Proline-Proline Dyad in the Fusion Peptide of the Murine ß-Coronavirus Spike Protein's S2 Domain Modulates Its Neuroglial Tropism.

The ß-Coronavirus mouse hepatitis virus (MHV-A59)-RSA59 has a patent stretch of fusion peptide (FP) containing two consecutive central prolines (PP) in the S2 domain of the Spike protein. Our previous studies compared the PP-containing fusogenic-demyelinating strain RSA59(PP) to its one proline-deleted mutant strain RSA59(P) and one proline-containing non-fusogenic non-demyelinating parental strain RSMHV2(P) to its one proline inserted mutant strain RSMHV2(PP). These studies highlighted the crucial role of PP in fusogenicity, hepato-neuropathogenesis, and demyelination. Computational studies combined with biophysical data indicate that PP at the center of the FP provides local rigidity while imparting global fluctuation to the Spike protein that enhances the fusogenic properties of RSA59(PP) and RSMHV2(PP). To elaborate on the understanding of the role of PP in the FP of MHV, the differential neuroglial tropism of the PP and P mutant strains was investigated. Comparative studies demonstrated that PP significantly enhances the viral tropism for neurons, microglia, and oligodendrocytes. PP, however, is not essential for viral tropism for either astroglial or oligodendroglial precursors or the infection of meningeal fibroblasts in the blood-brain and blood-CSF barriers. PP in the fusion domain is critical for promoting gliopathy, making it a potential region for designing antivirals for neuro-COVID therapy.

Two Consecutive Prolines in the Fusion Peptide of Murine ß-Coronavirus Spike Protein Predominantly Determine Fusogenicity and May Be Essential but Not Sufficient to Cause Demyelination.

Combined in silico, in vitro, and in vivo comparative studies between isogenic-recombinant Mouse-Hepatitis-Virus-RSA59 and its proline deletion mutant, revealed a remarkable contribution of centrally located two consecutive prolines (PP) from Spike protein fusion peptide (FP) in enhancing virus fusogenic and hepato-neuropathogenic potential. To deepen our understanding of the underlying factors, we extend our studies to a non-fusogenic parental virus strain RSMHV2 (P) with a single proline in the FP and its proline inserted mutant, RSMHV2 (PP). Comparative in vitro and in vivo studies between virus strains RSA59(PP), RSMHV2 (P), and RSMHV2 (PP) in the FP demonstrate that the insertion of one proline significantly resulted in enhancing the virus fusogenicity, spread, and consecutive neuropathogenesis. Computational studies suggest that the central PP in Spike FP induces a locally ordered, compact, and rigid structure of the Spike protein in RSMHV2 (PP) compared to RSMHV2 (P), but globally the Spike S2-domain is akin to the parental strain RSA59(PP), the latter being the most flexible showing two potential wells in the energy landscape as observed from the molecular dynamics studies. The critical location of two central prolines of the FP is essential for fusogenicity and pathogenesis making it a potential site for designing antiviral.

Azadirachta indica A. Juss bark extract and its Nimbin isomers restrict ß-coronaviral infection and replication.

Emerging mutations in the SARS-CoV-2 genome pose a challenge for vaccine development and antiviral therapy. The antiviral efficacy of Azadirachta indica bark extract (NBE) was assessed against SARS-CoV-2 and m-CoV-RSA59 infection. Effects of in vivo intranasal or oral NBE administration on viral load, inflammatory response, and histopathological changes were assessed in m-CoV-RSA59-infection. NBE administered inhibits SARS-CoV-2 and m-CoV-RSA59 infection and replication in vitro, reducing Envelope and Nucleocapsid gene expression. NBE ameliorates neuroinflammation and hepatitis in vivo by restricting viral replication and spread. Isolated fractions of NBE enriched in Nimbin isomers shows potent inhibition of m-CoV-RSA59 infection in vitro. In silico studies revealed that NBE could target Spike and RdRp of m-CoV and SARS-CoV-2 with high affinity. NBE has a triterpenoids origin that may allow them to competitively target panoply of viral proteins to inhibit mouse and different strains of human coronavirus infections, suggesting its potential as an antiviral against pan-ß-Coronaviruses.

Calcium and s100a1 protein balance in the brain-heart axis in diabetic male Wistar rats.

Objectives Calcium deregulation in diabetes mellitus (DM) is central to the brain-heart axis pathology. This has led to the use of medical plants in complementary medicine such as Amaranthus hypochondriacus (GA). The objective of the study was to establish the effects of grain amaranth feed supplementation on calcium, s100al protein and antioxidant levels on the brain-heart axis in diabetic male Wistar rats. Methods The study involved six groups (n=5) with DM being induced in 20 rats. To the diabetic rats, Group I received mixtard®, Group II was positive control, Groups III and IV received GA feed supplementation at 25 and 50%. In the nondiabetic rats (n=10), Group V received 50% grain amaranth while Group VI was the negative control. The brain and heart tissues were harvested after five weeks and processed using standard methods. Results Grain amaranth feed supplementation led to improved calcium levels in DM as compared to the positive control. This also led to increased s100a1, antioxidant levels in the brain-heart axis during DM. This then protected the tissues against oxidative damage, thus preserving tissue function and structure. Conclusions Grain amaranth's actions on calcium signaling subsequently affected s100a1 protein levels, leading to improved tissue function in diabetes.

Azadirachta indica A. Juss Ameliorates Mouse Hepatitis Virus-Induced Neuroinflammatory Demyelination by Modulating Cell-to-Cell Fusion in an Experimental Animal Model of Multiple Sclerosis.

Mouse hepatitis virus (MHV)-induced murine neuroinflammation serves as a model to study acute meningoencephalomyelitis, hepatitis, and chronic neuroinflammatory demyelination; which mimics certain pathologies of the human neurologic disease, multiple sclerosis (MS). MHV-induced acute neuroinflammation occurs due to direct glial cell dystrophy instigated by central nervous system (CNS)-resident microglia and astrocytes, in contrast to peripheral CD4+T cell-mediated myelin damage prevalent in the experimental autoimmune encephalomyelitis (EAE) model of MS. Viral envelope Spike glycoprotein-mediated cell-to-cell fusion is an essential mechanistic step for MHV-induced CNS pathogenicity. Although Azadirachta indica (Neem), a traditional phytomedicine, is known for its anti-inflammatory, anti-fungal, and spermicidal activities, not much is known about anti-neuroinflammatory properties of its bark (NBE) in MHV-induced acute neuroinflammation and chronic demyelination. Recombinant demyelinating MHV strain (RSA59) was preincubated with NBE to arrest the infection-initiation event, and its effect on viral replication, viral transcription, cytokine expression, and successive pathogenicity were investigated in vitro and in vivo. Virus-free Luciferase assay explained NBE's anti-virus-to-cell fusion activity in vitro. Intracranial inoculation of RSA59 preincubated with NBE into the mouse brain significantly reduces acute hepatitis, meningoencephalomyelitis, and chronic progressive demyelination. Additionally, NBE effectively restricts viral entry, dissemination in CNS, viral replication, viral transcription, and expression of the viral nucleocapsid and inflammatory cytokines. From mechanistic standpoints, RSA59 preincubated with NBE reduced viral entry, viral replication and cell-to-cell fusion, as a mode of viral dissemination. Moreover, intraperitoneal injection with NBE (25 mg/kg B.W.) into mice revealed a significant reduction in viral Nucleocapsid protein expression in vivo. Conclusively, A. indica bark extract may directly bind to the virus-host attachment Spike glycoprotein and suppresses MHV-induced neuroinflammation and neuropathogenesis by inhibiting cell-to-cell fusion and viral replication. Further studies will focus on combining bioanalytical assays to isolate potential NBE bioactive compound(s) that contribute towards the anti-viral activity of NBE.

Grain Amaranth Is Associated with Improved Hepatic and Renal Calcium Metabolism in Type 2 Diabetes Mellitus of Male Wistar Rats.

BACKGROUND: Dysregulation of calcium signaling is a hallmark of diabetes mellitus (DM) and grain amaranth (AG) has antidiabetic properties. Information on the mechanism of action of AG on blood, renal, and hepatic tissues is sparse, although it continues to be an important alternative medicinal plant in several developing countries. The objective of the study was to determine key changes in calcium levels and s100a1 protein levels and antioxidant and histopathologic changes in blood, renal, and hepatic tissues of male diabetic Wistar rats. MATERIALS AND METHODS: This was an experimental study in which 30 male Wistar rats were kept for 5 weeks (6 groups, N =5). Groups 1-IV had T2DM induced using Nicotinamide and Streptozotocin: Group I, Mixtard®; group II, positive control; group III, 25% AG; group IV, 50% AG. Furthermore, group V consisted of normal rats given 50% GA and group VI was negative control. Blood, renal, and hepatic tissues were collected and analyzed for calcium, s100a1 protein levels, and antioxidant and histopathological changes. RESULTS AND DISCUSSION: In blood, renal, and hepatic tissue, calcium and s100a1 levels were low during T2DM and these increased following AG supplementation. This was important for improved metabolic processes, thus leading to the low malondialdehyde (MDA) and glutathione peroxidase (GPx) activity in the tissues. Efficient antioxidant status was important for improved calcium signaling mechanisms, thus leading to improved tissue function and protection demonstrating the importance of AG as an alternative medicinal source through the calcium signaling pathway. CONCLUSION: Grain amaranth exerts its antidiabetic properties through improved calcium homeostasis in blood, kidney, and liver.

Combined Donepezil and Ethanolic Extract of Propolis Improved Memory Better Than Donepezil and Propolis Monotherapy in Wild Type Drosophila melanogaster.

BACKGROUND: Donepezil is the most common drug used in the treatment of disorders associated with memory loss, especially that in Alzheimer's disease. Healthy individuals however have continued to use it as a memory enhancer. This study was aimed at evaluating the combined therapy of donepezil and propolis on cognition in Drosophila melanogaster. Method. Drosophila melanogaster flies were divided into five groups and fed with the different treatment doses of ethanolic extract of propolis and donepezil as follows: normal food, propolis 250 mg/mL, propolis 50 mg/mL, donepezil 0.001M, and donepezil 0.001M/propolis 50 mg/mL added to their food. The flies were fed from larval stage for 30 days. The memory and learning tests were conducted after every 10 days to assess improvement with time. RESULTS: The results obtained showed that the combination of propolis with donepezil caused a remarkable improvement in both the short- and long-term memory. In addition, there was a dose dependent improvement with the administration of propolis. CONCLUSION: Propolis extract obtained from different parts of Uganda expressed cognitive improvement when coadministered with donepezil in wild type Drosophila melanogaster.

A study on visual, audio and tactile reaction time among medical students at Kampala International University in Uganda.

BACKGROUND: Reaction time (RT) is an indicator of neural activity, however, its variation due to visual (VRT), audio (ART) and tactile (TRT) in African medical students has not been investigated. The aim of the study was to determine relationships between VRT, ART and TRT amongst medical students in Uganda. MATERIALS AND METHODS: This was a cross sectional study, the body mass index (BMI) and RT (i.e. VRT, ART and TRT) were determined using weighing scale with standiometer and the catch a ruler experiment respectively. A questionnaire was administered to collect information on participant's lifestyle patterns and analysis was done using SPSS Version 20. RESULTS: The mean (± SEM) VRT, ART and TRT in the study were found to be 0.148 ± 0.002s, 0.141 ± 0.002s and 0.139 ± 0.003s respectively. A strong correlation between TRT and ART was found to exist in the youthful Ugandan medical student's population. Furthermore, significant differences in ART and VRT were observed with sex, although these were absent amongst preclinical and clinical students, showing the importance of sex in RT. CONCLUSION: The low VRT and ART in Ugandan medical students is indicative of a healthy somatosensory connectivity, thus of academic importance.

Memory Improvement Effect of Ethanol Garlic (A. sativum) Extract in Streptozotocin-Nicotinamide Induced Diabetic Wistar Rats Is Mediated through Increasing of Hippocampal Sodium-Potassium ATPase, Glutamine Synthetase, and Calcium ATPase Activities.

Studies suggest that garlic (A. sativum) improves memory dependent on the hippocampus. However, the effect of ethanol garlic extract on hippocampus Na+/K+ ATPase, Ca2+ ATPase, and glutamine synthetase (GS) activities as possible mechanisms in memory improvement in diabetic Wistar rats has not been reported. Twenty-four male Wistar rats weighing 200-250 g were divided into three groups with 8 rats each. Group (A), normal control rats, and Group (B), diabetic rats, received 1 ml of normal saline; diabetic rats in Group (C) received 1000 mg/kg of garlic extract orally for 21 days. Hyperglycemia was induced by a single intraperitoneal injection of streptozotocin 60 mg/kg followed by 120 mg/kg nicotinamide while extraction of garlic was done by cold maceration method. Memory was tested in all groups. After that, the rats were sacrificed, the brain was removed, and the hippocampi were carefully excised and then homogenized. Activities of Na+/K+ ATPase, calcium ATPase, and GS were analyzed from the homogenate. Results showed improvement in memory and a significant increase (P < 0.05) in hippocampus Na+/K+ ATPase, Ca2+ ATPase, and GS activities in diabetic rats treated with garlic extract. In conclusion, the increased activity of hippocampus Na+/K+ ATPase, calcium ATPase, and glutamine synthetase may account for the memory improvement.