Surveillance of omicron variant in Kangra District of Himachal Pradesh, India during the 3rd disease wave of COVID-19.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) wave has fluctuated erratically around the globe over the past three years of the pandemic, sometimes declining and at other times surging. The cases of infection in India have remained low, despite the continued surge of Omicron sub-lineages reported in a few countries. In this study, we determined the presence of the circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains in the population of Kangra District, Himachal Pradesh, India. METHODOLOGY: In vitro diagnostic real-time reverse transcriptase polymerase chain reaction (RT-qPCR) was performed using Tata MD CHECK RT-PCR Omisure kit (Tata Medical and Diagnostics Limited, Maharashtra, India), to detect the presence of Omicron in target samples. A total of 400 samples were analyzed in this study; 200 each for the second and third waves, respectively. The S gene target failure (SG-TF) and S gene mutation amplification (SG-MA) primer-probe sets were used. RESULTS: Our results corroborated that during the third wave, SG-MA amplification was noted, while amplification of SG-TF was not, and vice versa in the case of the second wave, indicating that all the tested patients were infected with the Omicron variant during the third wave, while Omicron was absent during the second wave. CONCLUSIONS: This study added more information about the prevalence of Omicron variants during the third wave in the chosen area, and it projected a use of in vitro RT-qPCR method for rapid prospective determination of the prevalence of the variant of concern (VOC) in developing countries with limited sequencing facility.

Combating the Progression of Novel Coronavirus SARS-CoV-2 Infectious Disease: Current State and Future Prospects in Molecular Diagnostics and Drug Discovery.

A highly infectious and life-threatening virus was first reported in Wuhan, China, in late 2019, and it rapidly spread all over the world. This novel virus belongs to the coronavirus family and is associated with severe acute respiratory syndrome (SARS), causing respiratory disease known as COVID-19. In March 2020, WHO has declared the COVID-19 outbreak a global pandemic. Its morbidity and mortality rates are swiftly rising day by day, with the situation becoming more severe and fatal for the comorbid population. Many COVID-19 patients are asymptomatic, but they silently spread the infection. There is a need for proper screening of infected patients to prevent the epidemic transmission of disease and for early curative interventions to reduce the risk of developing severe complications from COVID-19. To date, the diagnostic assays are of two categories, molecular detection of viral genetic material by real-time RTpolymerase chain reaction and serological test, which relies on detecting antiviral antibodies. Unfortunately, there are no effective prophylactics and therapeutics available against COVID-19. However, a few drugs have shown promising antiviral activity against it, and these presently are being referred for clinical trials, albeit FDA has issued an Emergency Use Authorization (EUA) for the emergency use of a few drugs for SARSCoV- 2 infection. This review provides an insight into current progress, challenges and future prospects of laboratory detection methods of COVID-19, and highlights the clinical stage of the major evidence-based drugs/vaccines recommended against the novel SARS-CoV-2 pandemic virus.

Environmentally relevant concentrations of Triclosan cause transcriptomic and biomolecular alterations in the hatchlings of Labeo rohita.

Suppression (p ≤ 0.05) of antioxidative/detoxification (except GPx and CYP3a) and cytoskeletal (except DHPR) genes but induction of metabolic (except for AST and TRY) and heat shock (except HSP60) genes of Labeo rohita hatchlings after 14 days of exposure to environmentally relevant concentrations of Triclosan (0.0063, 0.0126, 0.0252 and 0.06 mg/L) was followed by an increase (p ≤ 0.05) for most of the genes after 10 days recovery period. After recovery, LDH, ALT, CK, CHY, PA, HSP47 and DHPR declined, while SOD, CAT, GST, GR, GPx, CYP1a, CYP3a, AST, AChE, TRY, HSP60, HSP70, HSc71, HSP90 MLP-3, α-tropomyosin, desmin b and lamin b1 increased over exposure. Peak area of biomolecules (except 3290-3296, 2924-2925 and 2852-2855 cm-1) declined (p ≤ 0.01) more after recovery [except for an increase (p ≤ 0.01) at 1398-1401 cm-1]. CYP3a, CK, HSP90, MLP-3 and secondary structure of amide A are the most sensitive markers for the environmentally relevant concentrations of Triclosan.

Targeting Akt/NF-κB/p53 Pathway and Apoptosis Inducing Potential of 1,2-Benzenedicarboxylic Acid, Bis (2-Methyl Propyl) Ester Isolated from Onosma bracteata Wall. against Human Osteosarcoma (MG-63) Cells.

Onosma bracteata Wall. is an important medicinal and immunity-enhancing herbs. This plant is commonly used in the preparation of traditional Ayurvedic drugs to treat numerous diseases. Inspired by the medicinal properties of this plant, the present study aimed to investigate the antiproliferative potential and the primary molecular mechanisms of the apoptotic induction against human osteosarcoma (MG-63) cells. Among all the fractions isolated from O. bracteata, ethyl acetate fraction (Obea) showed good antioxidant activity in superoxide radical scavenging assay and lipid peroxidation assay with an EC50 value of 95.12 and 80.67 µg/mL, respectively. Silica gel column chromatography of ethyl acetate (Obea) fraction of O. bracteata yielded a pure compound, which was characterized by NMR, FTIR, and HR-MS analysis and was identified as 1,2-benzene dicarboxylic acid, bis (2-methyl propyl) ester (BDCe fraction). BDCe fraction was evaluated for the antiproliferative potential against human osteosarcoma MG-63, human neuroblastoma IMR-32, and human lung carcinoma A549 cell lines by MTT assay and exhibited GI50 values of 37.53 µM, 56.05 µM, and 47.12 µM, respectively. In MG-63 cells, the BDCe fraction increased the level of ROS and simultaneously decreased the mitochondria membrane potential (MMP) potential by arresting cells at the G0/G1 phase, suggesting the initiation of apoptosis. Western blotting analysis revealed the upregulation of p53, caspase3, and caspase9 while the expressions of p-NF-κB, p-Akt and Bcl-xl were decreased. RT-qPCR studies also showed upregulation in the expression of p53 and caspase3 and downregulation in the expression of CDK2, Bcl-2 and Cyclin E genes. Molecular docking analysis displayed the interaction between BDCe fraction with p53 (-151.13 kcal/mol) and CDK1 (-133.96 kcal/mol). The results of the present work suggest that the BDCe fraction has chemopreventive properties against osteosarcoma (MG-63) cells through the induction of cell cycle arrest and apoptosis via Akt/NF-κB/p53 pathways. This study contributes to the understanding of the utilization of BDCe fraction in osteosarcoma treatment.

Modulatory Effect of 4-(methylthio)butyl Isothiocyanate Isolated From Eruca Sativa Thell. on DMBA Induced Overexpression of Hypoxia and Glycolytic Pathway in Sprague-Dawley Female Rats.

4-(methylthio)butyl isothiocyanate (4-MTBITC) is a hydrolytic product from the plant Eruca sativa Thell. In the present study, we explored the anti-cancer effect of 4-MTBITC against 7,12-dimethylbenz [a] anthracene (DMBA) induced breast cancer. Hypoxic conditions were developed using a single dose of 60 mg/kg DMBA. Hepatic and renal parameters were increased along with antioxidants in cancer-bearing rats which were lowered with the treatment of 4-MTBITC. Further, it inhibited the up-regulation of glycolytic enzymes caused by DMBA. The hypoxia pathway was evaluated using RT-PCR and it was found that the 40 mg/kg doses of 4-MTBITC statistically lowered the expression of HIF-1α. Akt/mTOR signaling pathway was one of the major pathways involved in 4-MTBITC-induced cell growth arrest by western blotting. Amino acid profiling serum-free plasma revealed the downregulation of specific amino acids required for vital components of fast-growing cancer cells. 4-MTBITC reduced the levels of serine, arginine, alanine, asparagines, and glutamic acid. Histological examination also showed neoplastic growth following DMBA doses. 4-MTBITC treated rats showed less infiltration and normal physiology. Our findings for the first time demonstrated the potential therapeutic significance of 4-MTBITC on modulation of glycolytic enzymes and hypoxia pathway in female rats.

Genomic markers for the biological responses of Triclosan stressed hatchlings of Labeo rohita.

Triclosan (TCS) used commonly in pharmaceuticals and personal care products has become the most common pollutant in water. Three-day-old hatchlings of an indigenous fish, Labeo rohita, were given 96h exposure to a nonlethal (60 µg L-1) and two moderately lethal concentrations (67 and 97 µg L-1) of TCS and kept for 10 days of recovery for recording transcriptomic alterations in antioxidant/detoxification (SOD, GST, CAT, GPx, GR, CYP1a and CYP3a), metabolic (LDH, ALT and AST) and neurological (AchE) genes and DNA damage. The data were subjected to principal component analysis (PCA) for obtaining biomarkers for the toxicity of TCS. Hatchlings were highly sensitive to TCS (96h LC50 = 126 µg L-1 and risk quotient = 40.95), 96h exposure caused significant induction of CYP3a, AChE and ALT but suppression of all other genes. However, expression of all the genes increased significantly (except for a significant decline in ALT) after recovery. Concentration-dependent increase was also observed in DNA damage [Tail Length (TL), Tail Moment (TM), Olive Tail Moment (OTM) and Percent Tail DNA (TDNA)] after 96 h. The damage declined significantly over 96h values at 60 and 67 µg L-1 after recovery, but was still several times more than control. TCS elicited genomic alterations resulted in 5-11% mortality of exposed hatchlings during the recovery period. It is evident that hatchlings of L. rohita are a potential model and PCA shows that OTM, TL, TM, TDNA, SOD and GR (association with PC1 during exposure and recovery) are the biomarkers for the toxicity of TCS. Graphical abstract.

Perforator-based propeller flap for coverage of lower leg: Single centre experience.

This study was conducted to assess outcomes of propeller flaps for reconstruction of small- to medium-sized defects in the distal third of the leg. Of 53 lower third leg defects covered using the propeller flap, 43 survived without complications. Only minor complications were seen and no flap was lost completely. The propeller flap is thus a safe option for medium-sized defects of the lower leg.

Onosma bracteata Wall. induces G0/G1 arrest and apoptosis in MG-63 human osteosarcoma cells via ROS generation and AKT/GSK3ß/cyclin E pathway.

Onosma bracteata Wall. (Boraginaceae), commonly known as "gaozaban" is a highly valuable medicinal herb, useful in the treatment of body swellings, abdominal pain, eye-related problems, fever, and urinary calculi. The present study was performed to investigate the antioxidant properties of extract/fractions, viz. ethanol (Obeth) extract, hexane (Obhex) fraction, chloroform (Obcl) fraction, ethyl acetate (Obea) fraction, butanol (Obbu) fraction, and aqueous (Obaq) fraction isolated from O. bracteata. Obea fraction showed stronger free radical quenching ability in various antioxidant assays, as compared to the other fractions. Obea fraction with effective free radical-scavenging properties was further evaluated for the antiproliferative activity against human osteosarcoma MG-63, human neuroblastoma IMR-32, and human lung cancer A549 cell lines using MTT assay. Obea fraction showed strong cytotoxicity with GI50 value of 88.56, 101.61, and 112.7 µg/ml towards MG-63, IMR-32, and A549 cells respectively. Mechanistic studies revealed that Obea fraction in osteosarcoma MG-63 cells increased reactive oxygen species (ROS) level and reduced mitochondrial membrane potential. In the presence of Obea, the cells were found to be arrested in the G0/G1 phase in a dose-dependent manner which is also confirmed by the enhancement in the early apoptotic cell population in flow cytometer analysis. Western blotting demonstrated the decrease in expression of p-NFκB, COX-2, p-Akt, and Bcl-xL, whereas upregulation was observed in the expression of GSK-3ß, p53, caspase-3, and caspase-9 proteins. RT-qPCR studies revealed downregulation of Bcl-2, cyclin E, CDK2, and mortalin gene expression and upregulation in the expression of p53 genes. The antioxidant and cytotoxic potential of Obea was attributed to the presence of catechin, kaempferol, onosmin A, and epicatechin, as revealed by HPLC analysis. This is the first report regarding the antiproliferative potential of O. bracteata against osteosarcoma.

5-aminolevulinic acid regulates Krebs cycle, antioxidative system and gene expression in Brassica juncea L. to confer tolerance against lead toxicity.

Heavy metal pollution seriously impairs crop production and poses serious concerns for human health. Exogenous application of biomolecules has been efficiently tested for enhancing plant resistance to metal toxicity. Current study evaluates the possible effect of 5-aminolevulinic acid (ALA) in Brassica juncea L. seedlings subjected to lead (Pb) stress. Our results showed that shoot length, root length and chlorophyll contents were significantly recovered in Pb stressed seedlings after ALA application, accompanied by reduction in the Pb accumulation. Significant reduction in the contents of reactive oxygen species (ROS) like superoxide anion, hydrogen peroxide and malondialdehyde were also observed in ALA treated seedlings under Pb stress. Furthermore, we also noticed enhancement in the activities of antioxidative enzymes like superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), glutathione reductase (GR), glutathione-S-transferase (GST) and dehydroascorbate reductase (DHAR). We further noticed that ALA upregulated the expression of SOD (7.30 folds), POD (6.11 folds), CAT (3.52 folds), DHAR (6.42 folds), GR (6.04 folds), and GST (5.58 folds) under the Pb stress. However, RBOH1 (gene involved in ROS generation) and CHLASE (chlorophyllase) expressions were reduced in ALA treated seedlings grown under Pb stress (RBOH1 expression decreased to 3.44 from 6.50 fold and CHLASE expression decreased to 2.97 from 5.58 fold). Phenolic contents were increased in the presence of ALA and expression of genes like CHS (chalcone synthase; 7.50 fold) and PAL (phenylalanine ammonia lyase; 4.77 fold) was also stimulated by ALA under Pb stress. Furthermore, contents of the Krebs cycle metabolites (fumarate, succinate, malate and citrate) were also enhanced accompanied by upregulated expression of genes like CS (citrate Synthase; 8.13 fold), SUCLG1 (succinyl CoA ligase 1; 7.40 fold), SDH (succinate dehydrogenase; 5.10 fold) and FH (fumarate hydratase; 5.65 fold). In conclusion, current investigation revealed that ALA attenuated Pb toxicity by modulating the transcription patterns of key enzymes involved in plant defense system.

Assessment of collateral hand circulation by modified Allen's test in normal Indian subjects.

BACKGROUND: Allen's test (AT) and Modified Allen's Test (MAT) are used as screening methods for assessment of the hand circulation. Few people lack the dual blood supply of hand and are at risk of hand ischemia after any intervention on radial artery. The Purpose of the study was to assess the collateral circulation of hand using MAT in normal Indian subjects and in elderly population to know the prevalence of positivity of Allen's test. METHODS: 900 participants (1800 hands) were divided in two groups. Group I had participants with age <50 years and group II had participants with age ≥50 years. MAT was performed in all participants and results were compared between the two groups. RESULTS: In group I (n = 450, 900 hands), 313 were males and 137 were females, with mean age of 35.04 years. The relative percentages of a normal, equivocal, borderline and abnormal MAT were 77.8%, 16.6%, 3.7% and 1.6%, respectively. In group II (n = 450, 900 hands), 248 were males and 202 were females, with mean age of 60.4 years. The relative percentages of a normal, equivocal, borderline and abnormal MAT were 69.0%, 18.6%, 6.60% and 5.66%, respectively. A positive/abnormal test was significantly more common (5.66% Vs 1.66%, P < 0.00001) in older group. CONCLUSION: MAT is simple, time tested and non invasive test to assess collateral circulation of the hand. A negative MAT safely selects patients for radial artery harvest; however, if the test is positive and in older patients then a second objective test may be needed.

Antioxidant, Antiproliferative and Apoptosis-Inducing Efficacy of Fractions from Cassia fistula L. Leaves.

: Cassia fistula L. is a highly admirable traditional medicinal plant used for the treatment of various diseases and disorders. The present study was performed to divulge the antioxidant, antiproliferative, and apoptosis-inducing efficacy of fractions from C. fistula leaves. The hexane (CaLH fraction), chloroform (CaLC fraction), ethyl acetate (CaLE fraction), n-butanol (CaLB fraction), and aqueous (CaLA fraction) were sequentially fractionated from 80% methanolic (CaLM extract) of C. fistula leaves. The CaLE fraction was fractionated using column chromatography to yield a pure compound, which was characterized as Epiafzelechin (CFL1) based on 1H, 13C, and DEPT135 NMR. Among these fractions, CaLE and isolated CFL1 fractions exhibited an effective antioxidant potential in Ferric ion reducing power, (2,2'-azino-bis (3-ethylbenzothiazoline -6-sulfonic acid)) cation radical scavenging, and nitric oxide radical scavenging assays. Epiafzelechin was investigated for its antiproliferative effects against MG-63 (osteosarcoma), IMR-32 (neuroblastoma), and PC-3 (prostate adenocarcinoma), and was found to inhibit cell proliferation with a GI50 value of 8.73, 9.15, and 11.8 µM respectively. MG-63 cells underwent apoptotic cell death on treatment with Epiafzelechin as the cells showed the formation of apoptotic bodies, enhanced reactive oxygen species (ROS) generation, mitochondrial membrane depolarization along with an increase in early apoptotic cell population analyzed using Annexin V-FITC/PI double staining assay. Cells showed cell cycle arrest at the G0/G1 phase accompanied by a downregulation in the expression levels of p-Akt (Protein kinase B), p-GSK-3ß (Glycogen synthase kinase-3 beta), and Bcl-xl (B-cell lymphoma-extra large) proteins. RT-PCR (Real time-polymerase chain reaction) analysis revealed downregulation in the gene expression level of ß-catenin and CDK2 (cyclin-dependent kinases-2) while it upregulated the expression level of caspase-8 and p53 genes in MG-63 cells.

Molecular identification and phylogenetic relationship of Demodex mites based on mitochondrial 16S rDNA.

BACKGROUND AND OBJECTIVE: Demodex mites are tiny parasites that live around hair follicles of mammals. The two main species of Demodex i.e. Demodex folliculorum and Demodex brevis present in humans are found near the hair follicles of eyes. The present study was to understand the presence of Demodex mites in people suffering from blepharitis in Amritsar, Punjab. MATERIAL AND METHODS: Demodex mites samples present in blepharitis patients were isolated from the eyelashes. DNA was isolated from three mites and used for PCR amplification of mitochondrial (mt) 16S rDNA. The amplified PCR product were purified and used for molecular identification. RESULTS: The amplified mt16s rDNA product was sequenced and subjected to BLAST search in the NCBI database for molecular identification. The identified mite belongs to Demodex folliculorum species. The phylogenetic tree constructed by using mt16s rDNA sequence suggests that D. folliculorum is closer to D. canis than to D. brevis. CONCLUSION: All the three isolates belong to D. folliculorum and the mitochondrial DNA 16S rDNA partial sequence is applicable for phylogenetic relationship analysis.

Characterization of a cold-active, detergent-stable metallopeptidase purified from Bacillus sp. S1DI 10 using Response Surface Methodology.

The colder regions of Earth are inhabited by cold-adapted microorganisms designated as psychrophiles that are known to produce cold-active enzymes, such as peptidases, chaperones, lipases, cellulases, and phosphatases. These types of enzymes are a major part of the market of industrial enzymes. Bacteria isolated from water samples collected from the Chamba region in the Himalayas were screened for peptidase production using skim milk agar plates. Among the peptidase-producing bacteria isolated, 20% of the isolates exhibited fast growth and maximum zones of clearance, and thus, were used for further studies. The 16S rDNA sequence analysis of isolate S1DI 10 identified it as a Bacillus sp. The peptidase was cloned in pET28a vector and expressed in Escherichia coli BL21(DE3) and the His-tagged recombinant protein was purified using Ni-NTA column. The purified peptidase of SIDI 10 was found to be an alkaline, cold-active peptidase with optimal enzyme activity at 10°C and pH 8. An approach of one variable at a time was used to further study the effect of various metal ions, organic solvents and detergents on the peptidase enzyme. The peptidase activity was enhanced in the presence of Fe2+ and Mn2+ (metal ions), hexane (organic solvent), SDS- sodium dodecyl sulfate (anionic detergent) and Tween 80 (nonionic detergent). Response surface methodology (RSM) was used to determine the cumulative effect of these five variables. A 25 full factorial central composite design was applied for the five independent variables to determine the optimal combinations of these constituents at the maximum peptidase activity.

24-epibrassinolide stimulates imidacloprid detoxification by modulating the gene expression of Brassica juncea L.

BACKGROUND: Pesticides cause oxidative stress to plants and their residues persist in plant parts, which are a major concern for the environment as well as human health. Brassinosteroids (BRs) are known to protect plants from abiotic stress conditions including pesticide toxicity. The present study demonstrated the effects of seed-soaking with 24-epibrassinolide (EBR) on physiological responses of 10-day old Brassica juncea seedlings grown under imidacloprid (IMI) toxicity. RESULTS: In the seedlings raised from EBR-treated seeds and grown under IMI toxicity, the contents of hydrogen peroxide (H2O2) and superoxide anion (O.2-) were decreased, accompanied by enhanced activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), glutathione-S-transferase (GST), guaiacol peroxidase (POD) and the content of glutathione (GSH). As compared to controls, the gene expressions of SOD, CAT, GR, POD, NADH (NADH-ubiquinone oxidoreductase), CXE (carboxylesterase), GSH-S (glutathione synthase), GSH-T (glutathione transporter-1), P450 (cytochrome P450 monooxygenase) and GST1-3,5-6 were enhanced in the seedlings raised from EBR-treated seeds and grown in IMI supplemented substratum. However, expression of RBO (respiratory burst oxidase, the gene responsible for H2O2 production) was decreased in seedlings raised from EBR treated seeds and grown under IMI toxicity. Further, the EBR seed treatment decreased IMI residues by more than 38% in B. juncea seedlings. CONCLUSIONS: The present study revealed that EBR seed soaking can efficiently reduce oxidative stress and IMI residues by modulating the gene expression of B. juncea under IMI stress. In conclusion, exogenous EBR application can protect plants from pesticide phytotoxicity.

Pre-sowing Seed Treatment with 24-Epibrassinolide Ameliorates Pesticide Stress in Brassica juncea L. through the Modulation of Stress Markers.

The present experiment was designed to assess the effects of seed soaking with 24-epibrassinolide (EBR) on the physiology of Brassica juncea L. seedlings grown under imidacloprid (IMI) toxicity. Application of EBR increased the length of seedlings, dry weight, and pigment contents, polyphenols, total phenols, and organic acids under IMI toxicity. The expression of genes coding key enzymes of pigment, phenols, polyphenols, and organic acid biosynthetic pathways was also studied including CHLASE (chlorophyllase), PSY (phytoene synthase), CHS (chalcone synthase) and PAL (phenylalanine ammonialyase), CS (citrate synthase), SUCLG1 (succinyl Co-A ligase,), SDH (succinate dehydrogenase), FH (fumarate hydratase), MS (malate synthase). Multiple linear regression (MLR) analysis revealed that IMI application regressed negatively on seedling length, dry weight and total chlorophyll content. However, EBR seed treatment regressed positively on all the parameters studied. Moreover, interaction between IMI and EBR showed positive regression for growth parameters, content of pigments, total polyphenol, total phenol and malate, and expression of PSY and PAL. Negative interactions were noticed for the contents of fumarate, succinate and citrate, and expression of CHS and all genes studied related to organic acid metabolism. In conclusion, EBR enhanced the growth and contents of all studied metabolites by regulating the gene expression of B. juncea seedlings under IMI stress.