Angiotensin-converting enzymes 1 and 2 in the feces: presence and catalytic activity in the rat 2,4,6-trinitrobenzene sulfonic acid-induced model of colitis.

BACKGROUND AND AIM: Inflammatory bowel disease is challenging to diagnose. Fecal biomarkers offer noninvasive solutions. The renin-angiotensin-aldosterone system is implicated in intestinal inflammation. Angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) regulate its activity, but conflicting findings on these enzymes in colitis require further investigation. We aimed to assess ACE and ACE2 presence and activities in the feces, serum, and colon of 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced rats. METHODS: Colitis was induced in male rats by rectal instillation of a 21% ethanolic TNBS solution. After rats' sacrifice, colonic portions, serum, and feces were collected. ACE and ACE2 presence in the feces was analyzed by western Blot, and colonic and serum enzymes' concentrations were quantified using ELISA kits. ACE activity was assessed using Hippuryl-His-Leu and Z-Phe-His-Leu as substrates. ACE2 activity was assessed using Mca-APK (Dnp) as a substrate in the presence and absence of DX600 (ACE2 inhibitor). RESULTS: An ACE isoform of ~70 kDa was found only in the feces of TNBS-induced rats. ACE concentration was higher than that of ACE2 in the serum and the inflamed colon. ACE N-domain activity was higher than that of the C-domain in all matrices. ACE2 activity was higher in the feces of TNBS-induced animals compared to controls. CONCLUSION: A 70 kDa ACE isoform only detected in the feces of TNBS-induced rats may have translational relevance. ACE N-domain seems to play a significant role in regulating colonic lesions. Further research using human samples is necessary to validate these findings.

Profound Rhabdomyolysis and Viral Myositis Due to SARS-CoV-2: A Case Report.

The novel SARS-CoV-2 introduced several new inflammatory conditions including SARS-CoV-2-associated rhabdomyolysis and viral myositis. We present a 22-year-old man who noted a week of cough followed by myalgias, dark-colored urine, and decreased oral intake. He was found to have acute nontraumatic rhabdomyolysis after an acutely positive SARS-CoV-2 test. Initial creatine kinase (CK) level was above the reference range as were liver enzymes reflective of muscle breakdown. Treatment involved fluid resuscitation and pain control, with close monitoring of kidney, liver, and skeletal markers over five days of hospitalization till there was clinical and symptomatic improvement.

Angiotensin-Converting Enzyme (ACE) Inhibitor-Induced Angioedema in an African American Male With Coronavirus Disease 2019 (COVID-19).

Angioedema is a condition characterized by non-pitting swelling of the subcutaneous or submucosal tissues in particular the face, lips, and oral cavity. Angiotensin-converting enzyme (ACE) inhibitors are known to contribute to the development of angioedema by increasing the levels of bradykinin and its active metabolites. Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is hypothesized to contribute to the development of angioedema by modifying ACE II levels and further increasing the level of bradykinin in patients taking ACE inhibitors. African Americans may be at particular risk of developing angioedema with concomitant SARS-CoV-2 infection and ACE inhibitor use. This case involves a 31-year-old African American male diagnosed with coronavirus disease 2019 (COVID-19) who developed angioedema while taking an ACE inhibitor.

Rapid Detection of SARS-CoV-2 Variants Using an Angiotensin-Converting Enzyme 2-Based Surface-Enhanced Raman Spectroscopy Sensor Enhanced by CoVari Deep Learning Algorithms.

An integrated approach combining surface-enhanced Raman spectroscopy (SERS) with a specialized deep learning algorithm to rapidly and accurately detect and quantify SARS-CoV-2 variants is developed based on an angiotensin-converting enzyme 2 (ACE2)-functionalized AgNR@SiO2 array SERS sensor. SERS spectra with concentrations of different variants were collected using a portable Raman system. After appropriate spectral preprocessing, a deep learning algorithm, CoVari, is developed to predict both the viral variant species and concentrations. Using a 10-fold cross-validation strategy, the model achieves an average accuracy of 99.9% in discriminating between different virus variants and R2 values larger than 0.98 for quantifying viral concentrations of the three viruses, demonstrating the high quality of the detection. The limit of detection of the ACE2 SERS sensor is determined to be 10.472, 11.882, and 21.591 PFU/mL for SARS-CoV-2, SARS-CoV-2 B1, and CoV-NL63, respectively. The feature importance of virus classification and concentration regression in the CoVari algorithm are calculated based on a permutation algorithm, which showed a clear correlation to the biochemical origins of the spectra or spectral changes. In an unknown specimen test, classification accuracy can achieve >90% for concentrations larger than 781 PFU/mL, and the predicted concentrations consistently align with actual values, highlighting the robustness of the proposed algorithm. Based on the CoVari architecture and the output vector, this algorithm can be generalized to predict both viral variant species and concentrations simultaneously for a broader range of viruses. These results demonstrate that the SERS + CoVari strategy has the potential for rapid and quantitative detection of virus variants and potentially point-of-care diagnostic platforms.

Two Novel Angiotensin-Converting Enzyme (ACE) Inhibitory and ACE2 Upregulating Peptides from the Hydrolysate of Pumpkin (Cucurbita moschata) Seed Meal.

Pumpkin (Cucurbita moschata) seed meal (PSM), the major byproduct of pumpkin seed oil industry, was used to prepare angiotensin-converting enzyme (ACE) inhibitory and angiotensin-converting enzyme 2 (ACE2) upregulating peptides. These peptides were isolated and purified from the PSM hydrolysate prepared using Neutrase 5.0 BG by ultrafiltration, Sephadex G-15 column chromatography, and reversed-phase high-performance liquid chromatography. Two peptides with significant ACE inhibition activity were identified as SNHANQLDFHP and PVQVLASAYR with IC50 values of 172.07 and 90.69 µM, respectively. The C-terminal tripeptides of the two peptides contained Pro, Phe, and Tyr, respectively, and PVQVLASAYR also had Val in its N-terminal tripeptide, which was a favorable structure for ACE inhibition. Molecular docking results declared that the two peptides could interact with ACE through hydrogen bonds and hydrophobic interactions. Furthermore, the two peptides performed protective function on EA.hy926 cells by decreasing the secretion of endothelin-1, increasing the release of nitric oxide, and regulating the ACE2 activity. In vitro simulated gastrointestinal digestion showed the two peptides exhibited good stability against gastrointestinal enzyme digestion. In conclusion, PSM is a promising material for preparing antihypertensive peptides.

Susceptibility to recombinant SARS-CoV-2 spike protein entry in the lungs of high-fat diet-induced obese mice.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Obesity is a major risk factor for the development of COVID-19. Angiotensin-converting enzyme 2 (ACE2) is an essential receptor for cell entry of SARS-CoV-2. The receptor-binding domain of the S1 subunit (S1-RBD protein) in the SARS-CoV-2 spike glycoprotein binds to ACE2 on host cells, through which the virus enters several organs, including the lungs. Considering these findings, recombinant ACE2 might be utilized as a decoy protein to attenuate SARS-CoV-2 infection. Here, we examined whether obesity increases ACE2 expression in the lungs and whether recombinant ACE2 administration diminishes the entry of S1-RBD protein into lung cells. We observed that high-fat diet-induced obesity promoted ACE2 expression in the lungs by increasing serum levels of LPS derived from the intestine. S1-RBD protein entered the lungs specifically through ACE2 expressed in host lungs and that the administration of recombinant ACE2 attenuated this entry. We conclude that obesity makes hosts susceptible to recombinant SARS-CoV-2 spike proteins due to elevated ACE2 expression in lungs, and this model of administering S1-RBD protein can be applied to new COVID-19 treatments.

Why have SGLT2 Inhibitors Failed to Achieve the Desired Success in COVID-19?

The SARS-CoV-2 virus emerged towards the end of 2019 and caused a major worldwide pandemic lasting at least 2 years, causing a disease called COVID-19. SARS-CoV-2 caused a severe infection with direct cellular toxicity, stimulation of cytokine release, increased oxidative stress, disruption of endothelial structure, and thromboinflammation, as well as angiotensin-converting enzyme 2 (ACE2) down-regulation-mediated renin-angiotensin system (RAS) activation. In addition to glucosuria and natriuresis, sodium-glucose transport protein 2 (SGLT2) inhibitors (SGLT2i) cause weight loss, a decrease in glucose levels with an insulin-independent mechanism, an increase in erythropoietin levels and erythropoiesis, an increase in autophagy and lysosomal degradation, Na+/H+-changer inhibition, prevention of ischemia/reperfusion injury, oxidative stress and they have many positive effects such as reducing inflammation and improving vascular function. There was great anticipation for SGLT2i in treating patients with diabetes with COVID-19, but current data suggest they are not very effective. Moreover, there has been great confusion in the literature about the effects of SGLT2i on COVID-19 patients with diabetes . Various factors, including increased SGLT1 activity, lack of angiotensin receptor blocker co-administration, the potential for ketoacidosis, kidney injury, and disruptions in fluid and electrolyte levels, may have hindered SGLT2i's effectiveness against COVID-19. In addition, the duration of use of SGLT2i and their impact on erythropoiesis, blood viscosity, cholesterol levels, and vitamin D levels may also have played a role in their failure to treat the virus. This article aims to uncover the reasons for the confusion in the literature and to unravel why SGLT2i failed to succeed in COVID-19 based on some solid evidence as well as speculative and personal perspectives.

COVID-19 and Its Ophthalmic Manifestations: A Literature Review.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus discovered in late 2019 in Wuhan, Hubei Province, China. The virus has now developed into a full-scale global pandemic affecting hundreds of millions of people to date. A majority of cases present with nonspecific acute upper respiratory symptoms. A wide range of systemic symptoms has been reported, with some patients presenting with nonspecific extrapulmonary symptoms. Recently, there has been an increased association of COVID-19-positive patients presenting with ocular symptoms. As an increasing number of patients present with ophthalmic manifestations, recognizing these visual symptoms is of utmost importance. Some patients may present with ocular symptoms as the first indication of COVID-19 infection; quickly isolating and starting treatment can aid in stopping the spread of this novel coronavirus. This review will describe the current epidemiology and pathophysiology of SARS-CoV-2, emphasizing the ophthalmic manifestations and their clinical course progression. Further, we will be reporting on the growing number of rare ocular manifestations that have occurred in some COVID-19-positive patients, along with the route of transmission, specific manifestations, and the treatment methods for both these pulmonary and extrapulmonary symptoms, specifically the ocular manifestations.

Identification of structural origins of complex charge heterogeneity in therapeutic ACE2Fc fusion protein facilitated by free-flow isoelectric focusing.

Fc Fusion protein represents a versatile molecular platform with considerable potential as protein therapeutics of which the charge heterogeneity should be well characterized according to regulatory guidelines. Angiotensin-converting enzyme 2 Fc fusion protein (ACE2Fc) has been investigated as a potential neutralizing agent to various coronaviruses, including the lingering SARS-CoV-2, as this coronavirus must bind to ACE2 to allow for its entry into host cells. ACE2Fc, an investigational new drug developed by Henlius (Shanghai China), has passed the Phase I clinical trial, but its huge amount of charge isoforms and complicated charge heterogeneity posed a challenge to charge variant investigation in pharmaceutical development. We employed offline free-flow isoelectric focusing (FF-IEF) fractionation, followed by detailed characterization of enriched ACE2Fc fractions, to unveil the structural origins of charge heterogeneity in ACE2Fc expressed by recombinant CHO cells. We adopted a well-tuned 3-component separation medium for ACE2Fc fractionation, the highest allowable voltage to maximize the FF-IEF separation window and a mild Protein A elution method for preservation of protein structural integrity. Through peptide mapping and other characterizations, we revealed that the intricate profiles of ACE2Fc charge heterogeneity are mainly caused by highly sialylated multi-antenna N-glycosylation. In addition, based on fraction characterization and in silico glycoprotein model analysis, we discovered that the large acidic glycans at N36, N73, and N305 of ACE2Fc were able to decrease the binding activity towards Spike (S) protein of SARS-CoV-2. Our study exemplifies the value of FF-IEF in highly complex fusion protein characterization and revealed a quantitative sialylation-activity relationship in ACE2Fc.

Inhibitory Efficacy of Main Components of Scutellaria baicalensis on the Interaction between Spike Protein of SARS-CoV-2 and Human Angiotensin-Converting Enzyme II.

Blocking the interaction between the SARS-CoV-2 spike protein and the human angiotensin-converting enzyme II (hACE2) protein serves as a therapeutic strategy for treating COVID-19. Traditional Chinese medicine (TCM) treatments containing bioactive products could alleviate the symptoms of severe COVID-19. However, the emergence of SARS-CoV-2 variants has complicated the process of developing broad-spectrum drugs. As such, the aim of this study was to explore the efficacy of TCM treatments against SARS-CoV-2 variants through targeting the interaction of the viral spike protein with the hACE2 receptor. Antiviral activity was systematically evaluated using a pseudovirus system. Scutellaria baicalensis (S. baicalensis) was found to be effective against SARS-CoV-2 infection, as it mediated the interaction between the viral spike protein and the hACE2 protein. Moreover, the active molecules of S. baicalensis were identified and analyzed. Baicalein and baicalin, a flavone and a flavone glycoside found in S. baicalensis, respectively, exhibited strong inhibitory activities targeting the viral spike protein and the hACE2 protein, respectively. Under optimized conditions, virus infection was inhibited by 98% via baicalein-treated pseudovirus and baicalin-treated hACE2. In summary, we identified the potential SARS-CoV-2 inhibitors from S. baicalensis that mediate the interaction between the Omicron spike protein and the hACE2 receptor. Future studies on the therapeutic application of baicalein and baicalin against SARS-CoV-2 variants are needed.

A Biosensor Assay Based on Coiled-Coil-Mediated Human ACE2 Receptor Capture for the Analysis of Its Interactions with the SARS-CoV-2 Receptor Binding Domain.

Surface plasmon resonance (SPR)-based biosensing enables the characterization of protein-protein interactions. Several SPR-based approaches have been designed to evaluate the binding mechanism between the angiotensin-converting enzyme 2 (ACE2) receptor and the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein leading to a large range of kinetic and thermodynamic constants. This chapter describes a robust SPR assay based on the K5/E5 coiled-coil capture strategy that reduces artifacts. In this method, ACE2 receptors were produced with an E5-tag and immobilized as ligands in the SPR assay. This chapter details methods for high-yield production and purification of the studied proteins, functionalization of the sensor chip, conduction of the SPR assay, and data analysis.

Anti-COVID-19 Potential of Withaferin-A and Caffeic Acid Phenethyl Ester.

BACKGROUND: The recent COVID-19 (coronavirus disease 2019) pandemic triggered research on the development of new vaccines/drugs, repurposing of clinically approved drugs, and assessment of natural anti-COVID-19 compounds. Based on the gender difference in the severity of the disease, such as a higher number of men hospitalized and in intense care units, variations in sex hormones have been predicted to play a role in disease susceptibility. Cell surface receptors (Angiotensin-Converting Enzyme 2; ACE2 and a connected transmembrane protease serine 2- TMPSS2) are upregulated by androgens. Conversely, androgen antagonists have also been shown to lower ACE2 levels, implying their usefulness in COVID-19 management. OBJECTIVES: In this study, we performed computational and cell-based assays to investigate the anti- COVID-19 potential of Withaferin-A and Caffeic acid phenethyl ester, natural compounds from Withania somnifera and honeybee propolis, respectively. METHODS: Structure-based computational approach was adopted to predict binding stability, interactions, and dynamics of the two test compounds to three target proteins (androgen receptor, ACE2, and TMPRSS2). Further, in vitro, cell-based experimental approaches were used to investigate the effect of compounds on target protein expression and SARS-CoV-2 replication. RESULTS: Computation and experimental analyses revealed that (i) CAPE, but not Wi-A, can act as androgen antagonist and hence inhibit the transcriptional activation function of androgen receptor, (ii) while both Wi-A and CAPE could interact with ACE2 and TMPRSS2, Wi-A showed higher binding affinity, and (iii) combination of Wi-A and CAPE (Wi-ACAPE) caused strong downregulation of ACE2 and TMPRSS2 expression and inhibition of virus infection. CONCLUSION: Wi-A and CAPE possess multimodal anti-COVID-19 potential, and their combination (Wi-ACAPE) is expected to provide better activity and hence warrant further attention in the laboratory and clinic.

Chemical Composition of Thyme (Thymus vulgaris) Extracts, Potential Inhibition of SARS-CoV-2 Spike Protein-ACE2 Binding and ACE2 Activity, and Radical Scavenging Capacity.

Water and ethanol extracts of dried thyme (Thymus vulgaris) were analyzed for chemical composition, inhibition of the SARS-CoV-2 spike protein-ACE2 interaction, inhibition of ACE2 activity, and free radical scavenging capacity. Thirty-two compounds were identified in water extract (WE) and 27 were identified in ethanol extract (EE) of thyme through HPLC-MS. The WE (33.3 mg/mL) and EE (3.3 mg/mL) of thyme inhibited the spike protein-ACE2 interaction by 82.6 and 86.4%, respectively. The thyme WE at 5 mg/mL inhibited ACE2 activity by 99%, and the EE at 5 mg/mL inhibited ACE2 by 65.8%. Total phenolics were determined to be 38.9 and 8.8 mg of GAE/g in WE and EE, respectively. The HO• scavenging capacities were 1121.1 and 284.4 µmol of TE/g in WE and EE, respectively. The relative DPPH• scavenging capacities were 126.3 µmol TE/g in WE and 28.2 µmol TE/g in EE. The ABTS•+ scavenging capacities were 267.1 µmol TE/g in WE and 96.7 µmol TE/g in EE. The results suggested that the thyme extract could be potentially used to prevent SARS-CoV-2 infection and mitigate the complications from the infection.

Potential Antihypertensive Activity of the Aqueous Extract of Ammi visnaga and its Effect on ACE-2 in Rats.

AIMS: This work aimed to investigate the antihypertensive activity of Ammi visnaga. BACKGROUND: The aqueous extract of Ammi visnaga has traditionally been used to treat hypertension in Morocco. OBJECTIVE: The objective of this investigation was to evaluate the effect of Ammi visnaga aqueous extract (AVAE) on arterial blood pressure, systolic blood pressure (SBP), mean blood pressure (MBP), diastolic blood pressure (DBP), and heart rate (HR) in normotensive and hypertensive rats. In addition, the effect of the aqueous extract of Ammi visnaga on vasodilatation was assessed in isolated rat aortic rings with functional endothelium pre-contracted with epinephrine EP or KCl. METHODS: AVAE was obtained, and its antihypertensive ability was pharmacologically investigated in L-NAME hypertensive and normotensive rats. The rats received oral AVAE at two selected doses of 70 and 140 mg/kg for six hours (acute experiment) and seven days (sub-chronic). Thereafter, systolic, diastolic, mean arterial blood pressure and heart rate were evaluated. Moreover, the vasorelaxant activity of AESA was performed in thoracic aortic ring rats. In addition, the mechanisms of action involved in the vasorelaxant effect were studied. RESULTS: AVAE lowered blood pressure only in L-Name-induced hypertensive rats. Furthermore, AVAE (0.375-1.375 mg/ml) showed a vasodilator effect in isolated aortic rats. In addition, not all of the medications used in our study were responsible for the signaling pathway. As a result, additional pharmaceuticals are required to confirm the mechanism of this signaling pathway. CONCLUSION: The aqueous extract of Ammi visnaga exerts an interesting antihypertensive activity, which could be mediated through its vasorelaxant activity. The study supports its use as a medicinal plant against hypertension in Morocco.

Molecular insights to the anti-COVID-19 potential of α-, ß- and γ-cyclodextrins.

SARS-CoV-2 viral infection is regulated by the host cell receptors ACE2 and TMPRSS2, and therefore the effect of various natural and synthetic compounds on these receptors has recently been the subject of investigations. Cyclodextrins, naturally occurring polysaccharides derived from starch, are soluble in water and have a hydrophobic cavity at their center enabling them to accommodate small molecules and utilize them as carriers in the food, supplements, and pharmaceutical industries to improve the solubility, stability, and bioavailability of target compounds. In the current study, computational molecular simulations were used to investigate the ability of α-, ß- and γ-Cyclodextrins on human cell surface receptors. Cell-based experimental approaches, including expression analyses at mRNA and protein levels and virus replication, were used to assess the effect on receptor expression and virus infection, respectively. We found that none of the three CDs could dock effectively to human cell surface receptor ACE2 and viral protease Mpro (essential for virus replication). On the other hand, α- and ß-CD showed strong and stable interactions with TMPRSS2, and the expression of both ACE2 and TMPRSS2 was downregulated at the mRNA and protein levels in cyclodextrin (CD)-treated cells. A cell-based virus replication assay showed ∼20% inhibition by ß- and γ-CD. Taken together, the study suggested that (i) downregulation of expression of host cell receptors may not be sufficient to inhibit virus infection (ii) activity of the receptors and virus protein Mpro may play a critical and clinically relevant role, and hence (iii) newly emerging anti-Covid-19 compounds warrant multimodal functional analyses.Communicated by Ramaswamy H. Sarma.

Eye Washing Downregulated Angiotensin-Converting Enzyme 2 in Conjunctival Tissue Samples from Smokers.

This study aimed to (1) determine whether the expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 is increased in tobacco smokers, which potentially increases their susceptibility to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and (2) assess whether eye rinsing can reduce susceptibility. This prospective study included 20 eyes of 10 smokers and 18 eyes of nine healthy non-smokers (control) for reverse-transcription polymerase chain reaction. This study also included 28 eyes of 14 smokers and 16 eyes of eight healthy non-smokers (control) for enzyme-linked immunosorbent assay. Tear and impression cytology samples were collected from the right eye of each patient. The left eye was then rinsed for 30 s, and after 5 min, the tear and impression cytology samples were collected in the same manner. The expression of the ACE2 gene was significantly higher in the conjunctiva of smokers (n = 17; median 3.07 copies/ng of total RNA) than in those of non-smokers (n = 17; median 1.92 copies/ng of total RNA, p = 0.003). Further, mRNA expression and protein levels of ACE2 were weakly correlated in smokers (r = 0.49). ACE2 protein levels in Schirmer's strip samples were significantly reduced from 5051 to 3202 pg/mL after eye washing (n = 10; p = 0.001). Ocular surface cells are susceptible to SARS-CoV-2 infection. Smoking may be a risk factor for SARS-CoV-2 infection, and eye rinsing may reduce the risk of infection.

Structural insights into ACE2 interactions and immune activation of SARS-CoV-2 and its variants: an in-silico study.

The initial interaction between COVID-19 and the human body involves the receptor-binding domain (RBD) of the viral spike protein with the angiotensin-converting enzyme 2 (ACE2) receptor. Likewise, the spike protein can engage with immune-related proteins, such as toll-like receptors (TLRs) and pulmonary surfactant proteins A (SP-A) and D (SP-D), thereby triggering immune responses. In this study, we utilize computational methods to investigate the interactions between the spike protein and TLRs (specifically TLR2 and TLR4), as well as (SP-A) and (SP-D). The study is conducted on four variants of concern (VOC) to differentiate and identify common virus behaviours. An assessment of the structural stability of various variants indicates slight changes attributed to mutations, yet overall structural integrity remains preserved. Our findings reveal the spike protein's ability to bind with TLR4 and TLR2, prompting immune activation. In addition, our in-silico results reveal almost similar docking scores and therefore affinity for both ACE2-spike and TLR4-spike complexes. We demonstrate that even minor changes due to mutations in all variants, surfactant A and D proteins can function as inhibitors against the spike in all variants, hindering the ACE2-RBD interaction.Communicated by Ramaswamy H. Sarma.

Large-Scale Purification and Characterization of Recombinant Receptor-Binding Domain (RBD) of SARS-CoV-2 Spike Protein Expressed in Yeast.

SARS-CoV-2 spike protein is an essential component of numerous protein-based vaccines for COVID-19. The receptor-binding domain of this spike protein is a promising antigen with ease of expression in microbial hosts and scalability at comparatively low production costs. This study describes the production, purification, and characterization of RBD of SARS-CoV-2 protein, which is currently in clinical trials, from a commercialization perspective. The protein was expressed in Pichia pastoris in a large-scale bioreactor of 1200 L capacity. Protein capture and purification are conducted through mixed-mode chromatography followed by hydrophobic interaction chromatography. This two-step purification process produced RBD with an overall productivity of ~21 mg/L at >99% purity. The protein's primary, secondary, and tertiary structures were also verified using LCMS-based peptide mapping, circular dichroism, and fluorescence spectroscopy, respectively. The glycoprotein was further characterized for quality attributes such as glycosylation, molecular weight, purity, di-sulfide bonding, etc. Through structural analysis, it was confirmed that the product maintained a consistent quality across different batches during the large-scale production process. The binding capacity of RBD of spike protein was also assessed using human angiotensin-converting enzyme 2 receptor. A low binding constant range of KD values, ranging between 3.63 × 10-8 to 6.67 × 10-8, demonstrated a high affinity for the ACE2 receptor, revealing this protein as a promising candidate to prevent the entry of COVID-19 virus.

Meta-Analysis of the Mechanisms Underlying COVID-19 Modulation of Parkinson's Disease.

Coronavirus disease-19 (COVID-19) is caused by the infection of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The virus enters host cells through receptor-mediated endocytosis of angiotensin-converting enzyme-2 (ACE2), leading to systemic inflammation, also known as a "cytokine storm", and neuroinflammation. COVID-19's upstream regulator, interferon-gamma (IFNG), is downregulated upon the infection of SARS-CoV-2, which leads to the downregulation of ACE2. The neuroinflammation signaling pathway (NISP) can lead to neurodegenerative diseases, such as Parkinson's disease (PD), which is characterized by the formation of Lewy bodies made primarily of the α-synuclein protein encoded by the synuclein alpha (SNCA) gene. We hypothesize that COVID-19 may modulate PD progression through neuroinflammation induced by cytokine storms. This study aimed to elucidate the possible mechanisms and signaling pathways involved in COVID-19-triggered pathology associated with neurodegenerative diseases like PD. This study presents the analysis of the pathways involved in the downregulation of ACE2 following SARS-CoV-2 infection and its effect on PD progression. Through QIAGEN's Ingenuity Pathway Analysis (IPA), the study identified the NISP as a top-five canonical pathway/signaling pathway and SNCA as a top-five upstream regulator. Core Analysis was also conducted on the associated molecules between COVID-19 and SNCA to construct a network connectivity map. The Molecule Activity Predictor tool was used to simulate the infection of SARS-CoV-2 by downregulating IFNG, which leads to the predicted activation of SNCA, and subsequently PD, through a dataset of intermediary molecules. Downstream effect analysis was further used to quantify the downregulation of ACE2 on SNCA activation.

Hypoxia modifies levels of the SARS-CoV-2 cell entry proteins, angiotensin-converting enzyme 2, and furin in fetal human brain endothelial cells.

BACKGROUND: It is not known whether human fetal brain endothelial cells that form the blood-brain barrier express angiotensin-converting enzyme 2, transmembrane serine protease 2, and furin, which are SARS-CoV-2 cell entry proteins. Moreover, it is unclear whether hypoxia, commonly observed during severe maternal COVID-19, can modify their level of expression. We hypothesized that human fetal brain endothelial cells isolated from early- and midpregnancy brain microvessels express angiotensin-converting enzyme 2, transmembrane serine protease 2, and furin. Furthermore, we hypothesized that hypoxia modifies their expression levels in a gestational age- and time-of-exposure-dependent manner. OBJECTIVE: This study aimed to investigate whether early- and midpregnancy human fetal brain endothelial cells express angiotensin-converting enzyme 2, transmembrane serine protease 2, and furin SARS-CoV-2-associated cell entry proteins and to determine the effects of hypoxia on angiotensin-converting enzyme 2, transmembrane serine protease 2, and furin expression levels in human fetal brain endothelial cells. STUDY DESIGN: This was a prospective study where human fetal brain endothelial cells isolated from early-pregnancy (12.4±0.7 weeks of gestation) and midpregnancy (17.9±0.5 weeks of gestation) fetal brain microvessels (6 per group) were exposed to different oxygen tensions (20%, 5%, and 1% oxygen) for 6, 24, and 48 hours. Angiotensin-converting enzyme 2, transmembrane serine protease 2, and furin messenger RNA and protein levels and localization were assessed using quantitative polymerase chain reaction, Western blot testing, and immunofluorescence. RESULTS: Angiotensin-converting enzyme 2, transmembrane serine protease 2, and furin co-localize with the endothelial cell marker von Willebrand factor in human fetal brain endothelial cells isolated from early pregnancy and midpregnancy. In early pregnancy, TMPRSS2 messenger RNA expression was decreased by 5% oxygen compared with 20% oxygen after 6 hours of exposure (P<.05). In midpregnancy, 5% oxygen down-regulated ACE2 messenger RNA compared with 20% oxygen after 24 hours (P<.05). Furin messenger RNA expression was decreased under 5% and 1% oxygen compared with 20% oxygen (P<.05) after 24 hours. In midpregnancy, angiotensin-converting enzyme 2 protein levels were decreased under 5% and 1% oxygen (P<.001) after 24 hours. In contrast, furin protein levels were increased under 1% oxygen compared with 20% oxygen after 24 hours (P<.05). At 48 hours, 1% oxygen increased angiotensin-converting enzyme 2 protein levels compared with 20% oxygen (P<.01). CONCLUSION: Hypoxia modifies the expression of selected SARS-CoV-2 cell entry proteins in human fetal brain endothelial cells in a gestational age- and time-of-exposure-dependent manner. As severe COVID-19 may lead to maternal hypoxia, an altered expression of these proteins in the developing human blood-brain barrier could potentially lead to altered SARS-CoV-2 brain invasion and neurologic sequelae in neonates born to pregnancies complicated by SARS-CoV-2 infection.