th-cells were reduced in circulation vs controls. In BDL models, MLN CFU correlated with VCAN level. In vitro, VCAN enhanced T cell suppression and impaired migration which was reversed by CD44 blockade. In vivo VCAN knockdown reduced fibrosis and bacterial burden in MLN, while restoring Th-cell activation locally and systemically. Clinically, plasma VCAN levels were elevated in advanced cirrhosis patients and remained an independent predictor of 28-day sepsis-related mortality. ConclusionIncreased VCAN impairs T cell activation and migration in MLN, fostering immune suppression and bacterial persistence. Plasma VCAN levels serve as promising biomarker for MLN dysfunction and prognostic factor for predicting sepsis-related mortality in end-stage cirrhosis. What is already known on this topic - Enhanced gut bacterial translocation through mes-enteric lymph nodes (MLN) facilitates systemic bacterial spread and increases mortality in cirrhosis. The mechanisms underlying MLN failure to effectively contain bacterial spread during advanced cirrhosis remain largely unknown. What this study adds - The study unveils a critical role of lymph node fibrosis and elevated versican (VCAN) expression in causing deranged immune responses and bacterial clearance in MLN, increasing systemic bacterial load and immunosuppression. Most importantly, high plasma VCAN emerges as a prognostic biomarker for functional failure of MLN and 28-day mortality predictor in critically ill patients with cirrhosis. How this study might affect research, practice or policy - VCAN, representing enhanced MLN fibrosis and dysfunction, emerges as a predictive biomarker of adverse clinical out-comes in patients with advanced cirrhosis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/690350v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@369f4eorg.highwire.dtl.DTLVardef@14c232org.highwire.dtl.DTLVardef@d99b21org.highwire.dtl.DTLVardef@57ac95_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2025

Wnt7a is a Novel Lymphangiocrine Factor Driving Cholangiocyte Proliferation during Liver Regeneration

The significance of lymphatic vessels (LVs) and lymphatic endothelial cells (LyECs) during liver regeneration remains unexplored. We aimed to characterize contribution of LVs and LyECs in liver regeneration using two-thirds partial hepatectomy (PHx) models. An increased number and diameter of CD31+PDPN+ LVs was observed at day 2 and 5 post-PHx. Proteomic analysis of LyECs and ELISA studies in conditioned media of LyECs revealed that one of the secreted proteins, Wnt7a was uniquely expressed at day 2 post PHx. In vitro studies confirmed that Wnt7a is a potent stimulator of proliferation for both hepatocytes and cholangiocytes. In vivo inhibition of lymphangiogenesis and reduction in Wnt7a led to a decline in the percentage of CK19+ cholangiocytes and bile ducts. Western blot analysis showed that Wnt7a activated the downstream Frizzled 7 receptor and non-canonical planar-cell-polarity pathway to drive the proliferation of cholangiocytes. In vivo, Wnt7a loss-of-function experiments in zebrafish along with inhibitor studies in rat models of PHx further re-iterated clear links between Wnt7a and proliferation of bile ducts. Pro-regenerative effects of Wnt7a were demonstrated in a model of small-for-size syndrome (80% PHx), where administering recombinant Wnt7a enhanced both cholangiocyte and hepatocyte proliferation. Elevated Wnt7a levels were seen in donors of liver transplant patients at day 1 and 2 post-hepatectomy and Wnt7a also facilitated growth of human cholangiocyte organoids in vitro. Our findings uncover the novel role of Wnt7 as one of the key lymphangiocrine signals that facilitates proliferation of cholangiocytes during liver regeneration. HighlightsO_LINumber of functional lymphatic Vessels (LVs) and lymphangiogenesis is enhanced between day 2 and day 5 after partial hepatectomy (PHx) in rat models and liver transplant donors. C_LIO_LIWnt7a released from lymphatic endothelial cells (LyECs) acts as a potent lymphangiocrine factor, stimulating proliferation of hepatocytes and cholangiocytes in vitro, across both rodent and human models. C_LIO_LIInhibition of lymphagiogenesis during PHx transiently reduces number of cholangiocytes during early liver regeneration. C_LIO_LIWnt7a signals through Frizzled 7 receptor to activate p-AKT, p-JAK1 and p-STAT3 proteins in cholangiocytes. C_LIO_LIAdministration of recombinant Wnt7a in 80% PHx models induces proliferation of hepatocytes and bile ducts. C_LI

physiology↗

bioRxiv2025

Microbial Scl1 Activates TGFBR1 receptor kinase signalling to Drive Fibrosis-Inflammation Axis in Alcohol-Associated Liver Disease

Background and AimsAlcohol consumption alters gut microbiota, which can affect metabolism, immune regulation, and signalling pathways and lead to alcohol-associated liver disease (ALD). We investigated how alcohol-associated gut microbiota (AGMs) modulate liver kinome signalling to drive inflammation and fibrosis in ALD. MethodLiver kinome and metaproteome changes were studied in rats (n=6/group) colonized with stool of severe alcohol-related hepatitis (SAH) patients (SAH[->]healthy-rats) or healthy human donors (HD[->]ALD-rats). AGM-associated liver kinome changes were cross-correlated with bacterial genera. A bacterial protein; Streptococcal collagen-like protein 1 (Scl1) was identified with affinity for TGF{beta}R1. This was validated by molecular docking and immunoprecipitation-LCMS assay. Expression of Streptococcus pneumonia and pyogenes was done in stool of SAH patients (n=10). Also, Scl1 was quantified in patient stool (n=24) and liver tissue (n=19) samples. Validation was performed by assessing Scl1 level in plasma and Streptococcus level in stool samples before and after fecal microbiota transplantation (FMT) in SAH patients. ResultsStool metaproteomics showed significant increase in 10 bacterial genera (Streptococcus, Staphylococcus, and Clostridium) in SAH[->]Healthy-rats, mirroring changes seen in ALD-rats (FC>1.5, p<0.05). Clusters of Orthologous Groups analysis indicated increased post-translational modifications (PTMs) and decreased lipid metabolism in ALD-rats and SAH[->]Healthy-rats. Liver kinome profiling showed 85 upregulated kinases in SAH[->]Healthy-rats, with 34 overlapping with ALD-rats, associated with inflammation (Mapk14, Map3k10 and others), fibrosis (Tgfbr1, Igf1r, and others), lipid metabolism (Cdk14, Cdk18) and regeneration (Met, Bmpr1a and others). FMT from healthy human-donors to ALD-rats reversed the expression of Streptococcus (10-fold), Staphylococcus (3-fold), and Clostridium (5-fold), along with 18 kinases associated with inflammation (Btk, Camk4, Nuak1) and fibrosis (Tgfbr1, Col4a1, Fgfr2 and others). Strong association (r{superscript 2}>0.9, p<0.05) was seen between Streptococcus abundance and fibrosis and inflammation-related kinases. Level of Scl1 was significantly high in SAH and ALD-rat stool samples (FC>2, p<0.05). It showed strong affinity for TGF{beta}R1, as validated by molecular docking (>86%confidence) and immunoprecipitation assays (>100FC,p<0.05), indicating it as a potential ligand for TGF{beta}R1. Concordantly, expression of Scl1 was highest amongst all the known ligands for TGF{beta}R1 (p<0.05) suggesting Scl1 is a major contributor for TGF{beta}R1 activation and its downstream signalling in these patients. Following fecal microbiota transplantation, expression of Scl1 in plasma and Streptococcus levels in stool were significantly reduced by 2.1-folds and 1.9 folds respectively in SAH patients. Further, Scl1 levels SAH plasma is capable of predicting poor therapeutic response and 30-day mortality with high accuracy (AUC=0.92, cutoff >70 normalized abundance). ConclusionAlcohol induced gut dysbiosis alters the liver kinome, promoting inflammation, fibrosis, and lipid dysregulation. Streptococcal Scl1 is identified as a bacterial protein mimicking human collagen and capable of activating fibrotic signalling pathway through TGF{beta}R1in liver and is capable of predicting poor response in SAH. FMT from healthy donor restores microbial imbalance and harmful microbial-host interactions.

microbiology↗

bioRxiv2025

Liver Kinome Profiling Identifies PS1145 as a Potential Therapeutic molecule for amelioration of Systemic Inflammation in Alcohol-related Liver Disease

Background and aimsAlcohol-related liver disease (ALD) has high mortality due to systemic inflammation. We analysed liver and monocyte kinome profiles in a chronic ethanol-fed pre-clinical rat model to identify therapeutic targets that could mitigate inflammation in ALD. MethodKinome profile was performed in liver and circulating monocytes at baseline, and after 8,12,16,20 and 24-weeks of 40% ethanol administration. Pathway-specific inhibitors, including PS1145 (IKK-phosphorylation inhibitor), PH-797804 (MAPK14 inhibitor), resveratrol and prednisolone were tested for their anti-inflammatory effects in ALD-rats, PBMC from patients and NIAAA mouse model. ResultsKinome profiling identified 497 liver and 345 monocyte kinases in ALD rats (FDR<0.01). A time-dependent increase in MAPK14-associated kinases was observed in both tissues (FC>1.5, p<0.05). By 24 weeks, 172 liver and 48 monocyte kinases were significantly upregulated, particularly those linked to MyD88-TLR4, PI3K-Akt, TNF, TGF{beta}, and cellular senescence pathways. Key contributors included TGF{beta}R1, ROS-generating kinases, and IL-1-driven MAPK14 and IKK phosphorylation. Targeting this axis, PS1145 (an IKK inhibitor) suppressed NF{kappa}B activation and inflammation in THP1 and HepG2 cells, as well as PBMCs from healthy and SAH patients, outperforming PH797804, resveratrol, and prednisolone (p<0.05, FC>1.5). PS1145 significantly reduced IL-6, TNF, and NF{kappa}B, while increasing IL-10. In vivo, PS1145 treatment in the NIAAA mouse model markedly reduced hepatic steatosis, cellular stress and inflammatory pathways (cytokine signalling, IL-36 signalling and others) more effectively than standard therapies, Notably, it also downregulated IL36R, impairing TLR receptor dimerization, suggesting a dual mechanism of action (p<0.05) highlighting its therapeutic potential in ameliorating systemic inflammation in ALD. ConclusionOur study highlights the key role of MAPK14 and MYD88-TLR4 pathway kinases in driving systemic inflammation in SAH. The IKK inhibitor PS1145, by blocking both IKK phosphorylation and TLR dimerization, effectively suppresses inflammatory signalling and improves liver pathology, positioning it as a promising targeted therapy for ALD.

pharmacology and toxicology↗

bioRxiv2025

Metabolomic Profiling of Ashwagandha (Withania somnifera): Linking Bioactive Compounds to Functional Therapeutic Roles

IntroductionWithania somnifera (Ashwagandha) is a widely used medicinal plant with established roles in traditional systems of medicine. Known for diverse pharmacological effects, including neuroprotection, hepatoprotection, enhancement of male fertility, and anti-inflammatory activity. Despite its longstanding use, a deeper understanding of its phytochemical composition and bioactive mechanisms is required to bridge traditional knowledge with modern therapeutic applications. MethodsA comprehensive metabolomics profiling of Withania somnifera was performed to identify and quantify its bioactive secondary metabolites. Focus was placed on major classes such as alkaloids, phenolic compounds, and terpenoids. Known compounds were annotated and categorized based on their abundance, chemical class, and reported biological functions. Particular attention was given to compounds implicated in neuroprotection, liver health, reproductive support, and inflammation-modulation. ResultsThe analysis revealed a rich but low-abundance of known metabolites. Alkaloids such as Gentianaine (8.44%) and Sauroxine (2.94%), and phenolics like Mulberrofuran A (14.58%) and Sulphuretin (9.61%), were among the most prominent. These compounds are associated with antioxidative, anti-inflammatory, and neuroregenerative activities. Terpenoids including Pinocarvone and Ergosterol contributed to hepatoprotective and anti-inflammatory properties. Compounds such as Gomphrenin-I, Sitosterol, Pfaffic acid, beta-Carotene and others (>3%) supported spermatogenesis, while Embelin and Kievitone enhanced oxidative stress response. Although most identified compounds were individually present in low percentages (<0.30%), their collective activity suggests synergistic effects. ConclusionMetabolomic analysis of Withania somnifera confirms high abundance of bioactive secondary metabolites that have hepatoprotective, neuroprotective, anti-oxidant and spermatogenesis-enhancing effects. These findings justify protective and beneficial effect of Withania in promoting stress relief, energy, and overall wellness. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/659832v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1ae8e5forg.highwire.dtl.DTLVardef@105300corg.highwire.dtl.DTLVardef@1958320org.highwire.dtl.DTLVardef@d3ba13_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Chenodeoxycholic Acid Modulation via Bacteroides intestinalis AM1 underscores a Novel Approach in Acute Liver Failure

BackgroundAcute liver failure (ALF) is associated with rapid and progressive hepatocellular injury, and severe metabolic-microbial derangements. We investigated early metabolic markers of non-survival, and a potential microbial intervention using Bacteroides intestinalis-AM1, to improve outcomes in ALF. MethodPlasma metabolomics and meta-proteomics were performed in 40 ALF patients and 5 healthy controls (training cohort). A non-survival marker panel was identified and validated in 270 ALF patients (test cohort) using high resolution mass spectrometry and machine learning. It was functionally validated in acetaminophen-induced ALF mouse model. B. intestinalis-AM1 was used to study alteration of gut bacteria and amelioration of liver injury. ResultsALF non-survivors showed a distinct metabolomic signature with elevated primary bile acids {chenodeoxycholic acid (CDCA), cholic acid (CA)}, tryptophan, tyrosine, and enrichment of pathways linked to inflammation, cell death, and stress response (p<0.01, FDR<0.01, FC>1.5). Non-survivors had higher alpha/beta diversity (p<0.05) with increase in Proteobacteria, Firmicutes, Actinobacteria (p<0.05); functionally associated with energy, amino acid and xenobiotic metabolism (p<0.05). A gut microbiota derangement in converting primary to secondary bile acids was evident as CDCA and cytotoxic metabolites (4-(2-Amino phenyl)-2,4-dioxobutanoate, L-Tyrosine) were higher. Elevated CDCA (logFC>10) levels correlated with mortality in ALF patients as well as in mouse model. In the later, administration of B. intestinalis-AM1 bacteria, (10^9) reduced CDCA and CA levels by enhancing FXR, FGF15, SLC10A1 gene expression, attenuating inflammation (IL-1beta, TLR4-signalling), necroptosis, and modulating glutathione(oxidative-repair), tryptophan(inflammation), and histidine (tissue repair) metabolism. ConclusionHigh levels of chenodeoxycholic acid (CDCA) represent a poor prognostic indicator in ALF patients. B. intestinalis-AM1, a primary-to-secondary bile acid converter, effectively reduce CDCA levels, activated FXR, reduced inflammation and protected hepatocytes, highlighting its therapeutic potential in ALF.

microbiology↗

bioRxiv2023

Epigenetic mediated functional reprogramming of immune cells leads to HBsAg seroconversion in Hepatitis B Virus Reactivation patients

BackgroundHepatitis B virus (HBV) modulates epigenetic landscape by epigenetic regulators. HBsAg seroconversion is possible with immune activation, therefore we aimed to investigate epigenetic modulation in HBV reactivation (rHBV) for viral clearance and seroconversion. MethodsSixteen retrospectively collected rHBV patients [Seroconverters (SC, n=7, HBsAg loss and anti-HBs>10 IU/ml), non- seroconverters (NSC, n=9)], chronic hepatitis B treatment naive (nCHBV, n=7) patients and healthy controls (HC, n=7) were included in this study. Genome methylation, gene expression, plasma-cytokines, and immune cell profiling was analysed by Reduced Representation Bisulfite Sequencing (RRBS), QRT-PCR, multiplex-cytokine-bead array and flow-cytometry. ResultsrHBV patients having high HBV DNA and ALT showed epigenetic remodellers; KDM2B, NCOR2 and GATA6, immune and metabolic genes; TGF-{beta}, IL-6, IRF8, RPTOR, HK3 significantly (p<0.05) hypomethylated at specific CpG islands compared to nCHBV. TOX was hypomethylated in nCHBV suggesting immune-exhaustion. At-baseline, seroconverters showed hypomethylation of KDM2B, COX19, IRF8, TLR5 and hypermethylation of LAG3 compared to non-seroconverters. Further, in seroconverters at week-24, IL17RA, IFN-{gamma}, TGF-{beta}, and STAT5B (p<0.05) were additionally hypomethylated at specific CpG islands suggesting immune activation. Cytokine-bead analysis revealed increased IL-6 (p=0.009) and decreased LAG3 plasma levels (p=0.01) also imply on significantly differentiated HBV specific CD8, Tfh and Th1/17 cells in seroconverters at baseline and week-24. However, both nCHBV and non-seroconverters had consistent hypomethylation of LAG3 and TOX, which leads to immune exhaustion. ConclusionIn rHBV, seroconversion is driven by position specific CpG islands methylation in epigenetic remodellers, immune and metabolic genes. Immune metabolic reprograming is reflected by Th1/17 differentiation, extensive interleukin production for HBsAg seroconversion. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/554133v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@48e206org.highwire.dtl.DTLVardef@ef0fa7org.highwire.dtl.DTLVardef@ecb710org.highwire.dtl.DTLVardef@e6b34e_HPS_FORMAT_FIGEXP M_FIG C_FIG Lay summaryEpigenetic landscape in nCHBV depicts exhaustion and immune dysfunction. Out of many hypermethylated CpG islands of nCHBV, few become hypomethylated in rHBV and drives immune and metabolic reprogramming. This study provides insights into the cellular and molecular basis of epigenomic programs that regulate the differentiation and activation of immune cells leading to viral clearance and seroconversion. Targeting epigenetic mechanism could be promising strategy for the treatment of nCHBV and non-seroconverters.

immunology↗

bioRxiv2023

Hepatitis B virus x (HBx) protein increases cellular OCT3/4 and MYC and facilitates cellular reprogramming (of fibroblasts to iPSC)

HBx is a multifunctional protein coded by the Hepatitis B virus which is involved in various cellular processes such as proliferation, cell survival/apoptosis, and histone methylation. HBx was reported to be associated with liver cancer stem cells. The stemness inducing properties of HBx could also facilitate the generation of pluripotent stem cells from somatic cells. It is well established that somatic cells can be reprogrammed to induced pluripotent stem cells (iPSC) using a cocktail of transcription factors called Yamanakas factors (OCT4, SOX2, KLF4, and MYC). The reprogramming process proceeds step-by-step with reprogramming factor chromatin interactions, transcription and chromatin states changing during transitions. HBx is a broad spectrum trans-activator and therefore could facilitate these transitions. We electroporated low passage and high passage (difficult to reprogram) fibroblasts using Yamanakas factors (YF) with and without HBx and evaluated the reprogramming efficiency. We also investigated the tri-lineage and terminal differentiation potential of iPSC derived using HBx. We found, addition of HBx to YF, improves iPSC derivation, and increases the efficiency of iPSC generation from difficult or hard-to-reprogram samples such as high passage/senescent fibroblasts. Furthermore, we show that HBx can substitute the key transcription factor MYC in the YF cocktail to generate iPSC. Our results have practical value in improving the efficiency of pluripotent stem cell derivation from difficult to reprogram somatic cells in addition to providing some insights into the mechanisms of liver carcinogenesis in chronic hepatitis B. To conclude, HBx improves the reprogramming efficiency of YFs. HBx increases the cellular levels of OCT3/4 and MYC. Lay summaryHBx is a protein made by hepatitis B virus. Yamanakas Factors (YF) are a mixture of master-proteins which are used to convert adult cells to embryonic stem cells. We found that HBx protein can augment the efficiency of YF in generating these stem cells (concept summary figure).

cell biology↗

bioRxiv2023

Metabolic alterations unravel the materno fetal immune responses with disease severity in pregnant women infected with SARS-CoV-2

BackgroundPregnancy being immune compromised state, COVID-19 disease poses high risk of premature delivery and threat to fetus. Plasma metabolome regulates immune cellular responses and we aimed to analyze the plasma secretome, metabolome and immune cells in COVID-19 positive pregnant mother and cord blood. MethodsCOVID-19 RT-PCR positive pregnant females (n=112) asymptomatic (n=82), or with mild (n=21) or moderate (n=9) disease and control healthy pregnant (n=10) females were included. Mothers blood and cord blood (n=80) was analysed for untargeted metabolome profiling and plasma cytokines by high-resolution mass spectrometry (MS) and multiplex cytokine bead array. Immune scan in mothers was done using flow cytometry. ResultsIn asymptomatic SARS-CoV-2 infection, --the amino acid metabolic pathways such as glycine, serine, L-lactate and threonine metabolism was upregulated, riboflavin and tyrosine metabolism, downregulated. In mild to moderate disease, the pyruvate and NAD+ metabolism (energy metabolic pathways) were mostly altered. In addition to raised TNF-, IFN-, IFN-{gamma}, IL-6 cytokine storm, IL-9 was increased in both mothers and neonates. Pyruvate and NAD+ metabolic pathways along with IL-9 and IFN-{gamma} had impact on non-classical monocytes, increased CD4 T cells and B cells but depleted CD8+ T cells. Cord blood mimicked the mothers metabolomic profiles by showing altered valine, leucine, isoleucine, glycine, serine, threonine in asymptomatic and NAD+ and riboflavin metabolism in mild and moderate disease subjects. ConclusionsOur results demonstrate a graduated immune-metabolomic interplay in mother and fetus in pregnant females with different degrees of severity of COVID-19 disease. IL-9 and IFN- {gamma} regulated pyruvate, lactate TCA metabolism and riboflavin metabolism with context to disease severity are hall marks of this materno-fetal metabolome. HighlightsO_LISARS-CoV-2 infection alters energy consumption metabolic pathways during pregnancy. C_LIO_LIPregnant women with mild to moderate COVID-19 show increased energy demands, and consume stored glucose by upregulating pyruvate and NAD+ metabolism. C_LIO_LIIncreased TNF- and IL-9 in mild COVID-19 disease involve TCA cycle to produce lactate and consume stored glucose by up regulating pyruvate and nicotinamide and nicotinate metabolism. C_LIO_LIWith mild to moderate disease, raised IL-9 and TNF-, decreased riboflavin pathway, exhaustion of T and B cells cause pathogenesis. C_LIO_LICord blood mimics the metabolic profile of mothers peripheral blood, SARS- CoV-2 infection reshapes immune-metabolic profiles of mother-infant dyad. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=141 HEIGHT=200 SRC="FIGDIR/small/540101v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@657cc3org.highwire.dtl.DTLVardef@e1e76forg.highwire.dtl.DTLVardef@12153f2org.highwire.dtl.DTLVardef@10e5332_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Genomic perspectives of SARS CoV-2 in liver disease patients with its clinical correlation: A single centre retrospective study

BackgroundSevere Acute Respiratory Syndrome Coronavirus-2 (SARS CoV-2), is a causative agent of current global pandemic of Coronavirus disease-19 (COVID-19). Due to propagated outbreak and global vaccination drive an immense immunological selection pressure has been exerted on SARS CoV-2 leading to evolution of new variants. This study was performed to compare the mutational and clinical profile of liver disease patients infected with different variants of SARS CoV-2. MethodologyThis was a single-centre, retrospective, cohort study in which clinicogenomic analysis of liver disease (LD) patients infected with SARS CoV-2 was performed. Complete demographic and clinical details were retrieved from Hospital Information System (HIS). QC-threshold passed FASTA files containing sequences from COVID-19 patients (n=174) were compared with a reference genome of SARS-CoV-2 isolate named Wuhan-Hu-1 (NCBI Reference Sequence: NC_045512.2) for mutational analysis. ResultsOut of 232 finally analysed patients 137 (59.1%) were LD-CoV (+) and 95 (40.9%) were LD-CoV(-). LD patients with comorbidities were affected more with COVID-19 (p=0.002). On comparing the outcome in the terms of mortality, LD-CoV (+) had 2.29 times (OR 2.29, CI 95%, 1.25-4.29) higher of odds of succumbing to COVID-19 (p=0.006). Multivariate regression analysis revealed, abdominal distention (p=0.05), severe COVID-19 pneumonia (p=0.046) and the change in serum bilirubin levels (p=0.005) as well as Alkaline phosphatase (ALP) levels (p=0.003) to have an association with adverse outcome in LD patients with COVID-19. In Delta (22%) and Omicron (48%) groups, Spike gene harboured maximum mutations. On comparing the mutations between LD-CoV(+/D) and LD-CoV(+/O) a total of nine genes had more mutations in LD-CoV(+/O) whereas three genes had more mutations in LD-CoV(+/D). ConclusionWe concluded that LD patients are more susceptible to COVID-19 as compared to a healthy adult with associated adverse clinical outcomes in terms of mortality and morbidity. Therefore this special group should be given priority while devising and introducing new vaccination and vaccination policies. The infection with different variants did not result in different outcome in our group of patients.

microbiology↗

bioRxiv2020

Cloning of Human ABCB11 Gene in E. coli required the removal of an Intragenic Pribnow-Schaller Box before its Insertion into Genomic Safe Harbor AAVS1 Site using CRISPR Cas9

BackgroundGenomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesnt cause any foreseeable risk to the host. The AAVS1 site is the site which is disrupted upon integration of Adeno Associated Virus (AAV) and is considered a safe-harbor in human genome because about one third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man. Therefore, we chose to target AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in Progressive Familial Intra Hepatic Cholestasis Type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient. MethodsWe used CRISPR Cas9 a genome editing tool to insert ABCB11 gene at AAVS1 site in human cell-lines. ResultsWe found that human ABCB11 sequence has a "Pribnow- Schaller Box" which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli and the removal of the same was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool. We also found that ABCB11 protein has similarity with E. coli Endotoxin (Lipid A) Transporter MsbA. ConclusionWe inserted ABCB11 at AAVS1 site using CRISPR-Cas9, however, the frequency of homologous recombination was very low for this approach to be successful in-vivo (Figure: pictorial abstract). Pictorial Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/284125v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1ae72ddorg.highwire.dtl.DTLVardef@1f72ee8org.highwire.dtl.DTLVardef@128a226org.highwire.dtl.DTLVardef@adacfb_HPS_FORMAT_FIGEXP M_FIG ABCB11 gene (which codes the transporter of human bile salts) is targeted to AAVS1 site using a construct which has 5 and 3 overhangs which are homologous to the AAVS1 site. A Pribnow box was detected inside ABCB11 gene which allowed the gene to transcribe in E. Coli causing bacterial lysis probably through competitive replacement of a homologous transporter protein in E. Coli (E. coli Endotoxin (Lipid A) Transporter) MsbA, resulting in Lipid A (L) accumulation inside the bacteria. C_FIG

cell biology↗

bioRxiv2020

Multi-omics analysis of aspirin treatment response in mice provide molecular insights and targets linked to liver fibrosis regression.

Background & Aims Aspirin has potent anti-platelet activities and possibly helps regression of fibrosis. We investigated antifibrotic mechanisms of aspirin in the murine CCl4 model and in patients with hepatic fibrosis.Methods Multiomics analysis identified networks and molecular targets regulated by aspirin which were validated in murine model and in patients with liver fibrosis.Results Biochemical/histopathological changes and hepatic fibrosis were greater in CCl4-treated mice compared to CCl4-aspirin (CCl4+ASA) or control mice (p<0.05). In CCl4+ASA mice, integrated proteome-metabolome analysis showed an increase in autophagy, drug metabolism, glutathione and energy metabolism (p<0.05) and decrease in inflammatory pathways, arachidonic acid and butanoate metabolism (p<0.05). Global cross-correlation analysis linked fibrosis markers with protein-metabolite pathways (r2>0.5, p<0.05). Liver proteome enrichment for immune clusters using blood transcription module correlated with histidine and tryptophan metabolism (r2>0.5, p<0.05). Aspirin decreased Ryanodine-receptor-2 (RYR2;oxidative-stress), Arginase-1 (ARG-1;urea cycle), Arachidonate-5-lipoxygenase (ALOX5;leukotriene metabolism), and Kynurenine-3-monooxygenase (KMO;tryptophan metabolism; p<0.05) which correlated with reduction in α-SMA, PDGFR-β and degree of hepatic fibrosis (r2>0.75; p<0.05) in animal and human studies, and, in-vitro analysis. Aspirin modulated intracellular-calcium and oxidative-stress levels by reducing RYR2 expression in activated LX-2 cells. It modulated the liver microbiome and its functions which also correlated with ARG1, ALOX5, RYR2 expression (r2>0.5, p<0.05). Metaproteome analysis showed significant microbiome similarity at phylum level in murine liver tissues and fecal samples. Aspirin increased the abundance of Firmicutes (Ruminococcaceae, Lachnospiraceae, and Clostridiaceae) and their functionality, as assessed by glycerol-3-phosphate dehydrogenase (NAD(P)(+) and dTMP-kinase activity (p<0.05).Conclusions Aspirin demonstrates broad beneficial effects following oxidative injury, inflammation, and hepatic fibrosis. Aspirin induces distinctive hepatic proteome/metabolome and intrahepatic microbiome changes which are indicative of fibrosis regression and could be further explored as therapeutic targets.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗