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).

Circulating extracellular vesicles induce monocyte dysfunction and are associated with sepsis and high mortality in cirrhosis.

BACKGROUND: Sepsis is common in cirrhosis and is often a result of immune dysregulation. Specific stimuli and pathways of inter-cellular communications between immune cells in cirrhosis and sepsis are incompletely understood. Immune cell-derived extracellular vesicles (EV) were studied to understand mechanisms of sepsis in cirrhosis. METHODS: Immune cell-derived EV were measured in cirrhosis patients [Child-Turcotte-Pugh (Child) score A, n = 15; B n = 16; C n = 43 and Child-C with sepsis (n = 38)], and healthy controls (HC, n = 11). In vitro and in vivo functional relevance of EV in cirrhosis and associated sepsis was investigated. RESULTS: Monocyte, neutrophil and hematopoietic stem cells associated EV progressively increased with higher Child score (P < .001)and correlated with liver disease severity indices (r2  > 0.3, P < .001), which further increased in Child C sepsis than without sepsis(P < .001); monocyte EV showing the highest association with disease stage [P = .013; Odds ratio-4.14(1.34-12.42)]. A threshold level of monocyte EV of 53/µl predicted mortality in patients of Child C with sepsis [Odds ratio-6.2 (2.4-15.9), AUROC = 0.76, P < .01]. In vitro EV from cirrhotic with sepsis compared without sepsis, induced mobilization arrest in healthy monocytes within 4 hours (P = .004), reduced basal oxygen consumption rate (P < .001) and induced pro-inflammatory genes (P < .05). The septic-EV on adoptive transfer to C57/BL6J mice, induced sepsis-like condition within 24 h with leukocytopenia (P = .005), intrahepatic inflammation with increased CD11b + cells (P = .03) and bone marrow hyperplasia (P < .01). CONCLUSION: Extracellular vesicles induce functional impairment in circulating monocytes and contribute to the development and perpetuation of sepsis. High levels of monocyte EV correlate with mortality and can help early stratification of sicker patients.

Baseline Plasma Metabotype Correlates With Direct-Acting Antiviral Therapy Nonresponse for HCV in HIV-HCV Coinfected Patients.

Introduction: With the advent of direct-acting antiviral (DAA) therapy for HCV, the cure is achieved at similar rates among HIV-HCV coinfected patients as in HCV mono-infected patients. The present study evaluates host plasma metabolites as putative indicators in predicting the treatment response in baseline HIV-HCV patients. Methods: Non-cirrhotic HIV-HCV (N = 43) coinfected patients were treated with sofosbuvir and daclatasvir for 12 weeks. Plasma metabolite profiling of pre- and post-therapy was analyzed in 20/43 patients. Of the 20 selected, 10 (50%) attained the sustained viral response [(SVR) (responders)] as defined by the absence of HCV RNA at 12 weeks after the treatment, and 10 (50%) did not attain the cure for HCV (nonresponders). Results: A total of 563 features were annotated (metabolomic/spectral databases). Before therapy, 39 metabolites differentiated (FC ±1.5, p < 0.05) nonresponders from responders. Of these, 20 upregulated and 19 downregulated were associated with tryptophan metabolism, nicotinamide metabolism, and others. Post therapy, 62 plasma metabolites (12 upregulated and 50 downregulated, FC±1.5, p < 0.05) differentiated nonresponders from responders and highlighted a significant increase in the steroid and histidine metabolism and significant decrease in tryptophan metabolism and ascorbate and pyruvate metabolism in the nonresponders. Based on random forest and multivariate linear regression analysis, the baseline level of N-acetylspermidine (FC > 2, AUC = 0.940, Bfactor = -0.267) and 2-acetolactate (FC > 2, AUC = 0.880, Bfactor = -0.713) significantly differentiated between nonresponders from responders in HIV-HCV coinfected patients and was able to predict the failure of treatment response. Conclusion: Increased baseline levels of N-acetylspermidine and 2-acetolactate levels are associated with the likeliness of failure to attain the cure for HCV in HIV-HCV coinfected patients.