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1.
EMBO J ; 40(3): e106501, 2021 02 01.
Article in English | MEDLINE | ID: covidwho-1389834

ABSTRACT

Interferon-induced transmembrane proteins (IFITMs) restrict infections by many viruses, but a subset of IFITMs enhance infections by specific coronaviruses through currently unknown mechanisms. We show that SARS-CoV-2 Spike-pseudotyped virus and genuine SARS-CoV-2 infections are generally restricted by human and mouse IFITM1, IFITM2, and IFITM3, using gain- and loss-of-function approaches. Mechanistically, SARS-CoV-2 restriction occurred independently of IFITM3 S-palmitoylation, indicating a restrictive capacity distinct from reported inhibition of other viruses. In contrast, the IFITM3 amphipathic helix and its amphipathic properties were required for virus restriction. Mutation of residues within the IFITM3 endocytosis-promoting YxxФ motif converted human IFITM3 into an enhancer of SARS-CoV-2 infection, and cell-to-cell fusion assays confirmed the ability of endocytic mutants to enhance Spike-mediated fusion with the plasma membrane. Overexpression of TMPRSS2, which increases plasma membrane fusion versus endosome fusion of SARS-CoV-2, attenuated IFITM3 restriction and converted amphipathic helix mutants into infection enhancers. In sum, we uncover new pro- and anti-viral mechanisms of IFITM3, with clear distinctions drawn between enhancement of viral infection at the plasma membrane and amphipathicity-based mechanisms used for endosomal SARS-CoV-2 restriction.


Subject(s)
Antigens, Differentiation/genetics , COVID-19/genetics , Membrane Proteins/genetics , RNA-Binding Proteins/genetics , SARS-CoV-2 , Angiotensin-Converting Enzyme 2/genetics , Animals , Cell Line , Chlorocebus aethiops , Humans , Mice , Mutation , SARS-CoV-2/physiology , Serine Endopeptidases , Virus Internalization
2.
Int J Mol Sci ; 22(11)2021 May 29.
Article in English | MEDLINE | ID: covidwho-1389398

ABSTRACT

Trypsin-like proteases (TLPs) belong to a family of serine enzymes with primary substrate specificities for the basic residues, lysine and arginine, in the P1 position. Whilst initially perceived as soluble enzymes that are extracellularly secreted, a number of novel TLPs that are anchored in the cell membrane have since been discovered. Muco-obstructive lung diseases (MucOLDs) are characterised by the accumulation of hyper-concentrated mucus in the small airways, leading to persistent inflammation, infection and dysregulated protease activity. Although neutrophilic serine proteases, particularly neutrophil elastase, have been implicated in the propagation of inflammation and local tissue destruction, it is likely that the serine TLPs also contribute to various disease-relevant processes given the roles that a number of these enzymes play in the activation of both the epithelial sodium channel (ENaC) and protease-activated receptor 2 (PAR2). More recently, significant attention has focused on the activation of viruses such as SARS-CoV-2 by host TLPs. The purpose of this review was to highlight key TLPs linked to the activation of ENaC and PAR2 and their association with airway dehydration and inflammatory signalling pathways, respectively. The role of TLPs in viral infectivity will also be discussed in the context of the inhibition of TLP activities and the potential of these proteases as therapeutic targets.


Subject(s)
COVID-19/enzymology , Lung Diseases, Obstructive/enzymology , SARS-CoV-2/metabolism , Trypsin/metabolism , Animals , COVID-19/pathology , Epithelial Sodium Channels/metabolism , Humans , Lung Diseases, Obstructive/pathology , Receptor, PAR-2/metabolism
3.
Front Microbiol ; 11: 2014, 2020.
Article in English | MEDLINE | ID: covidwho-1389202

ABSTRACT

Electron microscopy is a powerful tool in the field of microbiology. It has played a key role in the rapid diagnosis of viruses in patient samples and has contributed significantly to the clarification of virus structure and function, helping to guide the public health response to emerging viral infections. In the present study, we used scanning electron microscopy (SEM) to study the infectious cycle of SARS-CoV-2 in Vero E6 cells and we controlled some key findings by classical transmission electronic microscopy (TEM). The replication cycle of the virus was followed from 1 to 36 h post-infection. Our results revealed that SARS-CoV-2 infected the cells through membrane fusion. Particles are formed in the peri-nuclear region from a budding of the endoplasmic reticulum-Golgi apparatus complex into morphogenesis matrix vesicae. New SARS-CoV-2 particles were expelled from the cells, through cell lysis or by fusion of virus containing vacuoles with the cell plasma membrane. Overall, this cycle is highly comparable to that of SARS-CoV. By providing a detailed and complete SARS-CoV-2 infectious cycle, SEM proves to be a very rapid and efficient tool compared to classical TEM.

4.
Cell Rep Med ; 2(6): 100321, 2021 06 15.
Article in English | MEDLINE | ID: covidwho-1253745

ABSTRACT

The pathogenesis of severe coronavirus disease 2019 (COVID-19) remains poorly understood. While several studies suggest that immune dysregulation plays a central role, the key mediators of this process are yet to be defined. Here, we demonstrate that plasma from a high proportion (93%) of critically ill COVID-19 patients, but not healthy controls, contains broadly auto-reactive immunoglobulin M (IgM) and less frequently auto-reactive IgG or IgA. Importantly, these auto-IgMs preferentially recognize primary human lung cells in vitro, including pulmonary endothelial and epithelial cells. By using a combination of flow cytometry, analytical proteome microarray technology, and lactose dehydrogenase (LDH)-release cytotoxicity assays, we identify high-affinity, complement-fixing, auto-reactive IgM directed against 260 candidate autoantigens, including numerous molecules preferentially expressed on the cellular membranes of pulmonary, vascular, gastrointestinal, and renal tissues. These findings suggest that broad IgM-mediated autoimmune reactivity may be involved in the pathogenesis of severe COVID-19, thereby identifying a potential target for therapeutic interventions.


Subject(s)
Autoantibodies/immunology , COVID-19/pathology , Immunoglobulin M/immunology , Autoantibodies/blood , COVID-19/immunology , COVID-19/virology , Cell Line , Complement C4/metabolism , Critical Illness , Humans , Immunoglobulin M/blood , Intensive Care Units , Lung/metabolism , Protein Array Analysis , Proteome/analysis , SARS-CoV-2/isolation & purification
5.
J Biol Chem ; 297(1): 100847, 2021 07.
Article in English | MEDLINE | ID: covidwho-1246014

ABSTRACT

The zoonotic transmission of highly pathogenic coronaviruses into the human population is a pressing concern highlighted by the ongoing SARS-CoV-2 pandemic. Recent work has helped to illuminate much about the mechanisms of SARS-CoV-2 entry into the cell, which determines host- and tissue-specific tropism, pathogenicity, and zoonotic transmission. Here we discuss current findings on the factors governing SARS-CoV-2 entry. We first reviewed key features of the viral spike protein (S) mediating fusion of the viral envelope and host cell membrane through binding to the SARS-CoV-2 receptor, angiotensin-converting enzyme 2. We then examined the roles of host proteases including transmembrane protease serine 2 and cathepsins in processing S for virus entry and the impact of this processing on endosomal and plasma membrane virus entry routes. We further discussed recent work on several host cofactors that enhance SARS-CoV-2 entry including Neuropilin-1, CD147, phosphatidylserine receptors, heparan sulfate proteoglycans, sialic acids, and C-type lectins. Finally, we discussed two key host restriction factors, i.e., interferon-induced transmembrane proteins and lymphocyte antigen 6 complex locus E, which can disrupt SARS-CoV-2 entry. The features of SARS-CoV-2 are presented in the context of other human coronaviruses, highlighting unique aspects. In addition, we identify the gaps in understanding of SARS-CoV-2 entry that will need to be addressed by future studies.


Subject(s)
COVID-19/metabolism , SARS-CoV-2/physiology , Virus Internalization , Animals , Basigin/genetics , Basigin/metabolism , COVID-19/genetics , COVID-19/virology , Host-Pathogen Interactions , Humans , Lectins, C-Type/genetics , Lectins, C-Type/metabolism , Neuropilin-1/genetics , Neuropilin-1/metabolism , Receptors, Virus/genetics , Receptors, Virus/metabolism , SARS-CoV-2/genetics
6.
Nan Fang Yi Ke Da Xue Xue Bao ; 41(4): 628-632, 2021 Apr 20.
Article in Chinese | MEDLINE | ID: covidwho-1219329

ABSTRACT

The high comorbidity between cardiovascular and metabolic diseases (CVMD) and coronavirus disease 2019 (COVID-19) and the consequent high mortality and the potential risk of cardiovascular damage have brought great challenges to the clinical diagnosis and treatment of the condition. The latest studies found that advanced age, immune function defects, inflammatory factor storms and oxidative stress damage all potentially contribute to the high comorbidity of the two. Direct virus invasion, myocardial oxygen supply and demand imbalance and vascular endothelial and coagulation dysfunction may be important mechanisms for cardiovascular injury in COVID-19 patients. In addition, the expression level of ACE2 (the cell membrane receptor of SARS-CoV-2) in various organs and the peripheral blood not only mediates the direct invasion and damage of the organs, but also participates in regulation of the balance of systematic inflammation and oxidative stress, thus affecting the susceptibility and outcomes of the patients. Herein we review the recent research progress in the comorbidity between COVID-19 and CVMD and explore the mechanisms of cardiovascular damage caused by SARS-CoV-2, thus to provide a theoretical basis for the clinical diagnosis and treatment of COVID-19 with underlying CVMD.


Subject(s)
COVID-19 , Cardiovascular Diseases , Metabolic Diseases , Cardiovascular Diseases/epidemiology , Comorbidity , Humans , Metabolic Diseases/complications , Metabolic Diseases/epidemiology , SARS-CoV-2
7.
mBio ; 12(3)2021 05 04.
Article in English | MEDLINE | ID: covidwho-1216782

ABSTRACT

The outbreak of SARS-CoV-2 infection has enormously impacted our lives. Clinical evidence has implicated the emergence of cytokine release syndrome as the prominent cause of mortality in COVID-19 patients. In this study, we observed massive elevation of plasma Galectin-9 (Gal-9) in COVID-19 patients compared to healthy controls (HCs). By using the receiver operating characteristic (ROC) curve, we found that a baseline of 2,042 pg/ml plasma Gal-9 can differentiate SARS-CoV-2-infected from noninfected individuals with high specificity/sensitivity (95%). Analysis of 30 cytokines and chemokines detected a positive correlation of the plasma Gal-9 with C-reactive protein (CRP) and proinflammatory cytokines/chemokines such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), IP-10, MIP-1α, and MCP-1 but an inverse correlation with transforming growth factor ß (TGF-ß) in COVID-19 patients. In agreement, we found enhanced production of IL-6 and TNF-α by monocytes and NK cells of COVID-19 patients once treated with the recombinant human Gal-9 in vitro Also, we observed that although the cell-membrane expression of Gal-9 on monocytes does not change in COVID-19 patients, those with higher Gal-9 expression exhibit an activated phenotype. Furthermore, we noted significant downregulation of surface Gal-9 in neutrophils from COVID-19 patients compared to HCs. Our further investigations indicated that immune activation following SARS-CoV-2 infection results in Gal-9 shedding from neutrophils. The strong correlation of Gal-9 with proinflammatory mediators suggests that inhibition of Gal-9 may severe as a therapeutic approach in COVID-19 infection. Besides, the plasma Gal-9 measurement may be used as a surrogate diagnostic biomarker in COVID-19 patients.IMPORTANCE The outbreak of SARS-CoV-2 infection has enormously impacted our lives. Clinical evidence has implicated the emergence of cytokine release syndrome as the prominent cause of mortality in COVID-19 patients. We observed substantial elevation of the plasma Galectin-9 (Gal-9) in COVID-19 patients compared to healthy controls. Gal-9 is an abundant protein in many immune and nonimmune cells. We found that Gal-9 detection assay can differentiate SARS-CoV-2-infected from noninfected individuals with a specificity/sensitivity of 95%. Importantly, we found a positive correlation of the plasma Gal-9 with a wide range of proinflammatory biomarkers in COVID-19 patients. In agreement, we found enhanced expression and production of such proinflammatory molecules by immune cells of COVID-19 patients once treated with Gal-9 in vitro Our results propose Gal-9 as an important contributing factor in cytokine release syndrome; therefore, Gal-9 inhibition may serve as a beneficial therapeutic approach by suppressing the hyperimmune activation in COVID-19 patients.


Subject(s)
Biomarkers/blood , COVID-19/blood , COVID-19/diagnosis , Cytokine Release Syndrome/blood , Cytokine Release Syndrome/physiopathology , Galectins/blood , Sex Factors , Adult , Age Factors , Aged , Aged, 80 and over , Female , Healthy Volunteers , Humans , Male , Middle Aged , ROC Curve , SARS-CoV-2
8.
Prog Lipid Res ; 82: 101099, 2021 04.
Article in English | MEDLINE | ID: covidwho-1201814

ABSTRACT

Despite encouraging progresses achieved in the management of viral diseases, efficient strategies to counteract infections are still required. The current global challenge highlighted the need to develop a rapid and cost-effective strategy to counteract the SARS-CoV-2 pandemic. Lipid metabolism plays a crucial role in viral infections. Viruses can use the host lipid machinery to support their life cycle and to impair the host immune response. The altered expression of mevalonate pathway-related genes, induced by several viruses, assures survival and spread in host tissue. In some infections, statins, HMG-CoA-reductase inhibitors, reduce cholesterol in the plasma membrane of permissive cells resulting in lower viral titers and failure to internalize the virus. Statins can also counteract viral infections through their immunomodulatory, anti-inflammatory and anti-thrombotic effects. Beyond statins, interfering with the mevalonate pathway could have an adjuvant effect in therapies aimed at mitigating endothelial dysfunction and deregulated inflammation in viral infection. In this review we depicted the historical and current evidence highlighting how lipid homeostasis and mevalonate pathway targeting represents a valid approach to rapidly neutralize viruses, focusing our attention to their potential use as effective targets to hinder SARS-CoV-2 morbidity and mortality. Pros and cons of statins and Mevalonate-pathway inhibitors have been also dissected.


Subject(s)
COVID-19/metabolism , Homeostasis , Lipid Metabolism , Mevalonic Acid/metabolism , COVID-19/drug therapy , COVID-19/virology , Humans , Hydroxymethylglutaryl-CoA Reductase Inhibitors/pharmacology , Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use , Hypolipidemic Agents/pharmacology , Hypolipidemic Agents/therapeutic use , Mevalonic Acid/antagonists & inhibitors , SARS-CoV-2/isolation & purification
9.
J Transl Med ; 19(1): 149, 2021 04 14.
Article in English | MEDLINE | ID: covidwho-1183544

ABSTRACT

BACKGROUND: Mesenchymal stem cells derived from human umbilical cord (hUC-MSCs) have immunomodulatory properties that are of interest to treat novel coronavirus disease 2019 (COVID-19). Leng et al. recently reported that hUC-MSCs derived from one donor negatively expressed Angiotensin-Converting Enzyme 2 (ACE2), a key protein for viral infection along with Transmembrane Serine Protease 2 (TMPRSS2). The purpose of this study was to quantify the expression of ACE2 and TMPRSS2 in hUC-MSCs lots derived from multiple donors using molecular-based techniques in order to demonstrate their inability to be a host to SARS-CoV-2. METHODS: Expression of ACE2 and TMPRSS2 was analyzed in 24 lots of hUC-MSCs derived from Wharton's jelly via quantitative polymerase chain reaction (qPCR), Western Blot, immunofluorescence and flow cytometry using 24 different donors. RESULTS: hUC-MSCs had significantly lower ACE2 (p = 0.002) and TMPRSS2 (p = 0.008) expression compared with human lung tissue homogenates in Western blot analyses. Little to no expression of ACE2 was observed in hUC-MSC by qPCR, and they were not observable with immunofluorescence in hUC-MSCs cell membranes. A negative ACE2 and TMPRSS2 population percentage of 95.3% ± 15.55 was obtained for hUC-MSCs via flow cytometry, with only 4.6% ACE2 and 29.5% TMPRSS2 observable positive populations. CONCLUSIONS: We have demonstrated negative expression of ACE2 and low expression of TMPRSS2 in 24 lots of hUC-MSCs. This has crucial implications for the design of future therapeutic options for COVID-19, since hUC-MSCs would have the ability to "dodge" viral infection to exert their immunomodulatory effects.


Subject(s)
COVID-19 , Mesenchymal Stem Cells , Angiotensin-Converting Enzyme 2 , Humans , Peptidyl-Dipeptidase A/genetics , SARS-CoV-2 , Serine Endopeptidases/genetics , Umbilical Cord
10.
Nature ; 594(7861): 88-93, 2021 06.
Article in English | MEDLINE | ID: covidwho-1171428

ABSTRACT

COVID-19 is a disease with unique characteristics that include lung thrombosis1, frequent diarrhoea2, abnormal activation of the inflammatory response3 and rapid deterioration of lung function consistent with alveolar oedema4. The pathological substrate for these findings remains unknown. Here we show that the lungs of patients with COVID-19 contain infected pneumocytes with abnormal morphology and frequent multinucleation. The generation of these syncytia results from activation of the SARS-CoV-2 spike protein at the cell plasma membrane level. On the basis of these observations, we performed two high-content microscopy-based screenings with more than 3,000 approved drugs to search for inhibitors of spike-driven syncytia. We converged on the identification of 83 drugs that inhibited spike-mediated cell fusion, several of which belonged to defined pharmacological classes. We focused our attention on effective drugs that also protected against virus replication and associated cytopathicity. One of the most effective molecules was the antihelminthic drug niclosamide, which markedly blunted calcium oscillations and membrane conductance in spike-expressing cells by suppressing the activity of TMEM16F (also known as anoctamin 6), a calcium-activated ion channel and scramblase that is responsible for exposure of phosphatidylserine on the cell surface. These findings suggest a potential mechanism for COVID-19 disease pathogenesis and support the repurposing of niclosamide for therapy.


Subject(s)
Anoctamins/antagonists & inhibitors , COVID-19/pathology , Cell Fusion , Drug Evaluation, Preclinical , Giant Cells/drug effects , SARS-CoV-2/drug effects , Spike Glycoprotein, Coronavirus/antagonists & inhibitors , Aged , Aged, 80 and over , Alveolar Epithelial Cells/drug effects , Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/virology , Animals , Anoctamins/metabolism , COVID-19/metabolism , COVID-19/virology , Calcium Signaling/drug effects , Cell Line , Chloride Channels/metabolism , Chlorocebus aethiops , Female , Giant Cells/metabolism , Giant Cells/virology , Humans , Lung/drug effects , Lung/pathology , Lung/virology , Male , SARS-CoV-2/metabolism , SARS-CoV-2/pathogenicity , Spike Glycoprotein, Coronavirus/metabolism , Virus Replication/drug effects
11.
Int J Mol Sci ; 22(6)2021 Mar 11.
Article in English | MEDLINE | ID: covidwho-1143517

ABSTRACT

The interactions at the atomic level between small molecules and the main components of cellular plasma membranes are crucial for elucidating the mechanisms allowing for the entrance of such small species inside the cell. We have performed molecular dynamics and metadynamics simulations of tryptophan, serotonin, and melatonin at the interface of zwitterionic phospholipid bilayers. In this work, we will review recent computer simulation developments and report microscopic properties, such as the area per lipid and thickness of the membranes, atomic radial distribution functions, angular orientations, and free energy landscapes of small molecule binding to the membrane. Cholesterol affects the behaviour of the small molecules, which are mainly buried in the interfacial regions. We have observed a competition between the binding of small molecules to phospholipids and cholesterol through lipidic hydrogen-bonds. Free energy barriers that are associated to translational and orientational changes of melatonin have been found to be between 10-20 kJ/mol for distances of 1 nm between melatonin and the center of the membrane. Corresponding barriers for tryptophan and serotonin that are obtained from reversible work methods are of the order of 10 kJ/mol and reveal strong hydrogen bonding between such species and specific phospholipid sites. The diffusion of tryptophan and melatonin is of the order of 10-7 cm2/s for the cholesterol-free and cholesterol-rich setups.


Subject(s)
1,2-Dipalmitoylphosphatidylcholine/chemistry , Cholesterol/chemistry , Dimyristoylphosphatidylcholine/chemistry , Melatonin/chemistry , Serotonin/chemistry , Tryptophan/chemistry , 1,2-Dipalmitoylphosphatidylcholine/metabolism , Cholesterol/metabolism , Dimyristoylphosphatidylcholine/metabolism , Hydrogen Bonding , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Melatonin/metabolism , Molecular Dynamics Simulation , Serotonin/metabolism , Solutions , Static Electricity , Thermodynamics , Tryptophan/metabolism , Water/chemistry
12.
J Physiol ; 599(11): 2851-2868, 2021 06.
Article in English | MEDLINE | ID: covidwho-1133113

ABSTRACT

KEY POINTS: We report a novel method for the transient expression of SARS-CoV-2 envelope (E) protein in intracellular organelles and the plasma membrane of mammalian cells and Xenopus oocytes. Intracellular expression of SARS-CoV-2 E protein increases intra-Golgi pH. By targeting the SARS-CoV-2 E protein to the plasma membrane, we show that it forms a cation channel, viroporin, that is modulated by changes of pH. This method for studying the activity of viroporins may facilitate screening for new antiviral drugs to identify novel treatments for COVID-19. ABSTRACT: The envelope (E) protein of coronaviruses such as SARS-CoV-1 is proposed to form an ion channel or viroporin that participates in viral propagation and pathogenesis. Here we developed a technique to study the E protein of SARS-CoV-2 in mammalian cells by directed targeting using a carboxyl-terminal fluorescent protein tag, mKate2. The wild-type SARS-CoV-2 E protein can be trafficked to intracellular organelles, notably the endoplasmic reticulum-Golgi intermediate complex, where its expression increases pH inside the organelle. We also succeeded in targeting SARS-CoV-2 E to the plasma membrane, which enabled biophysical analysis using whole-cell patch clamp recording in a mammalian cell line, HEK 293 cells, and two-electrode voltage clamp electrophysiology in Xenopus oocytes. The results suggest that the E protein forms an ion channel that is permeable to monovalent cations such as Na+ , Cs+ and K+ . The E current is nearly time- and voltage-independent when E protein is expressed in mammalian cells, and is modulated by changes of pH. At pH 6.0 and 7.4, the E protein current is activated, whereas at pH 8.0 and 9.0, the amplitude of E protein current is reduced, and in oocytes the inward E current fades at pH 9 in a time- and voltage-dependent manner. Using this directed targeting method and electrophysiological recordings, potential inhibitors of the E protein can be screened and subsequently investigated for antiviral activity against SARS-CoV-2 in vitro and possible efficacy in treating COVID-19.


Subject(s)
COVID-19 , SARS-CoV-2 , Animals , Cations , HEK293 Cells , Humans , Hydrogen-Ion Concentration
13.
FASEB J ; 35(4): e21384, 2021 04.
Article in English | MEDLINE | ID: covidwho-1132575

ABSTRACT

Novel coronary pneumonia (COVID-19) is a respiratory distress syndrome caused by a new type of coronavirus. Understanding the genetic basis of susceptibility and prognosis to COVID-19 is of great significance to disease prevention, molecular typing, prognosis, and treatment. However, so far, there have been only two genome-wide association studies (GWASs) on the susceptibility of COVID-19. Starting with these reported DNA variants, we found the genes regulated by these variants through cis-eQTL and cis-meQTL acting. We further did a series of bioinformatics analysis on these potential risk genes. The analysis shows that the genetic variants on EHF regulate the expression of its neighbor CAT gene via cis-eQTL. There was significant evidence that CAT and the SARS-CoV-2-related S protein binding protein ACE2 interact with each other. Intracellular localization results showed that CAT and ACE2 proteins both exists in the cell membrane and extracellular area and their interaction could have an impact on the cell invasion ability of S protein. In addition, the expression of these three genes showed a significant positive correlation in the lungs. Based on these results, we propose that CAT plays a crucial intermediary role in binding effectiveness of ACE2, thereby affecting the susceptibility to COVID-19.


Subject(s)
COVID-19 , Catalase , Gene Expression Regulation, Enzymologic , Genetic Predisposition to Disease , Polymorphism, Genetic , SARS-CoV-2/metabolism , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , COVID-19/genetics , COVID-19/metabolism , Catalase/biosynthesis , Catalase/genetics , Female , Genome-Wide Association Study , Humans , Male , Retrospective Studies , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism
14.
Int J Mol Sci ; 22(5)2021 Mar 05.
Article in English | MEDLINE | ID: covidwho-1129733

ABSTRACT

While there are various kinds of drugs for type 2 diabetes mellitus at present, in this review article, we focus on metformin which is an insulin sensitizer and is often used as a first-choice drug worldwide. Metformin mainly activates adenosine monophosphate-activated protein kinase (AMPK) in the liver which leads to suppression of fatty acid synthesis and gluconeogenesis. Metformin activates AMPK in skeletal muscle as well, which increases translocation of glucose transporter 4 to the cell membrane and thereby increases glucose uptake. Further, metformin suppresses glucagon signaling in the liver by suppressing adenylate cyclase which leads to suppression of gluconeogenesis. In addition, metformin reduces autophagy failure observed in pancreatic ß-cells under diabetic conditions. Furthermore, it is known that metformin alters the gut microbiome and facilitates the transport of glucose from the circulation into excrement. It is also known that metformin reduces food intake and lowers body weight by increasing circulating levels of the peptide hormone growth/differentiation factor 15 (GDF15). Furthermore, much attention has been drawn to the fact that the frequency of various cancers is lower in subjects taking metformin. Metformin suppresses the mechanistic target of rapamycin (mTOR) by activating AMPK in pre-neoplastic cells, which leads to suppression of cell growth and an increase in apoptosis in pre-neoplastic cells. It has been shown recently that metformin consumption potentially influences the mortality in patients with type 2 diabetes mellitus and coronavirus infectious disease (COVID-19). Taken together, metformin is an old drug, but multifaceted mechanisms of action of metformin have been unraveled one after another in its long history.


Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Metformin/pharmacology , Autophagy/drug effects , COVID-19/complications , COVID-19/mortality , Diabetes Mellitus, Type 2/complications , Diabetes Mellitus, Type 2/etiology , Diabetes Mellitus, Type 2/mortality , Gastrointestinal Microbiome/drug effects , Humans , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Intracellular Signaling Peptides and Proteins/drug effects , Intracellular Signaling Peptides and Proteins/metabolism
15.
Glob Health Med ; 3(2): 67-72, 2021 Apr 30.
Article in English | MEDLINE | ID: covidwho-1128387

ABSTRACT

Angiotensin converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2), two receptors on the cell membrane of bronchial epithelial cells, are indispensable for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. ACE2 receptor is increased among aged, males, and smokers. As smoking upsurges ACE2 expression, chronic obstructive pulmonary disease (COPD) patients are prone to SARS-CoV-2 infection, and are at a higher risk for severe forms of COVID-19 (coronavirus disease 2019) once infected. The expression of ACE2 and TMPRSS2 in asthma patients is identical (or less common) to that of healthy participants. ACE2 especially, tends to be low in patients with strong atopic factors and in those with poor asthma control. Therefore, it could be speculated that asthma patients are not susceptible to COVID-19. Epidemiologically, asthma patients are less likely to suffer from COVID-19, and the number of hospitalized patients due to exacerbation of asthma in Japan is also clearly reduced during the COVID-19 pandemic; therefore, they are not aggravating factors for COVID-19. Related academic societies in Japan and abroad still lack clear evidence regarding asthma treatment during the COVID-19 pandemic, and recommend that regular treatment including biologics for severe patients be continued.

16.
Exp Eye Res ; 205: 108527, 2021 04.
Article in English | MEDLINE | ID: covidwho-1116639

ABSTRACT

The purpose of this study was to evaluate the expression of the SARS-CoV-2 receptors ACE2 and TMPRSS2 in an immortalized human conjunctival epithelial cell line and in healthy human conjunctiva excised during ocular surgery, using Western blot, confocal microscopy and immunohistochemistry. The Western blot showed that ACE2 and TMPRSS2 proteins were expressed in human immortalized conjunctival cells, and this was confirmed by confocal microscopy images, that demonstrated a marked cellular expression of the viral receptors and their co-localization on the cell membranes. Healthy conjunctival samples from 11 adult patients were excised during retinal detachment surgery. We found the expression of ACE2 and TMPRSS2 in all the conjunctival surgical specimens analyzed and their co-localization in the superficial conjunctival epithelium. The ACE2 Western blot levels and immunofluorescence staining for ACE2 were variable among specimens. These results suggest the susceptibility of the conjunctival epithelium to SARS-CoV-2 infection, even though with a possible interindividual variability.


Subject(s)
COVID-19/genetics , Conjunctiva/metabolism , Epithelial Cells/metabolism , Gene Expression Regulation , Peptidyl-Dipeptidase A/genetics , Serine Endopeptidases/genetics , COVID-19/metabolism , COVID-19/pathology , Epithelial Cells/pathology , Humans , Immunohistochemistry , Peptidyl-Dipeptidase A/biosynthesis , RNA/genetics , RNA/metabolism , SARS-CoV-2 , Serine Endopeptidases/biosynthesis
17.
Comput Struct Biotechnol J ; 19: 1379-1390, 2021.
Article in English | MEDLINE | ID: covidwho-1103817

ABSTRACT

The type 2 coronavirus causes severe acute respiratory syndrome (SARS-CoV-2) and produces pneumonia with pulmonary alveolar collapse. In some cases it also causes sepsis and septic shock. There is no specific treatment for coronavirus disease 2019 (COVID-19). Vitamin C (Vit C), Vitamin E (Vit E), N-acetylcysteine (NAC) and Melatonin (MT) increase the intracellular content of GSH, kidnap free radicals and protect DNA, proteins in the cytosol and lipids in cell membranes. Pentoxifylline (Px) has anti-inflammatory activities. Here we evaluate the effect of Vit C, Vit E, NAC, and MT plus Px in COVID-19 patients with moderate and severe pneumonia. 110 patients of either sex were included. They were divided into five groups with 22 patients each. Group 1 received Vit C + Px, group 2 Vit E + Px, group 3 NAC + Px, group 4 MT + Px, and group 5 only Px. Oxidative stress (OS) markers such as lipid peroxidation (LPO) levels, total antioxidant capacity (TAC) and nitrites (NO2 -) were evaluated in plasma. The antioxidant therapy improved the survival scores including the Sequential Organ Failure Assessment (SOFA), the Acute Physiology and chronic Health Evaluation II (Apache II), the Simplified Acute Physiology Score II (SAPS II), the Critical Illness Risk Score, Launched during COVID-19 crisis (COVIDGRAM) and the Glasgow Coma Scale (GCS). We found that LPO (p≤0.04) and inflammation markers such as interleukin-6 (IL-6, p≤ 0.01), C reactive protein (CRP, p ≤ 0.01) and procalcitonin (PCT, p ≤ 0.05) were elevated. TAC (p ≤ 0.03) and NO2 - (p ≤ 0.04) found themselves diminished in diminished in COVID-19 patients upon admission to the hospital. The different antioxidants reversed this alteration at the end of the treatment. The treatment with antioxidant supplements such as Vit C, E, NAC, and MT plus Px could decelerate the aggressive and lethal development of COVID-19. Antioxidant therapy can be effective in this pandemia since it improves the survival scores including SOFA, Apache II, SAPS II, COVIDGRAM, GCS by lowering the LPO, IL-6, CRP, PCT and increasing systemic TAC and NO2 -.

18.
Biophys J ; 120(6): 1105-1119, 2021 03 16.
Article in English | MEDLINE | ID: covidwho-1103746

ABSTRACT

Cell penetration after recognition of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus by the ACE2 receptor and the fusion of its viral envelope membrane with cellular membranes are the early steps of infectivity. A region of the Spike protein of the virus, identified as the "fusion peptide" (FP), is liberated at its N-terminal site by a specific cleavage occurring in concert with the interaction of the receptor-binding domain of the Spike. Studies have shown that penetration is enhanced by the required binding of Ca2+ ions to the FPs of coronaviruses, but the mechanisms of membrane insertion and destabilization remain unclear. We have predicted the preferred positions of Ca2+ binding to the SARS-CoV-2-FP, the role of Ca2+ ions in mediating peptide-membrane interactions, the preferred mode of insertion of the Ca2+-bound SARS-CoV-2-FP, and consequent effects on the lipid bilayer from extensive atomistic molecular dynamics simulations and trajectory analyses. In a systematic sampling of the interactions of the Ca2+-bound peptide models with lipid membranes, SARS-CoV-2-FP penetrated the bilayer and disrupted its organization only in two modes involving different structural domains. In one, the hydrophobic residues F833/I834 from the middle region of the peptide are inserted. In the other, more prevalent mode, the penetration involves residues L822/F823 from the LLF motif, which is conserved in CoV-2-like viruses, and is achieved by the binding of Ca2+ ions to the D830/D839 and E819/D820 residue pairs. FP penetration is shown to modify the molecular organization in specific areas of the bilayer, and the extent of membrane binding of the SARS-CoV-2 FP is significantly reduced in the absence of Ca2+ ions. These findings provide novel mechanistic insights regarding the role of Ca2+ in mediating SARS-CoV-2 fusion and provide a detailed structural platform to aid the ongoing efforts in rational design of compounds to inhibit SARS-CoV-2 cell entry.


Subject(s)
Calcium/metabolism , Cell Membrane/metabolism , Recombinant Fusion Proteins/metabolism , SARS-CoV-2/metabolism , Amino Acid Sequence , Cell Membrane Permeability , Membrane Lipids/chemistry , Molecular Dynamics Simulation , Pressure , Probability , Protein Stability , Recombinant Fusion Proteins/chemistry , Water/chemistry
19.
Physiol Rep ; 9(3): e14744, 2021 02.
Article in English | MEDLINE | ID: covidwho-1080461

ABSTRACT

During postnatal development, colostrum and breastmilk are sequentially the first sources of nutrition with protein components and bioactive molecules that confer protection and immunostimulatory function to the gut. Caseins, whey proteins, secretory immunoglobulin A (sIgA), mucins, tryptophan, and growth factors are among milk-borne elements that are directly important in the control of mucosa development and protection. Consequently, breastfeeding is associated with the low incidence of gastrointestinal inflammation and with the decrease in respiratory diseases during postnatal period. The novel coronavirus (SARS-CoV-2) binds to angiotensin II-converting enzyme (ACE2) on the cell membrane, allowing virus entrance, replication, and host commitment. ACE2 is expressed by different cell types, which include ciliated cells in the lungs and enterocytes in the intestine. Such cells are highly active in metabolism, as they internalize molecules to be processed and used by the organism. The disruption of ACE2 impairs leads to intestinal inflammation and decreased synthesis of serotonin, affecting motility. By reviewing the effects of SARS-CoV-2 in the gastrointestinal and respiratory tracts in infants, and gut responses to breastfeeding interruption, we suggest that it is important to maintain breastfeeding during SARS-CoV-2 infection, as it might be essential to protect newborns from gastrointestinal-associated disorders and relieve disease symptoms.


Subject(s)
Breast Feeding/trends , COVID-19/metabolism , COVID-19/prevention & control , COVID-19/transmission , Female , Humans
20.
Nat Commun ; 12(1): 961, 2021 02 11.
Article in English | MEDLINE | ID: covidwho-1078585

ABSTRACT

The global spread of SARS-CoV-2 is posing major public health challenges. One feature of SARS-CoV-2 spike protein is the insertion of multi-basic residues at the S1/S2 subunit cleavage site. Here, we find that the virus with intact spike (Sfull) preferentially enters cells via fusion at the plasma membrane, whereas a clone (Sdel) with deletion disrupting the multi-basic S1/S2 site utilizes an endosomal entry pathway. Using Sdel as model, we perform a genome-wide CRISPR screen and identify several endosomal entry-specific regulators. Experimental validation of hits from the CRISPR screen shows that host factors regulating the surface expression of angiotensin-converting enzyme 2 (ACE2) affect entry of Sfull virus. Animal-to-animal transmission with the Sdel virus is reduced compared to Sfull in the hamster model. These findings highlight the critical role of the S1/S2 boundary of SARS-CoV-2 spike protein in modulating virus entry and transmission and provide insights into entry of coronaviruses.


Subject(s)
COVID-19/virology , CRISPR-Cas Systems , Genome-Wide Association Study , Host-Pathogen Interactions , SARS-CoV-2/physiology , Virus Internalization , A549 Cells , Angiotensin-Converting Enzyme 2/genetics , Angiotensin-Converting Enzyme 2/metabolism , Animals , COVID-19/genetics , Chlorocebus aethiops , Disease Models, Animal , Endosomes/virology , HeLa Cells , Humans , Mesocricetus , Serine Endopeptidases , Spike Glycoprotein, Coronavirus/metabolism , Vero Cells
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