Single-Cell Chemical Proteomics (SCCP) Interrogates the Timing and Heterogeneity of Cancer Cell Commitment to Death.

Chemical proteomics studies the effects of drugs upon a cellular proteome. Due to the complexity and diversity of tumors, the response of cancer cells to drugs is also heterogeneous, and thus, proteome analysis at the single-cell level is needed. Here, we demonstrate that single-cell proteomics techniques have become quantitative enough to tackle the drug effects on target proteins, enabling single-cell chemical proteomics (SCCP). Using SCCP, we studied here the time-resolved response of individual adenocarcinoma A549 cells to anticancer drugs methotrexate, camptothecin, and tomudex, revealing the early emergence of cellular subpopulations committed and uncommitted to death. As a novel and useful approach to exploring the heterogeneous response to drugs of cancer cells, SCCP may prove to be a breakthrough application for single-cell proteomics.

DiagnoProt: a tool for discovery of new molecules by mass spectrometry.

MOTIVATION: Around 75% of all mass spectra remain unidentified by widely adopted proteomic strategies. We present DiagnoProt, an integrated computational environment that can efficiently cluster millions of spectra and use machine learning to shortlist high-quality unidentified mass spectra that are discriminative of different biological conditions. RESULTS: We exemplify the use of DiagnoProt by shortlisting 4366 high-quality unidentified tandem mass spectra that are discriminative of different types of the Aspergillus fungus. AVAILABILITY AND IMPLEMENTATION: DiagnoProt, a demonstration video and a user tutorial are available at http://patternlabforproteomics.org/diagnoprot . CONTACT: andrerfsilva@gmail.com or paulo@pcarvalho.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Naturally occurring dipeptide from elite controllers with dual anti-HIV-1 mechanism.

BACKGROUND: Enhanced levels of a dipeptide, WG-am, have been reported among elite controllers - patients who spontaneously control their HIV-1 infection. This study aimed to evaluate anti-HIV-1 activity and mechanism of action of WG-am. METHODS: Drug sensitivity assays in TZM.bl cells, PBMCs and ACH-2 cells using WT and mutated HIV-1 strainswere performed to evaluate the antiviral mechanism of WG-am. Mass spectrometry-based proteomics and Real-time PCR analysis of reverse transcription steps were performed to unravel the second anti-HIV-1 mechanism of WG-am. RESULTS: The data suggest that WG-am binds to the CD4 binding pocket of HIV-1 gp120 and blocks its binding to the host cell receptors. Additionally, the time course assay showed that WG-am also inhibited HIV-1 at 4-6 hours post-infection, suggesting a second antiviral mechanism. Drug sensitivity assays under acidic wash conditions confirmed the ability of WG-am to internalise into the host cell in an HIV independent manner. Proteomic studies showed a clustering of all samples treated with WG-am independent of the number of doses or presence or absence of HIV-1. Differentially expressed proteins due to the WG-am treatment indicated an effect on HIV-1 reverse transcription, which was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). CONCLUSION: Naturally occurring in HIV-1 elite controllers, WG-am stands out as a new kind of antiviral compound with two independent inhibitory mechanisms of action on HIV-1 replication. WG-am halts HIV-1 entry to the host cell by binding to HIV-1 gp120, thereby blocking the binding of HIV-1 to the host cell. WG-am also exerts a post-entry but pre-integration antiviral effect related to RT-activity.

Single Cell Proteomics Using Multiplexed Isobaric Labeling for Mass Spectrometric Analysis.

Single cell proteomics is an emerging field of bioanalysis allowing one to capture proteome profiles of isolated single cells, which is expected to yield additional biological information in comparison with bulk cell analysis. Mass spectrometry-based methods provide unbiased analysis of detectable proteins limited only by technical parameters, such as sensitivity, which necessitates the development of best-practice workflows. Here, we describe the entire experimental design of single cell proteome analysis, exemplified by cultured A549 lung adenocarcinoma cells treated with an anti-cancer drug (methotrexate) and utilizing tandem mass tag (TMTpro™) labeling strategy for mass spectrometric data acquisition.

Multi-omics insights into host-viral response and pathogenesis in Crimean-Congo hemorrhagic fever viruses for novel therapeutic target.

The pathogenesis and host-viral interactions of the Crimean-Congo hemorrhagic fever orthonairovirus (CCHFV) are convoluted and not well evaluated. Application of the multi-omics system biology approaches, including biological network analysis in elucidating the complex host-viral response, interrogates the viral pathogenesis. The present study aimed to fingerprint the system-level alterations during acute CCHFV-infection and the cellular immune responses during productive CCHFV-replication in vitro. We used system-wide network-based system biology analysis of peripheral blood mononuclear cells (PBMCs) from a longitudinal cohort of CCHF patients during the acute phase of infection and after one year of recovery (convalescent phase) followed by untargeted quantitative proteomics analysis of the most permissive CCHFV-infected Huh7 and SW13 cells. In the RNAseq analysis of the PBMCs, comparing the acute and convalescent-phase, we observed system-level host's metabolic reprogramming towards central carbon and energy metabolism (CCEM) with distinct upregulation of oxidative phosphorylation (OXPHOS) during CCHFV-infection. Upon application of network-based system biology methods, negative coordination of the biological signaling systems like FOXO/Notch axis and Akt/mTOR/HIF-1 signaling with metabolic pathways during CCHFV-infection were observed. The temporal quantitative proteomics in Huh7 showed a dynamic change in the CCEM over time and concordant with the cross-sectional proteomics in SW13 cells. By blocking the two key CCEM pathways, glycolysis and glutaminolysis, viral replication was inhibited in vitro. Activation of key interferon stimulating genes during infection suggested the role of type I and II interferon-mediated antiviral mechanisms both at the system level and during progressive replication.

Crimean-Congo hemorrhagic fever (CCHF) is an emerging disease that is increasingly spreading to new populations. The condition is now endemic in almost 30 countries in sub-Saharan Africa, South-Eastern Europe, the Middle East and Central Asia. CCHF is caused by a tick-borne virus and can cause uncontrolled bleeding. It has a mortality rate of up to 40%, and there are currently no vaccines or effective treatments available. All viruses depend entirely on their hosts for reproduction, and they achieve this through hijacking the molecular machinery of the cells they infect. However, little is known about how the CCHF virus does this and how the cells respond. To understand more about the relationship between the cell's metabolism and viral replication, Neogi, Elaldi et al. studied immune cells taken from patients during an infection and one year later. The gene activity of the cells showed that the virus prefers to hijack processes known as central carbon and energy metabolism. These are the main regulator of the cellular energy supply and the production of essential chemicals. By using cancer drugs to block these key pathways, Neogi, Elaldi et al. could reduce the viral reproduction in laboratory cells. These findings provide a clearer understanding of how the CCHF virus replicates inside human cells. By interfering with these processes, researchers could develop new antiviral strategies to treat the disease. One of the cancer drugs tested in cells, 2-DG, has been approved for emergency use against COVID-19 in some countries. Neogi, Elaldi et al. are now studying this further in animals with the hope of reaching clinical trials in the future.

Transcription of genes involved in sulfolipid and polyacyltrehalose biosynthesis of Mycobacterium tuberculosis in experimental latent tuberculosis infection.

The Influence of trehalose-based glycolipids in the virulence of Mycobacterium tuberculosis (Mtb) is recognised; however, the actual role of these cell-wall glycolipids in latent infection is unknown. As an initial approach, we determined by two-dimensional thin-layer chromatography the sulfolipid (SL) and diacyltrehalose/polyacyltrehalose (DAT/PAT) profile of the cell wall of hypoxic Mtb. Then, qRT-PCR was extensively conducted to determine the transcription profile of genes involved in the biosynthesis of these glycolipids in non-replicating persistent 1 (NRP1) and anaerobiosis (NRP2) models of hypoxia (Wayne model), and murine models of chronic and progressive pulmonary tuberculosis. A diminished content of SL and increased amounts of glycolipids with chromatographic profile similar to DAT were detected in Mtb grown in the NRP2 stage. A striking decrease in the transcription of mmpL8 and mmpL10 transporter genes and increased transcription of the pks (polyketidesynthase) genes involved in SL and DAT biosynthesis were detected in both the NRP2 stage and the murine model of chronic infection. All genes were found to be up-regulated in the progressive disease. These results suggest that SL production is diminished during latent infection and the DAT/PAT precursors can be accumulated inside tubercle bacilli and are possibly used in reactivation processes.