The relationship between MMP-2 rs243865, MMP-9 rs398242 and CXCL-12 rs1801157 gene polymorphisms with Japanese encephalitis disease and disease outcome in North Indian population.

BACKGROUND & OBJECTIVES: Japanese encephalitis virus (JEV) is one of the most important causes of acute and uncontrolled inflammatory disease in Asia. Matrix metalloproteinases (MMPs) and chemokines play a detrimental role in the host response to JE disease, aetiology, and disease outcome. Evidently, MMPs are widely circulated in the brain and regulate various process including microglial activation, inflammation, blood-brain barrier disruption as well as affects central nervous system (CNS). The present study was to assess the association of single nucleotide polymorphisms of MMP-2, MMP-9 and chemokine (CXCL-12/SDF1-3') in the north Indian population. METHODS: We performed case-control study comprising of 125 patients and 125 healthy controls in north Indian population. Genomic DNA was extracted from whole blood and gene polymorphism have been determined by PCR-RFLP method. RESULTS: MMP-2, MMP-9 and CXCL-12 gene was not significantly associated with JE disease, but homozygous (T/T) genotype of MMP-2 was statically associated with disease outcome (p=0.05, OR=0.110). A/G and G/G genotype of CXCL-12 was significantly associated with severity of disease. (p=0.032, OR=5.500, p=0.037, OR= 9.167). The serum level of MMP-2 was observed significantly increased in JE patients with homozygous (T/T) genotype whereas increased MMP-9 level was associated with heterozygous genotype. INTERPRETATION & CONCLUSION: MMP-2, MMP-9 and CXCL-12 gene polymorphism were not associated with JE susceptibility, but MMP-2 may be contributed to disease protection. CXCL-12 was associated with disease severity. In our concern this is the first report from northern India.

Differential expression of circulating microRNAs in serum: Potential biomarkers to track Japanese encephalitis virus infection.

Japanese encephalitis is one of the serious vector-borne viral encephalitis diseases found worldwide and poses a major threat to public health. Most Japanese encephalitis virus (JEV) infections are subclinical; only 1: 250 to 1:1000 infected persons develop clinical presentations. Delay in proper diagnosis of JE affects the timeliness of treatment initiation and increases the mortality rate in patients. Therefore, there is an extreme need to develop potential biomarkers, which might improve the diagnosis and can become the basis for development of new therapeutics. The microRNAs (miRNAs/or miRs) are small noncoding RNAs of 17-24 nucleotides that are known to regulate about 60% of human genes. Although miRNAs have been found to regulate various aspects of innate and adaptive immune responses, less information on circulating miRNAs in JE is known. The study of JEV infected human serum miRNAs will provide novel information for the diagnosis of JE as well as for the improvement of disease outcome. Total RNA, including miRNA, was extracted from serum followed by the complementary DNA (cDNA) synthesis by using sequence-specific primers. cDNA was amplified using target-specific TaqMan MicroRNA Assay. Real-time polymerase chain reaction data was normalized using both exogenous (cel-miR-39) and endogenous (hsa-miR-93) controls. We have found significantly altered expression of miR-155 and miR-21 in serum of JEV infected patients as compared to healthy controls, revealing their role as a a noninvasive biomarker in JE. A significant correlation between miRNAs and JE was observed that offers the basis for miRNAs to serve as a new component to develop possible therapeutic strategies for JE in near future.

Pro-inflammatory and anti-inflamatory cytokine genes polymorphisms and susceptibility to Japanese encephalitis disease in the North Indian population.

BACKGROUND: Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in many regions of Asia. Cytokines, including pro-inflammatory and anti-inflammatory are key regulators playing a detrimental role in the host response to JE infection, pathogenesis and disease outcome. Evidently, the host's cytokine response is genetically determined, representing the complexity of interindividual differences regarding immune response to viral infection. The current study assesses the association of single nucleotide polymorphisms of classical interleukin IL-1ß and IL-10 with JEV susceptibility and disease severity in north Indian population. METHODS: We performed a case-control study using 85 JE patients and 85 healthy controls. Polymorphisms in the IL-1ß (-511 C/T) and IL-10 (-1082 A/G) genes were genotyped using PCR-RFLP. All continuous variables were expressed as mean ± standard deviation, and categorical variables were expressed in percentage. RESULTS: The mRNA level of IL-1ß and IL-10 were found significantly increased in JE patients. In severe JE patients, IL-1ß mRNA level was significantly higher with heterozygous (C/T) and homozygous (C/C) genotype compared to wild (T/T) genotype and mRNA level of IL-10 was higher in heterozygous genotype (A/G) compared to wild genotype (A/A). The C/T and C/C genotypes of IL-1ß were significantly associated with higher risk of JE infection (p < 0.05, OR = 7.25 and 4.40) whereas, the A/G genotype of IL-10 was associated with a reduced risk of JEV infection (p < 0.05, OR = 0.30). The C allele of IL-1ß was associated with fever and neck stiffness (p < 0.05) and CT genotype was associated with disease severity and worse outcomes in JE patients. Along with this, IL-10 polymorphism was found associated with fever, and AG genotype was found to be associated with worse disease outcomes such as neurological sequelae (p < 0.05). CONCLUSION: Mutant allele and genotype at IL-1ß (-511 C/T) and IL-10 (-1082 A/G) gene polymorphism show increased expression of IL-1ß and IL-10 in JE patients which contribute to disease severity as well as adverse outcomes of disease. Overall this is the first report from northern India, which shows the association of IL-1ß and IL-10 polymorphisms with JEV infection.

Steroidal lactones from Withania somnifera effectively target Beta, Gamma, Delta and Omicron variants of SARS-CoV-2 and reveal a decreased susceptibility to viral infection and perpetuation: a polypharmacology approach.

Prevention from disease is presently the cornerstone of the fight against COVID-19. With the rapid emergence of novel SARS-CoV-2 variants, there is an urgent need for novel or repurposed agents to strengthen and fortify the immune system. Existing vaccines induce several systemic and local side-effects that can lead to severe consequences. Moreover, elevated cytokines in COVID-19 patients with cancer as co-morbidity represent a significant bottleneck in disease prognosis and therapy. Withania somnifera (WS) and its phytoconstituent(s) have immense untapped immunomodulatory and therapeutic potential and the anticancer potential of WS is well documented. To this effect, WS methanolic extract (WSME) was characterized using HPLC. Withanolides were identified as the major phytoconstituents. In vitro cytotoxicity of WSME was determined against human breast MDA-MB-231 and normal Vero cells using MTT assay. WSME displayed potent cytotoxicity against MDA-MB-231 cells (IC50: 66 µg/mL) and no effect on Vero cells in the above range. MD simulations of Withanolide A with SARS-CoV-2 main protease and spike receptor-binding domain as well as Withanolide B with SARS-CoV spike glycoprotein and SARS-CoV-2 papain-like protease were performed using Schrödinger. Stability of complexes followed the order 6M0J-Withanolide A > 6W9C-Withnaolide B > 5WRG-Withanolide B > 6LU7-Withanolide A. Maximum stable interaction(s) were observed between Withanolides A and B with SARS-CoV-2 and SARS-CoV spike glycoproteins, respectively. Withanolides A and B also displayed potent binding to pro-inflammatory markers viz. serum ferritin and IL-6. Thus, WS phytoconstituents have the potential to be tested further in vitro and in vivo as novel antiviral agents against COVID-19 patients having cancer as a co-morbidity. Supplementary Information: The online version contains supplementary material available at 10.1007/s40203-023-00184-y.

Virtual screening of phytoconstituents from miracle herb nigella sativa targeting nucleocapsid protein and papain-like protease of SARS-CoV-2 for COVID-19 treatment.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel etiological agent of coronavirus disease 2019 (COVID-19). Nigella sativa, commonly known as black seed or black cumin, has been a historical and traditional plant since thousands of years. Based on their therapeutic efficacy, the chief components of terpenoids and flavonoids were selected from N. sativa seeds and seed oil. This study was designed to check the antiviral efficacy of N. sativa main phytoconstituents against five potential targets of SARS-CoV-2 using in silico structure-based virtual screening approach. Out of twenty five phytocomponents, ten components showed best binding affinity against two viral proteins viz. N-terminal RNA binding domain (NRBD; PDB ID: 6M3M) of nucleocapsid protein and papain-like protease (PL-PRO; PDB ID: 6W9C) of SARS-CoV-2 using AutoDock 4.2.6, AutoDock Vina and iGEMDOCK. PASS analyses of all ten phytocomponents using Lipinski's Rule of five showed promising results. Further, druglikeness and toxicity assessment using OSIRIS Data Warrior v5.2.1 software exhibited the feasibility of phytocomponents as drug candidates with no predicted toxicity. Molecular dynamics simulation study of NRBD of SARS-CoV-2 nucleocapsid protein-alpha-spinasterol complex and PL-PRO-cycloeucalenol complex displayed strong stability at 300 K. Both these complexes exhibited constant root mean square deviation (RMSDs) of protein side chains and Cα atoms throughout the simulation run time. Interestingly, PL-PRO and NRBD are key proteins in viral replication, host cell immune evasion and viral assembly. Thus, NRBD and PL-PRO have the potential to serve as therapeutic targets for N. sativa phytoconstituents in drug discovery process against COVID-19.

Anticancer potential of Phoenix dactylifera L. seed extract in human cancer cells and pro-apoptotic effects mediated through caspase-3 dependent pathway in human breast cancer MDA-MB-231 cells: an in vitro and in silico investigation.

BACKGROUND: Phoenix dactylifera L. has a diverse set of pharmacological properties due to its distinct phytochemical profile. The purpose of this study was to investigate the anticancer potential of Phoenix dactylifera seed extract (PDSE) in human breast cancer MDA-MB-231 and MCF-7 cells, as well as liver cancer HepG2 cells, and to investigate the anticancer efficacy in triple-negative MDA-MB-231 cells, followed by in silico validation of the molecular interaction between active components of PDSE and caspase-3, an apoptosis executioner protein . METHODS: In this study, human cancer cell lines were cultured and subsequently treated with 10 to 100 µg/mL of PDSE. MTT test was performed to determine the cell viability, MMP was measured using fluorescent probe JC-1, nuclear condensation was determined by Hoechst 33258 dye, Annexin V-FITC & PI staining and cell cycle analysis were evaluated through flow cytometer, and apoptotic markers were detected using western blotting. The bioactive agents in PDSE were identified using high-performance liquid chromatography (HPLC) analysis. The binding affinity was validated using molecular docking tools AutoDock Vina and iGEMDOCK v2.1. RESULTS: Cell viability data indicated that PDSE inhibited cell proliferation in both breast cancer cells and liver cancer cells. MDA-MB-231 cells showed maximum growth inhibition with an IC50 value of 85.86 µg/mL for PDSE. However, PDSE did not show any significant toxicity against the normal Vero cell line. PDSE induced MMP loss and formation of apoptotic bodies, enhanced late apoptosis at high doses and arrested cells in the S phase of cell cycle. PDSE activated the enzymatic activity of cleaved caspase-3 and caused the cleavage of poly-ADB ribose polymerase (PARP) protein. PDSE upregulated pro-apoptotic Bax protein markedly but  no significant effect on tumor suppressor protein p53, while it downregulated the anti-apoptotic Bcl-2 protein expression. HPLC analysis showed the presence of rutin and quercetin bioactive flavonols in ethanolic extract of PDS. Interestingly, both active components revealed a strong binding interaction with amino acid residues of caspase-3 (PDB ID: 2XYP; Hetero 4-mer - A2B2) protein. CONCLUSION: PDS could serve as a potential medicinal source for apoptotic cell death in human breast cancer cells and, thus, could be used as a promising and crucial candidate in anticancer drug development. This study warrants further in vivo research, followed by clinical investigation.

Prophylactic and therapeutic potential of selected immunomodulatory agents from Ayurveda against coronaviruses amidst the current formidable scenario: an in silico analysis.

There is currently a dearth of specific therapies to treat respiratory infections caused by the three related species of coronaviruses viz. SARS-CoV-2, SARS-CoV and MERS-CoV. Prevention from disease is currently the safest and most convenient alternative available. The present study aimed to evaluate the preventive and therapeutic effect of fifteen phytoconstituents from medicinal plants of Ayurveda against coronaviruses by in silico screening. All the phytoconstituents exhibited rapid GI absorption and bioavailability and most of them had no toxicity versus reference drug chloroquine. BAS analyses revealed that most of the phytocomponents had favorable bioactivity scores towards biological target proteins. Principal component analysis revealed that most of the phytoconstituents fell close to chloroquine in 3D projection of chemical space. Affinity of phytoconstituents towards SARS-CoV-2 spike protein-human ACE2 complex decreased as isomeldenin > tinosporaside > EGCG whereas in case of unbound ACE2, the strength of binding followed the order isomeldenin > tinosporaside > ellagic acid. Towards SARS-CoV-2 main and papain-like proteases, the affinity decreased as isomeldenin > EGCG > tinosporaside and EGCG > tinosporaside > isomeldenin, respectively. Most phytoconstituents displayed significant binding kinetics to the selected protein targets than chloroquine. SAR analysis revealed that isomeldenin, tinosporaside, EGCG and ellagic acid bind to viral spike glycoproteins via H-bond, Pi-Pi, Pi-sigma and Pi-alkyl type interactions. Molecular dynamics simulation of isomeldenin and EGCG with SARS-CoV and SARS-CoV-2 spike glycoproteins exhibited low deviations throughout the 100 ns simulation indicating good stability and compactness of the protein-ligand complexes. Thus, the above four phytoconstituents have the potential to emerge as prophylactic and therapeutic agents against coronaviruses if investigated further in vitro and in vivo.

Interaction of Bioactive Compounds of Moringa oleifera Leaves with SARS-CoV-2 Proteins to Combat COVID-19 Pathogenesis: a Phytochemical and In Silico Analysis.

Novel SARS-CoV-2 claimed a large number of human lives. The main proteins for viral entry into host cells are SARS-CoV-2 spike glycoprotein (PDB ID: 6VYB) and spike receptor-binding domain bound with ACE2 (spike RBD-ACE2; PDB ID: 6M0J). Currently, specific therapies are lacking globally. This study was designed to investigate the bioactive components from Moringa oleifera leaf (MOL) extract by gas chromatography-mass spectroscopy (GC-MS) and their binding interactions with spike glycoprotein and spike RBD-ACE2 protein through computational analysis. GC-MS-based analysis unveiled the presence of thirty-seven bioactive components in MOL extract, viz. polyphenols, fatty acids, terpenes/triterpenes, phytosterols/steroids, and aliphatic hydrocarbons. These bioactive phytoconstituents showed potential binding with SARS-CoV-2 spike glycoprotein and spike RBD-ACE2 protein through the AutoDock 4.2 tool. Further by using AutoDock 4.2 and AutoDock Vina, the top sixteen hits (binding energy ≥ - 6.0 kcal/mol) were selected, and these might be considered as active biomolecules. Moreover, molecular dynamics simulation was determined by the Desmond module. Interestingly two biomolecules, namely ß-tocopherol with spike glycoprotein and ß-sitosterol with spike RBD-ACE2, displayed the best interacting complexes and low deviations during 100-ns simulation, implying their strong stability and compactness. Remarkably, both ß-tocopherol and ß-sitosterol also showed the drug- likeness with no predicted toxicity. In conclusion, these findings suggested that both compounds ß-tocopherol and ß-sitosterol may be developed as anti-SARS-CoV-2 drugs. The current findings of in silico approach need to be optimized using in vitro and clinical studies to prove the effectiveness of phytomolecules against SARS-CoV-2.

Structural interactions of phytoconstituent(s) from cinnamon, bay leaf, oregano, and parsley with SARS-CoV-2 nucleocapsid protein: A comparative assessment for development of potential antiviral nutraceuticals.

SARS-CoV-2 has been responsible for causing 6,218,308 deaths globally till date and has garnered worldwide attention. The lack of effective preventive and therapeutic drugs against SARS-CoV-2 has further worsened the scenario and has bolstered research in the area. The N-terminal and C-terminal RNA binding domains (NTD and CTD) of SARS-CoV-2 nucleocapsid protein represent attractive therapeutic drug targets. Naturally occurring compounds are an excellent source of novel drug candidates due to their structural diversity and safety. Ten major bioactive compounds were identified in ethanolic extract (s) of Cinnamomum zeylanicum, Cinnamomum tamala, Origanum vulgare, and Petroselinum crispum using HPLC and their cytotoxic potential was determined against cancer and normal cell lines by MTT assay to ascertain their biological activity in vitro. To evaluate their antiviral potential, the binding efficacy to NTD and CTD of SARS-CoV-2 nucleocapsid protein was determined using in silico biology tools. In silico assessment of the phytocomponents revealed that most of the phytoconstituents displayed a druglike character with no predicted toxicity. Binding affinities were in the order apigenin > catechin > apiin toward SARS-CoV-2 nucleocapsid NTD. Toward nucleocapsid CTD, the affinity decreased as apigenin > cinnamic acid > catechin. Remdesivir displayed lesser affinity with NTD and CTD of SARS-CoV-2 nucleocapsid proteins than any of the studied phytoconstituents. Molecular dynamics (MD) simulation results revealed that throughout the 100 ns simulation, SARS-CoV-2 nucleocapsid protein NTD-apigenin complex displayed greater stability than SARS-CoV-2 nucleocapsid protein NTD-cinnamic acid complex. Hence, apigenin, catechin, apiin and cinnamic acid might prove as effective prophylactic and therapeutic candidates against SARS-CoV-2, if examined further in vitro and in vivo. PRACTICAL APPLICATIONS: Ten major bioactive compounds were identified in the extract(s) of four medicinally important plants viz. Cinnamomum zeylanicum, Cinnamomum tamala, Origanum vulgare and Petroselinum crispum using HPLC and their biological activity was also evaluated against cancer and normal cell lines. Interestingly, while all extract(s) wielded significant cytotoxicity against cancer cells, no significant toxicity was found against normal cells. The outcome of the results prompted evaluation of the antiviral potential of the ten bioactive compounds using in silico biology tools. The present study emphasizes on the application of computational approaches to understand the binding interaction and efficacy of the ten bioactive compounds from the above plants with SARS-CoV-2 nucleocapsid protein N-terminal and C-terminal RNA binding domains in preventing and/or treating COVID-19 using in silico tools. Druglikeness and toxicity profiles of the compounds were carried out to check the therapeutic application of the components. Additionally, molecular dynamics (MD) simulation was performed to check the stability of ligand-protein complexes. The results provided useful insights into the structural binding interaction(s) that can be exploited for the further development of potential antiviral agents targeting SARS-CoV-2 especially since no specific therapy is still available to combat the rapidly evolving virus and the existing treatment is more or less symptomatic which makes search for novel antiviral agents all the more necessary and crucial.

Cytotoxicity of Moringa oleifera fruits on human liver cancer and molecular docking analysis of bioactive constituents against caspase-3 enzyme.

Moringa oleifera is an excellent source of phenolics and flavonoids comprise various pharmacological activities. The fourth widespread leading cause of the patients' death is liver cancer. This study was formulated to perform the antiproliferative activity of Moringa oleifera fruit (MOF) extract on human liver cancer HepG2 cells and computational validation of cell death. HepG2 cell line was treated with 25, 50, 75, 100, and 200 µg/ml of MOF extract for 48 hr, and antiproliferative activity was analyzed using MTT assay, nuclear condensation, annexin V-FITC/PI double stain, ROS generation, and apoptosis executioner enzyme caspase-3. MOF extract reduced the cell viability significantly (p Ë‚ .05) by increasing cellular apoptosis which was confirmed by annexin V-FITC/PI staining assay. In addition, MOF stimulated intracellular ROS production and subsequently induced caspase-3 activity depending upon dose. In silico analysis revealed the good binding interaction between amino acid residues of caspase-3 (PDB ID: 1GFW) protein and selected active constituents of MOF. PASS analyses of the phytoconstituents showed no violation of Lipinski's rule of five. Analysis of drug-likeness and toxicity measurement exhibited drug-like candidates with no predicted toxicity. In conclusion, this study showed the potential anticancer activity of MOF extract which may be valuable source for anticancer drug development. PRACTICAL APPLICATIONS: Moringa oleifera fruit extract induced the anti-proliferative activity against human hepatocellular carcinoma HepG2 cells through ROS-mediated apoptosis and activation of caspase-3 enzyme. Structure-based virtual screening study between bioactive components of Moringa oleifera fruits and apoptosis executioner caspase-3 enzyme has validated the anti-proliferative activity of Moringa oleifera fruit extract. Interestingly, active phytoconstituents of Moringa oleifera fruit exhibited drug-like candidates with no predicted toxicity. Thus, Moringa oleifera fruit could be used as valuable source for anticancer drug development against human liver cancer with relatively non-toxic to healthy cells.

Cytostatic and Anti-tumor Potential of Ajwa Date Pulp against Human Hepatocellular Carcinoma HepG2 Cells.

Ajwa dates (Phoenix dactylifera L.) are used by traditional therapeutic practitioners for several health benefits but most remain to be scientifically validated. In this study, we evaluated the apoptosis-inducing effect of ethanolic extract of Ajwa date pulp (ADP) on human hepatocellular carcinoma (HCC) HepG2 cells. High performance liquid chromatography analysis revealed the presence of polysaccharide ß-D-glucan in ADP extract. Treated HCC cells revealed morphological characteristics of apoptosis under phase contrast microscopy. MTT assay demonstrated significant (p < 0.05) dose- and time-dependent inhibition of HCC cell growth. HCC cells were found to be in late apoptotic stage on treatment with higher doses of ADP extract as depicted by acridine orange/ethidium bromide and Annexin V-FITC/PI double stain. Importantly, ADP extract increased the reactive oxygen species level and decreased the mitochondrial membrane potential in treated HCC cells. Flow cytometry analysis demonstrated that ADP extract induced elevation of S and G2/M phases of cell cycle. Moreover, ADP extract induced apoptosis in HCC cells independent of tumor suppressor genes viz. CHEK2, ATM and TP53. Interestingly, ADP extract did not display any significant effect on normal cell line Vero. This study provides validation that ADP extract can be considered as a safe and natural potential drug candidate against human liver cancer.