Redox imbalance in COVID-19 pathophysiology.

BACKGROUND: The pathophysiologic significance of redox imbalance is unquestionable as numerous reports and topic reviews indicate alterations in redox parameters during corona virus disease 2019 (COVID-19). However, a more comprehensive understanding of redox-related parameters in the context of COVID-19-mediated inflammation and pathophysiology is required. METHODS: COVID-19 subjects (n = 64) and control subjects (n = 19) were enrolled, and blood was drawn within 72 h of diagnosis. Serum multiplex assays and peripheral blood mRNA sequencing was performed. Oxidant/free radical (electron paramagnetic resonance (EPR) spectroscopy, nitrite-nitrate assay) and antioxidant (ferrous reducing ability of serum assay and high-performance liquid chromatography) were performed. Multivariate analyses were performed to evaluate potential of indicated parameters to predict clinical outcome. RESULTS: Significantly greater levels of multiple inflammatory and vascular markers were quantified in the subjects admitted to the ICU compared to non-ICU subjects. Gene set enrichment analyses indicated significant enhancement of oxidant related pathways and biochemical assays confirmed a significant increase in free radical production and uric acid reduction in COVID-19 subjects. Multivariate analyses confirmed a positive association between serum levels of VCAM-1, ICAM-1 and a negative association between the abundance of one electron oxidants (detected by ascorbate radical formation) and mortality in COVID subjects while IL-17c and TSLP levels predicted need for intensive care in COVID-19 subjects. CONCLUSION: Herein we demonstrate a significant redox imbalance during COVID-19 infection affirming the potential for manipulation of oxidative stress pathways as a new therapeutic strategy COVID-19. However, further work is requisite for detailed identification of oxidants (O2•-, H2O2 and/or circulating transition metals such as Fe or Cu) contributing to this imbalance to avoid the repetition of failures using non-specific antioxidant supplementation.

Network pharmacology and experimental validation identify the potential mechanism of sophocarpine for COVID-19.

Introduction. Coronavirus disease 2019 (COVID-19) has caused a serious threat to public health worldwide, and there is currently no effective therapeutic strategy for treating COVID-19.Hypothesis/Gap Statement. We propose that sophocarpine (SOP) might have potential therapeutic effects on COVID-19 through inhibiting the cytokine storm and the nuclear factor NF-κB signalling pathway.Aim. The objective was to elucidate the potential mechanism of SOP against COVID-19 through a network pharmacology analysis and its experimental validation.Methodology. The BATMAN-TCM database was used to identify the therapeutic targets of SOP, while the GeneCards and DisGeNET databases were used to identify the targets related to COVID-19. A protein-protein interaction (PPI) network was constructed from the STRING and analysed using Cytoscape software. Gene ontology (GO), Kyoto Encyclopaedia of Genes and Genomes (KEGG) and disease ontology (DO) enrichment analyses of the co-targets were performed using Metascape. Autodock 4.2.6 and Pymol software were applied for molecular docking. Levels of the proinflammatory cytokines IL-6, TNFα and IL-1ß were measured by ELISA, while mRNA expression levels of intercellular adhesion molecule 1 (ICAM-1), vascular endothelial growth factor A (VEGFA) and IFN gamma (IFNG) were detected by real-time quantitative reverse transcription PCR. The protein levels of the molecules involved in the NF-κB signalling pathway were validated by western blot analysis.Results. A total of 65 co-targets of SOP and COVID-19 were determined. GO and KEGG enrichment analyses suggested that SOP affected COVID-19 by regulating the IL-17 signalling pathway, TNF signalling pathway and other signalling pathways. The PPI network and molecular docking showed that p65, ICAM-1 and VEGFA were key targets of SOP against COVID-19 and the underlying mechanism was validated in A549 cells in vitro. SOP attenuated the LPS-induced production of TNF-α and IL-6 and downregulated the LPS-induced mRNA expression of ICAM-1, VEGFA and IFNG. Mechanistically, SOP pretreatment inhibited the phosphorylation of p65 and facilitated the activation of Nrf2.Conclusions. SOP has a potential therapeutic effect on COVID-19 through multiple pathways and targets, and inhibits the production of pro-inflammatory cytokines and molecules involved in the NF-κB signalling pathway.

Endothelial cells: potential novel regulators of renal inflammation.

Substantial evidence has supported the role of endothelial cell (EC) activation and dysfunction in the development of hypertension, chronic kidney disease (CKD), and lupus nephritis (LN). In both humans and experimental models of hypertension, CKD, and LN, ECs become activated and release potent mediators of inflammation including cytokines, chemokines, and reactive oxygen species that cause EC dysfunction, tissue damage, and fibrosis. Factors that activate the endothelium include inflammatory cytokines, mechanical stretch, and pathological shear stress. These signals can activate the endothelium to promote upregulation of adhesion molecules, such as intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, which promote leukocyte adhesion and migration to the activated endothelium. More importantly, it is now recognized that some of these signals may in turn promote endothelial antigen presentation through major histocompatibility complex II. In this review, we will consider in-depth mechanisms of endothelial activation and the novel mechanism of endothelial antigen presentation. Moreover, we will discuss these proinflammatory events in renal pathologies and consider possible new therapeutic approaches to limit the untoward effects of endothelial inflammation in hypertension, CKD, and LN.

Endothelial Cell-Activating Antibodies in COVID-19.

OBJECTIVE: While endothelial dysfunction has been implicated in the widespread thromboinflammatory complications of COVID-19, the upstream mediators of endotheliopathy remain, for the most part, unknown. This study was undertaken to identify circulating factors contributing to endothelial cell activation and dysfunction in COVID-19. METHODS: Human endothelial cells were cultured in the presence of serum or plasma from 244 patients hospitalized with COVID-19 and plasma from 100 patients with non-COVID-19-related sepsis. Cell adhesion molecules (E-selectin, vascular cell adhesion molecule 1, and intercellular adhesion molecule 1 [ICAM-1]) were quantified using in-cell enzyme-linked immunosorbent assay. RESULTS: Serum and plasma from COVID-19 patients increased surface expression of cell adhesion molecules. Furthermore, levels of soluble ICAM-1 and E-selectin were elevated in patient serum and correlated with disease severity. The presence of circulating antiphospholipid antibodies was a strong marker of the ability of COVID-19 serum to activate endothelium. Depletion of total IgG from antiphospholipid antibody-positive serum markedly reduced the up-regulation of cell adhesion molecules. Conversely, supplementation of control serum with patient IgG was sufficient to trigger endothelial activation. CONCLUSION: These data are the first to indicate that some COVID-19 patients have potentially diverse antibodies that drive endotheliopathy, providing important context regarding thromboinflammatory effects of autoantibodies in severe COVID-19.

The Pathogenesis of COVID-19 Myocardial Injury: An Immunohistochemical Study of Postmortem Biopsies.

Rationale: Myocardial injury associates significantly and independently with mortality in COVID-19 patients. However, the pathogenesis of myocardial injury in COVID-19 remains unclear, and cardiac involvement by SARS-CoV-2 presents a major challenge worldwide. Objective: This histological and immunohistochemical study sought to clarify the pathogenesis and propose a mechanism with pathways involved in COVID-19 myocardial injury. Methods and Results: Postmortem minimally invasive autopsies were performed in six patients who died from COVID-19, and the myocardium samples were compared to a control group (n=11). Histological analysis was performed using hematoxylin-eosin and toluidine blue staining. Immunohistochemical (IHC) staining was performed using monoclonal antibodies against targets: caspase-1, caspase-9, gasdermin-d, ICAM-1, IL-1ß, IL-4, IL-6, CD163, TNF-α, TGF-ß, MMP-9, type 1 and type 3 collagen. The samples were also assessed for apoptotic cells by TUNEL. Histological analysis showed severe pericardiocyte interstitial edema and higher mast cells counts per high-power field in all COVID-19 myocardium samples. The IHC analysis showed increased expression of caspase-1, ICAM-1, IL-1ß, IL-6, MMP-9, TNF-α, and other markers in the hearts of COVID-19 patients. Expression of caspase-9 did not differ from the controls, while gasdermin-d expression was less. The TUNEL assay was positive in all the COVID-19 samples supporting endothelial apoptosis. Conclusions: The pathogenesis of COVID-19 myocardial injury does not seem to relate to primary myocardiocyte involvement but to local inflammation with associated interstitial edema. We found heightened TGF-ß and interstitial collagen expression in COVID-affected hearts, a potential harbinger of chronic myocardial fibrosis. These results suggest a need for continued clinical surveillance of patients for myocardial dysfunction and arrythmias after recovery from the acute phase of COVID-19.

Rapid and Effective Vitamin D Supplementation May Present Better Clinical Outcomes in COVID-19 (SARS-CoV-2) Patients by Altering Serum INOS1, IL1B, IFNg, Cathelicidin-LL37, and ICAM1.

BACKGROUND: We aimed to establish an acute treatment protocol to increase serum vitamin D, evaluate the effectiveness of vitamin D3 supplementation, and reveal the potential mechanisms in COVID-19. METHODS: We retrospectively analyzed the data of 867 COVID-19 cases. Then, a prospective study was conducted, including 23 healthy individuals and 210 cases. A total of 163 cases had vitamin D supplementation, and 95 were followed for 14 days. Clinical outcomes, routine blood biomarkers, serum levels of vitamin D metabolism, and action mechanism-related parameters were evaluated. RESULTS: Our treatment protocol increased the serum 25OHD levels significantly to above 30 ng/mL within two weeks. COVID-19 cases (no comorbidities, no vitamin D treatment, 25OHD <30 ng/mL) had 1.9-fold increased risk of having hospitalization longer than 8 days compared with the cases with comorbidities and vitamin D treatment. Having vitamin D treatment decreased the mortality rate by 2.14 times. The correlation analysis of specific serum biomarkers with 25OHD indicated that the vitamin D action in COVID-19 might involve regulation of INOS1, IL1B, IFNg, cathelicidin-LL37, and ICAM1. CONCLUSIONS: Vitamin D treatment shortened hospital stay and decreased mortality in COVID-19 cases, even in the existence of comorbidities. Vitamin D supplementation is effective on various target parameters; therefore, it is essential for COVID-19 treatment.

The Role of Endothelial Related Circulating Biomarkers in COVID-19. A Systematic Review and Meta-analysis.

BACKGROUND: Several studies have revealed the link between Coronavirus Disease 2019 (COVID-19) and endothelial dysfunction. To better understand the global pattern of this relationship, we conducted a meta-analysis on endothelial biomarkers related to COVID-19 severity. METHODS: We systematically searched the literature up to March 10, 2021, for studies investigating the association between COVID-19 severity and the following endothelial biomarkers: Intercellular Adhesion Molecule 1 (ICAM-1), Vascular Cell Adhesion Molecule 1 (VCAM-1), E-selectin, P-selectin, Von Willebrand Factor Antigen (VWFAg), soluble Thrombomodulin (sTM), Mid-regional pro-adrenomedullin (MR-proADM), and Angiopoietin-2 (Ang-2). Pooled estimates and mean differences (PMD) for each biomarker were reported. RESULTS: A total of 27 studies (n=2213 patients) were included. Critically ill patients presented with higher levels of MR-proADM (PMD: 0.71 nmol/L, 95% CI: 0.22 to 1.20 nmol/L, p=0.02), E-selectin (PMD: 13,32 pg/ml, 95% CI: 4,89 to 21,75 pg/ml, p=0.008), VCAM-1 (PMD: 479 ng/ml, 95% CI: 64 to 896 ng/ml, p=0.03), VWF-Ag (PMD: 110.5 IU/dl, 95% CI: 44.8 to 176.1 IU/dl, p=0.04) and Ang-2 (PMD: 2388 pg/ml, 95% CI: 1121 to 3655 pg/ml, p=0.003), as compared to non-critically ill ones. ICAM-1, P-selectin and thrombomodulin did not differ between the two groups (p>0.05). CONCLUSION: Endothelial biomarkers display significant heterogeneity in COVID-19 patients, with higher MR-proADM, E-selectin, VCAM-1, VWF-Ag, and Ang-2 levels being associated with increased severity. These findings strengthen the evidence on the key role of endothelial dysfunction in disease progress.

Suppressing STAT3 activity protects the endothelial barrier from VEGF-mediated vascular permeability.

Vascular permeability triggered by inflammation or ischemia promotes edema, exacerbates disease progression and impairs tissue recovery. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular permeability. VEGF plays an integral role in regulating vascular barrier function physiologically and in pathologies, including cancer, stroke, cardiovascular disease, retinal conditions and COVID-19-associated pulmonary edema, sepsis and acute lung injury. Understanding temporal molecular regulation of VEGF-induced vascular permeability will facilitate developing therapeutics to inhibit vascular permeability, while preserving tissue-restorative angiogenesis. Here, we demonstrate that VEGF signals through signal transducer and activator of transcription 3 (STAT3) to promote vascular permeability. We show that genetic STAT3 ablation reduces vascular permeability in STAT3-deficient endothelium of mice and VEGF-inducible zebrafish crossed with CRISPR/Cas9-generated Stat3 knockout zebrafish. Intercellular adhesion molecule 1 (ICAM-1) expression is transcriptionally regulated by STAT3, and VEGF-dependent STAT3 activation is regulated by JAK2. Pyrimethamine, an FDA-approved antimicrobial agent that inhibits STAT3-dependent transcription, substantially reduces VEGF-induced vascular permeability in zebrafish, mouse and human endothelium. Collectively, our findings suggest that VEGF/VEGFR-2/JAK2/STAT3 signaling regulates vascular barrier integrity, and inhibition of STAT3-dependent activity reduces VEGF-induced vascular permeability. This article has an associated First Person interview with the first author of the paper.

Identification of COVID-19 and Dengue Host Factor Interaction Networks Based on Integrative Bioinformatics Analyses.

Background: The outbreak of Coronavirus disease 2019 (COVID-19) has become an international public health crisis, and the number of cases with dengue co-infection has raised concerns. Unfortunately, treatment options are currently limited or even unavailable. Thus, the aim of our study was to explore the underlying mechanisms and identify potential therapeutic targets for co-infection. Methods: To further understand the mechanisms underlying co-infection, we used a series of bioinformatics analyses to build host factor interaction networks and elucidate biological process and molecular function categories, pathway activity, tissue-specific enrichment, and potential therapeutic agents. Results: We explored the pathologic mechanisms of COVID-19 and dengue co-infection, including predisposing genes, significant pathways, biological functions, and possible drugs for intervention. In total, 460 shared host factors were collected; among them, CCL4 and AhR targets were important. To further analyze biological functions, we created a protein-protein interaction (PPI) network and performed Molecular Complex Detection (MCODE) analysis. In addition, common signaling pathways were acquired, and the toll-like receptor and NOD-like receptor signaling pathways exerted a significant effect on the interaction. Upregulated genes were identified based on the activity score of dysregulated genes, such as IL-1, Hippo, and TNF-α. We also conducted tissue-specific enrichment analysis and found ICAM-1 and CCL2 to be highly expressed in the lung. Finally, candidate drugs were screened, including resveratrol, genistein, and dexamethasone. Conclusions: This study probes host factor interaction networks for COVID-19 and dengue and provides potential drugs for clinical practice. Although the findings need to be verified, they contribute to the treatment of co-infection and the management of respiratory disease.

SARS-CoV-2 Spike Protein Induces Paracrine Senescence and Leukocyte Adhesion in Endothelial Cells.

Increased mortality in COVID-19 cases is often associated with microvascular complications. We have recently shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein promotes an inflammatory cytokine interleukin 6 (IL-6)/IL-6R-induced trans signaling response and alarmin secretion. Virus-infected or spike-transfected human epithelial cells exhibited an increase in senescence, with a release of senescence-associated secretory phenotype (SASP)-related inflammatory molecules. Introduction of the bromodomain-containing protein 4 (BRD4) inhibitor AZD5153 to senescent epithelial cells reversed this effect and reduced SASP-related inflammatory molecule release in TMNK-1 or EAhy926 (representative human endothelial cell lines), when cells were exposed to cell culture medium (CM) derived from A549 cells expressing SARS-CoV-2 spike protein. Cells also exhibited a senescence phenotype with enhanced p16, p21, and senescence-associated ß-galactosidase (SA-ß-Gal) expression and triggered SASP pathways. Inhibition of IL-6 trans signaling by tocilizumab and inhibition of inflammatory receptor signaling by the Bruton's tyrosine kinase (BTK) inhibitor zanubrutinib, prior to exposure of CM to endothelial cells, inhibited p21 and p16 induction. We also observed an increase in reactive oxygen species (ROS) in A549 spike-transfected and endothelial cells exposed to spike-transfected CM. ROS generation in endothelial cell lines was reduced after treatment with tocilizumab and zanubrutinib. Cellular senescence was associated with an increased level of the endothelial adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1), which have in vitro leukocyte attachment potential. Inhibition of senescence or SASP function prevented VCAM-1/ICAM-1 expression and leukocyte attachment. Taken together, we identified that human endothelial cells exposed to cell culture supernatant derived from SARS-CoV-2 spike protein expression displayed cellular senescence markers, leading to enhanced leukocyte adhesion. IMPORTANCE The present study was aimed at examining the underlying mechanism of extrapulmonary manifestations of SARS-CoV-2 spike protein-associated pathogenesis, with the notion that infection of the pulmonary epithelium can lead to mediators that drive endothelial dysfunction. We utilized SARS-CoV-2 spike protein expression in cultured human hepatocytes (Huh7.5) and pneumocytes (A549) to generate conditioned culture medium (CM). Endothelial cell lines (TMNK-1 or EAhy926) treated with CM exhibited an increase in cellular senescence markers by a paracrine mode and led to leukocyte adhesion. Overall, the link between these responses in endothelial cell senescence and a potential contribution to microvascular complication in productively SARS-CoV-2-infected humans is implicated. Furthermore, the use of inhibitors (BTK, IL-6, and BRD4) showed a reverse effect in the senescent cells. These results may support the selection of potential adjunct therapeutic modalities to impede SARS-CoV-2-associated pathogenesis.

Safety of inhaled ivermectin as a repurposed direct drug for treatment of COVID-19: A preclinical tolerance study.

INTRODUCTION: SARS-CoV-2 replication in cell cultures has been shown to be inhibited by ivermectin. However, ivermectin's low aqueous solubility and bioavailabilityhinders its application in COVID-19 treatment. Also, it has been suggested that best outcomes for this medication can be achieved via direct administration to the lung. OBJECTIVES: This study aimed at evaluating the safety of a novel ivermectin inhalable formulation in rats as a pre-clinical step. METHODS: Hydroxy propyl-ß-cyclodextrin(HP-ß-CD) was used to formulate readily soluble ivermectin lyophilized powder. Adult male rats were used to test lung toxicity for ivermectin-HP-ß-CD formulations in doses of 0.05, 0.1, 0.2, 0.4 and 0.8 mg/kg for 3 successive days. RESULTS: The X-ray diffraction for lyophilized ivermectin-HP-ß-CD revealed its amorphous structure that increased drug aqueous solubility 127-fold and was rapidly dissolved within 5 s in saline.Pulmonary administration of ivermectin-HP-ß-CD in dosesof 0.2, 0.4 and 0.8 mg/kgshowed dose-dependent increase in levels of TNF-α, IL-6, IL-13 and ICAM-1 as well as gene expression of MCP-1, protein expression of PIII-NP and serum levels of SP-D paralleled by reduction in IL-10. Moreover, lungs treated with ivermectin (0.2 mg/kg) revealed mild histopathological alterations, while severe pulmonary damage was seen in rats treated with ivermectin at doses of 0.4 and 0.8 mg/kg. However, ivermectin-HP-ß-CD formulation administered in doses of 0.05 and 0.1 mg/kg revealed safety profiles. CONCLUSION: The safety of inhaledivermectin-HP-ß-CD formulation is dose-dependent. Nevertheless, use of low doses(0.05 and 0.1 mg/kg) could be considered as a possible therapeutic regimen in COVID-19 cases.

HA of H1N1 enhanced the expression of ICAM-1 and IL-6 in HUVECs and pathological injury in the lungs in mice.

OBJECTIVE: Both COVID-19 and influenza are viral respiratory tract infections and the epidemics of viral respiratory tract infections remain highly prevalent with lethal consequences in susceptible individuals. Expression of ICAM-1 on vascular endothelium recruits leukocytes which initiates inflammation. IL-6 induces ICAM-1. Both ICAM-1 and IL-6 can be enhanced in influenza virus infection and COVID-19 patients. Besides initiation of virus entry host cells, whether HA alone, instead of whole virus, of influenza has the effects on expression of ICAM-1 and IL-6 in vascular endothelium with injury in the lungs, remains to be demonstrated. METHODS: RT-qPCR and Western blot as well as histopathologic examination were used to examine mRNA and protein of ICAM-1 and IL-6 as well as pathological injury in the lung tissues, respectively. RESULTS: After incubation of the Human Umbilical Vein Endothelial Cells (HUVECs) with HA of H1N1 for 24 h, the mRNA and protein of ICAM-1 and IL-6 in HUVECs were increased in group of 5 µg/ml concentration with statistical significance (p < 0.05). Pathological injury in lung tissues of the mice was shown 12 h after tail intravenous injection with 100 µl of HA (50 µg/ml and 100 µg/ml in normal saline), including widened alveolar spaces with angiotelectasis in alveolar wall, alveolar luminal and interstitial inflammatory infiltrates, alveolar luminal erythrocyte effusion. CONCLUSIONS: HA alone, instead of whole H1N1 virus, induced more expression of ICAM-1 and IL-6, two molecules involving in pathological and inflammatory responses, in HUVECs and pathological injury in lung tissues of the mice. This knowledge provides a new HA-targeted potential direction for prevention and treatment of disease related to H1N1 infection.

SARS-CoV-2 proteins regulate inflammatory, thrombotic and diabetic responses in human arterial fibroblasts.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for many pathological processes, including altered vascular disease development, dysfunctional thrombosis and a heightened inflammatory response. However, there is limited work to determine the underlying cellular responses induced by exposure to SARS-CoV-2 structural proteins. Thus, our objective was to investigate how human arterial adventitial fibroblasts inflammation, thrombosis and diabetic disease markers are altered in response to Spike, Nucleocapsid and Membrane-Envelope proteins. We hypothesized that after a short-term exposure to SARS-CoV-2 proteins, adventitial fibroblasts would have a higher expression of inflammatory, thrombotic and diabetic proteins, which would support a mechanism for altered vascular disease progression. After incubation, the expression of gC1qR, ICAM-1, tissue factor, RAGE and GLUT-4 was significantly up-regulated. In general, the extent of expression was different for each SARS-CoV-2 protein, suggesting that SARS-CoV-2 proteins interact with cells through different mechanisms. Thus, SARS-CoV-2 protein interaction with vascular cells may regulate vascular disease responses.

Integrin Activation Enables Sensitive Detection of Functional CD4+ and CD8+ T Cells: Application to Characterize SARS-CoV-2 Immunity.

We have previously shown that conformational change in the ß2-integrin is a very early activation marker that can be detected with fluorescent multimers of its ligand intercellular adhesion molecule (ICAM)-1 for rapid assessment of antigen-specific CD8+ T cells. In this study, we describe a modified protocol of this assay for sensitive detection of functional antigen-specific CD4+ T cells using a monoclonal antibody (clone m24 Ab) specific for the open, high-affinity conformation of the ß2-integrin. The kinetics of ß2-integrin activation was different on CD4+ and CD8+ T cells (several hours vs. few minutes, respectively); however, m24 Ab readily stained both cell types 4-6 h after antigen stimulation. With this protocol, we were able to monitor ex vivo effector and memory CD4+ and CD8+ T cells specific for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), and hepatitis B virus (HBV) in whole blood or cryopreserved peripheral blood mononuclear cells (PBMCs) of infected or vaccinated individuals. By costaining ß2-integrin with m24 and CD154 Abs, we assessed extremely low frequencies of polyfunctional CD4+ T cell responses. The novel assay used in this study allows very sensitive and simultaneous screening of both CD4+ and CD8+ T cell reactivities, with versatile applicability in clinical and vaccination studies.

Conus venom fractions inhibit the adhesion of Plasmodium falciparum erythrocyte membrane protein 1 domains to the host vascular receptors.

Using high-throughput BioPlex assays, we determined that six fractions from the venom of Conus nux inhibit the adhesion of various recombinant PfEMP-1 protein domains (PF08_0106 CIDR1α3.1, PF11_0521 DBL2ß3, and PFL0030c DBL3X and DBL5e) to their corresponding receptors (CD36, ICAM-1, and CSA, respectively). The protein domain-receptor interactions permit P. falciparum-infected erythrocytes (IE) to evade elimination in the spleen by adhering to the microvasculature in various organs including the placenta. The sequences for the main components of the fractions, determined by tandem mass spectrometry, yielded four T-superfamily conotoxins, one (CC-Loop-CC) with I-IV, II-III connectivity and three (CC-Loop-CXaaC) with a I-III, II-IV connectivity. The 3D structure for one of the latter, NuxVA = GCCPAPLTCHCVIY, revealed a novel scaffold defined by double turns forming a hairpin-like structure stabilized by the two disulfide bonds. Two other main fraction components were a miniM conotoxin, and a O2-superfamily conotoxin with cysteine framework VI/VII. This study is the first one of its kind suggesting the use of conotoxins for developing pharmacological tools for anti-adhesion adjunct therapy against malaria. Similarly, mitigation of emerging diseases like AIDS and COVID-19, can also benefit from conotoxins as inhibitors of protein-protein interactions as treatment. BIOLOGICAL SIGNIFICANCE: Among the 850+ species of cone snail species there are hundreds of thousands of diverse venom exopeptides that have been selected throughout several million years of evolution to capture prey and deter predators. They do so by targeting several surface proteins present in target excitable cells. This immense biomolecular library of conopeptides can be explored for potential use as therapeutic leads against persistent and emerging diseases affecting non-excitable systems. We aim to expand the pharmacological reach of conotoxins/conopeptides by revealing their in vitro capacity to disrupt protein-protein and protein-polysaccharide interactions that directly contribute to pathology of Plasmodium falciparum malaria. This is significant for severe forms of malaria, which might be deadly even after treated with current parasite-killing drugs because of persistent cytoadhesion of P. falciparum infected erythrocytes even when parasites within red blood cells are dead. Anti-adhesion adjunct drugs would de-sequester or prevent additional sequestration of infected erythrocytes and may significantly improve survival of malaria patients. These results provide a lead for further investigations into conotoxins and other venom peptides as potential candidates for anti-adhesion or blockade-therapies. This study is the first of its kind and it suggests that conotoxins can be developed as pharmacological tools for anti-adhesion adjunct therapy against malaria. Similarly, mitigation of emerging diseases like AIDS and COVID-19, can also benefit from conotoxins as potential inhibitors of protein-protein interactions as treatment.

SARS-CoV-2 attachment to host cells is possibly mediated via RGD-integrin interaction in a calcium-dependent manner and suggests pulmonary EDTA chelation therapy as a novel treatment for COVID 19.

SARS-CoV-2 is a highly contagious virus that has caused serious health crisis world-wide resulting into a pandemic situation. As per the literature, the SARS-CoV-2 is known to exploit humanACE2 receptors (similar toprevious SARS-CoV-1) for gaining entry into the host cell for invasion, infection, multiplication and pathogenesis. However, considering the higher infectivity of SARS-CoV-2 along with the complex etiology and pathophysiological outcomes seen in COVID-19 patients, it seems that there may be an alternate receptor for SARS-CoV-2. I performed comparative protein sequence analysis, database based gene expression profiling, bioinformatics based molecular docking using authentic tools and techniques for unveiling the molecular basis of high infectivity of SARS-CoV-2 as compared to previous known coronaviruses. My study revealed that SARS-CoV-2 (previously known as 2019-nCoV) harbors a RGD motif in its receptor binding domain (RBD) and the motif is absent in all other previously known SARS-CoVs. The RGD motif is well known for its role in cell-attachment and cell-adhesion. My hypothesis is that the SARS-CoV-2 may be (via RGD) exploiting integrins, that have high expression in lungs and all other vital organs, for invading host cells. However, an experimental verification is required. The expression of ACE2, which is a known receptor for SARS-CoV-2, was found to be negligible in lungs. I assume that higher infectivity of SARS-CoV-2 could be due to this RGD-integrin mediated acquired cell-adhesive property. Gene expression profiling revealed that expression of integrins is significantly high in lung cells, in particular αvß6, α5ß1, αvß8 and an ECM protein, ICAM1. The molecular docking experiment showed the RBD of spike protein binds with integrins precisely at RGD motif in a similar manner as a synthetic RGD peptide binds to integrins as found by other researchers. SARS-CoV-2 spike protein has a number of phosphorylation sites that can induce cAMP, PKC, Tyr signaling pathways. These pathways either activate calcium ion channels or get activated by calcium. In fact, integrins have calcium & metal binding sites that were predicted around and in vicinity of RGD-integrin docking site in our analysis which suggests that RGD-integrins interaction possibly occurs in calcium-dependent manner. The higher expression of integrins in lungs along with their previously known high binding affinity (~KD = 4.0 nM) for virus RGD motif could serve as a possible explanation for high infectivity of SARS-CoV-2. On the contrary, human ACE2 has lower expression in lungs and its high binding affinity (~KD = 15 nM) for spike RBD alone could not manifest significant virus-host attachment. This suggests that besides human ACE2, an additional or alternate receptor for SARS-CoV-2 is likely to exist. A highly relevant evidence never reported earlier which corroborate in favor of RGD-integrins mediated virus-host attachment is an unleashed cytokine storm which causes due to activation of TNF-α and IL-6 activation; and integrins role in their activation is also well established. Altogether, the current study has highlighted possible role of calcium and other divalent ions in RGD-integrins interaction for virus invasion into host cells and suggested that lowering divalent ion in lungs could avert virus-host cells attachment.

Elevated Expression of Serum Endothelial Cell Adhesion Molecules in COVID-19 Patients.

In a retrospective study of 39 COVID-19 patients and 32 control participants in China, we collected clinical data and examined the expression of endothelial cell adhesion molecules by enzyme-linked immunosorbent assays. Serum levels of fractalkine, vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), and vascular adhesion protein-1 (VAP-1) were elevated in patients with mild disease, dramatically elevated in severe cases, and decreased in the convalescence phase. We conclude the increased expression of endothelial cell adhesion molecules is related to COVID-19 disease severity and may contribute to coagulation dysfunction.