The brain reacting to COVID-19: analysis of the cerebrospinal fluid proteome, RNA and inflammation.

Patients with COVID-19 can have a variety of neurological symptoms, but the active involvement of central nervous system (CNS) in COVID-19 remains unclear. While routine cerebrospinal fluid (CSF) analyses in patients with neurological manifestations of COVID-19 generally show no or only mild inflammation, more detailed data on inflammatory mediators in the CSF of patients with COVID-19 are scarce. We studied the inflammatory response in paired CSF and serum samples of patients with COVID-19 (n = 38). Patients with herpes simplex virus encephalitis (HSVE, n = 10) and patients with non-inflammatory, non-neurodegenerative neurological diseases (n = 28) served as controls. We used proteomics, enzyme-linked immunoassays, and semiquantitative cytokine arrays to characterize inflammatory proteins. Autoantibody screening was performed with cell-based assays and native tissue staining. RNA sequencing of long-non-coding RNA and circular RNA was done to study the transcriptome. Proteomics on single protein level and subsequent pathway analysis showed similar yet strongly attenuated inflammatory changes in the CSF of COVID-19 patients compared to HSVE patients with, e.g., downregulation of the apolipoproteins and extracellular matrix proteins. Protein upregulation of the complement system, the serpin proteins pathways, and other proteins including glycoproteins alpha-2 and alpha-1 acid. Importantly, calculation of interleukin-6, interleukin-16, and CXCL10 CSF/serum indices suggest that these inflammatory mediators reach the CSF from the systemic circulation, rather than being produced within the CNS. Antibody screening revealed no pathological levels of known neuronal autoantibodies. When stratifying COVID-19 patients into those with and without bacterial superinfection as indicated by elevated procalcitonin levels, inflammatory markers were significantly (p < 0.01) higher in those with bacterial superinfection. RNA sequencing in the CSF revealed 101 linear RNAs comprising messenger RNAs, and two circRNAs being significantly differentially expressed in COVID-19 than in non-neuroinflammatory controls and neurodegenerative patients. Our findings may explain the absence of signs of intrathecal inflammation upon routine CSF testing despite the presence of SARS-CoV2 infection-associated neurological symptoms. The relevance of blood-derived mediators of inflammation in the CSF for neurological COVID-19 and post-COVID-19 symptoms deserves further investigation.

Alterations in platelet proteome signature and impaired platelet integrin αIIbß3 activation in patients with COVID-19.

BACKGROUND: Patients with COVID-19 are at increased risk of thrombosis, which is associated with altered platelet function and coagulopathy, contributing to excess mortality. OBJECTIVES: To characterize the mechanism of altered platelet function in COVID-19 patients. METHODS: The platelet proteome, platelet functional responses, and platelet-neutrophil aggregates were compared between patients hospitalized with COVID-19 and healthy control subjects using tandem mass tag proteomic analysis, Western blotting, and flow cytometry. RESULTS: COVID-19 patients showed a different profile of platelet protein expression (858 altered of the 5773 quantified). Levels of COVID-19 plasma markers were enhanced in the platelets of COVID-19 patients. Gene ontology pathway analysis demonstrated that the levels of granule secretory proteins were raised, whereas those of platelet activation proteins, such as the thrombopoietin receptor and protein kinase Cα, were lowered. Basally, platelets of COVID-19 patients showed enhanced phosphatidylserine exposure, with unaltered integrin αIIbß3 activation and P-selectin expression. Agonist-stimulated integrin αIIbß3 activation and phosphatidylserine exposure, but not P-selectin expression, were decreased in COVID-19 patients. COVID-19 patients had high levels of platelet-neutrophil aggregates, even under basal conditions, compared to controls. This association was disrupted by blocking P-selectin, demonstrating that platelet P-selectin is critical for the interaction. CONCLUSIONS: Overall, our data suggest the presence of 2 platelet populations in patients with COVID-19: one of circulating platelets with an altered proteome and reduced functional responses and another of P-selectin-expressing neutrophil-associated platelets. Platelet-driven thromboinflammation may therefore be one of the key factors enhancing the risk of thrombosis in COVID-19 patients.

[Proteomic saliva assay in patients with COVID-19]. / Proteomnyi analiz slyuny u patsientov s COVID-19.

THE AIM OF THE STUDY: Was to perform proteomic saliva assay in order to reveal mechanisms of the oral pathology caused by COVID-19. MATERIALS AND METHODS: Proteomic analysis was performed to compare saliva proteins profile in healthy individuals (10 samples) and patients with COVID-19 (30 samples). RESULTS: The obtained results of the saliva samples study in patients with COVID-19 indicate activation in the oral tissues the pathways of the cell renewal, apoptosis, DNA exchange processes and chromatin remodelling; there are also marked signs of immune response reactivation and immunostimulation. CONCLUSION: Of all the proteins presented, the saliva of patients with COVID-19 33 proteins have an intersection with GO-annotated proteins of inflammation and epithelial cornification.

Proteomic Profile of Procoagulant Extracellular Vesicles Reflects Complement System Activation and Platelet Hyperreactivity of Patients with Severe COVID-19.

Background: Extracellular vesicles (EVs) are a valuable source of biomarkers and display the pathophysiological status of various diseases. In COVID-19, EVs have been explored in several studies for their ability to reflect molecular changes caused by SARS-CoV-2. Here we provide insights into the roles of EVs in pathological processes associated with the progression and severity of COVID-19. Methods: In this study, we used a label-free shotgun proteomic approach to identify and quantify alterations in EV protein abundance in severe COVID-19 patients. We isolated plasma extracellular vesicles from healthy donors and patients with severe COVID-19 by size exclusion chromatography (SEC). Then, flow cytometry was performed to assess the origin of EVs and to investigate the presence of circulating procoagulant EVs in COVID-19 patients. A total protein extraction was performed, and samples were analyzed by nLC-MS/MS in a Q-Exactive HF-X. Finally, computational analysis was applied to signify biological processes related to disease pathogenesis. Results: We report significant changes in the proteome of EVs from patients with severe COVID-19. Flow cytometry experiments indicated an increase in total circulating EVs and with tissue factor (TF) dependent procoagulant activity. Differentially expressed proteins in the disease groups were associated with complement and coagulation cascades, platelet degranulation, and acute inflammatory response. Conclusions: The proteomic data reinforce the changes in the proteome of extracellular vesicles from patients infected with SARS-CoV-2 and suggest a role for EVs in severe COVID-19.

Exploring COVID-19 pathogenesis on command-line: A bioinformatics pipeline for handling and integrating omics data.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first identified in late 2019 in Wuhan, China, and has proven to be highly pathogenic, making it a global public health threat. The immediate need to understand the mechanisms and impact of the virus made omics techniques stand out, as they can offer a holistic and comprehensive view of thousands of molecules in a single experiment. Mastering bioinformatics tools to process, analyze, integrate, and interpret omics data is a powerful knowledge to enrich results. We present a robust and open access computational pipeline for extracting information from quantitative proteomics and transcriptomics public data. We present the entire pipeline from raw data to differentially expressed genes. We explore processes and pathways related to mapped transcripts and proteins. A pipeline is presented to integrate and compare proteomics and transcriptomics data using also packages available in the Bioconductor and providing the codes used. Cholesterol metabolism, immune system activity, ECM, and proteasomal degradation pathways increased in infected patients. Leukocyte activation profile was overrepresented in both proteomics and transcriptomics data. Finally, we found a panel of proteins and transcripts regulated in the same direction in the lung transcriptome and plasma proteome that distinguish healthy and infected individuals. This panel of markers was confirmed in another cohort of patients, thus validating the robustness and functionality of the tools presented.

A high-throughput yeast display approach to profile pathogen proteomes for MHC-II binding.

T cells play a critical role in the adaptive immune response, recognizing peptide antigens presented on the cell surface by major histocompatibility complex (MHC) proteins. While assessing peptides for MHC binding is an important component of probing these interactions, traditional assays for testing peptides of interest for MHC binding are limited in throughput. Here, we present a yeast display-based platform for assessing the binding of tens of thousands of user-defined peptides in a high-throughput manner. We apply this approach to assess a tiled library covering the SARS-CoV-2 proteome and four dengue virus serotypes for binding to human class II MHCs, including HLA-DR401, -DR402, and -DR404. While the peptide datasets show broad agreement with previously described MHC-binding motifs, they additionally reveal experimentally validated computational false positives and false negatives. We therefore present this approach as able to complement current experimental datasets and computational predictions. Further, our yeast display approach underlines design considerations for epitope identification experiments and serves as a framework for examining relationships between viral conservation and MHC binding, which can be used to identify potentially high-interest peptide binders from viral proteins. These results demonstrate the utility of our approach to determine peptide-MHC binding interactions in a manner that can supplement and potentially enhance current algorithm-based approaches.

Strategy for Microscale Extraction and Proteome Profiling of Peripheral Blood Mononuclear Cells.

Peripheral blood mononuclear cells (PBMCs) play vital roles in physiological and pathological processes and represent a rich source for disease monitoring. Typical molecular profiling on PBMCs involves the sorting of cell subsets and thus requires a large volume of peripheral blood (PB), which impedes the clinical practicability of omics tools in PBMC measurements. It would be clinically invaluable to develop a convenient approach for preparing PBMCs from small volumes of PB and for deep proteome profiling of PBMCs. To this end, here, we designed an apparatus (PBMC-mCap) for microscale enrichment and proteome analysis of PBMCs, which pushed the needed PB volume from the normal 2 mL or higher to 100 µL or lower, comparable to the volume of a drop of finger blood. A PBMC-specific mass spectra library containing 8869 proteins and 121,956 peptides was further built, which, in combination with the optimized data-independent acquisition strategy, helped to identify 6000 and 6500 proteins from PBMCs with 100 µL and 1 mL of PB as initial materials, respectively. Further application of the strategy for PBMC proteomes revealed a steady difference between gender (male vs female) and upon stimulus (COVID-19 vaccination). For the latter, we observed differentially expressed genes and pathways involving the activation of immune cells, including the NF-κB pathway, inflammation response, and antiviral response. Our strategy for the proteome analysis of microscale PBMCs may provide a convenient clinical toolkit for disease diagnosis and healthy state monitoring.

Proteomic Analysis of Human Sputum for the Diagnosis of Lung Disorders: Where Are We Today?

The identification of markers of inflammatory activity at the early stages of pulmonary diseases which share common characteristics that prevent their clear differentiation is of great significance to avoid misdiagnosis, and to understand the intrinsic molecular mechanism of the disorder. The combination of electrophoretic/chromatographic methods with mass spectrometry is currently a promising approach for the identification of candidate biomarkers of a disease. Since the fluid phase of sputum is a rich source of proteins which could provide an early diagnosis of specific lung disorders, it is frequently used in these studies. This report focuses on the state-of-the-art of the application, over the last ten years (2011-2021), of sputum proteomics in the investigation of severe lung disorders such as COPD; asthma; cystic fibrosis; lung cancer and those caused by COVID-19 infection. Analysis of the complete set of proteins found in sputum of patients affected by these disorders has allowed the identification of proteins whose levels change in response to the organism's condition. Understanding proteome dynamism may help in associating these proteins with alterations in the physiology or progression of diseases investigated.

Clinical applications of plasma proteomics and peptidomics: Towards precision medicine.

In the context of precision medicine, disease treatment requires individualized strategies based on the underlying molecular characteristics to overcome therapeutic challenges posed by heterogeneity. For this purpose, it is essential to develop new biomarkers to diagnose, stratify, or possibly prevent diseases. Plasma is an available source of biomarkers that greatly reflects the physiological and pathological conditions of the body. An increasing number of studies are focusing on proteins and peptides, including many involving the Human Proteome Project (HPP) of the Human Proteome Organization (HUPO), and proteomics and peptidomics techniques are emerging as critical tools for developing novel precision medicine preventative measures. Excitingly, the emerging plasma proteomics and peptidomics toolbox exhibits a huge potential for studying pathogenesis of diseases (e.g., COVID-19 and cancer), identifying valuable biomarkers and improving clinical management. However, the enormous complexity and wide dynamic range of plasma proteins makes plasma proteome profiling challenging. Herein, we summarize the recent advances in plasma proteomics and peptidomics with a focus on their emerging roles in COVID-19 and cancer research, aiming to emphasize the significance of plasma proteomics and peptidomics in clinical applications and precision medicine.

2 deoxy-D-glucose augments the mitochondrial respiratory chain in heart.

2-Deoxy-D-glucose (2DG) has recently received emergency approval for the treatment of COVID-19 in India, after a successful clinical trial. SARS-CoV-2 infection of cultured cells is accompanied by elevated glycolysis and decreased mitochondrial function, whereas 2DG represses glycolysis and stimulates respiration, and restricts viral replication. While 2DG has pleiotropic effects on cell metabolism in cultured cells it is not known which of these manifests in vivo. On the other hand, it is known that 2DG given continuously can have severe detrimental effects on the rodent heart. Here, we show that the principal effect of an extended, intermittent 2DG treatment on mice is to augment the mitochondrial respiratory chain proteome in the heart; importantly, this occurs without vacuolization, hypertrophy or fibrosis. The increase in the heart respiratory chain proteome suggests an increase in mitochondrial oxidative capacity, which could compensate for the energy deficit caused by the inhibition of glycolysis. Thus, 2DG in the murine heart appears to induce a metabolic configuration that is the opposite of SARS-CoV-2 infected cells, which could explain the compound's ability to restrict the propagation of the virus to the benefit of patients with COVID-19 disease.

Proteome-wide Mendelian randomization identifies causal links between blood proteins and severe COVID-19.

In November 2021, the COVID-19 pandemic death toll surpassed five million individuals. We applied Mendelian randomization including >3,000 blood proteins as exposures to identify potential biomarkers that may indicate risk for hospitalization or need for respiratory support or death due to COVID-19, respectively. After multiple testing correction, using genetic instruments and under the assumptions of Mendelian Randomization, our results were consistent with higher blood levels of five proteins GCNT4, CD207, RAB14, C1GALT1C1, and ABO being causally associated with an increased risk of hospitalization or respiratory support/death due to COVID-19 (ORs = 1.12-1.35). Higher levels of FAAH2 were solely associated with an increased risk of hospitalization (OR = 1.19). On the contrary, higher levels of SELL, SELE, and PECAM-1 decrease risk of hospitalization or need for respiratory support/death (ORs = 0.80-0.91). Higher levels of LCTL, SFTPD, KEL, and ATP2A3 were solely associated with a decreased risk of hospitalization (ORs = 0.86-0.93), whilst higher levels of ICAM-1 were solely associated with a decreased risk of respiratory support/death of COVID-19 (OR = 0.84). Our findings implicate blood group markers and binding proteins in both hospitalization and need for respiratory support/death. They, additionally, suggest that higher levels of endocannabinoid enzymes may increase the risk of hospitalization. Our research replicates findings of blood markers previously associated with COVID-19 and prioritises additional blood markers for risk prediction of severe forms of COVID-19. Furthermore, we pinpoint druggable targets potentially implicated in disease pathology.

COVIDomics: The Proteomic and Metabolomic Signatures of COVID-19.

Omics-based technologies have been largely adopted during this unprecedented global COVID-19 pandemic, allowing the scientific community to perform research on a large scale to understand the pathobiology of the SARS-CoV-2 infection and its replication into human cells. The application of omics techniques has been addressed to every level of application, from the detection of mutations, methods of diagnosis or monitoring, drug target discovery, and vaccine generation, to the basic definition of the pathophysiological processes and the biochemical mechanisms behind the infection and spread of SARS-CoV-2. Thus, the term COVIDomics wants to include those efforts provided by omics-scale investigations with application to the current COVID-19 research. This review summarizes the diverse pieces of knowledge acquired with the application of COVIDomics techniques, with the main focus on proteomics and metabolomics studies, in order to capture a common signature in terms of proteins, metabolites, and pathways dysregulated in COVID-19 disease. Exploring the multiomics perspective and the concurrent data integration may provide new suitable therapeutic solutions to combat the COVID-19 pandemic.

Pathogen-Host Interaction Repertoire at Proteome and Posttranslational Modification Levels During Fungal Infections.

Prevalence of fungal diseases has increased globally in recent years, which often associated with increased immunocompromised patients, aging populations, and the novel Coronavirus pandemic. Furthermore, due to the limitation of available antifungal agents mortality and morbidity rates of invasion fungal disease remain stubbornly high, and the emergence of multidrug-resistant fungi exacerbates the problem. Fungal pathogenicity and interactions between fungi and host have been the focus of many studies, as a result, lots of pathogenic mechanisms and fungal virulence factors have been identified. Mass spectrometry (MS)-based proteomics is a novel approach to better understand fungal pathogenicities and host-pathogen interactions at protein and protein posttranslational modification (PTM) levels. The approach has successfully elucidated interactions between pathogens and hosts by examining, for example, samples of fungal cells under different conditions, body fluids from infected patients, and exosomes. Many studies conclude that protein and PTM levels in both pathogens and hosts play important roles in progression of fungal diseases. This review summarizes mass spectrometry studies of protein and PTM levels from perspectives of both pathogens and hosts and provides an integrative conceptual outlook on fungal pathogenesis, antifungal agents development, and host-pathogen interactions.

Proteomic and metabolomic profiling of urine uncovers immune responses in patients with COVID-19.

The utility of the urinary proteome in infectious diseases remains unclear. Here, we analyzed the proteome and metabolome of urine and serum samples from patients with COVID-19 and healthy controls. Our data show that urinary proteins effectively classify COVID-19 by severity. We detect 197 cytokines and their receptors in urine, but only 124 in serum using TMT-based proteomics. The decrease in urinary ESCRT complex proteins correlates with active SARS-CoV-2 replication. The downregulation of urinary CXCL14 in severe COVID-19 cases positively correlates with blood lymphocyte counts. Integrative multiomics analysis suggests that innate immune activation and inflammation triggered renal injuries in patients with COVID-19. COVID-19-associated modulation of the urinary proteome offers unique insights into the pathogenesis of this disease. This study demonstrates the added value of including the urinary proteome in a suite of multiomics analytes in evaluating the immune pathobiology and clinical course of COVID-19 and, potentially, other infectious diseases.

Next generation plasma proteome profiling of COVID-19 patients with mild to moderate symptoms.

BACKGROUND: COVID-19 has caused millions of deaths globally, yet the cellular mechanisms underlying the various effects of the disease remain poorly understood. Recently, a new analytical platform for comprehensive analysis of plasma protein profiles using proximity extension assays combined with next generation sequencing has been developed, which allows for multiple proteins to be analyzed simultaneously without sacrifice on accuracy or sensitivity. METHODS: We analyzed the plasma protein profiles of COVID-19 patients (n = 50) with mild and moderate symptoms by comparing the protein levels in newly diagnosed patients with the protein levels in the same individuals after 14 days. FINDINGS: The study has identified more than 200 proteins that are significantly elevated during infection and many of these are related to cytokine response and other immune-related functions. In addition, several other proteins are shown to be elevated, including SCARB2, a host cell receptor protein involved in virus entry. A comparison with the plasma protein response in patients with severe symptoms shows a highly similar pattern, but with some interesting differences. INTERPRETATION: The study presented here demonstrates the usefulness of "next generation plasma protein profiling" to identify molecular signatures of importance for disease progression and to allow monitoring of disease during recovery from the infection. The results will facilitate further studies to understand the molecular mechanism of the immune-related response of the SARS-CoV-2 virus. FUNDING: This work was financially supported by Knut and Alice Wallenberg Foundation.

SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis.

COVID-19-induced "acute respiratory distress syndrome" (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyze pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single-cell genomics, immunohistology, and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS.

Impacts of p97 on Proteome Changes in Human Cells during Coronaviral Replication.

Human coronavirus (HCoV) similar to other viruses rely on host cell machinery for both replication and to spread. The p97/VCP ATPase is associated with diverse pathways that may favor HCoV replication. In this study, we assessed the role of p97 and associated host responses in human lung cell line H1299 after HCoV-229E or HCoV-OC43 infection. Inhibition of p97 function by small molecule inhibitors shows antiviral activity, particularly at early stages of the virus life cycle, during virus uncoating and viral RNA replication. Importantly, p97 activity inhibition protects human cells against HCoV-induced cytopathic effects. The p97 knockdown also inhibits viral production in infected cells. Unbiased quantitative proteomics analyses reveal that HCoV-OC43 infection resulted in proteome changes enriched in cellular senescence and DNA repair during virus replication. Further analysis of protein changes between infected cells with control and p97 shRNA identifies cell cycle pathways for both HCoV-229E and HCoV-OC43 infection. Together, our data indicate a role for the essential host protein p97 in supporting HCoV replication, suggesting that p97 is a therapeutic target to treat HCoV infection.

Selectivity aspects of activity-based (chemical) probes.

Selective chemical modulators are ideal tools to study the function of a protein. Yet, the poor ligandability of many proteins has hampered the development of specific chemical probes for numerous protein classes. Tools, such as covalent inhibitors and activity-based protein profiling, have enhanced our understanding of thus-far difficult-to-target proteins and have enabled correct assessment of the selectivity of small-molecule modulators. This also requires deeper knowledge of compound and target site reactivity, evaluation of binding to noncovalent targets and protein turnover. The availability of highly selective chemical probes, the evolution of activity-based probes, and the development of profiling methods will open a new era of drugging the undruggable proteome.

The proximal proteome of 17 SARS-CoV-2 proteins links to disrupted antiviral signaling and host translation.

Viral proteins localize within subcellular compartments to subvert host machinery and promote pathogenesis. To study SARS-CoV-2 biology, we generated an atlas of 2422 human proteins vicinal to 17 SARS-CoV-2 viral proteins using proximity proteomics. This identified viral proteins at specific intracellular locations, such as association of accessary proteins with intracellular membranes, and projected SARS-CoV-2 impacts on innate immune signaling, ER-Golgi transport, and protein translation. It identified viral protein adjacency to specific host proteins whose regulatory variants are linked to COVID-19 severity, including the TRIM4 interferon signaling regulator which was found proximal to the SARS-CoV-2 M protein. Viral NSP1 protein adjacency to the EIF3 complex was associated with inhibited host protein translation whereas ORF6 localization with MAVS was associated with inhibited RIG-I 2CARD-mediated IFNB1 promoter activation. Quantitative proteomics identified candidate host targets for the NSP5 protease, with specific functional cleavage sequences in host proteins CWC22 and FANCD2. This data resource identifies host factors proximal to viral proteins in living human cells and nominates pathogenic mechanisms employed by SARS-CoV-2.