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The SARS-CoV-2 main protease (Mpro) is essential for viral replication because it is responsible for the processing of most of the non-structural proteins encoded by the virus. Inhibition of Mpro prevents viral replication and therefore constitutes an attractive antiviral strategy. We set out to develop a high-throughput Mpro enzymatic activity assay using fluorescently labeled peptide substrates. A library of fluorogenic substrates of various lengths, sequences and dye/quencher positions was prepared and tested against full length SARS-CoV-2 Mpro enzyme for optimal activity. The addition of buffers containing strongly hydrated kosmotropic anion salts, such as citrate, from the Hofmeister series significantly boosted the enzyme activity and enhanced the assay detection limit, enabling the ranking of sub-nanomolar inhibitors without relying on the low-throughput Morrison equation method. By comparing cooperativity in citrate or non-citrate buffer while titrating the Mpro enzyme concentration, we found full positive cooperativity of Mpro with citrate buffer at less than one nanomolar (nM), but at a much higher enzyme concentration (∼320 nM) with non-citrate buffer. In addition, using a tight binding Mpro inhibitor, we confirmed there was only one active catalytical site in each Mpro monomer. Since cooperativity requires at least two binding sites, we hypothesized that citrate facilitates dimerization of Mpro at sub-nanomolar concentration as one of the mechanisms enhances Mpro catalytic efficiency. This assay has been used in high-throughput screening and structure activity relationship (SAR) studies to support medicinal chemistry efforts. IC50 values determined in this assay correlates well with EC50 values generated by a SARS-CoV-2 antiviral assay after adjusted for cell penetration.

Over the last 5 years, IR-MALDESI-MS (Infrared Matrix-Assisted Laser Desorption Electrospray Ionization Mass Spectrometry) has been demonstrated for use in a range of high-throughput biochemical and cellular assays with remarkable sample acquisition rates up to 22 Hz for a single 384-well assay plate. With such high single plate acquisition rates, the rate limiting step becomes how fast subsequent plates can be presented to the MS for analysis. To make this transfer as fast as possible while maintaining safe operation in a laboratory environment, we developed a collaborative robotic plate transfer system (CRPTS) that combines a 6-axis robot with dual plate grippers, a 7th axis conveyor stage, and a 420-plate capacity sample loading window. As a demonstration of the throughput and flexibility of CRPTS, we performed a biochemical assay that monitored the oxidation of tris(2-carboxyethyl)phosphine (TCEP) to screen for nuisance compounds. Using continuous and step motion scan profiles, we analyzed 158,799 compounds contained in 448 assay plates over the course of 12.5 h (Z-Factor=0.87) and 17.5 h (Z-factor=0.99), respectively. Extrapolating these results enables the screening of a million compounds within 6–7 working days.

Background: Thoracolumbar interfascial plane (TLIP) block is a novel regional anesthetic technique that can provide effective postoperative analgesia in patients undergoing lumbar spine surgeries. This block targets the dorsal rami of thoracolumbar nerves as they pass through the paraspinal muscles. Multilevel spine surgeries are often prolonged, requiring extended duration of postoperative analgesia. Objective: To compare the duration and quality of postoperative analgesia of TLIP block when given with and without dexamethasone as an additive to bupivacaine. Methodology: This was a prospective, double-blinded randomized study done on 20 patients undergoing two- or three-level lumbar spine surgeries. Ten patients in group C received 20 ml of 0.25% bupivacaine alone, whereas 10 patients in Group D received 8 mg dexamethasone along with 20 ml of bupivacaine for TLIP block before skin incision. Time for first analgesic request and pain scores using Wong–Baker Faces Pain Rating Scale were measured postoperatively. Results: The mean duration of sensory block was longer in group D when compared to group C (502.00 ± 95.13 vs. 383.5 ± 54.53 min with a P value of 0.001). The amount of rescue analgesic requirement was also higher in group C compared to group D. Conclusion: Dexamethasone, as an additive to bupivacaine, prolongs the duration and improves the quality of TLIP block for lumbar spine surgeries.

Abstract Anti-IL17A therapies have proven effective for numerous inflammatory diseases including psoriasis, axial spondylitis and psoriatic arthritis. Modulating and/or antagonizing protein–protein interactions of IL17A cytokine binding to its cell surface receptors with oral therapies offers the promise to bring forward biologics-like efficacy in a pill to patients. We used an NMR-based fragment screen of recombinant IL17A to uncover starting points for small molecule IL17A antagonist discovery. By examining chemical shift perturbations in 2D [1H, 13C-HSQC] spectra of isotopically labeled IL17A, we discovered fragments binding the cytokine at a previously undescribed site near the IL17A C-terminal region, albeit with weak affinity (> 250 µM). Importantly this binding location was distinct from previously known chemical matter modulating cytokine responses. Subsequently through analog screening, we identified related compounds that bound symmetrically in this novel site with two copies. From this observation we employed a linking strategy via structure-based drug design and obtained compounds with increased binding affinity (

Objective: To explore the anticoagulant, antiplatelet and antioxidant activities of protein extract of kenaf seed (PEKS). Methods: Sodium dodecyl sulphate polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography techniques were employed for protein characterization. Antioxidant activity of PEKS was assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The protective effect of PEKS on sodium nitrite (NaNO2) induced oxidative stress was evaluated using the in vitro red blood cell model, while the effect of PEKS on diclofenac-induced oxidative stress was examined in vivo in rats. Platelet-rich plasma and platelet-poor plasma were used for anticoagulant and antiplatelet activities of PEKS. Results: PEKS revealed similar protein bands on SDS-PAGE under reduced and non-reduced conditions. Several acidic proteins were present in native PAGE. PEKS showed antioxidant properties by scavenging DPPH with an IC50 of 24.58 μg. PEKS exhibited a protective effect on NaNO2 induced oxidative stress in red blood cells by restoring the activity of stress markers. In addition, PEKS alleviated diclofenac-induced tissue damage of the liver, kidney, and small intestine. PEKS showed an anticoagulant effect in both in vivo and in vitro experiments by enhancing normal clotting time. PEKS did not affect prothrombin time but increase activated partial thromboplastin time. Furthermore, PEKS inhibited adenosine diphosphate and epinephrine-induced platelet aggregation. Conclusions: PEKS protects tissues from oxidative stress and exhibits antithrombotic activity.

Oxidative stress and chronic inflammation interplay with the pathogenesis of cancer. Breast cancer in women is the burning issue of this century, despite chemotherapy and magnetic therapy. The management of secondary complications triggered by post-chemotherapy poses a great challenge. Thus, identifying target-specific drugs with anticancer potential without secondary complications is a challenging task for the scientific community. It is possible that green technology has been employed in a greater way in order to fabricate nanoparticles by amalgamating plants with medicinal potential with metal oxide nanoparticles that impart high therapeutic properties with the least toxicity. Thus, the present study describes the synthesis of Titanium dioxide nanoparticles (TiO2 NPs) using aqueous Terenna asiatica fruit extract, with its antioxidant, anti-inflammatory and anticancer properties. The characterisation of TiO2 NPs was carried out using a powdered X-ray diffractometer (XRD), Fourier transform infrared (FTIR), scanning electron microscopy (SEM), energy-dispersive X-ray diffraction (EDX), high-resolution transmission electron microscopy (HR-TEM), dynamic light scattering (DLS), and zeta-potential. TiO2 NPs showed their antioxidant property by scavenging 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals in a dose-dependent manner with an IC50 value of 80.21 µg/µL. To ascertain the observed antioxidant potential of TiO2 NPs, red blood cells (RBC) were used as an in vitro model system. Interestingly, TiO2 NPs significantly ameliorated all the stress parameters, such as lipid peroxidation (LPO), protein carbonyl content (PCC), total thiol (TT), superoxide dismutase (SOD), and catalase (CAT) in sodium nitrite (NaNO2)-induced oxidative stress, in RBC. Furthermore, TiO2 NPs inhibited RBC membrane lysis and the denaturation of both egg and bovine serum albumin, significantly in a dose-dependent manner, suggesting its anti-inflammatory property. Interestingly, TiO2 NPs were found to kill the MCF-7 cells as a significant decrease in cell viability of the MCF-7 cell lines was observed. The percentage of growth inhibition of the MCF-7 cells was compared to that of untreated cells at various doses (12.5, 25, 50, 100, and 200 µg/mL). The IC50 value of TiO2 NPs was found to be (120 µg/mL). Furthermore, the Annexin V/PI staining test was carried out to confirm apoptosis. The assay indicated apoptosis in cancer cells after 24 h of exposure to TiO2 NPs (120 µg/mL). The untreated cells showed no significant apoptosis in comparison with the standard drug doxorubicin. In conclusion, TiO2 NPs potentially ameliorate NaNO2-induced oxidative stress in RBC, inflammation and MCF-7 cells proliferation.

The more than 1.5 billion people who live in South Asia are correctly viewed not as a single large population but as many small endogamous groups. We assembled genome-wide data from over 2,800 individuals from over 260 distinct South Asian groups. We identified 81 unique groups, 14 of which had estimated census sizes of more than 1 million, that descend from founder events more extreme than those in Ashkenazi Jews and Finns, both of which have high rates of recessive disease due to founder events. We identified multiple examples of recessive diseases in South Asia that are the result of such founder events. This study highlights an underappreciated opportunity for decreasing disease burden among South Asians through discovery of and testing for recessive disease-associated genes.

The present study describes the green biofunctional synthesis of magnesium oxide (MgO) nanoparticles using the aqueous Tarenna asiatica fruit extract. The characterization of Tarenna asiatica fruit extract MgO nanoparticles (TAFEMgO NPs) was achieved by X-ray powder diffraction, UV-Vis spectroscopy, FTIR, TEM, SEM, and energy-dispersive X-ray diffraction. TAFEMgO NPs scavenged the DPPH free radicals with an IC50 value of 55.95 μg/μL, and it was highly significant compared to the standard. To authenticate the observed antioxidant potential of TAFEMgO NPs, oxidative stress was induced in red blood cells (RBC) using sodium nitrite (NaNO2). Interestingly, TAFEMgO NPs ameliorated the RBC damage from oxidative stress by significantly restoring the stress parameters, such as the protein carbonyl content (PCC), lipid peroxidation (LPO), total thiol (TT), super-oxide dismutase (SOD), and catalase (CAT). Furthermore, oxidative stress was induced in-vivo in Sprague Dawley female rats using diclofenac (DFC). TAFEMgO NPs normalized the stress parameters in-vivo and minimized the oxidative damage in tissues. Most importantly, TAFEMgO NPs restored the function and architecture of the damaged livers, kidneys, and small intestines by regulating biochemical parameters. TAFEMgO NPs exhibited an anticoagulant effect by increasing the clotting time from 193 s in the control to 885 s in the platelet rich plasma. TAFEMgO NPs prolonged the formation of the clot process in the activated partial thromboplastin time and the prothrombin time, suggest the effective involvement in both intrinsic and extrinsic clotting pathways of the blood coagulation cascade. TAFEMgO NPs inhibited adenosine di-phosphate (ADP)-induced platelet aggregation. TAFEMgO NPs did not show hemolytic, hemorrhagic, and edema-inducing properties at the tested concentration of 100 mg/kgbody weight, suggesting its non-toxic property. In conclusion, TAFEMgO NPs mitigates the sodium nitrite (NaNO2)- and diclofenac (DFC)-induced stress due to oxidative damage in both in vitro and in vivo experimental models.

Mucopolysaccharidosis VI (MPS VI) is an autosomal recessive inborn error of metabolism caused by mutations in the arylsulfatase B gene (ARSB) and consequent deficient activity of ARSB, a lysosomal enzyme. We present here the results of a study undertaken to identify the mutations in ARSB in MPS VI patients in India. Around 160 ARSB mutations, of which just 4 are from India, have been reported in the literature. Our study covered nine MPS VI patients from eight families. Both familial mutations were found in seven families, and only one mutation was found in one family. Seven mutations were found — four novel (p.G38_G40del3, p.C91R, p.L98R and p.R315P), two previously reported from India (p.D53N and p.W450C), and one reported from outside India (p.R160Q). One mutation, p.W450C, was present in two families, and the other six mutations were present in one family each. Analysis of the molecular structure of the enzyme revealed that most of these mutations either cause loss of an active site residue or destabilize the structure of the enzyme. The only previous study on mutations in ARSB in Indian MPS VI patients, by Kantaputra et al. 2014 [1], reported four novel mutations of which two (p.D53N and p.W450C) were found in our study as well. Till date, nine mutations have been reported from India, through our study and the Kantaputra study. Eight out of these nine mutations have been found only in India. This suggests that the population studied by us might have its own typical set of mutations, with other populations equally likely to have their own set of mutations.

Seeds of flax or linseed (Linum usitatissimum L.) are important nutraceutical foods with antioxidant, anti-inflammatory, estrogenic, laxative, and antibacterial properties. Flaxseed oil and seeds are the richest vegetarian source of omega-3 fatty acids. Consumption of flaxseeds helps in prevention and control of cardiovascular disease, neurodegenerative disorders, obesity, diabetes mellitus, polycystic ovary syndrome, gout, liver and kidney dysfunction, oxidative stress-related diseases, post-menopausal symptoms, osteoporosis, irritable bowel syndrome, dry eye disease, cystic fibrosis, diarrhea, and cancer, particularly of the mammary and prostate gland cancer. Of late, flaxseed is gaining more importance not only because of its industrial values but also due to its nutraceutical and pharmaceutical properties. The literature review was performed using PubMed, Scopus, PubMed Central, Google Scholar, and Web of Science from 1995 onwards. Data was also obtained from websites/books/book chapters. [ABSTRACT FROM AUTHOR]

Introduction: Propofol and isoflurane are commonly used in general anaesthesia. Both the drugs are neither neuroprotective nor neurotoxic. In clinical settings, inhaled anaesthetics like isoflurane are frequently used. But, it has been claimed that isoflurane anaesthesia would be a factor inducing cognitive impairment. Propofol is metabolised quickly, primarily in the liver, and its by products are inert. After the initial dose, the half-life of propofol is 2 to 8 minutes, and even with prolonged infusions, propofol promotes quick recovery. Aim: To evaluate the effects of propofol and isoflurane on postoperative recovery patterns in patients receiving general anaesthesia and to determine how they affect cognitive function and memory. Materials and Methods: A double-blinded randomised clinical study was conducted at the Department of Anaesthesia, RL Jalappa Hospital and Research Centre, Tamaka, Kolar, Karnataka, India, during the period from January 2022 to March 2022. In the present study, 60 patients of between age 50-90 years were included. Patients were split into two groups: group A received an intravenous infusion of propofol, and group B received isoflurane. Patients in both groups had their cognitive ability and memory tested before surgery. In the present study, baseline Mean Atrial Pressure (MAP), Heart Rate (HR), Pulse Oximetry (SpO2) and Ramsay Sedation score were comparable in both groups. The novel variables, such as surgery types, duration, and medications were evaluated in both groups. Mini Mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) were assessed one hour before and four hours after surgery. Comparison of a continuous variable across the groups was performed using the Student's t-test or Mann-Whitney U test, depending on the normality of the distribution. A comparison of categorical variables across the two study groups was made using the Chi-square test. A p-value of <0.05 was considered statistically significant. Results: Age group of 50-60 years were more in both group A (66.7%), and group B (70%). In Group A, most participants were females (56.7%), and in group B, majority were males (60%). In group A, the postoperative assessment showed a mean MoCA score of 25.6±1.52; in group B postoperative assessment showed a mean MoCA score of 24.17±1.46 with a p-value of 0.001, which was statistically significant. The postoperative assessment showed a mean MMSE score of 26.3±1.58 in group A and in group B, the postoperative assessment showed a mean MMSE score of 24.9±1.4 with a p-value of 0.001, which was statistically significant. Conclusion: The current results imply that postoperative delirium is more frequently present after isoflurane anaesthesia than after propofol anaesthesia. [ABSTRACT FROM AUTHOR]

High level of exogenous ROS in the circulation affects RBC membrane integrity which facilitates the generation of endogenous RBC ROS, implicated in series of physiological changes primarily associated with thrombosis and vital tissue damage. Although, Pennisetum glaucum (pearl millet) stores abundance of proteins, their therapeutic potential is least explored. Thus, the purpose of this study is to examine the role of Pennisetum Glaucum Protein Extract (PGE) on oxidative stress induced cell/tissue damage and thrombosis. In this investigation, protein characterization was done by using SDS-PAGE, Native-PAGE, PAS-staining and HPLC. In-vitro oxidative stress was induced in RBC using sodium nitrite. While, in-vivo oxidative stress was induced in experimental rats using diclofenac. Stress markers and biochemical parameters were evaluated. Role of PGE on thrombosis was assessed by using, in-vitro plasma recalcification time, activated partial thromboplastin time, prothrombin time, mouse tail bleeding time (In-vivo) and platelet aggregation. PGE revealed varied range of molecular weight proteins on SDS-PAGE. PGE normalized the sodium nitrite induced oxidative damage of RBC and diclofenac induced oxidative damage in liver, kidney and small intestine. PGE exhibited anticoagulant effect by increasing the coagulation time of both PRP and PPP and mouse tail bleeding time. Furthermore, PGE prolonged the clotting time of only APTT but did not affect PT. PGE inhibited agonists ADP and epinephrine induced platelet aggregation. Our findings suggest, PGE could be a better contender in the management of oxidative stress and its associated diseases. PGE Pennisetum Glaucum protein Extract APPT Activated Partial Thromboplastin Time PT Prothrombin Time ROS Reactive Oxygen Species PRP Platelet Rich Plasma PPP Platelet Poor Plasma SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis PAS Periodic Acid-schiff Staining OD Optical Density INR International Normalized Ratio PBS Phosphate Buffered Saline SOD Superoxide Dismutase TCA Trichloro Acetatic Acid DTNB Di-Thio-bis-NitroBenzoic acid SGOT Serum Glutamate Oxaloacetate Transaminase SGPT Serum Glutamate Pyruvate Transaminase ALP Alkaline Phosphatase DFC Diclofenac Syl Silymarin MED Minimum Edema Dose MHD Minimum Hemorrhagic Dose [ABSTRACT FROM AUTHOR]