SARS-CoV-2 NSP14 governs mutational instability and assists in making new SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the rapidly evolving RNA virus behind the COVID-19 pandemic, has spawned numerous variants since its 2019 emergence. The multifunctional Nonstructural protein 14 (NSP14) enzyme, possessing exonuclease and messenger RNA (mRNA) capping capabilities, serves as a key player. Notably, single and co-occurring mutations within NSP14 significantly influence replication fidelity and drive variant diversification. This study comprehensively examines 120 co-mutations, 68 unique mutations, and 160 conserved residues across NSP14 homologs, shedding light on their implications for phylogenetic patterns, pathogenicity, and residue interactions. Quantitative physicochemical analysis categorizes 3953 NSP14 variants into three clusters, revealing genetic diversity. This research underscoresthe dynamic nature of SARS-CoV-2 evolution, primarily governed by NSP14 mutations. Understanding these genetic dynamics provides valuable insights for therapeutic and vaccine development.

Hippocampal Microglia Activation Induced by Acute Pancreatic Injury in Rats.

BACKGROUND: Acute pancreatitis is an inflammation of the pancreatic glandular parenchyma that causes injury with or without the destruction of pancreatic acini. Clinical and experimental evidence suggest that certain systemic proinflammatory mediators may be responsible for initiating the fundamental mechanisms involved in microglial reactivity. Here, we investigated the possible repercussions of acute pancreatitis (AP) on the production of inflammatory mediators in the brain parenchyma focusing on microglial activation in the hippocampus. METHODS: The acute pancreatic injury in rats was induced by a pancreas ligation surgical procedure (PLSP) on the splenic lobe, which corresponds to approximately 10% of total mass of the pancreas. Blood samples were collected via intracardiac puncture for the measurement of serum amylase. After euthanasia, frozen or paraffin-embedded brains and pancreas were analyzed using qRT-PCR or immunohistochemistry, respectively. RESULTS: Immunohistochemistry assays showed a large number of Iba1 and PU.1-positive cells in the CA1, CA3, and dentate gyrus (DG) regions of the hippocampus of the PLSP group. TNF-α mRNA expression was significantly higher in the brain from PLSP group. NLRP3 inflammasome expression was found to be significantly increased in the pancreas and brain of rats of the PLSP group. High levels of BNDF mRNA were found in the rat brain of PLSP group. In contrast, NGF mRNA levels were significantly higher in the control group versus PLSP group. CONCLUSION: Our findings suggest that AP has the potential to induce morphological changes in microglia consistent with an activated phenotype.

Ultrasound biomicroscopy in the quantification of brain perfusion parameters of a rat stroke model: Analysis of contrast agent bolus kinetic dynamics.

BACKGROUND: Ischemic stroke represents a significant global health concern, necessitating thorough investigations and the utilization of stroke animal models to explore novel treatment modalities and diagnostic imaging techniques. NEW METHOD: Ultrasound biomicroscopy (BMU), operating at a center frequency of 21 MHz, along with ultrasound contrast agents (UCAs), was used to quantify microcirculation cerebral blood flow in a rat model of ischemic stroke. The microcirculation parameters were derived from time intensity curve (TIC) plots obtained based on UCA-bolus kinetics. RESULTS: Semiquantitative perfusion-related parameters were assessed. The TIC curves showed differences in amplitude when compared intra-animal between the left and right sides, and three situations were observed: normal perfusion, hypoperfusion, and nonperfusion. ROC analysis of delays between the left and right time intensity peak (TIP) for regions of interest (ROIs) in the control and stroke-hypoperfusion groups revealed an optimal cutpoint of 0.39 s to indicate when hypoperfusion is occurring in rats, with a sensitivity of 93.33 % and a specificity of 80 %. COMPARISON WITH EXISTING METHOD(S): Ultrasound perfusion imaging through the temporal bone window has been clinically applied to stroke patients using a UCA bolus for TIC analysis. TIC parameters were correlated with MRI- and CT-based measurements. CONCLUSIONS: This investigation quantified cerebral blood flow in a rat model of ischemic stroke by measuring microcirculation parameters. The study demonstrated the efficacy of this approach as a valuable tool for conducting preclinical studies.

Would New SARS-CoV-2 Variants Change the War against COVID-19?

The scientific, private, and industrial sectors use a wide variety of technological platforms available to achieve protection against SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), including vaccines. However, the virus evolves continually into new highly virulent variants, which might overcome the protection provided by vaccines and may re-expose the population to infections. Mass vaccinations should be continued in combination with more or less mandatory non-pharmaceutical interventions. Therefore, the key questions to be answered are: (i) How to identify the primary and secondary infections of SARS-CoV-2? (ii) Why are neutralizing antibodies not long-lasting in both cases of natural infections and post-vaccinations? (iii) Which are the factors responsible for this decay in neutralizing antibodies? (iv) What strategy could be adapted to develop long-term herd immunity? (v) Is the Spike protein the only vaccine target or is a vaccine cocktail better?

Non-uniform aspects of the SARS-CoV-2 intraspecies evolution reopen question of its origin.

Several hypotheses have been presented on the origin of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from its identification as the agent causing the current coronavirus disease 19 (COVID-19) pandemic. So far, no solid evidence has been found to support any hypothesis on the origin of this virus, and the issue continue to resurface over and over again. Here we have unfolded a pattern of distribution of several mutations in the SARS-CoV-2 proteins in 24 geo-locations across different continents. The results showed an evenly uneven distribution of the unique protein variants, distinct mutations, unique frequency of common conserved residues, and mutational residues across these 24 geo-locations. Furthermore, ample mutations were identified in the evolutionarily conserved invariant regions in the SARS-CoV-2 proteins across almost all geo-locations studied. This pattern of mutations potentially breaches the law of evolutionary conserved functional units of the beta-coronavirus genus. These mutations may lead to several novel SARS-CoV-2 variants with a high degree of transmissibility and virulence. A thorough investigation on the origin and characteristics of SARS-CoV-2 needs to be conducted in the interest of science and for the preparation of meeting the challenges of potential future pandemics.

An issue of concern: unique truncated ORF8 protein variants of SARS-CoV-2.

Open reading frame 8 (ORF8) shows one of the highest levels of variability among accessory proteins in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the causative agent of Coronavirus Disease 2019 (COVID-19). It was previously reported that the ORF8 protein inhibits the presentation of viral antigens by the major histocompatibility complex class I (MHC-I), which interacts with host factors involved in pulmonary inflammation. The ORF8 protein assists SARS-CoV-2 in evading immunity and plays a role in SARS-CoV-2 replication. Among many contributing mutations, Q27STOP, a mutation in the ORF8 protein, defines the B.1.1.7 lineage of SARS-CoV-2, engendering the second wave of COVID-19. In the present study, 47 unique truncated ORF8 proteins (T-ORF8) with the Q27STOP mutations were identified among 49,055 available B.1.1.7 SARS-CoV-2 sequences. The results show that only one of the 47 T-ORF8 variants spread to over 57 geo-locations in North America, and other continents, which include Africa, Asia, Europe and South America. Based on various quantitative features, such as amino acid homology, polar/non-polar sequence homology, Shannon entropy conservation, and other physicochemical properties of all specific 47 T-ORF8 protein variants, nine possible T-ORF8 unique variants were defined. The question as to whether T-ORF8 variants function similarly to the wild type ORF8 is yet to be investigated. A positive response to the question could exacerbate future COVID-19 waves, necessitating severe containment measures.

The importance of accessory protein variants in the pathogenicity of SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) is caused by the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS- CoV-2) with an estimated fatality rate of less than 1%. The SARS-CoV-2 accessory proteins ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10 possess putative functions to manipulate host immune mechanisms. These involve interferons, which appear as a consensus function, immune signaling receptor NLRP3 (NLR family pyrin domain-containing 3) inflammasome, and inflammatory cytokines such as interleukin 1ß (IL-1ß) and are critical in COVID-19 pathology. Outspread variations of each of the six accessory proteins were observed across six continents of all complete SARS-CoV-2 proteomes based on the data reported before November 2020. A decreasing order of percentage of unique variations in the accessory proteins was determined as ORF3a > ORF8 > ORF7a > ORF6 > ORF10 > ORF7b across all continents. The highest and lowest unique variations of ORF3a were observed in South America and Oceania, respectively. These findings suggest that the wide variations in accessory proteins seem to affect the pathogenicity of SARS-CoV-2.

Periodically aperiodic pattern of SARS-CoV-2 mutations underpins the uncertainty of its origin and evolution.

Various lineages of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have contributed to prolongation of the Coronavirus Disease 2019 (COVID-19) pandemic. Several non-synonymous mutations in SARS-CoV-2 proteins have generated multiple SARS-CoV-2 variants. In our previous report, we have shown that an evenly uneven distribution of unique protein variants of SARS-CoV-2 is geo-location or demography-specific. However, the correlation between the demographic transmutability of the SARS-CoV-2 infection and mutations in various proteins remains unknown due to hidden symmetry/asymmetry in the occurrence of mutations. This study tracked how these mutations are emerging in SARS-CoV-2 proteins in six model countries and globally. In a geo-location, considering the mutations having a frequency of detection of at least 500 in each SARS-CoV-2 protein, we studied the country-wise percentage of invariant residues. Our data revealed that since October 2020, highly frequent mutations in SARS-CoV-2 have been observed mostly in the Open Reading Frame (ORF) 7b and ORF8, worldwide. No such highly frequent mutations in any of the SARS-CoV-2 proteins were found in the UK, India, and Brazil, which does not correlate with the degree of transmissibility of the virus in India and Brazil. However, we have found a signature that SARS-CoV-2 proteins were evolving at a higher rate, and considering global data, mutations are detected in the majority of the available amino acid locations. Fractal analysis of each protein's normalized factor time series showed a periodically aperiodic emergence of dominant variants for SARS-CoV-2 protein mutations across different countries. It was noticed that certain high-frequency variants have emerged in the last couple of months, and thus the emerging SARS-CoV-2 strains are expected to contain prevalent mutations in the ORF3a, membrane, and ORF8 proteins. In contrast to other beta-coronaviruses, SARS-CoV-2 variants have rapidly emerged based on demographically dependent mutations. Characterization of the periodically aperiodic nature of the demographic spread of SARS-CoV-2 variants in various countries can contribute to the identification of the origin of SARS-CoV-2.

COVID-19 Vaccines and Thrombosis-Roadblock or Dead-End Street?

Two adenovirus-based vaccines, ChAdOx1 nCoV-19 and Ad26.COV2.S, and two mRNA-based vaccines, BNT162b2 and mRNA.1273, have been approved by the European Medicines Agency (EMA), and are invaluable in preventing and reducing the incidence of coronavirus disease-2019 (COVID-19). Recent reports have pointed to thrombosis with associated thrombocytopenia as an adverse effect occurring at a low frequency in some individuals after vaccination. The causes of such events may be related to SARS-CoV-2 spike protein interactions with different C-type lectin receptors, heparan sulfate proteoglycans (HSPGs) and the CD147 receptor, or to different soluble splice variants of the spike protein, adenovirus vector interactions with the CD46 receptor or platelet factor 4 antibodies. Similar findings have been reported for several viral diseases after vaccine administration. In addition, immunological mechanisms elicited by viral vectors related to cellular delivery could play a relevant role in individuals with certain genetic backgrounds. Although rare, the potential COVID-19 vaccine-induced immune thrombotic thrombocytopenia (VITT) requires immediate validation, especially in risk groups, such as the elderly, chronic smokers, and individuals with pre-existing incidences of thrombocytopenia; and if necessary, a reformulation of existing vaccines.

Cryopreserved Rat Thyroid Autotransplantation in the Treatment of Postoperative Hypothyroidism.

To verify the viability and functionality of cryopreserved thyroid autotransplantation in rats who underwent total thyroidectomy in the treatment of postoperative hypothyroidism. Thirty-two Wistar rats were randomly assigned into groups (G) with eight animals each: control (CG); simulation (SG); hypothyroidism (HTG) and transplanted (TG). At the beginning and in the 13th week of the experiment, serum levels of total T3, free T4, TSH and calcium were determined. In both the first and 14th weeks, scintigraphic examinations, 99m-Tc pertechnetate radioisotope biodistribution and histopathology were performed. In the 14th week, the expression of proliferating cell nuclear antigen (PCNA) and cellular apoptosis (caspase-3) were also evaluated. In the 13th week, the transplanted animals had normal serum levels of total T3 and free T4. TSH levels showed a tendency towards normality. In the 14th week, scintigraphic exams displayed graft isotopic uptake in all animals in the TG group. Histological examinations 13 weeks after transplantation showed the viability and functionality of thyroid follicles. PCNA revealed significant immunoreactivity of the graft (p < 0.001) when the TG was compared to the CG. There was no difference between CG and TG considering the expression of activated caspase-3. The experimental study confirmed the viability and functionality of thyroid autotransplantation implanted in skeletal muscle with evidence of cell proliferation without cellular apoptosis. This surgical strategy was effective in the treatment of postoperative hypothyroidism.

Urgent Need for Field Surveys of Coronaviruses in Southeast Asia to Understand the SARS-CoV-2 Phylogeny and Risk Assessment for Future Outbreaks.

Phylogenetic analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is focused on a single isolate of bat coronaviruses (bat CoVs) which does not adequately represent genetically related coronaviruses (CoVs) [...].

Notable sequence homology of the ORF10 protein introspects the architecture of SARS-CoV-2.

The current Coronavirus Disease 19 (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) shows similar pathology to MERS and SARS-CoV, with a current estimated fatality rate of 1.4%. Open reading frame 10 (ORF10) is a unique SARS-CoV-2 accessory protein, which contains eleven cytotoxic T lymphocyte (CTL) epitopes each of nine amino acids in length. Twenty-two unique SARS-CoV-2 ORF10 variants have been identified based on missense mutations found in sequence databases. Some of these mutations are predicted to decrease the stability of ORF10 in silico physicochemical and structural comparative analyses were carried out on SARS-CoV-2 and Pangolin-CoV ORF10 proteins, which share 97.37% amino acid (aa) homology. Though there is a high degree of ORF10 protein similarity of SARS-CoV-2 and Pangolin-CoV, there are differences of these two ORF10 proteins related to their sub-structure (loop/coil region), solubility, antigenicity and shift from strand to coil at aa position 26 (tyrosine). SARS-CoV-2 ORF10, which is apparently expressed in vivo since reactive T cell clones are found in convalescent patients should be monitored for changes which could correlate with the pathogenesis of COVID-19.

A unique view of SARS-CoV-2 through the lens of ORF8 protein.

Immune evasion is one of the unique characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) attributed to its ORF8 protein. This protein modulates the adaptive host immunity through down-regulation of MHC-1 (Major Histocompatibility Complex) molecules and innate immune responses by surpassing the host's interferon-mediated antiviral response. To understand the host's immune perspective in reference to the ORF8 protein, a comprehensive study of the ORF8 protein and mutations possessed by it have been performed. Chemical and structural properties of ORF8 proteins from different hosts, such as human, bat, and pangolin, suggest that the ORF8 of SARS-CoV-2 is much closer to ORF8 of Bat RaTG13-CoV than to that of Pangolin-CoV. Eighty-seven mutations across unique variants of ORF8 in SARS-CoV-2 can be grouped into four classes based on their predicted effects (Hussain et al., 2021) [1]. Based on the geo-locations and timescale of sample collection, a possible flow of mutations was built. Furthermore, conclusive flows of amalgamation of mutations were found upon sequence similarity analyses and consideration of the amino acid conservation phylogenies. Therefore, this study seeks to highlight the uniqueness of the rapidly evolving SARS-CoV-2 through the ORF8.

The structural basis of accelerated host cell entry by SARS-CoV-2†.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the pandemic coronavirus disease 2019 (COVID-19) that exhibits an overwhelming contagious capacity over other human coronaviruses (HCoVs). This structural snapshot describes the structural bases underlying the pandemic capacity of SARS-CoV-2 and explains its fast motion over respiratory epithelia that allow its rapid cellular entry. Based on notable viral spike (S) protein features, we propose that the flat sialic acid-binding domain at the N-terminal domain (NTD) of the S1 subunit leads to more effective first contact and interaction with the sialic acid layer over the epithelium, and this, in turn, allows faster viral 'surfing' of the epithelium and receptor scanning by SARS-CoV-2. Angiotensin-converting enzyme 2 (ACE-2) protein on the epithelial surface is the primary entry receptor for SARS-CoV-2, and protein-protein interaction assays demonstrate high-affinity binding of the spike protein (S protein) to ACE-2. To date, no high-frequency mutations were detected at the C-terminal domain of the S1 subunit in the S protein, where the receptor-binding domain (RBD) is located. Tight binding to ACE-2 by a conserved viral RBD suggests the ACE2-RBD interaction is likely optimal. Moreover, the viral S subunit contains a cleavage site for furin and other proteases, which accelerates cell entry by SARS-CoV-2. The model proposed here describes a structural basis for the accelerated host cell entry by SARS-CoV-2 relative to other HCoVs and also discusses emerging hypotheses that are likely to contribute to the development of antiviral strategies to combat the pandemic capacity of SARS-CoV-2.

Possible Transmission Flow of SARS-CoV-2 Based on ACE2 Features.

Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that is engendering the severe coronavirus disease 2019 (COVID-19) pandemic. The spike (S) protein receptor-binding domain (RBD) of SARS-CoV-2 binds to the three sub-domains viz. amino acids (aa) 22-42, aa 79-84, and aa 330-393 of ACE2 on human cells to initiate entry. It was reported earlier that the receptor utilization capacity of ACE2 proteins from different species, such as cats, chimpanzees, dogs, and cattle, are different. A comprehensive analysis of ACE2 receptors of nineteen species was carried out in this study, and the findings propose a possible SARS-CoV-2 transmission flow across these nineteen species.

Protective effect of adipose tissue-derived mesenchymal stromal cells in an experimental model of high-risk colonic anastomosis.

BACKGROUND: Dehiscence of intestinal anastomosis results in high morbidity and mortality. The aim of this study was to investigate the effects of locally administered adipose tissue-derived mesenchymal stromal cells in a model of high-risk colonic anastomosis in rats. METHODS: Seven days after induction of colitis with 2,4,6-trinitrobenzene sulfonic acid, Wistar rats were submitted to a transection of the descending colon followed by end-to-end anastomosis and were then treated with 2×106 adipose tissue-derived mesenchymal stromal cells (from the preperitoneal fat) or an acellular culture solution instilled onto the surface of the anastomosis. At day 14, after macroscopic survey of the abdominal cavity, the anastomotic area was submitted to histologic and immunohistochemical analysis, evaluation of myeloperoxidase activity, fibrosis, epithelial integrity, NF-κ B activation, expression of inflammatory cytokines, and extracellular matrix-related genes. RESULTS: Anastomotic leakage and mortality associated with high-risk anastomosis decreased with treatment with adipose tissue-derived mesenchymal stromal cells (P < .03). Application of adipose tissue-derived mesenchymal stromal cells resulted in lower histologic scores (P = .011), decreased deposition of collagen fibers (P = .003), preservation of goblet cells (P = .033), decreased myeloperoxidase activity (P = .012), decreased accumulation of CD4+ T-cells (P = .014) and macrophages (P = .011) in the lamina propria, a decrease in the number of apoptotic cells (P = .008), and the activation of NF-κ B (P = .036). Overexpression of IL-17, TNF-α , IFN-γ, and metalloproteinases in the acellular culture solution-treated, high-risk anastomosis group decreased (P < .05) to near normal values with adipose tissue-derived mesenchymal stromal cells treatment. CONCLUSION: Improvements in outcomes of a high-risk colonic anastomosis with adipose tissue-derived mesenchymal stromal cells therapy reflect the immunomodulatory activity and healing effect of these cells, even after just topical administration and reinforces their use in future translational research.

Rat models of high risk colorectal anastomoses 1.

PURPOSE: To evaluate the most frequent surgical techniques of high-risk colorectal anastomoses in rats. METHODS: Wistar rats were enrolled in three different models comprising inflammatory (TNBS enema), vascular (portal vein occlusion) or obstructive (a non-ischemic constricting ring) mechanisms associated with colonic anastomosis that had accomplished after these former lesions. Histological analyses (Hematoxylin and eosin and Picrosirius red) were performed. RESULTS: All anastomoses techniques were associated with risk factors and had complications, mainly anastomotic leakage. In Study 1, the use of a pharmacological agent, trinitrobenzene sulfonic acid (TNBS) mimicked an inflammatory bowel disease such as Crohn's disease with 50% of anastomosis leakage, the higher percentage among all models tested. In Study 2, after portal ischemia followed by reperfusion it was observed a dense neutrophil infiltrate in the midst of necrotic tissue and fibrin at the anastomotic site and 5 days after the anastomosis, no collagen was produced. In Study 3, 5 days after the mechanical obstruction some denuded areas of epithelium with marked oedema of mucosa and submucosa were seen, at the anastomotic site and anastomosis group showed some reduction of collagen density when compared with Control/Sham group. CONCLUSION: All the experimental surgical techniques tested in rats were associated with high-risk colorectal anastomoses and were useful to study colonic anastomotic healing and intestinal leakage.

Rat models of high risk colorectal anastomoses

Abstract Purpose: To evaluate the most frequent surgical techniques of high-risk colorectal anastomoses in rats. Methods: Wistar rats were enrolled in three different models comprising inflammatory (TNBS enema), vascular (portal vein occlusion) or obstructive (a non-ischemic constricting ring) mechanisms associated with colonic anastomosis that had accomplished after these former lesions. Histological analyses (Hematoxylin and eosin and Picrosirius red) were performed. Results: All anastomoses techniques were associated with risk factors and had complications, mainly anastomotic leakage. In Study 1, the use of a pharmacological agent, trinitrobenzene sulfonic acid (TNBS) mimicked an inflammatory bowel disease such as Crohn's disease with 50% of anastomosis leakage, the higher percentage among all models tested. In Study 2, after portal ischemia followed by reperfusion it was observed a dense neutrophil infiltrate in the midst of necrotic tissue and fibrin at the anastomotic site and 5 days after the anastomosis, no collagen was produced. In Study 3, 5 days after the mechanical obstruction some denuded areas of epithelium with marked oedema of mucosa and submucosa were seen, at the anastomotic site and anastomosis group showed some reduction of collagen density when compared with Control/Sham group. Conclusion: All the experimental surgical techniques tested in rats were associated with high-risk colorectal anastomoses and were useful to study colonic anastomotic healing and intestinal leakage.

Cholecystectomy during ceftriaxone therapy. A translational study with a new rabbit model

Abstract Purpose: To evaluate the actual incidence of both microlithiasis and acute cholecystitis during treatment with intravenous ceftriaxone in a new rabbit model. Methods: New Zealand rabbits were treated with intravenous ceftriaxone or saline for 21 days. Ultrasound monitoring of the gallbladder was performed every seven days until the 21st day when histopathology, immunohistochemistry for proliferating cell nuclear antigen (PCNA), pro-caspase-3 and CD68, liver enzyme biochemistry, and chromatography analysis of the bile and sediments were also performed. Results: All animals treated with ceftriaxone developed acute cholecystitis, confirmed by histopathology (P<0.05) and biliary microlithiasis, except one that exhibited sediment precipitation. In the group treated with ceftriaxone there was an increase in pro-caspase-3, gamma-glutamyl transpeptidase concentration, PCNA expression and in the number of cells positive for anti-CD68 (P<0.05). In the ceftriaxone group, the cholesterol and lecithin concentrations increased in the bile and a high concentration of ceftriaxone was found in the microlithiasis. Conclusion: Ceftriaxone administered intravenously at therapeutic doses causes a high predisposition for lithogenic bile formation and the development of acute lithiasic cholecystitis.