Biomolecular map of albumin identifies signatures of severity and early mortality in acute liver failure.

BACKGROUND & AIMS: Acute liver failure (ALF) is associated with high mortality. Alterations in albumin structure and function have been shown to correlate with outcomes in cirrhosis. We undertook a biomolecular analysis of albumin to determine its correlation with hepatocellular injury and early mortality in ALF. METHODS: Altogether, 225 participants (200 patients with ALF and 25 healthy controls [HC]) were enrolled. Albumin was purified from the baseline plasma of the training cohort (ALF, n = 40; survivors, n = 8; non-survivors, n = 32; and HC, n = 5); analysed for modifications, functionality, and bound multi-omics signatures; and validated in a test cohort (ALF, n = 160; survivors, n = 53; non-survivors, n = 107; and HC, n = 20). RESULTS: In patients with ALF, albumin is more oxidised and glycosylated with a distinct multi-omics profile than that in HC, more so in non-survivors (p <0.05). In non-survivors, albumin was more often bound (p <0.05, false discovery rate <0.01) to proteins associated with inflammation, advanced glycation end product, metabolites linked to arginine, proline metabolism, bile acid, and mitochondrial breakdown products. Increased bacterial taxa (Listeria, Clostridium, etc.) correlated with lipids (triglycerides [4:0/12:0/12:0] and phosphatidylserine [39:0]) and metabolites (porphobilinogen and nicotinic acid) in non-survivors (r2 >0.7). Multi-omics signature-based probability of detection for non-survival was >90% and showed direct correlation with albumin functionality and clinical parameters (r2 >0.85). Probability-of-detection metabolites built on the top five metabolites, namely, nicotinic acid, l-acetyl carnitine, l-carnitine, pregnenolone sulfate, and N-(3-hydroxybutanoyl)-l-homoserine lactone, showed diagnostic accuracy of 98% (AUC 0.98, 95% CI 0.95-1.0) and distinguish patients with ALF predisposed to early mortality (log-rank <0.05). On validation using high-resolution mass spectrometry and five machine learning algorithms in test cohort 1 (plasma and paired one-drop blood), the metabolome panel showed >92% accuracy/sensitivity and specificity for prediction of mortality. CONCLUSIONS: In ALF, albumin is hyperoxidised and substantially dysfunctional. Our study outlines distinct 'albuminome' signatures capable of distinguishing patients with ALF predisposed to early mortality or requiring emergency liver transplantation. IMPACTS AND IMPLICATIONS: Here, we report that the biomolecular map of albumin is distinct and linked to severity and outcome in patients with acute liver failure (ALF). Detailed structural, functional, and albumin-omics analysis in patients with ALF led to the identification and classification of albumin-bound biomolecules, which could segregate patients with ALF predisposed to early mortality. More importantly, we found albumin-bound metabolites indicative of mitochondrial damage and hyperinflammation as a putative indicator of <30-day mortality in patients with ALF. This preclinical study validates the utility of albuminome analysis for understanding the pathophysiology and development of poor outcome indicators in patients with ALF.

Modification Patterns of Urinary Albumin Correlates With Serum Albumin and Outcome in Severe Alcoholic Hepatitis.

BACKGROUND AND AIMS: Albumin modifications and deranged functions are well documented in serum of severe alcoholic hepatitis (SAH). We investigated whether urinary albumin (u-Alb) can serve as surrogate marker of circulatory albumin phenotype, functionality, and could predict outcome in SAH patients. PATIENTS AND METHODS: Baseline serum and urine samples from 100 SAH, 20 alcoholic cirrhosis, and 20 healthy controls were subjected to u-Alb, ischemia modified albumin (IMA), IMA to albumin ratio (IMAr), advanced oxidation protein products, advanced glycation end-products, albumin-binding capacity determination. In addition, SAH urinary samples were also analyzed at day 4 and day 7 to predict nonresponse to corticosteroid therapy. RESULTS: Urine and serum levels of IMA, advanced oxidation protein products and advanced glycation end-products were higher (P<0.05) in SAH versus alcoholic cirrhosis and healthy controls. IMAr was low in urine but high in serum of SAH (P<0.05). Albumin-binding capacity was lower (P<0.05) in both urinary and serum albumin of SAH. Urinary and serum albumin parameters showed direct correlation, whereas IMAr showed inverse correlation (cc>0.2, P<0.05). Baseline u-Alb level was significantly higher in SAH, and was correlated directly with corticosteroid treatment outcome and 12-month mortality in SAH. Baseline u-Alb showed an area under the receivers operating curve analysis of 0.7 and a hazard ratio of 1.23 for prediction of 12-month mortality in SAH. Baseline u-Alb level >9.0 mg/dL was associated with reduced 12-month survival in SAH (log rank <0.01). CONCLUSIONS: u-Alb modifications are reflective of serum albumin modifications. Further baseline u-Alb levels could be exploited to predict steroid response and mortality in SAH patients.

Molecular Ellipticity of Circulating Albumin-Bilirubin Complex Associates With Mortality in Patients With Severe Alcoholic Hepatitis.

BACKGROUND & AIMS: Hyperbilirubinemia and hypoalbuminemia are features of hepatic dysfunction that associate with disease severity. This is because hepatic insufficiency causes hypoalbuminemia, which indirectly increases the circulating levels of free bilirubin. Circular dichroism (CD) spectroscopy can be used to quantify the molecular ellipticity (ME) of the albumin-bilirubin complex, and might associate with the severity or outcome of severe alcoholic hepatitis (SAH). METHODS: We performed a cross-sectional study of 265 patients with SAH admitted in the Department of Hepatology, Institute of Liver and Biliary Sciences in New Delhi, India from January 2014 through January 2016. Blood samples were collected and patients were followed for 12 months or death. The molar ratios of bilirubin: albumin and albumin-bilirubin complexes were determined for a discovery cohort (30 patients who survived the study period and 60 patients who did not survive) and compared with those of 60 patients with alcoholic cirrhosis and 30 healthy individuals (controls). Optical activities of albumin-bilirubin complexes in blood samples were determined by CD spectroscopy and compared among groups. Findings were validated in a separate cohort of 150 patients with SAH from the same institute. We studied the correlation between ME and albumin binding capacity (ABiC). RESULTS: The molar ratio of bilirubin: albumin was higher in patients with SAH than with alcoholic cirrhosis or controls (P < .05). Patients with SAH had different CD spectra and higher ME than the other groups (P < .01); ME correlated with model for end-stage liver disease score (with and without Na) and discriminant function (r2 > .3; P < .01). ME values above a cut off of 1.84 mdeg predicted 3-month mortality in patients with SAH with an area under receiver operating characteristic curve of 0.87 (95% CI, 0.79-0.95), a 77% positive predictive value, and a 90% negative predictive value. The hazard ratio and concordance index of ME values for 3-month mortality in patients with SAH was 10% higher than the hazard ratio and concordance index of model for end-stage liver disease score. In patients with SAH, there was an inverse correlation between ME and ABiC (r2 > 0.7; P < .01). We observed a significant reduction in ABiC with increasing levels of bilirubin in vitro prepared albumin-bilirubin complex. CONCLUSION: In a cross-sectional study of patients with SAH, we associated ME of the albumin-bilirubin complex, measured by CD spectroscopy, with outcomes of patients with SAH. Increased loading of bilirubin on albumin could explain reduced albumin function. Bilirubin removal by albumin dialysis might benefit patients with SAH.

Hyperoxidized albumin modulates neutrophils to induce oxidative stress and inflammation in severe alcoholic hepatitis.

Albumin is a potent scavenger of reactive oxygen species (ROS). However, modifications in albumin structure may reduce its antioxidant properties and modulate its immune-regulatory functions. We examined alterations in circulating albumin in severe alcoholic hepatitis (SAH) patients and their contribution to neutrophil activation, intracellular stress, and alteration in associated molecular pathways. Albumin modifications and plasma oxidative stress were assessed in SAH patients (n = 90), alcoholic cirrhosis patients (n = 60), and healthy controls (n = 30) using liquid chromatography/mass spectrometry and spectrophotometry. Activation and intracellular ROS were measured in healthy neutrophils after treatment with purified albumin from the study groups. Gene expression of SAH neutrophils was analyzed and compared to gene expression from healthy neutrophils after stimulation with purified albumin from SAH patient plasma. SAH-albumin showed the highest albumin oxidative state (P < 0.05) and prominent alteration as human nonmercaptalbumin 2 (P < 0.05). Plasma oxidative stress (advanced oxidative protein product) was higher in SAH versus alcoholic cirrhosis patients and healthy controls (P < 0.05). Neutrophil gelatinase-associated lipocalin, myeloperoxidase, and intracellular ROS levels were highest in SAH-albumin-treated neutrophils (P < 0.05). Genes associated with neutrophil activation, ROS production, intracellular antioxidation, and leukocyte migration plus genes for proinflammatory cytokines and various toll-like receptors were overexpressed in SAH neutrophils compared to healthy neutrophils (P < 0.05). Expression of the above-mentioned genes in SAH-albumin-stimulated healthy neutrophils was comparable with SAH patient neutrophils, except for genes associated with apoptosis, endoplasmic reticulum stress, and autophagy (P < 0.05). CONCLUSIONS: In patients with SAH, there is a significant increase in albumin oxidation, and albumin acts as a pro-oxidant; this promotes oxidative stress and inflammation in SAH patients through activation of neutrophils. (Hepatology 2017;65:631-646).

Bone marrow stem cells and their niche components are adversely affected in advanced cirrhosis of the liver.

UNLABELLED: Bone marrow (BM) is a reservoir for immune and hematopoietic cells and critical for tissue repair and regeneration. All of these functions are severely altered in cirrhosis. We investigated the cellular and functional state of BM in cirrhosis patients. We studied the histological, cellular, and molecular changes in BM of cirrhosis patients (n = 168) and controls (n = 44). Hematopoietic stem cells (HSCs) and associated niche cells, mesenchymal stem cells, Schwann cells, neural fibers, and endothelial cells were evaluated by immunohistochemistry. Cytokines and growth factors were analyzed in peripheral blood and BM plasma. Cirrhotic BM showed an inverse correlation between cluster of differentiation 34+HSCs and Model of End-Stage Liver Disease (ρ = -0.582, P < 0.001) and Child's scores (P < 0.038). BMs of cirrhosis patients with higher Model of End-Stage Liver Disease (>15) showed significantly decreased HSCs, mesenchymal stem cells, Schwann cells, and neural fibers; increased interleukin-1ß (P = 0.004), tumor necrosis factor-α (P = 0.040), and interferon-γ (P = 0.03); and decreased oncostatin M (P = 0.04), stem cell factor (P = 0.05), and stromal cell-derived factor 1 (P = 0.03) compared to those with lower Model of End-Stage Liver Disease scores (≤15). The cluster of differentiation 34+ cell population was a predictor for the development of sepsis (P < 0.001), and per unit loss increased the probability of sepsis by 16%. Cirrhosis patients with fewer HSCs had lower hemoglobin (P = 0.05) and platelet counts (P = 0.05) and showed early graft dysfunction. CONCLUSIONS: Increasing severity of cirrhosis causes derangement of the hematopoietic niche and loss of HSCs, contributing to the hematological and immunological dysfunctions and reduced potential for regeneration; restoring BM functions could provide new therapeutic options in cirrhosis. (Hepatology 2016;64:1273-1288).

Genetic signatures coupled with lineage shift characterise endemic evolution of Dengue virus serotype 2 during 2015 outbreak in Delhi, India.

OBJECTIVE: In 2015, New Delhi witnessed a massive outbreak of Dengue virus (DENV) resulting in high morbidity and mortality. We report the molecular characterisation of the dominant circulating DENV strain to understand its evolution and dispersal. MATERIALS AND METHODS: DENV infections were diagnosed by detection of IgM/NS1 antigen, and serotyping was performed by C-PrM PCR. Envelope gene was amplified, and variation(s) in envelope gene were analysed. Phylogenetic tree construction, time-based phylogeny and origin of DENV were analysed. Site-specific selection pressure of envelope gene variants was analysed. RESULTS: Confirmed DENV infection was observed in 11.34% (32 of 282) cases, while PCR positivity for C-PrM region was observed in 54.16% (13 of 24) of NS1 antigen-positive cases. All samples belonged to serotype 2 and cosmopolitan genotype. Phylogenetic analysis using envelope gene revealed segregation of cosmopolitan genotype strains into specific lineages. The Indian strains clustered separately forming a distinct monophyletic lineage (lineage III) with a signature amino acid substitution viz., I162V and R288K. Selection pressure analysis revealed that 215D, 288R and 304K were positively selected sites. The rate of nucleotide substitution was 6.93 × 10-4 substitutions site-1 year-1 with time to most common ancestor was around 10 years with JX475906 (Hyderabad strain) and JN030345 (Singapore strain) as its most probable ancestor. CONCLUSION: We observed evolution of a distinct lineage of DENV-2 strains on the Indian subcontinent with possible changes in endemic circulating dengue strains that might give rise to more pathogenic strains.

Impact of family history of metabolic traits on severity of non-alcoholic steatohepatitis related cirrhosis: A cross-sectional study.

BACKGROUND & AIMS: Familial aggregation of metabolic traits with fatty liver disease is well documented. However, there is scarcity of data regarding such association with non-alcoholic steatohepatitis (NASH)-related cirrhosis. This study was aimed to explore the association of family history of metabolic traits with severity of cirrhosis. METHODS: In a cross-sectional study, all consecutive patients with NASH-related cirrhosis presenting to our tertiary care centre were included. Family history, personal history, demographic characteristics, medical history, anthropometric measurements and laboratory data were recorded. RESULTS: Of the 1133 cirrhotics (68.1% males, age 51.4±10.9 years); 779 (68.8%) had family history for metabolic traits. These patients had lower age at diagnosis (45.4±10.6 vs 49.6±11.2 years), higher Child-Turcotte-Pugh (CTP) score (7.8±1.9 vs 6.6±1.5), higher model for end stage liver disease (MELD) score (12.9±6.1 vs 10.9±4.1) and more incidence of decompensation in the form of ascites (46.3% vs 25.7%), jaundice (12.1% vs 6.2%) and hepatic encephalopathy (26.1% vs 11.0%). Patients with family and personal history of metabolic traits, had an increased risk of an early diagnosis of cirrhosis at<45 years of age (OR: 3.1, 95% CI 2.1-4.4), CTP≥10 (OR: 4.6, 95% CI 2.3-9.1), MELD>15 (OR: 6.6, 95% CI 3.8-11.5) with ≥1 features of decompensation (OR: 4.2, 95% CI 2.9-6.1). Family history of diabetes alone, also had higher risk of cirrhosis with MELD>15 (OR: 4.3, 95% CI 2.4-5.3, P<.001). CONCLUSION: Family and personal history of metabolic traits are associated with early age at diagnosis of cirrhosis with more severity and decompensation and so, has a prognostic importance in NASH-related cirrhotics.

Study of Carnitine/Acylcarnitine and Amino Acid Profile in Children and Adults With Acute Liver Failure.

OBJECTIVES: Fatty acid oxidation defects (FAODs) may underlie or modify the course of acute liver failure (ALF). Overall significance of carnitine/acylcarnitine and amino acid profile in ALF is similarly undetermined. Thus, this study was undertaken to study the abnormalities in carnitine/acylcarnitine and amino acid profile in ALF. METHODS: A prospective study was performed including all patients with ALF, and detailed evaluation including metabolic testing was done. RESULTS: A total of 55 patients (33 pediatric and 22 adult patients) were included in the study. Three patients (a 1-year 6-month-old child, a 13-year-old adolescent, and a 21-year-old adult, ie, 5.5% of all) were identified for the study with underlying metabolic etiology, that is, carnitine palmitoyl transferase-1 deficiency, based on the abnormal carnitine/acylcarnitine profile. Almost three-fourths of patients (78%) had evidence of serum hyperaminoacidemia. Thirty-one patients (56%) had evidence of abnormal carnitine/acylcarnitine profile with predominant abnormality being low free carnitine (C0). Higher levels of serum tyrosine (P = 0.002) and lower levels of serum C0 (P = 0.032) in children and higher levels of serum phenyalanine (P = 0.047) in adults predicted poor outcome (death/liver transplant) on univariate analysis. CONCLUSIONS: FAODs are not uncommon in ALF with a suggested prevalence of approximately 5.5%. FAODs can cause ALF or modify the natural course of ALF caused by other etiologies. Serum hyperaminoacidemia and low serum free carnitine may predict poor outcome in patients with acute liver failure.

Dysregulated iron homeostasis is strongly associated with multiorgan failure and early mortality in acute-on-chronic liver failure.

UNLABELLED: Acute-on-chronic liver failure (ACLF) is an ailment with high incidence of multiorgan failure (MOF) and consequent mortality. Dysregulated iron homeostasis and macrophage dysfunction are linked to increased incidence of MOF. We investigated whether a panel of circulating iron-regulating proteins are associated with development of MOF and can predict 15- or 30-day mortality in ACLF patients. One hundred twenty patients with ACLF, 20 patients with compensated cirrhosis, and 20 healthy controls were studied. Relative protein expression profiling was performed in the derivative cohort and confirmed in the validation cohort. A panel of iron regulators and indices were determined. Multiparametric flow cytometry for quantitation of labile iron pool (LIP) was performed. Validation studies confirmed lower serum transferrin (Tf) and ceruloplasmin levels in ACLF and ACLF-MOF, compared to patients with cirrhosis and controls (P < 0.01). Serum iron and ferritin levels were markedly elevated (P < 0.001; P < 0.05) and hepcidin levels were lower (P < 0.001) in ACLF patients with MOF than those without and other groups (P < 0.001). Percentage Tf saturation (%SAT) was higher in ACLF-MOF (39.2%; P < 0.001) and correlated with poor outcome (hazard ratio: 6.970; P < 0.01). Intracellular LIP indices were significantly elevated in the subsets of circulating macrophages in ACLF-MOF, compared to other groups (P < 0.01). Whereas expression of iron-regulatory genes was markedly down-regulated, genes related to endoplasmic reticulum stress, apoptosis, and inflammation were up-regulated in ACLF patients, compared to patients with cirrhosis. Severe dysregulation of autophagy mechanisms was also observed in the former. CONCLUSIONS: Iron metabolism and transport are severely deranged in ACLF patients and more so in those with MOF. %SAT, circulating hepcidin, and LIP in macrophages correlate with disease severity and %SAT could be used for early prognostication in ACLF patients.

Identification and full-length molecular characterization of rare hepatitis C virus genotype 5a from India.

This report presents a molecular characterization of the complete genome of a rare hepatitis C virus (HCV) genotype (GT5a) from India. Sequence homology of full genome revealed that the strain belonged to HCV GT5a. To trace the origin of this virus and to understand its evolutionary pattern, a phylogenetic reconstruction was carried out on full HCV genome sequences using Bayesian coalescent methods. The phylogenetic tree reconstruction revealed genotypic divergence, with formation of distinct clades. This analysis revealed that HCV genotype 5 might have originated from HCV genotype 3, as they have a recent common ancestor.

Fecal microbiota transplantation compared with prednisolone in severe alcoholic hepatitis patients: a randomized trial.

BACKGROUND: Severe alcoholic hepatitis (SAH) has high 90-day mortality. Prednisolone therapy has shown modest survival benefits over placebo at 28 but not 90 days. Fecal microbial transplantation (FMT) has shown promise in these patients. We compared the efficacy and safety of the two therapies in SAH patients. METHODS: Steroid eligible SAH patients were randomized in an open-label study to prednisolone (n = 60) 40 mg/day for 28 days (assessed at day-7 for continuation) or healthy donor FMT (n = 60) through naso-duodenal tube, daily for seven days. Primary outcome of study was day-90 survival. RESULTS: Patients in prednisolone and FMT arms were comparable at baseline (discriminant function score 65 ± 16.2 and 68 ± 14, MELD score 17.1 and 16.5, respectively). Of 120 patients, 112 [prednisolone-57; FMT-55] completed trial. As per intention-to-treat analysis, 90-day survival was achieved by 56.6% (34/60) patients in prednisolone and 75% (45/60) in FMT group (p = 0.044, FMT HR = 0.528, 95%CI 0.279-0.998). Secondary outcome of 28-day survival [78.33% (47/60) and 88.33% (53/60) (p = 0.243, FMT HR = 0.535, 95%CI 0.213-1.34)] with comparable severity scores over time between both arms. Infections accounted for 11 (19.3%) and 2 (3.6%) deaths in prednisolone and FMT groups, respectively (p = 0.01). Path-tracing showed a slow establishment of microbiota and alpha diversity (Shannon index) improvement by day-28 (p = 0.029). FMT resulted in 23 new taxa by day-28, reduction from baseline in pathogenic taxa [Campylobacter (19-fold, p = 0.035), anaerobes (Parcubacteria, Weisella and Leuconostocaceae)], and increase of Alphaproteobacteria [~ sevenfold, p = 0.047] and Thaumarcheota (known ammonia oxidizer, p = 0.06). Lachnospiraceae (p = 0.008), Prevotella and Viellonella communities in gut favored survival (p < 0.05). CONCLUSION: In severe alcoholic hepatitis, FMT is safe and improves 90-day survival and reduces infections by favorably modulating microbial communities. It can be a useful alternative to prednisolone therapy.

Glp-1 analog, liraglutide, ameliorates hepatic steatosis and cardiac hypertrophy in C57BL/6J mice fed a Western diet.

The aims of this study were designed to determine whether liraglutide, a long-acting glucagon-like peptide, could reverse the adverse effects of a diet high in fat that also contained trans-fat and high-fructose corn syrup (ALIOS diet). Specifically, we examined whether treatment with liraglutide could reduce hepatic insulin resistance and steatosis as well as improve cardiac function. Male C57BL/6J mice were pair fed or fed ad libitum either standard chow or the ALIOS diet. After 8 wk the mice were further subdivided and received daily injections of either liraglutide or saline for 4 wk. Hyperinsulinemic-euglycemic clamp studies were performed after 6 wk, revealing hepatic insulin resistance. Glucose tolerance and insulin resistance tests were performed at 8 and 12 wk prior to and following liraglutide treatment. Liver pathology, cardiac measurements, blood chemistry, and RNA and protein analyses were performed. Clamp studies revealed hepatic insulin resistance after 6 wk of ALIOS diet. Liraglutide reduced visceral adiposity and liver weight (P < 0.001). As expected, liraglutide improved glucose and insulin tolerance. Liraglutide improved hypertension (P < 0.05) and reduced cardiac hypertrophy. Surprisingly, liver from liraglutide-treated mice had significantly higher levels of fatty acid binding protein, acyl-CoA oxidase II, very long-chain acyl-CoA dehydrogenase, and microsomal triglyceride transfer protein. We conclude that liraglutide reduces the harmful effects of an ALIOS diet by improving insulin sensitivity and by reducing lipid accumulation in liver through multiple mechanisms including, transport, and increase ß-oxidation.

Adiponectin inhibits leptin signalling via multiple mechanisms to exert protective effects against hepatic fibrosis.

Adiponectin is protective against hepatic fibrosis, whereas leptin promotes fibrosis. In HSCs (hepatic stellate cells), leptin signals via a JAK2 (Janus kinase 2)/STAT3 (signal transducer and activator of transcription 3) pathway, producing effects that enhance ECM (extracellular matrix) deposition. SOCS-3 (suppressor of cytokine signalling-3) and PTP1B (protein tyrosine phosphatase 1B) are both negative regulators of JAK/STAT signalling, and recent studies have demonstrated a role for adiponectin in regulating SOCS-3 expression. In the present study we investigate mechanisms whereby adiponectin dampens leptin signalling and prevents excess ECM production. We treated culture-activated rat HSCs with recombinant adiponectin, leptin, both or neither, and also treated adiponectin knockout (Ad-/-) and wild-type mice with leptin and/or carbon tetrachloride (CCl4) or saline. We analyse JAK2 and Ob-Rb (long form of the leptin receptor) phosphorylation, and PTP1B expression and activity. We also explore potential mechanisms through which adiponectin regulates SOCS-3-Ob-Rb association. Adiponectin inhibits leptin-stimulated JAK2 activation and Ob-Rb phosphorylation in HSCs, whereas both were increased in Ad-/- mice. Adiponectin stimulates PTP1B expression and activity in vitro, whereas PTP1B expression was lower in Ad-/-mice than in wild-type mice. Adiponectin also promotes SOCS-3-Ob-R association and blocks leptin-stimulated formation of extracellular TIMP-1 (tissue inhibitor of metalloproteinases-1)-MMP-1 (matrix metalloproteinase-1) complexes in vitro. These results suggest two novel mechanisms whereby adiponectin inhibits hepatic fibrosis: (i) by promoting binding of SOCS-3 to Ob-Rb, and (ii) by stimulating PTP1B expression and activity, thus inhibiting JAK2/STAT3 signalling at multiple points.

Protocol for global proteome, virome, and metaproteome profiling of respiratory specimen (VTM) in COVID-19 patient by LC-MS/MS-based analysis.

In this protocol, we describe global proteome profiling for the respiratory specimen of COVID-19 patients, patients suspected with COVID-19, and H1N1 patients. In this protocol, details for identifying host, viral, or bacterial proteome (Meta-proteome) are provided. Major steps of the protocol include virus inactivation, protein quantification and digestion, desalting of peptides, high-resolution mass spectrometry (HRMS)-based analysis, and downstream bioinformatics analysis. For complete details on the use and execution of this profile, please refer to Maras et al. (2021).

Global metabolome profiling of COVID-19 respiratory specimen using high-resolution mass spectrometry (HRMS).

Here we describe a protocol for identifying metabolites in respiratory specimens of patients that are SARS-CoV-2 positive, SARS-CoV-2 negative, or H1N1 positive. This protocol provides step-by-step instructions on sample collection from patients, followed by metabolite extraction. We use ultra-high-pressure liquid chromatography (UHPLC) coupled with high-resolution mass spectrometry (HRMS) for data acquisition and describe the steps for data analysis. The protocol was standardized with specific customization for SARS-CoV-2-containing respiratory specimens. For complete details on the use and execution of this protocol, please refer to Maras et al. (2021).

Defects in energy metabolism are associated with functional exhaustion of bone marrow mesenchymal stem cells in cirrhosis.

OBJECTIVES: Cellular and functional exhaustion of bone marrow mesenchymal stem cells (BM-MSC) is significantly associated with the loss of HSCs and hepatic osteodystrophy in cirrhosis. The molecular mechanisms underlying the dysfunction of BM-MSCs are not well understood. We investigated the underlying mechanisms of cellular and functional exhaustion of BM-MSCs in cirrhosis. METHODS: The MSCs were isolated retrospectively from bone marrow of decompensated alcoholic cirrhosis patients {(Trial registration: ClinicalTrials.gov NCT01902511) (n=10; MELD=16.2±2.3; CTP=8.7±2.3)} and age and gender-matched healthy controls (n=8). Global gene expression profile of healthy bone marrow MSCs (hBM-MSCs) and cirrhosis patients BM-MSCs (cBM-MSCs) were done by mRNA sequencing. XFe24-bioanalyzer analyzed the bioenergetic potential of cells. Level of different cytokines and growth factors in BM-plasma and MSCs secretome were analyzed by Luminex-based bead array. RESULTS: Analysis of differentially expressed genes showed significant (P<0.01) up-regulation of genes associated with ubiquitination and catabolism of proteins; TNF signaling, insulin resistance, and down-regulation of genes associated with DNA repair, protein processing, cell cycle, and mitochondrial respiration in cBM-MSCs in comparison to hBM-MSCs. Compared to hBM-MSCs, cBM-MSCs showed a significant defect in glycolysis due to insulin resistance and poor glucose uptake (P=0.002). This led to compromised self-renewal capacity and cellular loss of MSCs in cirrhosis. cBM-MSCs also showed a significant impairment in Oxidative phosphorylation (OXPHOS) due to mitochondrial dysfunction leading to defects in the osteogenic differentiation with early aging and senescence. CONCLUSION: Compromised energy metabolism due to inflammatory and metabolic stress-induced insulin resistance underlies the cellular and functional exhaustion of BM-MSCs in cirrhosis.

Adiponectin antagonizes the oncogenic actions of leptin in hepatocellular carcinogenesis.

UNLABELLED: Obesity is rapidly becoming a pandemic and is associated with increased carcinogenesis. Obese populations have higher circulating levels of leptin in contrast to low concentrations of adiponectin. Hence, it is important to evaluate the dynamic role between adiponectin and leptin in obesity-related carcinogenesis. Recently, we reported the oncogenic role of leptin including its potential to increase tumor invasiveness and migration of hepatocellular carcinoma (HCC) cells. In the present study we investigated whether adiponectin could antagonize the oncogenic actions of leptin in HCC. We employed HCC cell lines HepG2 and Huh7, the nude mice-xenograft model of HCC, and immunohistochemistry data from tissue-microarray to demonstrate the antagonistic role of adiponectin on the oncogenic actions of leptin. Adiponectin treatment inhibited leptin-induced cell proliferation of HCC cells. Using scratch-migration and electric cell-substrate impedance-sensing-based migration assays, we found that adiponectin inhibited leptin-induced migration of HCC cells. Adiponectin treatment effectively blocked leptin-induced invasion of HCC cells in Matrigel invasion assays. Although leptin inhibited apoptosis in HCC cells, we found that adiponectin treatment induced apoptosis even in the presence of leptin. Analysis of the underlying molecular mechanisms revealed that adiponectin treatment reduced leptin-induced Stat3 and Akt phosphorylation. Adiponectin also increased suppressor of cytokine signaling (SOCS3), a physiologic negative regulator of leptin signal transduction. Importantly, adiponectin significantly reduced leptin-induced tumor burden in nude mice. In HCC samples, leptin expression significantly correlated with HCC proliferation as evaluated by Ki-67, whereas adiponectin expression correlated significantly with increased disease-free survival and inversely with tumor size and local recurrence. CONCLUSION: Collectively, these data demonstrate that adiponectin has the molecular potential to inhibit the oncogenic actions of leptin by blocking downstream effector molecules.