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1.
Cell Rep Med ; 2(5): 100287, 2021 05 18.
Article in English | MEDLINE | ID: covidwho-1683718

ABSTRACT

Mechanisms underlying severe coronavirus disease 2019 (COVID-19) disease remain poorly understood. We analyze several thousand plasma proteins longitudinally in 306 COVID-19 patients and 78 symptomatic controls, uncovering immune and non-immune proteins linked to COVID-19. Deconvolution of our plasma proteome data using published scRNA-seq datasets reveals contributions from circulating immune and tissue cells. Sixteen percent of patients display reduced inflammation yet comparably poor outcomes. Comparison of patients who died to severely ill survivors identifies dynamic immune-cell-derived and tissue-associated proteins associated with survival, including exocrine pancreatic proteases. Using derived tissue-specific and cell-type-specific intracellular death signatures, cellular angiotensin-converting enzyme 2 (ACE2) expression, and our data, we infer whether organ damage resulted from direct or indirect effects of infection. We propose a model in which interactions among myeloid, epithelial, and T cells drive tissue damage. These datasets provide important insights and a rich resource for analysis of mechanisms of severe COVID-19 disease.

2.
Processes ; 10(2):322, 2022.
Article in English | MDPI | ID: covidwho-1674762

ABSTRACT

ADAMTS13 is a hemostatic enzyme that breaks down pro-thrombotic ultra-large multimers of von Willebrand factor (VWF). The deficiency of ADAMTS13 increases VWF-mediated thrombogenic potential and may lead to thrombotic thrombocytopenic purpura (TTP). Recently, clinical studies have shown the development of acquired TTP after COVID-19 infection and a correlation between low ADAMTS13 plasma levels and increased mortality. As a result, investigating ADAMTS13 as a potential recombinant therapeutic is of broad interest in the field of hematology. ADAMTS13 is considered challenging to purify in its biologically active state. Current purification methods utilize immobilized metal ions, which can interfere with ADAMTS13 metalloprotease activity. For this reason, we optimized an alternative strategy to isolate milligram quantities of highly active recombinant ADAMTS13 (rADAMTS13) from conditioned media after exogenous expression in human cell line, HEK293. HEK293 cells stably expressing C-terminal V5-His-tagged ADAMTS13 were grown in two parallel systems, culture bottles and flasks, for identifying an optimal cultivation strategy. Subsequently, we employed anion exchange followed by anti-V5-tag affinity chromatography to purify rADAMTS13, and extracted rADAMTS13 of high specific activity while preserving its native post-translational modifications. In addition, this process has been optimized and scaled up to produce active rADAMTS13 at levels sufficient for laboratory-scale structural, enzymatic, and biochemical studies.

3.
Nat Med ; 27(3): 546-559, 2021 03.
Article in English | MEDLINE | ID: covidwho-1319033

ABSTRACT

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.


Subject(s)
COVID-19/epidemiology , COVID-19/genetics , Host-Pathogen Interactions/genetics , SARS-CoV-2/physiology , Sequence Analysis, RNA/statistics & numerical data , Single-Cell Analysis/statistics & numerical data , Virus Internalization , Adult , Aged , Aged, 80 and over , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/virology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , COVID-19/virology , Cathepsin L/genetics , Cathepsin L/metabolism , Datasets as Topic/statistics & numerical data , Demography , Female , Gene Expression Profiling/statistics & numerical data , Humans , Lung/metabolism , Lung/virology , Male , Middle Aged , Organ Specificity/genetics , Respiratory System/metabolism , Respiratory System/virology , Sequence Analysis, RNA/methods , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Single-Cell Analysis/methods
4.
Cell Rep Med ; 2(5): 100287, 2021 05 18.
Article in English | MEDLINE | ID: covidwho-1213572

ABSTRACT

Mechanisms underlying severe coronavirus disease 2019 (COVID-19) disease remain poorly understood. We analyze several thousand plasma proteins longitudinally in 306 COVID-19 patients and 78 symptomatic controls, uncovering immune and non-immune proteins linked to COVID-19. Deconvolution of our plasma proteome data using published scRNA-seq datasets reveals contributions from circulating immune and tissue cells. Sixteen percent of patients display reduced inflammation yet comparably poor outcomes. Comparison of patients who died to severely ill survivors identifies dynamic immune-cell-derived and tissue-associated proteins associated with survival, including exocrine pancreatic proteases. Using derived tissue-specific and cell-type-specific intracellular death signatures, cellular angiotensin-converting enzyme 2 (ACE2) expression, and our data, we infer whether organ damage resulted from direct or indirect effects of infection. We propose a model in which interactions among myeloid, epithelial, and T cells drive tissue damage. These datasets provide important insights and a rich resource for analysis of mechanisms of severe COVID-19 disease.

5.
JMIR Med Educ ; 7(2): e27877, 2021 Apr 21.
Article in English | MEDLINE | ID: covidwho-1195984

ABSTRACT

Telehealth has become an increasingly important part of health care delivery, with a dramatic rise in telehealth visits during the COVID-19 pandemic. Telehealth visits will continue to be a part of care delivery after the pandemic subsides, and it is important that medical students receive training in telehealth skills to meet emerging telehealth competencies. This paper describes strategies for successfully integrating medical students into telehealth visits in the ambulatory setting based on existing literature and the extensive experience of the authors teaching and learning in the telehealth environment.

6.
Nat Med ; 27(3): 546-559, 2021 03.
Article in English | MEDLINE | ID: covidwho-1114717

ABSTRACT

Angiotensin-converting enzyme 2 (ACE2) and accessory proteases (TMPRSS2 and CTSL) are needed for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cellular entry, and their expression may shed light on viral tropism and impact across the body. We assessed the cell-type-specific expression of ACE2, TMPRSS2 and CTSL across 107 single-cell RNA-sequencing studies from different tissues. ACE2, TMPRSS2 and CTSL are coexpressed in specific subsets of respiratory epithelial cells in the nasal passages, airways and alveoli, and in cells from other organs associated with coronavirus disease 2019 (COVID-19) transmission or pathology. We performed a meta-analysis of 31 lung single-cell RNA-sequencing studies with 1,320,896 cells from 377 nasal, airway and lung parenchyma samples from 228 individuals. This revealed cell-type-specific associations of age, sex and smoking with expression levels of ACE2, TMPRSS2 and CTSL. Expression of entry factors increased with age and in males, including in airway secretory cells and alveolar type 2 cells. Expression programs shared by ACE2+TMPRSS2+ cells in nasal, lung and gut tissues included genes that may mediate viral entry, key immune functions and epithelial-macrophage cross-talk, such as genes involved in the interleukin-6, interleukin-1, tumor necrosis factor and complement pathways. Cell-type-specific expression patterns may contribute to the pathogenesis of COVID-19, and our work highlights putative molecular pathways for therapeutic intervention.


Subject(s)
COVID-19/epidemiology , COVID-19/genetics , Host-Pathogen Interactions/genetics , SARS-CoV-2/physiology , Sequence Analysis, RNA/statistics & numerical data , Single-Cell Analysis/statistics & numerical data , Virus Internalization , Adult , Aged , Aged, 80 and over , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/virology , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/pathology , COVID-19/virology , Cathepsin L/genetics , Cathepsin L/metabolism , Datasets as Topic/statistics & numerical data , Demography , Female , Gene Expression Profiling/statistics & numerical data , Humans , Lung/metabolism , Lung/virology , Male , Middle Aged , Organ Specificity/genetics , Respiratory System/metabolism , Respiratory System/virology , Sequence Analysis, RNA/methods , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Single-Cell Analysis/methods
7.
chemRxiv; 2021.
Preprint in English | ChemRxiv | ID: ppcovidwho-6298

ABSTRACT

The COVID-19 pandemic and related shutdowns have caused changes in everyday activities for many people, and signs of those changes are present in the chemical signatures of sewage sludge produced during the pandemic. We analyzed primary sewage sludge samples from a wastewater treatment plant in New Haven, CT USA collected between March 19 and June 30, 2020. This time period encompassed the first wave of the COVID-19 pandemic, the initial statewide stay at home order, and the first phase of reopening. We used liquid chromatography coupled with high resolution mass spectrometry and targeted and suspect screening strategies to identify contaminants in the sludge. We and found evidence of increasing opioid, cocaine, and antidepressant use, as well as upward trends in chemicals used in disinfectants and sunscreens during the study period. Benzotriazole, an anti-corrosion chemical associated with traffic pollution, decreased through the stay-at-home period, and increased during reopening. Hydroxychloroquine, a drug that received significant attention for its potential to treat COVID-19, had elevated concentrations in the week following the implementation of the United States Emergency Use Authorization. Our results directly relate to nationwide reports of increased demand for fentanyl, antidepressants, and other medications, as well as reports of increased drug overdose deaths during the pandemic. Though wastewater surveillance during the pandemic has largely focused on measuring SARS-CoV-2 RNA concentrations, chemical analysis can also show trends that are important for revealing the public and environmental health effects of the pandemic.

8.
Cell ; 181(5): 1016-1035.e19, 2020 05 28.
Article in English | MEDLINE | ID: covidwho-100497

ABSTRACT

There is pressing urgency to understand the pathogenesis of the severe acute respiratory syndrome coronavirus clade 2 (SARS-CoV-2), which causes the disease COVID-19. SARS-CoV-2 spike (S) protein binds angiotensin-converting enzyme 2 (ACE2), and in concert with host proteases, principally transmembrane serine protease 2 (TMPRSS2), promotes cellular entry. The cell subsets targeted by SARS-CoV-2 in host tissues and the factors that regulate ACE2 expression remain unknown. Here, we leverage human, non-human primate, and mouse single-cell RNA-sequencing (scRNA-seq) datasets across health and disease to uncover putative targets of SARS-CoV-2 among tissue-resident cell subsets. We identify ACE2 and TMPRSS2 co-expressing cells within lung type II pneumocytes, ileal absorptive enterocytes, and nasal goblet secretory cells. Strikingly, we discovered that ACE2 is a human interferon-stimulated gene (ISG) in vitro using airway epithelial cells and extend our findings to in vivo viral infections. Our data suggest that SARS-CoV-2 could exploit species-specific interferon-driven upregulation of ACE2, a tissue-protective mediator during lung injury, to enhance infection.


Subject(s)
Alveolar Epithelial Cells/metabolism , Enterocytes/metabolism , Goblet Cells/metabolism , Interferon Type I/metabolism , Nasal Mucosa/cytology , Peptidyl-Dipeptidase A/genetics , Adolescent , Alveolar Epithelial Cells/immunology , Angiotensin-Converting Enzyme 2 , Animals , Betacoronavirus/physiology , COVID-19 , Cell Line , Cells, Cultured , Child , Coronavirus Infections/virology , Enterocytes/immunology , Goblet Cells/immunology , HIV Infections/immunology , Humans , Influenza, Human/immunology , Interferon Type I/immunology , Lung/cytology , Lung/pathology , Macaca mulatta , Mice , Mycobacterium tuberculosis , Nasal Mucosa/immunology , Pandemics , Peptidyl-Dipeptidase A/metabolism , Pneumonia, Viral/virology , Receptors, Virus/genetics , SARS-CoV-2 , Serine Endopeptidases/metabolism , Single-Cell Analysis , Tuberculosis/immunology , Up-Regulation
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