Increases in expression of carbohydrate sulfotransferases CHST11 and CHST15 and decline in N-acetylgalactosamine-4-sulfatase (Arylsulfatase B, ARSB) require phospho-p38-MAPK following exposure to SARS-CoV-2 spike protein receptor binding domain in human a

Objective: Immunohistochemistry of post-mortem lung tissue from Covid-19 patients with diffuse alveolar damage demonstrated marked increases in chondroitin sulfate and CHST15 and decline in N-acetylgalactosamine-4-sulfatase. Studies were undertaken to identify the mechanisms involved in these effects. Method(s): Human primary small airway epithelial cells (PCS 301-010;ATCC) were cultured and exposed to the SARSCoV- 2 spike protein receptor binding domain (SPRBD;AA: Lys310-Leu560;Amsbio). Expression of the spike protein receptor, angiotensin converting enzyme 2 (ACE2), was enhanced by treatment with Interferon-beta. Promoter activation, DNA-binding, RNA silencing, QPCR, Western blots, ELISAs, and specific enzyme inhibitors were used to elucidate the underlying molecular mechanisms. Result(s): Treatment of the cultured cells by the SPRBD led to increased CHST15 and CHST11 expression and decline in ARSB expression. Sulfotransferase activity, total chondroitin sulfate, and sulfated glycosaminoglycan (GAG) content were increased. Phospho-T180/T182-p38-MAPK and phospho- S423/S425-Smad3 were required for the activation of the CHST15 and CHST11 promoters. Inhibition by SB203580, a phospho-p38 MAPK inhibitor, and by SIS3, a Smad3 inhibitor, blocked the CHST15 and CHST11 promoter activation. SB203580 reversed the SPRBD-induced decline in ARSB expression, but SIS3 had no effect on ARSB expression or promoter activation. Phospho-p38 MAPK was shown to reduce retinoblastoma protein (RB) S807/S811 phosphorylation and increase RB S249/T252 phosphorylation. E2F-DNA binding declined following exposure to SPRBD, and SB203580 reversed this effect. This indicates a mechanism by which SPRBD, phospho-p38 MAPK, E2F, and RB can regulate ARSB expression and thereby impact on chondroitin 4-sulfate and dermatan sulfate and molecules that bind to these sulfated GAGs, including Interleukin-8, bone morphogenetic protein-4, galectin-3 and SHP-2 (Src homology region 2-containing protein tyrosine phosphatase 2). Conclusion(s): The enzyme ARSB is required for the degradation of chondroitin 4-sulfate and dermatan sulfate, and accumulation of these sulfated GAGs can contribute to lung pathophysiology, as evident in Covid-19. Some effects of the SPRBD may be attributable to unopposed Angiotensin II, when Ang1-7 counter effects are diminished due to binding of ACE2 with the SARS-CoV-2 spike protein and reduced production of Ang1-7. Aberrant cell signaling and activation of the phospho-p38 MAPK and Smad3 pathways increase CHST15 and CHST11 production, which can contribute to increased chondroitin sulfate in infected cells. Decline in ARSB may occur as a consequence of effects of phospho-p38 MAPK on RB phosphorylation and E2F1 availability. Decline in ARSB and the resulting impaired degradation of sulfated GAGs have profound consequences on cellular metabolic, signaling, and transcriptional events. Funding is VA Merit Award.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Angiotensin-converting enzyme 2 (ACE2) in COVID-19 and other diseases.

The coronavirus SARS-CoV-2 was detected in isolates of pneumonia patients in January 2020. The virus cannot multiply extracellularly but requires access to the cells of a host organism. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as a receptor, to which it docks with its spikes. ACE2 belongs to the renin angiotensin system (RAS), whose inhibitors have been used for years against high blood pressure. Renin is an endopeptidase that is predominantly formed in the juxtaglomerular apparatus of the kidney and cleaves the decapeptide angiotensin I (Ang I) from angiotensinogen. Through the angiotensin-converting enzyme (ACE), another 2 C-terminal amino acids are removed from Ang I, so that finally the active octapeptide angiotensin II (Ang II) is formed. The biological effect of Ang II via the angiotensin II receptor subtype 1 (AT1-R) consists of vasoconstriction, fibrosis, proliferation, inflammation, and thrombosis formation. ACE2 is a peptidase that is a homolog of ACE. ACE2 is predominantly expressed by pulmonary alveolar epithelial cells in humans and has been detected in arterial and venous endothelial cells. In contrast to the dicarboxy-peptidase ACE, ACE2 is a monocarboxypeptidase that cleaves only one amino acid from the C-terminal end of the peptides. ACE2 can hydrolyze the nonapeptide Ang-(1-9) from the decapeptide Ang I and the heptapeptide Ang-(1-7) from the octapeptide Ang II. Ang-(1-7) acts predominantly antagonistically (vasodilatory, anti-fibrotic, anti-proliferative, anti-inflammatory, anti-thrombogenetically) via the G protein-coupled Mas receptor to the AT1-R-mediated effects of Ang II. In the pathogenesis of COVID-19 infection, it is therefore assumed that there is an imbalance due to overstimulation of the AT1 receptor in conjunction with a weakening of the biological effects of the Mas receptor.Copyright © 2022 Dustri-Verlag Dr. K. Feistle.

Mastocytosis and COVID19 infection and vaccination: A pediatric series

Introduction: Mastocytosis encompasses a heterogeneous group of diseases characterized by an accumulation of clonal mast cells (MC) in the skin and/or internal organs, and symptoms of MC activation. This MC activation can be elucidated by several factors, including infections or vaccination. Objective(s): We present our experience with COVID infection and vaccination in a series of 133 patients with pediatric mastocytosis. Method(s): Between January 1998 and December 2022, 133 pediatric patients have been referred to our hospital owing to clinically suspected MC disorder, mainly with mastocytosis in the skin. The final diagnoses of mastocytosis were established by the presence of typical skin lesions together with an increase of MC numbers in a biopsy from lesional skin or activating KIT mutations in lesional skin tissue. Serum baseline tryptase and total immunoglobulin E levels were measured, and patients underwent a comprehensive allergy workup to confirm atopic status and history of anaphylaxis. Regarding vaccination, REMA's (Spanish Network on Mastocytosis) protocol was followed. Result(s): 13 patients with COVID infection were identified, of which 25 (56,8%) were female and 0% had symptoms of MC activation. All of them had an asymptomatic or mild course of COVID infection. None of the patients experimented MC activation symptoms during viral illness. Regarding COVID vaccination, all patients received premedication with antihistamine 60 minutes prior vaccination. No one experimented immediate reactions and only one patient (0,75%) referred worsening of MC activation symptoms (baseline pruritus, urtication and brain fog) only after the first doses, recovering without changes in his treatment (oral cromoglycate and antihistamine) in two months. Discussion(s): Although MC have been implicated in the pathogenesis of cytokine storm in COVID19, there is no clinical evidence of SARSCoV- 2-induced MC activation, perhaps related to the fact that bone marrow MC lack angiotensin-converting enzyme 2 receptors.

Experience of Using Remdesivir in Patients with Novel Coronavirus Infection.

Timely, effective, and safe antiviral therapy in COVID-19 patients reduces complications, disability and mortality rates. The greatest concern with remdesivir is the risk of drug-induced liver injury, including in patients whose liver function is compromised by COVID-19. The aim of the study was to investigate the efficacy and safety of remdesivir in patients with confirmed SARSCoV-2 infection who had been admitted to an infectious diseases hospital in the Volgograd region in March 2022. Material(s) and Method(s): the authors carried out an open, non-randomised, single-arm study using medical records of 234 patients who had been diagnosed with "U07.1 COVID-19, virus identified" and prescribed remdesivir upon admission. The effectiveness of therapy was evaluated using two criteria: the need for oxygen supplementation or ventilatory support, or mortality. The authors conducted the evaluation on days 7, 14, and 28 using the six-point ordinal severity scale by Y. Wang et al. The safety of therapy was assessed on the basis of complaints and changes in laboratory findings. Result(s): for the patients prescribed remdesivir at admission, the 7-day mortality rate was 3.0%, the 14-day mortality rate was 5.6%, and the 28-day mortality rate was 7.3%. With the exception of a patient with myocardial infarction, all the patients who had been hospitalised with mild COVID-19 and prescribed remdesivir did not require oxygen therapy and/or transfer to intensive care and were discharged following recovery. The patients with moderate to severe COVID-19 had the 14-day mortality rate of 6.4% and the 28-day mortality rate of 8.6%. 17 patients (7.2%) discontinued remdesivir prematurely for various reasons, including adverse drug reactions. Remdesivir therapy of 5-10 days was associated with an increase in ALT activity by 2.7 +/- 0.8 times in 15.9% of patients with mild COVID-19, by 3.8 +/- 1.8 times in 20.4% of patients with moderately severe COVID-19, and by 4.8 +/- 2.7 times in 24% (12/50) of patients with severe COVID-19. In two patients (0.9%), the increase exceeded 10-fold the upper limit of normal. Conclusion(s): the obtained results support recommending remdesivir to patients with mild, moderate and severe COVID-19, including those with moderately elevated baseline activity of hepatic transaminases.Copyright © NEICON ISP LLC. All rights reserved.

Low-level laser therapy for treatment of severe acute respiratory syndrome 2 infection

SARS-CoV-2 is a new threat to public health due to its increased transmissibility and immune evasion. Angiotensin-converting enzyme 2 (ACE2) plays a critical role in SARS-CoV-2 infection as its serve as the virus's major entry receptor in humans. Vaccines have been authorized for emergency use to control the current pandemic and they have greatly reduced the spread of SARS-CoV-2 and mortality rates, nevertheless this coronavirus has shown the ability to endure crucial mutations that increases its infectivity which makes it likely that the virus will continue to mutate and disseminate. There is a need to find and introduce alternative and effective methods of controlling SARS-CoV-2. Notably, low-level laser therapy (LLLT) is a method of exposing cells or tissue to low levels of red and near infrared light which has a high success rate for treatment of other ailments. The aim of the study is to determine for the first time, the effects of LLLT on SARS-CoV-2 infected HEK293/ACE2 cells and compare them to uninfected ones. Both infected and uninfected HEK293/ACE2 cells were irradiated at a wavelength of 640 nm, at different doses. Then, the effects of laser irradiation on the cells and the virus were evaluated using luciferase, cytotoxicity, and cell viability assays. Preliminary results showed that irradiated uninfected cells had no changes in cell viability and cytotoxicity, while there were changes in irradiated infected cells. In addition, laser irradiation caused cell membrane damage in infected cells. Lastly, uninfected irradiated cells showed no luciferase activity while laser irradiation reduced luciferase activity in infected cells. © 2023 SPIE.

QSAR and DFT Studies of Some Tacrine-Hydroxamate Derivatives as Inhibitors of Cholinesterase (AChEs) in the Treatment of Alzheimer's Disease

Introduction: This work was devoted to an in silico investigation conducted on twenty-eight Tacrine-hydroxamate derivatives as a potential treatment for Alzheimer's disease using DFT and QSAR modeling techniques. Method(s): The data set was randomly partitioned into a training set (22 compounds) and a test set (6 compounds). Then, fourteen models were built and were used to compute the predicted pIC50 of compounds belonging to the test set. Result(s): Al built models were individualy validated using both internal and external validation methods, including the Y-Randomization test and Golbraikh and Tropsha's model acceptance criteria. Then, one model was selected for its higher R2, R2test, and Q2cv values (R2 = 0.768, R2adj = 0.713, MSE = 0.304, R2test=0.973, Q2cv = 0.615). From these outcomes, the activity of the studied compounds toward the main protease of Cholinesterase (AChEs) seems to be influenced by 4 descriptors, i.e., the total dipole moment of the molecule (mu), number of rotatable bonds (RB), molecular topology radius (MTR) and molecular topology polar surface area (MTPSA). The effect of these descriptors on the activity was studied, in particular, the increase in the total dipole moment and the topological radius of the molecule and the reduction of the rotatable bond and topology polar surface area increase the activity. Conclusion(s): Some newly designed compounds with higher AChEs inhibitory activity have been designed based on the best-proposed QSAR model. In addition, ADMET pharmacokinetic properties were carried out for the proposed compounds, the toxicity results indicate that 7 molecules are nontoxic.Copyright © 2023 Bentham Science Publishers.

Scientific rationale of Indian AYUSH Ministry advisory for COVID-19 prevention, prophylaxis, and immunomodulation

The current outbreak of COVID-19 is caused by the SARS-CoV-2 virus that has affected > 210 countries. Various steps are taken by different countries to tackle the current war-like health situation. In India, the Ministry of AYUSH released a self-care advisory for immunomodulation measures during the COVID-19 and this review article discusses the detailed scientific rationale associated with this advisory. Authors have spotted and presented in-depth insight of advisory in terms of immunomodulatory, antiviral, antibacterial, co-morbidity associated actions, and their probable mechanism of action. Immunomodulatory actions of advised herbs with no significant adverse drug reaction/toxicity strongly support the extension of advisory for COVID-19 prevention, prophylaxis, mitigations, and rehabilitation capacities. This advisory also emphasized Dhyana (meditation) and Yogasanas as a holistic approach in enhancing immunity, mental health, and quality of life. The present review may open-up new meadows for research and can provide better conceptual leads for future researches in immunomodulation, antiviral-development, psychoneuroimmunology, especially for COVID-19.Copyright © 2021, Institute of Korean Medicine, Kyung Hee University.

Characteristics and Clinical Implications of Anti-IFN-alpha cytokine antibodies in partial Recombinase Activating Gene Deficiency patients before and during the COVID-19 Pandemic

Introduction: With the onset of the COVID-19 pandemic, there was increased attention on anti- IFN-alpha autoantibodies and its correlation with severe clinical outcomes in a large group of patients. However, this correlation has not been extensively investigated in patients with partial Recombinase Activating Gene Deficiency (pRD) who are known to have increased prevalence of anti- IFN-alpha autoantibodies. Therefore, there is a need to assess the presence of anti- IFN-alpha antibodies in pRD patients before and after the COVID-19 pandemic and explore the relationship between anti- IFN-alpha antibody presence and clinical outcomes. Method(s): Sera was collected from the whole blood after informed consent and Enzyme-Linked Immunosorbent Assay was conducted to confirm the presence of IgG-specific anti- IFN-alpha autoantibodies. Positive samples were determined as OD values above 3 standard deviations of the healthy donor OD mean. Result(s): Our cohort included both adult (n = 13) and pediatric (n = 9) patients with variants in RAG1 and RAG2. Eleven patients (50%) out of the 22 showed elevated anti- IFN-alpha autoantibodies levels. Five patients (23%) were defined as low positive for anti- IFN-alpha autoantibodies, and 6 patients had no autoantibody titers. Of the 22 patients, 16 were symptomatic with infectious and non-infectious complications including recurrent viral and/or bacterial infections, autoimmune cytopenias, and lymphoproliferation. Ten (63%) of the symptomatic patients demonstrated high anti-IFN-alpha autoantibodies titers. Of the 11 patients with no or low neutralizing anti- IFN-alpha autoantibodies levels, 5 were asymptomatic. In temporal comparison, 16 samples were collected pre-COVID-19 pandemic;8 samples were collected during the pandemic, 2 of which belonged to patients with samples collected before and during the pandemic. In the pre-pandemic cohort, 66% had anti- IFN-alpha autoantibodies. Conversely, during the COVID-19 pandemic, 89% had anti- IFN-alpha autoantibodies. Of note, one patient who had neutralizing anti- IFN-alpha autoantibodies remained positive both before and during the pandemic despite HSCT. Patient also had a SARS-CoV-2 infection in summer of 2022 with a mild clinical course. Conclusions & Next Steps: We observed persistence of anti-IFN-alpha autoantibodies in our cohort post-pandemic and even post-HSCT. It is unclear whether the presence of anti-cytokine antibodies are risk factor for severe COVID-19.Copyright © 2023 Elsevier Inc.

Exploration of potential mechanism of SARS-CoV-2 induced immune inflammatory response leading to progression of atherosclerosis and prediction of traditional Chinese medicine for preventing and treating based on bioinformatics.

Objective To explore the core targets and important pathways of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) induced atherosclerosis (AS) progression from the perspective of immune inflammation, so as to predict the potential prevention and treatment of traditional Chinese medicine (TCM). Methods Microarray data were obtained from the Gene Expression Omnibus (GEO) database for coronavirus disease 2019 (COVID-19) patients and AS patients, and the "limmar" and "Venn" packages were used to screen out the common differentially expressed genes (DEGs) genes in both diseases. The gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses were performed on the common DEGs to annotate their functions and important pathways. The two gene sets were scored for immune cells and immune function to assess the level of immune cell infiltration. The protein-protein interaction (PPI) network was constructed by STRING database, and the CytoHubba plug-in of Cytoscape was used to identify the hub genes. Two external validation datasets were introduced to validate the hub genes and obtain the core genes. Immuno-infiltration analysis and gene set enrichment analysis (GSEA) were performed on the core genes respectively. Finally the potential TCM regulating the core genes were predicted by Coremine Medical database. Results A total of 7898 genes related to COVID-19, 471 genes related to AS progression;And 51 common DEGs, including 32 highly expressed genes and 19 low expressed genes were obtained. GO and KEGG analysis showed that common DEGs, which were mainly localized in cypermethrin-encapsulated vesicles, platelet alpha particles, phagocytic vesicle membranes and vesicles, were involved in many biological processes such as myeloid differentiation factor 88 (MyD88)-dependent Toll-like receptor signaling pathway transduction, interleukin-8 (IL-8) production and positive regulation, IL-6 production and positive regulation to play a role in regulating nicotinamide adenine dinucleotide phosphate oxidase activity, Toll-like receptor binding and lipopeptide and glycosaminoglycan binding through many biological pathways, including Toll-like receptor signaling pathways, neutrophil extracellular trap formation, complement and coagulation cascade reactions. The results of immune infiltration analysis demonstrated the state of immune microenvironment of COVID-19 and AS. A total of 5 hub genes were obtained after screening, among which Toll-like receptor 2 (TLR2), cluster of differentiation 163 (CD163) and complement C1q subcomponent subunit B (C1QB) genes passed external validation as core genes. The core genes showed strong correlation with immune process and inflammatory response in both immune infiltration analysis and GSEA enrichment analysis. A total of 35 TCMs, including Chuanxiong (Chuanxiong Rhizoma), Taoren (Persicae Semen), Danggui (Angelicae Sinensis Radix), Huangqin (Scutellariae Radix), Pugongying (Taraxaci Herba), Taizishen (Pseudostellariae Radix), Huangjing (Polygonati Rhizoma), could be used as potential therapeutic agents. Conclusion TLR2, CD163 and C1QB were the core molecules of SARS-CoV-2-mediated immune inflammatory response promoting AS progression, and targeting predicted herbs were potential drugs to slow down AS progression in COVID-19 patients.Copyright © 2023 Editorial Office of Chinese Traditional and Herbal Drugs. All rights reserved.

Research Article Molecular Docking and Drug Likeness Prediction of New Potent Sars Cov-2 Main Protease Inhibitors

The novel corona virus whose outbreak took place in December 2019 continues to spread at a rapid rate worldwide. The Main protease (Mpro) plays critical role in the SARS-CoV-2 life cycle through virus replication and transcription process making it as an attractive drug target. Herein, molecular docking study followed by drug-Likeness prediction, were performed in order to identify new potent Mpro inhibitors. Indeed, molecular docking of 1880 compounds into the Mpro active site reveals compounds S1 and S2 as promising inhibitors of this enzyme with binding energy of -39,22 KJ/mol, -36.27 KJ/mol respectively. These two compounds were also predicted to have satisfying drug likeness properties, indicating that they might be promising lead compounds for further anti-SARS CoV-2 drug research.Copyright © KESS All rights reserved.

Germline rare variants of lectin pathway genes predispose to asymptomatic SARS-CoV-2 infection in elderly individuals

Background/Objectives: Emerging evidence suggests that complement system infection-dependent hyperactivation may worsen COVID-19 outcome. We investigated the role of predicted high impact variants -referred as Qualifying Variants (QVs) -of complement system genes in predisposing asymptomatic COVID-19 in elderly individuals, known to be more susceptible to severe disease. Method(s): Exploiting Whole-Exome Sequencing (WES) data and 56 complement system genes, we performed a gene-based collapsing test between 164 asymptomatic subjects (age >= 60 y.o.) and 56,885 European individuals from the gnomAD database. We replicated this test comparing the same asymptomatic individuals with 147 hospitalized COVID-19 patients. Result(s): We found an enrichment of QVs in three genes (MASP1, COLEC10 and COLEC11), which belong to the lectin pathway, in the asymptomatic cohort. Moreover, individuals with QVs showed lower serum levels of Masp1 and of prothrombin activity compared to controls while no differences were observed for CH50 and AH50 levels that measure the activity of classical and alternative complement pathways, respectively. Finally, integrative analyses of genome-wide association study and expression quantitative loci traits data showed a correlation between polymorphisms associated with asymptomatic COVID-19 and decreased expression of MASP1, COLEC11 and COLEC10 genes in lung tissue. Conclusion(s): This study suggests that rare genetic variants can protect from severe COVID-19 by mitigating the activation of lectin pathway and prothrombin activity.

Functioning of the ADAMTS-13/vWF axis and its clinical significance

Advances in biology have allowed us to substantially deepen our knowledge about hemostasis functioning both in health and disease. ADAMTS-13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) and von Willebrand factor (vWF) are components of the hemostasis system, which physiological interaction holds an important place in maintaining homeostasis. ADAMTS-13 is a metalloproteinase mainly acting to release vWF fragments into the blood plasma, as well as regulating its activity by cleaving ultra-large vWF multimers (UL-vWF) into smaller and less active forms. The study of such factors is of great clinical importance, since a decrease in ADAMTS-13 activity and an increase in vWF level can be predictors of microcirculatory disorders that play an important role in developing multiple organ failure. However, very few and fully contradictory studies devoted to the physiological aspects of the ADAMTS-13/vWF axis functioning in the mother-fetus system are available, therefore requiring to be further investigated.Copyright © 2023 Russian Journal of Forensic Medicine. All rights reserved.

Production and characterisation of a SARSCoV-2 S-protein RBD homodimer with increased avidity for specific antibodies

Monitoring of the proportion of immune individuals and the effectiveness of vaccination in a population involves evaluation of several important parameters, including the level of virus-neutralising antibodies. In order to combat the COVID-19 pandemic, it is essential to develop approaches to detecting SARS-CoV-2 neutralising antibodies by safe, simple and rapid methods that do not require live viruses. To develop a test system for enzyme-linked immunosorbent assay (ELISA) that detects potential neutralising antibodies, it is necessary to obtain a highly purified recombinant receptor-binding domain (RBD) of the spike (S) protein with high avidity for specific antibodies. The aim of the study was to obtain and characterise a SARSCoV-2 S-protein RBD homodimer and a recombinant RBD-expressing cell line, as well as to create an ELISA system for detecting potential neutralising antibodies. Material(s) and Method(s): the genetic construct was designed in silico. To generate a stable producer cell line, the authors transfected CHO-S cells, subjected them to antibiotic pressure, and selected the optimal clone. To isolate monomeric and homodimeric RBD forms, the authors purified the recombinant RBD by chromatographic methods. Further, they analysed the activity of the RBD forms by Western blotting, bio-layer interferometry, and indirect ELISA. The analysis involved monoclonal antibodies GamXRH19, GamP2C5, and h6g3, as well as serum samples from volunteers vaccinated with Gam-COVID-Vac (Sputnik V) and unvaccinated ones. Result(s): the authors produced the CHO-S cell line for stable expression of the recombinant SARS-CoV-2 S-protein RBD. The study demonstrated the recombinant RBD's ability to homodimerise after fed-batch cultivation of the cell line for more than 7 days due to the presence of unpaired cysteines. The purified recombinant RBD yield from culture broth was 30-50 mg/L. Monomeric and homodimeric RBD forms were separated using gel-filtration chromatography and characterised by their ability to interact with specific monoclonal antibodies, as well as with serum samples from vaccinated volunteers. The homodimeric recombinant RBD showed increased avidity for both monoclonal and immune sera antibodies. Conclusion(s): the homodimeric recombinant RBD may be more preferable for the analysis of levels of antibodies to the receptor-binding domain of the SARS-CoV-2 S protein.Copyright © 2023 Authors. All rights reserved.

Design of a Laboratory Developed Assay to Aid in the Detection of Sars-Cov-2 Virus Rna in Oropharyngeal and Nasopharyngeal Swabs Adapted from the Cdc Protocol

Intro: SARS-CoV-2 is a single-strand enveloped RNA virus belonging to the family Coronaviridae. It was first recognized in late 2019 as causing COVID-19, and later declared a pandemic. The development of this assay aided in the detection of positive cases early in the pandemic which in turn facilitated the isolation of infected individuals to minimize the spread. Method(s): The SARS-CoV-2 RNA detection by real time RT-PCR is a molecular in vitro diagnostic test that aids in the detection and diagnosis of SARS-CoV-2 in nasopharyngeal and oropharyngeal specimens. This test is based on nucleic acid extraction and amplification technology and uses oligonucleotide primers and dual-labeled hydrolysis probes. RNA is isolated and purified from specimens using the Abbott m2000sp. This technology uses magnetic particles to capture and purify the RNA. The bound RNA is eluted and transferred to a 96 deep-well plate and is ready for amplification. The master mix is prepared manually and is added to a PCR plate together with the extracted RNA. The RNA is reverse transcribed to cDNA and subsequently amplified in the Abbott m2000rt. In this process, the probe anneals to a specific target sequence located between the forward and reverse primers. During the extension step of the PCR cycle, the 5' nuclease activity of Taq polymerase degrades the probe, causing the reporter dye molecules to be cleaved from their respective probes, increasing the fluorescence intensity. Fluorescence intensity is monitored at each PCR cycle on the Abbott m2000rt instrument. Finding(s): The clinical evaluation was performed by testing patient samples in a blinded fashion. The performance of SARS-CoV-2 Assay was established using 60 clinical specimens. The positive and negative percent agreements were analyzed by comparing the SARS-CoV-2 Assay results to Seegene's AllplexTM 2019-nCoV which showed 100% concordance. Conclusion(s): This assay demonstrated accuracy and reproducibility for the detection of SARS-CoV-2.Copyright © 2023

Enhanced fatty acid oxidation through Metformin and Baicalin as therapy for COVID-19 and associated inflammatory states in lung and kidney

Progressive respiratory failure is the primary cause of death in the coronavirus disease 2019 (COVID-19) pandemic. It is the final outcome of the acute respiratory distress syndrome (ARDS), characterized by an initial exacerbated inflammatory response and ultimate tissue scarring. Energy balance may be crucial for the recovery of clinical COVID-19. Hence, we asked if two key pathways involved in energy generation, AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) signaling and fatty acid oxidation (FAO) could be beneficial. We tested the drugs Metformin (AMPk activator) and Baicalin (Cpt1A activator) in different experimental models mimicking COVID-19 associated inflammation in lung and kidney. We also studied two different cohorts of COVID19 patients that had been previously treated with Metformin. These drugs ameliorated lung damage in an ARDS animal model, while activation of AMPK/ACC signaling increased mitochondrial function and decreased TGF-beta-induced fibrosis, apoptosis and inflammation markers in lung epithelial cells. Similar results were observed with two new indole derivatives IND6 and IND8 with AMPK activating capacity. Consistently, a reduced stay in the intensive care unit was observed in COVID-19 patients previously exposed to Metformin. Baicalin also reduced kidney fibrosis in two animal models of kidney injury, another key target of COVID-19, while in vitro both drugs improved mitochondrial function and prevented TGF-beta-induced renal epithelial cell dedifferentiation. Our results support that strategies based on energy supply may prove useful in the prevention of COVID-19-induced lung and renal damage.Copyright © 2023

Human Anti-Ace2 Monoclonal Antibodies as Pan-Sarbecovirus Prophylactic Agents

Background: Human monoclonal antibodies from convalescent individuals that target the SARS-CoV-2 spike protein have been deployed as therapeutics against SARS-CoV-2. However, nearly all of these antibodies have been rendered obsolete by SARS-CoV-2 variants that evolved to resist similar, naturally occurring antibodies. Moreover, Most SARS-CoV-2 specific antibodies are inactive against divergent sarbecoviruses Methods: By immunizing mice that carry human immunoglobulin variable gene segments we generated a suite of fully human monoclonal antibodies that bind the human ACE2 receptor (hACE2) rather than the viral spike protein and were engineered to lack effector functions such as ADCC. Result(s): These ACE2 binding antibodies block infection by all hACE2 binding sarbecoviruses, including emergent SARS-CoV-2 variants, with a potency that of the most potent spike binding therapeutic antibodies. Structural and biochemical analyses revealed that the antibodies target an hACE2 epitope that engages SARS-CoV-2 spike. Importantly, the antibodies do not inhibit hACE2 enzymatic activity, nor do they induce ACE depletion from cell surfaces. The antibodies exhibit favorable pharmacology in human ACE2 knock in mice and provide near complete protection of hACE2 knock-in mice against SARS-CoV-2 infection. Conclusion(s): ACE2 binding antibodies should be useful prophylactic and treatment agents against any current and future SARS-CoV-2 variants, as well as hACE2-binding sarbecoviruses that might emerge as future pandemic threats.

Ace2 Substrate Cleavage Effect Associated with Covid-19 Patient Plasma Immunoglobulin

Background: Many mechanisms responsible for COVID-19 pathogenesis are well-known, but COVID-19 includes features with unclear pathogenesis, like autonomic dysregulation, coagulopathies, and high levels of inflammation. The SARS-CoV-2 spike protein receptor binding domain (RBD) receptor is angiotensin converting enzyme 2 (ACE2). We hypothesized that some COVID-19 patients may develop immunoglobulins (Igs) that have negative molecular image of RBD sufficiently similar to ACE2 to yield ACE2-like catalytic activity - ACE2-like 'abzymes'. Method(s): To explore this hypothesis, we studied 67 patients hospitalized with COVID-19 who had disodium ethylenediaminetetraacetate (EDTA) anticoagulated plasma samples available, obtained about 7 days after admission. We used commercially available fluorometric ACE2 assays (Abcam), and a SpectraMax M5 microplate reader (Molecular Devices), measuring Relative Fluorescent Unit (RFU, Ex/Em = 320/420 nm;RFU) in a kinetic mode every 20 min at 37C. ACE2 inhibitor provided in the assay kit was used for additional controls. In some control experiments, we added Zn2+ to plasma, or conducted serial dilutions to decrease Zn2+. To deplete Igs, we passed plasma samples through a 0.45 mum filter to remove large particles, then passed the material through 100kDa cut-off ultrafiltration membrane (PierceTM) columns, and finally used protein A/G Magnetic Beads (Life Technologies) to specifically deplete Ig, removing >99.99% of Ig as assessed with a human IgG ELISA Kit (Abcam). Result(s): ACE2 is a metalloprotease that requires Zn2+ for activity. However, we found that the plasma of 11 of the 67 patients could cleave a synthetic ACE2 peptide substrate, even though the plasma samples were collected using EDTA anticoagulant. When we spiked plasma with synthetic ACE2, no ACE2 substrate cleavage activity was observed unless Zn2+ was added, or the plasma was diluted to decrease EDTA concentration. After processing samples by size exclusion and protein A/G adsorption, the plasma samples did not cleave the ACE2 substrate peptide. Conclusion(s): The data suggest that some patients with COVID-19 develop Igs with activity capable of cleaving synthetic ACE2 substrate. Since abzymes can exhibit promiscuous substrate specificities compared to the enzyme whose active site image they resemble, and since proteolytic cascades regulate physiologic processes, anti-RBD abzymes may contribute to some otherwise obscure features of COVID-19 pathogenesis. (Figure Presented).

The use of alanine, free carnitine and IGFBP-1 as potential biomarkers for glycogen storage disease type I

Background: Glycogen Storage Disease Ia (GSDIa) and Ib (GSDIb) are inborn errors of carbohydrate metabolism due to a deficiency of glucose-6-phosphatase (G6Pase) or glucose-6-phosphate translocase (G6PT), respectively. Consuming prescribed amounts of uncooked cornstarch (UCCS) to prevent hypoglycemia is the standard of care for GSDIa and GSDIb. Patients followed in our GSD Program are admitted to the hospital annually for evaluation of their metabolic control by measuring glucose and lactate levels and revising treatment regimens accordingly. Lack of bed space due to the COVID-19 pandemic has created a need for alternate markers of metabolic control as lactate measurements are unreliable in the outpatient setting. This research aims to identify alternative biomarkers to show degree of metabolic control in individuals with GSDI. Method(s): A retrospective chart review was conducted on 45 adults and children with GSDI using data from January 1, 2014 toMay 6, 2021. Plasma alanine and free carnitine levels were compared with laboratory reference ranges. Results from the three tests were not available on every subject. Plasma alanine was evaluated on 24 subjects (16-GSDIa, 8-GSDIb) and free carnitine was evaluated on 25 subjects (17-GSDIa, 8-GSDIb). Result(s): Alanine levels in subjects with GSDIa ranged from 378 to 786 umol/L, while alanine levels in subjects with GSDIb ranged from 254 to 506 umol/L (reference range = 103-528 umol/L). Free carnitine levels ranged from26 to 72 umol/L in subjects with GSDIa and from 44 to 90 umol/L in subjects with GSDIb (reference range = 19-55 umol/L). Conclusion(s): Our analysis showed that plasma alanine and free carnitine have potential to be used as biomarkers of metabolic control. For plasma alanine, there seemed to be differences between subjects with GSDIa and GSDIb, as the majority of subjects with GSDIa had elevations in plasma alanine, while subjects with GSDIb did not. Elevated plasma alanine levels indicate lactic acidosis. For GSDIb, we hypothesize that there may be some type of G6Pase enzyme activity that occurs outside of the endoplasmic reticulum. When looking at both groups, free carnitine levels were mostly elevated. This indicates that there could be inhibition of fatty acid oxidation.Copyright © 2022 Elsevier Inc. All rights reserved.

cGAS-STING PATHWAY LIMITS SARS-CoV-2 REPLICATION IN ACE2+ AIRWAY CELL LINES

Background: We previously screened 10 human lung and upper airway cell lines expressing variable levels of endogenous ACE2/TMPRSS2. We found that H522 human lung adenocarcinoma cells supported SARS-CoV-2 replication independent of ACE2, whereas the ACE2 positive cell lines were not permissive to infection. Type I/III interferons (IFNs) potently restrict SARS-CoV-2 replication through the actions of hundreds of interferon-stimulated genes (ISGs) that are upregulated upon IFN signaling. Here we report that a number of ACE2 positive airway cell lines are unable to support SARS-CoV-2 replication due to basal activation of the cGAS-STING DNA sensing pathway and subsequent upregulation of IFNs and ISGs which restrict SARS-CoV-2 replication. Method(s): SARS-CoV-2 WT strain 2019-nCoV/USA-WA1/2020 viral replication was detected through analysis of cell associated RNA. RNA sequencing was used to study the basal level of genes in the type-I IFN pathway in the 10 cell lines, which was further validated by western blotting and qRT-PCR. A panel of 5 cell lines, with varying expression levels of ACE2 and TMPRSS2, were pre-treated with Ruxolitinib, a JAK1/2 inhibitor. A siRNA-mediated screen was used to determine the molecular basis of basally high expression of ISGs in cell lines. CRISPR knockout of IFN-alpha receptor and cGAS-STING pathway components was conducted in parallel Results: Here we show that higher basal levels of IFN pathway activity underlie the inability of ACE2+ cell lines to support virus replication. Importantly, this IFN-induced block can be overcome by chemical inhibition and genetic disruption of the IFN signaling pathway or by ACE2 overexpression, suggesting that one or more saturable ISGs underlie the lack of permissivity of these cells. Ruxolitinib treatment increased SARS-CoV-2 RNA levels by nearly 3 logs in OE21 and SCC25. Furthermore, the baseline activation of the STING-cGAS pathway accounts for the high ISG levels and genetic disruption of the cGAS-STING pathway enhances levels by nearly 2 and 3 logs of virus replication in the two separate ACE2+ cell line models respectively. Conclusion(s): Our findings demonstrate that cGAS-STING-dependent activation of IFN-mediated innate immunity underlies the inability of ACE2+ airway cell lines to support SARS-CoV-2 replication. Our study highlights that in addition to ACE2, basal activation of cGAS-STING pathway, IFNs and ISGs may play a key role in defining SARS-CoV-2 cellular tropism and may explain the complex SARS-CoV- 2 pathogenesis in vivo.

Controversial role of neutrophilic granulocytes in the immunopathogenesis of COVID-19: correlation with imbalance of pro-inflammatory neutrophil-associated cytokines and interferon alpha deficiency.

Neutrophilic granulocytes (NG) are the main drivers of pathological inflammation in COVID-19. Objective. To specify the mechanisms of immunopathogenesis of COVID-19 based on a comparative immunological study of the number and phenotype of CD16+SD62L+CD11b+CD63- and CD16+SD62L+CD11b+CD63+ subsets with an assessment of their effector functions against changing profile of NG-associated cytokines IL-8, IL-18, IL-17A, VEGF-A, IFNalpha, and IFNgamma. Patients and methods. In patients with moderate-to-severe and severe COVID-19, we determined IL-1beta, TNFalpha, IL-6, IL-8, IL-18, IL-17A, VEGF-A, IFNalpha, and IFNgamma (ELISA), the phenotype of CD16+SD62L+CD11b+CD63- and CD16+SD62L+CD11b+CD63+ subsets, NF-kappaB-NG (CYTOMICS FC500), phagocytically active NG (%), neutrophil extracellular traps (NETs), NG in apoptosis, and the activity of NADPH oxidase. Results. In COVID-19 against the background of IFNalpha and IFNgamma production blockade and high levels of NG-associated IL-8, IL-18, IL-17A, VEGF-A, a reduction in the number of mature and functionally active CD16brightSD62LbrightCD11bbrightCD63-NG subsets was revealed, as well as an increase in the number of CD16dimSD62LdimSD11bbrightCD63-NG subsets with an immunosuppressive phenotype and CD16brightSD62LbrightSD11bbrightCD63bright-NG subsets with high cytotoxic activity and ability to form NETs, a decrease in the percentage of phagocytically active NG and an increase in the activity of NADPH oxidase, NETs, and NG in apoptosis. Conclusion. IFNalpha deficiency provokes a hyperergic response of NG-associated cytokines, which leads to the formation of uncontrolled immune inflammation involving NG subsets with an immunosuppressive and cytotoxic phenotype, exacerbating the course of COVID-19. The use of recombinant IFNalpha-2b with antioxidants (Viferon) in the early stages of the disease can help to restore immune homeostasis, normalize the level of NG-associated cytokines, reduce NERTs, and achieve good clinical efficacy.Copyright © 2022, Dynasty Publishing House. All rights reserved.