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1.
Metabolomics ; 18(1): 6, 2021 12 20.
Article in English | MEDLINE | ID: covidwho-1588749

ABSTRACT

INTRODUCTION: The diagnosis of COVID-19 is normally based on the qualitative detection of viral nucleic acid sequences. Properties of the host response are not measured but are key in determining outcome. Although metabolic profiles are well suited to capture host state, most metabolomics studies are either underpowered, measure only a restricted subset of metabolites, compare infected individuals against uninfected control cohorts that are not suitably matched, or do not provide a compact predictive model. OBJECTIVES: Here we provide a well-powered, untargeted metabolomics assessment of 120 COVID-19 patient samples acquired at hospital admission. The study aims to predict the patient's infection severity (i.e., mild or severe) and potential outcome (i.e., discharged or deceased). METHODS: High resolution untargeted UHPLC-MS/MS analysis was performed on patient serum using both positive and negative ionization modes. A subset of 20 intermediary metabolites predictive of severity or outcome were selected based on univariate statistical significance and a multiple predictor Bayesian logistic regression model was created. RESULTS: The predictors were selected for their relevant biological function and include deoxycytidine and ureidopropionate (indirectly reflecting viral load), kynurenine (reflecting host inflammatory response), and multiple short chain acylcarnitines (energy metabolism) among others. Currently, this approach predicts outcome and severity with a Monte Carlo cross validated area under the ROC curve of 0.792 (SD 0.09) and 0.793 (SD 0.08), respectively. A blind validation study on an additional 90 patients predicted outcome and severity at ROC AUC of 0.83 (CI 0.74-0.91) and 0.76 (CI 0.67-0.86). CONCLUSION: Prognostic tests based on the markers discussed in this paper could allow improvement in the planning of COVID-19 patient treatment.


Subject(s)
COVID-19/blood , Chromatography, Liquid/methods , Metabolomics/methods , Tandem Mass Spectrometry/methods , Aged , Biomarkers/blood , Female , Humans , Male , Middle Aged , Prognosis , SARS-CoV-2 , Severity of Illness Index
2.
Life Sci Alliance ; 5(1)2022 01.
Article in English | MEDLINE | ID: covidwho-1515726

ABSTRACT

Understanding pathways that might impact coronavirus disease 2019 (COVID-19) manifestations and disease outcomes is necessary for better disease management and for therapeutic development. Here, we analyzed alterations in sphingolipid (SL) levels upon infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 infection induced elevation of SL levels in both cells and sera of infected mice. A significant increase in glycosphingolipid levels was induced early post SARS-CoV-2 infection, which was essential for viral replication. This elevation could be reversed by treatment with glucosylceramide synthase inhibitors. Levels of sphinganine, sphingosine, GA1, and GM3 were significantly increased in both cells and the murine model upon SARS-CoV-2 infection. The potential involvement of SLs in COVID-19 pathology is discussed.


Subject(s)
COVID-19/metabolism , Disease Models, Animal , Sphingolipids/metabolism , Virus Replication/physiology , Animals , COVID-19/prevention & control , COVID-19/virology , Chlorocebus aethiops , Chromatography, Liquid/methods , Dioxanes/pharmacology , Gangliosides/blood , Gangliosides/metabolism , Glucosyltransferases/antagonists & inhibitors , Glucosyltransferases/metabolism , Humans , Mass Spectrometry/methods , Mice, Transgenic , Pyrrolidines/pharmacology , SARS-CoV-2/drug effects , SARS-CoV-2/physiology , Sphingolipids/blood , Sphingosine/analogs & derivatives , Sphingosine/blood , Sphingosine/metabolism , Vero Cells , Virus Replication/drug effects
3.
Sci Rep ; 11(1): 21725, 2021 11 05.
Article in English | MEDLINE | ID: covidwho-1504567

ABSTRACT

SARS-CoV-2 enters the intestine by the spike protein binding to angiotensin-converting enzyme 2 (ACE2) receptors in enterocyte apical membranes, leading to diarrhea in some patients. Early treatment of COVID-19-associated diarrhea could relieve symptoms and limit viral spread within the gastrointestinal (GI) tract. Diosmectite, an aluminomagnesium silicate adsorbent clay with antidiarrheal effects, is recommended in some COVID-19 management protocols. In rotavirus models, diosmectite prevents pathogenic effects by binding the virus and its enterotoxin. We tested the trapping and anti-inflammatory properties of diosmectite in a SARS-CoV-2 model. Trapping effects were tested in Caco-2 cells using spike protein receptor-binding domain (RBD) and heat-inactivated SARS-CoV-2 preparations. Trapping was assessed by immunofluorescence, alone or in the presence of cells. The effect of diosmectite on nuclear factor kappa B (NF-kappaB) activation and CXCL10 secretion induced by the spike protein RBD and heat-inactivated SARS-CoV-2 were analyzed by Western blot and ELISA, respectively. Diosmectite bound the spike protein RBD and SARS-CoV-2 preparation, and inhibited interaction of the spike protein RBD with ACE2 receptors on the Caco-2 cell surface. Diosmectite exposure also inhibited NF-kappaB activation and CXCL10 secretion. These data provide direct evidence that diosmectite can bind SARS-CoV-2 components and inhibit downstream inflammation, supporting a mechanistic rationale for consideration of diosmectite as a management option for COVID-19-associated diarrhea.


Subject(s)
COVID-19/drug therapy , Chemokine CXCL10/metabolism , NF-kappa B p50 Subunit/metabolism , SARS-CoV-2 , Silicates/chemistry , Adsorption , Aluminum Compounds/chemistry , Angiotensin-Converting Enzyme 2/metabolism , Anti-Inflammatory Agents , Binding Sites , Caco-2 Cells , Chromatography, Liquid , Clay , Diarrhea/etiology , Diarrhea/therapy , Enterocytes/metabolism , Gastroenterology , Humans , Magnesium Compounds/chemistry , Mass Spectrometry , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding/drug effects , Protein Domains , Rotavirus , Silicates/metabolism
4.
J Am Soc Nephrol ; 32(1): 86-97, 2021 01.
Article in English | MEDLINE | ID: covidwho-1496654

ABSTRACT

BACKGROUND: Cultured cell lines are widely used for research in the physiology, pathophysiology, toxicology, and pharmacology of the renal proximal tubule. The lines that are most appropriate for a given use depend upon the genes expressed. New tools for transcriptomic profiling using RNA sequencing (RNA-Seq) make it possible to catalog expressed genes in each cell line. METHODS: Fourteen different proximal tubule cell lines, representing six species, were grown on permeable supports under conditions specific for the respective lines. RNA-Seq followed standard procedures. RESULTS: Transcripts expressed in cell lines variably matched transcripts selectively expressed in native proximal tubule. Opossum kidney (OK) cells displayed the highest percentage match (45% of proximal marker genes [TPM threshold =15]), with pig kidney cells (LLC-PK1) close behind (39%). Lower-percentage matches were seen for various human lines, including HK-2 (26%), and lines from rodent kidneys, such as NRK-52E (23%). Nominally, identical OK cells from different sources differed substantially in expression of proximal tubule markers. Mapping cell line transcriptomes to gene sets for various proximal tubule functions (sodium and water transport, protein transport, metabolic functions, endocrine functions) showed that different lines may be optimal for experimentally modeling each function. An online resource (https://esbl.nhlbi.nih.gov/JBrowse/KCT/) has been created to interrogate cell line transcriptome data. Proteomic analysis of NRK-52E cells confirmed low expression of many proximal tubule marker proteins. CONCLUSIONS: No cell line fully matched the transcriptome of native proximal tubule cells. However, some of the lines tested are suitable for the study of particular metabolic and transport processes seen in the proximal tubule.


Subject(s)
Cell Culture Techniques/methods , Kidney Tubules, Proximal/metabolism , Transcriptome , Animals , Biological Transport , Cell Line , Chromatography, Liquid , Gene Expression Profiling , Humans , Internet , Mice , Opossums , Proteomics , RNA-Seq , Rats , Sequence Analysis, RNA , Species Specificity , Swine , Tandem Mass Spectrometry
5.
Sci Rep ; 11(1): 20866, 2021 10 21.
Article in English | MEDLINE | ID: covidwho-1479816

ABSTRACT

A causal relationship between plasma ceramide concentration and respiratory distress symptoms in COVID-19 patients is inferred. In this study, plasma samples of 52 individuals infected with COVID-19 were utilized in a lipidomic analysis. Lipids belonging to the ceramide class exhibited a 400-fold increase in total plasma concentration in infected patients. Further analysis led to the demonstration of concentration dependency for severe COVID-19 respiratory symptoms in a subclass of ceramides. The subclasses Cer(d18:0/24:1), Cer(d18:1/24:1), and Cer(d18:1/22:0) were shown to be increased by 48-, 40-, and 33-fold, respectively, in infected plasma samples and to 116-, 91- and 50-fold, respectively, in plasma samples with respiratory distress. Hence, monitoring plasma ceramide concentration, can be a valuable tool for measuring effects of therapies on COVID-19 respiratory distress patients.


Subject(s)
COVID-19/blood , COVID-19/complications , Ceramides/blood , Respiratory Distress Syndrome/blood , Respiratory Distress Syndrome/complications , Adult , Aged , Aged, 80 and over , Chromatography, Liquid , Drug Design , Female , Humans , Ions , Lipids/chemistry , Male , Metabolomics , Middle Aged , Principal Component Analysis , Software , Tandem Mass Spectrometry , Virus Diseases , Young Adult
6.
Amino Acids ; 53(10): 1569-1588, 2021 Oct.
Article in English | MEDLINE | ID: covidwho-1449969

ABSTRACT

The pandemic of the coronavirus disease (COVID-19) caused by SARS-CoV-2 affects millions of people worldwide. There are still many unknown aspects to this infection which affects the whole world. In addition, the potential impacts caused by this infection are still unclear. Amino acid metabolism, in particular, contains significant clues in terms of the development and prevention of many diseases. Therefore, this study aimed to compare amino acid profile of COVID-19 and healthy subject. In this study, the amino acid profiles of patients with asymptomatic, mild, moderate, and severe/critical SARS-CoV-2 infection were scanned with LC-MS/MS. The amino acid profile encompassing 30 amino acids in 142 people including 30 control and 112 COVID-19 patients was examined. 20 amino acids showed significant differences when compared to the control group in COVID-19 patient groups with different levels of severity in the statistical analyses conducted. It was detected that the branched-chain amino acids (BCAAs) changed in correlation with one another, and L-2-aminobutyric acid and L-phenylalanine had biomarker potential for COVID-19. Moreover, it was concluded that L-2-aminobutyric acid could provide prognostic information about the course of the disease. We believe that a new viewpoint will develop regarding the diagnosis, treatment, and prognosis as a result of the evaluation of the serum amino acid profiles of COVID-19 patients. Determining L-phenylalanine and L-2-aminobutyric levels can be used in laboratories as a COVID-19-biomarker. Also, supplementing COVID patients with taurine and BCAAs can be beneficial for treatment protocols.


Subject(s)
Amino Acids/blood , COVID-19/blood , SARS-CoV-2/metabolism , Adult , Aged , Aged, 80 and over , Biomarkers/blood , COVID-19/diagnosis , Chromatography, Liquid , Female , Humans , Male , Mass Spectrometry , Middle Aged , Prognosis
7.
J Pharm Biomed Anal ; 205: 114319, 2021 Oct 25.
Article in English | MEDLINE | ID: covidwho-1406301

ABSTRACT

The aim of this study was to develop the first quantitative serological test for anti-SARS-CoV-2 antibodies in human serum with liquid chromatography - quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Other assays, mostly immunoassays, are only qualitative or semi-quantitative, and hence, actual antibody concentrations after SARS-CoV-2 infection are unknown. In our assay, anti-SARS-CoV-2 antibodies were isolated with spike protein subunit 1 (S1) coupled to magnetic beads. IgG1 signature peptide GPSVFPLAPSSK was selected for quantification using ipilimumab calibration standards and SILuMAb K1 as the stable-isotope labeled internal standard. The anti-SARS-CoV-2 IgG1 calibration range was from 1.35 to 135 nM. Inter-assay accuracies were between 98.8%- 107% with inter-assay precisions between 8.37%- 13.5% measured at 3 concentration levels on three separate occasions. Anti-SARS-CoV-2 IgG1 antibodies were quantified in PCR-positive patients with mild to severe symptoms. IgM signature peptide DGFFGVPR was detected in patients that recently recovered from COVID-19. A unique and quantitative LC-QTOF-MS method to quantify anti-SARS-CoV-2 IgG1 in serum was successfully developed and its clinical applicability has been demonstrated.


Subject(s)
COVID-19 , SARS-CoV-2 , Chromatography, Liquid , Humans , Immunoglobulin G , Tandem Mass Spectrometry
8.
J Am Soc Mass Spectrom ; 32(4): 1116-1125, 2021 Apr 07.
Article in English | MEDLINE | ID: covidwho-1397839

ABSTRACT

The metabolism of vitamin D3 includes a parallel C-3 epimerization pathway-in addition to the standard metabolic processes for vitamin D3-reversing the stereochemical configuration of the -OH group at carbon-3 (ß→α). While the biological function of the 3α epimer has not been elucidated yet, the additional species cannot be neglected in the analytical determination of vitamin D3, as it has the potential to introduce analytical errors if not properly accounted for. Recently, some inconsistent mass spectral behavior was seen for the 25-hydroxyvitamin D3 (25(OH)D3) epimers during quantification using electrospray LC-MS/MS. The present work extends that of Flynn et al. ( Ann. Clin. Biochem. 2014, 51, 352-559) and van den Ouweland et al. ( J. Chromatogr. B 2014, 967, 195-202), who reported larger electrospray ionization response factors for the 3α epimer of 25(OH)D3 in human serum samples as compared to the regular 3ß variant. The present work was concerned with the mechanistic reasons for these differences. We used a combination of electrospray ionization, atmospheric pressure chemical ionization, and density functional theory calculations to uncover structural dissimilarities between the epimers. A plausible mechanism is described based on intramolecular hydrogen bonding in the gas phase, which creates a small difference of proton affinities between the epimers. More importantly, this mechanism allows the explanation of the different ionization efficiencies of the epimers based on kinetic control of the ionization process, where ionization initially takes place at the hydroxyl group with subsequent proton transfer to a basic carbon atom. The barrier for this transfer differs between the epimers and is in direct competition with H2O elimination from the protonated hydroxyl group. The "hidden" site of high gas phase basicity was revealed through computational calculations and appears to be inaccessible via direct protonation.


Subject(s)
Calcifediol/blood , Chromatography, Liquid/methods , Tandem Mass Spectrometry/methods , Calcifediol/chemistry , Density Functional Theory , Gases , Molecular Structure , Protons , Solvents , Stereoisomerism
9.
Viruses ; 13(9)2021 08 30.
Article in English | MEDLINE | ID: covidwho-1390783

ABSTRACT

The ongoing COVID-19 pandemic exemplifies the general need to better understand viral infections. The positive single-strand RNA genome of its causative agent, the SARS coronavirus 2 (SARS-CoV-2), encodes all viral enzymes. In this work, we focused on one particular methyltransferase (MTase), nsp16, which, in complex with nsp10, is capable of methylating the first nucleotide of a capped RNA strand at the 2'-O position. This process is part of a viral capping system and is crucial for viral evasion of the innate immune reaction. In light of recently discovered non-canonical RNA caps, we tested various dinucleoside polyphosphate-capped RNAs as substrates for nsp10-nsp16 MTase. We developed an LC-MS-based method and discovered four types of capped RNA (m7Gp3A(G)- and Gp3A(G)-RNA) that are substrates of the nsp10-nsp16 MTase. Our technique is an alternative to the classical isotope labelling approach for the measurement of 2'-O-MTase activity. Further, we determined the IC50 value of sinefungin to illustrate the use of our approach for inhibitor screening. In the future, this approach may be an alternative technique to the radioactive labelling method for screening inhibitors of any type of 2'-O-MTase.


Subject(s)
COVID-19/virology , Methyltransferases/metabolism , SARS-CoV-2/enzymology , Viral Nonstructural Proteins/metabolism , Viral Regulatory and Accessory Proteins/metabolism , Chromatography, Liquid , Gene Expression Regulation, Viral , Humans , Mass Spectrometry , Methylation , Methyltransferases/genetics , RNA Caps , RNA, Viral/genetics , SARS-CoV-2/genetics , Substrate Specificity , Viral Nonstructural Proteins/genetics , Viral Regulatory and Accessory Proteins/genetics
10.
J Pharm Biomed Anal ; 206: 114356, 2021 Nov 30.
Article in English | MEDLINE | ID: covidwho-1386097

ABSTRACT

In light of the recent global pandemic, Molnupiravir (MPV) or EIDD-2801, developed for the treatment of patients with uncomplicated influenza, is now being trialled for the treatment of infections caused by highly pathogenic coronaviruses, including COVID-19. A sensitive LC-MS/MS method was developed and validated for the simultaneous quantification of MPV and its metabolite ß-d-N4-hydroxycytidine (NHC) in human plasma and saliva. The analytes were extracted from the matrices by protein precipitation using acetonitrile. This was followed by drying and subsequently injecting the reconstituted solutions onto the column. Chromatographic separation was achieved using a polar Atlantis C18 column with gradient elution of 1 mM Ammonium acetate in water (pH4.3) and 1 mM Ammonium acetate in acetonitrile. Analyte detection was conducted in negative ionisation mode using SRM. Analysis was performed using stable isotopically labelled (SIL) internal standards (IS). The m/z transitions were: MPV (328.1→126.0), NHC (258.0→125.9) and MPV-SIL (331.0→129.0), NHC-SIL (260.9→128.9). Validation was over a linear range of 2.5-5000 ng/ml for both plasma and saliva. Across four different concentrations, precision and accuracy (intra- and inter-day) were 15%; and recovery of both analytes from plasma and saliva was between 95% and 100% and 65-86% respectively. Clinical pharmacokinetic studies are underway utilising this method for determination of MPV and its metabolite in patients with COVID-19 infection.


Subject(s)
COVID-19 , Saliva , Chromatography, Liquid , Cytidine/analogs & derivatives , Humans , Hydroxylamines , Reproducibility of Results , SARS-CoV-2 , Tandem Mass Spectrometry
11.
J Proteome Res ; 19(11): 4398-4406, 2020 11 06.
Article in English | MEDLINE | ID: covidwho-1387124

ABSTRACT

Presentation of antigenic peptides by MHCI is central to cellular immune responses against viral pathogens. While adaptive immune responses versus SARS-CoV-2 can be of critical importance to both recovery and vaccine efficacy, how protein antigens from this pathogen are processed to generate antigenic peptides is largely unknown. Here, we analyzed the proteolytic processing of overlapping precursor peptides spanning the entire sequence of the S1 spike glycoprotein of SARS-CoV-2, by three key enzymes that generate antigenic peptides, aminopeptidases ERAP1, ERAP2, and IRAP. All enzymes generated shorter peptides with sequences suitable for binding onto HLA alleles, but with distinct specificity fingerprints. ERAP1 was the most efficient in generating peptides 8-11 residues long, the optimal length for HLA binding, while IRAP was the least efficient. The combination of ERAP1 with ERAP2 greatly limited the variability of peptide sequences produced. Less than 7% of computationally predicted epitopes were found to be produced experimentally, suggesting that aminopeptidase processing may constitute a significant filter to epitope presentation. These experimentally generated putative epitopes could be prioritized for SARS-CoV-2 immunogenicity studies and vaccine design. We furthermore propose that this in vitro trimming approach could constitute a general filtering method to enhance the prediction robustness for viral antigenic epitopes.


Subject(s)
Aminopeptidases/metabolism , Antigens, Viral , Epitopes , Spike Glycoprotein, Coronavirus , Antigens, Viral/chemistry , Antigens, Viral/metabolism , Chromatography, Liquid , Epitopes/chemistry , Epitopes/metabolism , HEK293 Cells , HLA Antigens/chemistry , HLA Antigens/metabolism , Humans , Peptides/analysis , Peptides/chemistry , Peptides/metabolism , Proteomics/methods , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Tandem Mass Spectrometry
12.
Anal Chem ; 93(38): 12889-12898, 2021 09 28.
Article in English | MEDLINE | ID: covidwho-1379296

ABSTRACT

REGEN-COV is a cocktail of two human IgG1 monoclonal antibodies (REGN10933 + REGN10987) that targets severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and has shown great promise to reduce the SARS-CoV-2 viral load in COVID-19 patients enrolled in clinical studies. A liquid chromatography-multiple reaction monitoring-mass spectrometry (LC-MRM-MS)-based method, combined with trypsin and rAspN dual enzymatic digestion, was developed for the determination of total REGN10933 and total REGN10987 concentrations in several hundreds of pharmacokinetic (PK) serum samples from COVID-19 patients participating in phase I, II, and III clinical studies. The performance characteristics of this bioanalytical assay were evaluated with respect to linearity, accuracy, precision, selectivity, specificity, and analyte stability before and after enzymatic digestion. The developed LC-MRM-MS assay has a dynamic range from 10 to 2000 µg/mL antibody drug in the human serum matrix, which was able to cover the serum drug concentration from day 0 to day 28 after drug administration in two-dose groups for the clinical PK study of REGEN-COV. The concentrations of REGEN-COV in the two-dose groups measured by the LC-MRM-MS assay were comparable to the concentrations measured by a fully validated electrochemiluminescence (ECL) immunoassay.


Subject(s)
COVID-19 , Antibodies, Monoclonal , Chromatography, Liquid , Humans , SARS-CoV-2 , Tandem Mass Spectrometry
13.
Anal Bioanal Chem ; 413(29): 7215-7227, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1375628

ABSTRACT

Glycosylation analysis of viral glycoproteins contributes significantly to vaccine design and development. Among other benefits, glycosylation analysis allows vaccine developers to assess the impact of construct design or producer cell line choices for vaccine production, and it is a key measure by which glycoproteins that are produced for use in vaccination can be compared to their native viral forms. Because many viral glycoproteins are multiply glycosylated, glycopeptide analysis is a preferrable approach for mapping the glycans, yet the analysis of glycopeptide data can be cumbersome and requires the expertise of an experienced analyst. In recent years, a commercial software product, Byonic, has been implemented in several instances to facilitate glycopeptide analysis on viral glycoproteins and other glycoproteomics data sets, and the purpose of the study herein is to determine the strengths and limitations of using this software, particularly in cases relevant to vaccine development. The glycopeptides from a recombinantly expressed trimeric S glycoprotein of the SARS-CoV-2 virus were first analyzed using an expert-based analysis strategy; subsequently, analysis of the same data set was completed using Byonic. Careful assessment of instances where the two methods produced different results revealed that the glycopeptide assignments from Byonic contained more false positives than true positives, even when the data were assessed using a 1% false discovery rate. The work herein provides a roadmap for removing the spurious assignments that Byonic generates, and it provides an assessment of the opportunity cost for relying on automated assignments for glycopeptide data sets from viral glycoproteins.


Subject(s)
Glycopeptides/metabolism , Spike Glycoprotein, Coronavirus/metabolism , Algorithms , Amino Acid Sequence , Chromatography, Liquid/methods , Spike Glycoprotein, Coronavirus/chemistry , Tandem Mass Spectrometry/methods
14.
Anal Bioanal Chem ; 413(26): 6503-6511, 2021 Nov.
Article in English | MEDLINE | ID: covidwho-1370381

ABSTRACT

We describe a rapid liquid chromatography/tandem mass spectrometry (LC-MS/MS) method for the direct detection and quantitation of SARS-CoV-2 nucleoprotein in gargle solutions and saliva. The method is based on a multiple-reaction monitoring (MRM) mass spectrometry approach with a total cycle time of 5 min per analysis and allows the detection and accurate quantitation of SARS-CoV-2 nucleoprotein as low as 500 amol/µL. We improved the sample preparation protocol of our recent piloting SARS-CoV-2 LC-MS study regarding sensitivity, reproducibility, and compatibility with a complementary reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) analysis of the same sample. The aim of this work is to promote diagnostic tools that allow identifying and monitoring SARS-CoV-2 infections by LC-MS/MS methods in a routine clinical environment.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , SARS-CoV-2/isolation & purification , Saliva/virology , Tandem Mass Spectrometry/methods , COVID-19 Testing/economics , Chromatography, Liquid/economics , Chromatography, Liquid/methods , Coronavirus Nucleocapsid Proteins/analysis , Coronavirus Nucleocapsid Proteins/isolation & purification , Humans , Limit of Detection , Phosphoproteins/analysis , Phosphoproteins/isolation & purification , Reproducibility of Results , Specimen Handling , Tandem Mass Spectrometry/economics , Time Factors
15.
J Gen Virol ; 102(8)2021 08.
Article in English | MEDLINE | ID: covidwho-1368372

ABSTRACT

Infectious bronchitis virus (IBV) is an economically important coronavirus, causing damaging losses to the poultry industry worldwide as the causative agent of infectious bronchitis. The coronavirus spike (S) glycoprotein is a large type I membrane protein protruding from the surface of the virion, which facilitates attachment and entry into host cells. The IBV S protein is cleaved into two subunits, S1 and S2, the latter of which has been identified as a determinant of cellular tropism. Recent studies expressing coronavirus S proteins in mammalian and insect cells have identified a high level of glycosylation on the protein's surface. Here we used IBV propagated in embryonated hens' eggs to explore the glycan profile of viruses derived from infection in cells of the natural host, chickens. We identified multiple glycan types on the surface of the protein and found a strain-specific dependence on complex glycans for recognition of the S2 subunit by a monoclonal antibody in vitro, with no effect on viral replication following the chemical inhibition of complex glycosylation. Virus neutralization by monoclonal or polyclonal antibodies was not affected. Following analysis of predicted glycosylation sites for the S protein of four IBV strains, we confirmed glycosylation at 18 sites by mass spectrometry for the pathogenic laboratory strain M41-CK. Further characterization revealed heterogeneity among the glycans present at six of these sites, indicating a difference in the glycan profile of individual S proteins on the IBV virion. These results demonstrate a non-specific role for complex glycans in IBV replication, with an indication of an involvement in antibody recognition but not neutralisation.


Subject(s)
Coronavirus/physiology , Polysaccharides/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/metabolism , Alkaloids/chemistry , Alkaloids/pharmacology , Amino Acid Sequence , Animals , Binding Sites , Cells, Cultured , Chromatography, Liquid , Computational Biology/methods , Coronavirus/drug effects , Coronavirus Infections/veterinary , Gene Expression Regulation, Viral , Glycosylation/drug effects , Infectious bronchitis virus/physiology , Models, Molecular , Molecular Conformation , Molecular Weight , Neutralization Tests , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Polysaccharides/chemistry , Poultry Diseases/virology , Protein Transport , Spectrometry, Mass, Electrospray Ionization , Spike Glycoprotein, Coronavirus/genetics , Structure-Activity Relationship , Virus Replication/drug effects
16.
J Proteomics ; 248: 104354, 2021 09 30.
Article in English | MEDLINE | ID: covidwho-1364279

ABSTRACT

Porcine rotavirus (PoRV), particularly group A, is one of the most important swine pathogens, causing substantial economic losses in the animal husbandry industry. To improve understanding of host responses to PoRV infection, we applied isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to quantitatively identify the differentially expressed proteins in PoRV-infected IPEC-J2 cells and confirmed the differentially accumulated proteins (DAPs) expression differences by performing RT-qPCR and Western blot analysis. Herein, in PoRV- and mock-infected IPEC-J2 cells, relative quantitative data were identified for 4724 proteins, 223 of which were DAPs (125 up-accumulated and 98 down-accumulated). Bioinformatics analyses further revealed that a majority of the DAPs are involved in numerous crucial biological processes and signaling pathways, such as metabolic process, immune system process, amino acid metabolism, energy metabolism, immune system, MHC class I peptide loading complex, Hippo signaling pathway, Th1 and Th2 cell differentiation, antigen processing and presentation, and tubule bicarbonate reclamation. The cellular localization prediction analysis indicated that these DAPs may be located in the Golgi apparatus, nucleus, peroxisomal, cytoplasm, mitochondria, extracellular, plasma membrane, and endoplasmic reticulum (ER). Expression levels of three up-accumulated (VAMP4, IKBKE, and TJP3) or two down-accumulated (SOD3 and DHX9) DAPs upon PoRV infection, were further validated by RT-qPCR and Western blot analysis. Collectively, this work is the first time to investigate the protein profile of PoRV-infected IPEC-J2 cells using quantitative proteomics; these findings provide valuable information to better understand the mechanisms underlying the host responses to PoRV infection in piglets. SIGNIFICANCE: The proteomics analysis of this study uncovered the target associated with PoRV-induced innate immune response or cellular damage, and provided relevant insights into the molecular functions, biological processes, and signaling pathway in these targets. Out of these 223 DAPs, the expression levels of three up-accumulated (VAMP4, IKBKE, and TJP3) and two down-accumulated (SOD3 and DHX9) DAPs upon PoRV infection, have been further validated using RT-qPCR and Western blot analysis. These outcomes could uncover how PoRV manipulated the cellular machinery, which could further our understanding of PoRV pathogenesis in piglets.


Subject(s)
Proteome , Rotavirus , Animals , Cell Line , Chromatography, Liquid , Epithelial Cells , Swine , Tandem Mass Spectrometry
17.
J Chromatogr B Analyt Technol Biomed Life Sci ; 1181: 122884, 2021 Sep 01.
Article in English | MEDLINE | ID: covidwho-1364212

ABSTRACT

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) vaccines are the most promising approach to control the COVID-19 pandemic. There are eminent needs to develop robust analytical methods to ensure quality control, as well as to evaluate the long-term efficacy and safety of vaccine. Although in vivo animal tests, such as serum-based ELISA, have been commonly used for quality control of vaccines, these methods have poor precision, are labor intensive, and require the availability of expensive, specific antibodies. Thus, there is growing interest to develop robust bioanalytical assays as alternatives for qualitative and quantitative evaluation of complex vaccine antigens. In this study, a liquid chromatography tandem mass spectrometry method was developed using optimized unique peptides for simultaneous determination of spike (S) and nucleocapsid (N) protein. Method sensitivity, linearity, repeatability, selectivity, and recovery were evaluated. The amount of S and N proteins in 9 batches of inactivated COVID-19 vaccines were quantified, and their compositions relative to total protein content were consistent. We believe this method can be applied for quality evaluation of other S and/or N protein based COVID-19 vaccine, and could be extended to other viral vector, and protein subunit-based vaccines.


Subject(s)
COVID-19 Vaccines/analysis , Chromatography, Liquid/methods , Coronavirus Nucleocapsid Proteins/analysis , SARS-CoV-2/chemistry , Spike Glycoprotein, Coronavirus/analysis , Tandem Mass Spectrometry/methods , COVID-19/virology , Humans , Quality Control , Vaccines, Inactivated/analysis
18.
Se Pu ; 39(7): 679-685, 2021 Jul 08.
Article in Chinese | MEDLINE | ID: covidwho-1362622

ABSTRACT

The novel coronavirus disease 2019 (COVID-19) outbreak has brought to light unprecedented challenges to global public health security. Researchers have devoted their efforts to in-depth research on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to bring the epidemic under control as rapidly as possible. Among the many areas of burgeoning SARS-CoV-2 related research, various analytical technologies have been applied to the advancement of virus detection, and development of vaccines and innovative therapies. Separation technologies with the merits of simple operation, high separation efficiency, and high selectivity, have become widely used and are key to progress in life science, medicine, pharmaceutical discovery and development, and other fields. Separation technologies have played an irreplaceable role in the isolation, detection, diagnosis, treatment, and prevention of this novel coronavirus. In this review, an overview of the relevant literature is presented from ISI Web of Science spanning Jan. 1st, 2020-Dec. 31, 2020, using "SARS-CoV-2" or "COVID-19" as keywords. The top 20 research directions are summarized, based on papers published in high impact international journals (e. g. Nature, Science, and Cell). Incorporating the impact of published papers, this review summarizes the primary separation technologies applied in these coronavirus studies, and discusses contributions of the following six technologies: affinity chromatography and size exclusion chromatography, liquid chromatography, magnetic bead separation technology, centrifugal technology, micro/nano-separation technology, and electrophoresis. First, affinity chromatography and size exclusion chromatography are discussed, which are the most frequently used protein purification techniques in Nature, Science, and Cell. The SARS-CoV-2 related proteins purified by affinity chromatography and size exclusion chromatography are summarized, and their applications in coronavirus transmission, infection mechanisms, and drug screening are introduced. Subsequently, high performance liquid chromatography (HPLC) is introduced, which is mainly employed for assessing the purity of candidate drugs. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) incorporates the strengths of HPLC and MS, offering both high separation efficiency and structural analysis capabilities with extended applications. LC-MS/MS has been applied to characterization of the binding of SARS-CoV-2 related proteins to potential inhibitors, and to metabolic analyses of candidate drugs. In SARS-CoV-2 nucleic acid tests, magnetic bead separation technology plays a crucial role in the separation of novel coronaviruses. In combination with other analytical techniques, magnetic bead separation technology can be applied to cytological analyses and immunological detection by functionalization of bead surfaces. Centrifugal technology is undoubtedly the most basic separation technology. It has been employed in almost all SARS-CoV-2 related researches. By controlling centrifugation speed, centrifugal technology can rapidly isolate virus particles or cultured cells from complex samples. Micro-nano separation technologies, such as microfluidics, offer advantages including small size, low sample consumption, rapid diffusion, and large surface area. In general, microfluidic technologies are often used in combination with other technologies to realize highly sensitive detection of SARS-CoV-2 related proteins. Finally, the applications of electrophoresis are introduced, which commonly engages in the analysis of polymerase chain reaction (PCR) products. In novel coronavirus studies, the application of electrophoresis has been relatively limited but has potential with further development to contribute significantly to future research. In conclusion, this review summarizes the contributions of six primary separation technologies to novel coronavirus studies, including epidemic detection and prevention, analyzes the main problems facing coronavirus detection efforts, and discusses the role of separation technologies in addressing these problems, with the aim of providing references for broader application of separation technologies.


Subject(s)
COVID-19/diagnosis , Epidemics/prevention & control , SARS-CoV-2/isolation & purification , Technology/trends , COVID-19/epidemiology , Centrifugation , Chromatography, Liquid , Humans , Magnetics , Tandem Mass Spectrometry
19.
J Proteome Res ; 20(10): 4667-4680, 2021 10 01.
Article in English | MEDLINE | ID: covidwho-1351919

ABSTRACT

Severe coronavirus disease 2019 (COVID-19) infection may lead to lung injury, multi-organ failure, and eventually death. Cytokine storm due to excess cytokine production has been associated with fatality in severe infections. However, the specific molecular signatures associated with the elevated immune response are yet to be elucidated. We performed a mass-spectrometry-based proteomic and metabolomic analysis of COVID-19 plasma samples collected at two time points. Using Orbitrap Fusion LC-MS/MS-based label-free proteomic analysis, we identified around 10 significant proteins, 32 significant peptides, and 5 metabolites that were dysregulated at the severe time points. Few of these proteins identified by quantitative proteomics were validated using the multiple reaction monitoring (MRM) assay. Integrated pathway analysis using distinct proteomic and metabolomic signatures revealed alterations in complement and coagulation cascade, platelet aggregation, myeloid leukocyte activation pathway, and arginine metabolism. Further, we highlight the role of leukocyte activation and arginine metabolism in COVID-19 pathogenesis and targeting these pathways for COVID-19 therapeutics.


Subject(s)
COVID-19 , Proteomics , Chromatography, Liquid , Humans , Leukocytes , Longitudinal Studies , SARS-CoV-2 , Tandem Mass Spectrometry
20.
Forensic Sci Int ; 321: 110743, 2021 Apr.
Article in English | MEDLINE | ID: covidwho-1347605

ABSTRACT

Chemsex and slamsex represent a serious public health concern that has to be considered by both clinical and forensic toxicologists. Indeed, such practices appear to carry a significant degree of risk, including acute intoxication. Here we report the case of the intoxication of a 31-year-old male involving 3-methylmethcathinone (3-MMC) and gamma-hydroxybutyrate (GHB) during a slamsex session. In addition, we conducted a review of further cases. The 31-year-old man was admitted to the emergency department for severe impaired consciousness following the administration of psychoactive substances during a chemsex party. The detection and determination of 3-MMC and GHB concentrations were achieved using liquid chromatography-tandem mass spectrometry. 3-MMC and GHB blood concentrations were 177 ng/mL and 131 mg/L, respectively. Further, an English and French exhaustive literature search was performed using several different electronic databases without any limiting period in order to identify all published case reports detailing chemsex/slamsex-related (fatal and nonfatal) intoxications. Nine publications detailing chemsex/slamsex-related intoxication cases have been published (between 2016 and 2020). These articles reported an overall of 13 cases, all involving men with a mean age of 39.1±9.8 years. The outcome was fatal in only 6 cases. 4-MEC and GHB were the two predominant drugs identified. However, given the rapid emergence of novel NPSs in the global market as well as the ease with which they can be accessed through the Internet, toxicological laboratories have to be ready to face new patterns of intoxications resulting from chemsex/slamsex.


Subject(s)
Drug Overdose , Methamphetamine/analogs & derivatives , Psychotropic Drugs , Sexual Behavior , Sodium Oxybate , Adult , Chromatography, Liquid , Forensic Toxicology , Humans , Male , Methamphetamine/adverse effects , Methamphetamine/analysis , Psychotropic Drugs/adverse effects , Psychotropic Drugs/analysis , Sodium Oxybate/adverse effects , Sodium Oxybate/analysis , Substance Abuse Detection , Tandem Mass Spectrometry
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