Search

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.

Bio-materials in the nano size are promising high-value products with diverse applications. The green synthesis of lignin nanoparticles has opened a novel method to utilize lignin in advanced uses. In the present study lignin nanoparticles with dimensions of about 35nm were prepared from alkaline lignin extract directly in the moist condition. By rapid acid precipitation using methane sulphonic acid, accompanied by mild ultrasonication. The size, surface morphology, and thermal properties of the lignin nanoparticles were studied using different techniques such as; DLS, FTIR, XRD, UV, SEM, TGA, and DTA. [ABSTRACT FROM AUTHOR]

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 FROM AUTHOR]

Background: Thrombosis is the key event that obstructs the flow of blood throughout the circulatory system, leading to stroke, myocardial infarction and severe cardiovascular complications. Currently, available antithrombotic drugs trigger several life-threatening side effects., Introduction: Antithrombotic agents from natural sources devoid of adverse effects are grabbing high attention. In our previous study, we reported the antioxidant, anticoagulant and antiplatelet properties of kenaf seed protein extract. Therefore, in the current study, purification and characterization of cysteine protease from kenaf seed protein extract responsible for potential antithrombotic activity was undertaken., Methods: Purification of KSCP (Kenaf Seed Cysteine Protease) was carried out using gel permeation and ion exchange column chromatography. The purity of the enzyme was evaluated by SDS PAGE (Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis). RP-HPLC (Reverse Phase High-Performance Liquid Chromatography), MALDI-TOF (Matrix-Assisted Laser Desorption Ionization Time-Of-Flight) and CD (Circular Dichroism techniques) were employed for its characterization. Proteolytic, fibrinolytic and kinetic study was done using spectroscopy. Plasma recalcification time, Prothrombin Time (PT), Thrombin clotting time (TCT), Activated Partial Thromboplastin Time (APTT), bleeding time and platelet aggregation studies were carried out for antithrombotic activity of KSCP., Result: A single sharp band of KSCP was observed under both reduced and non-reduced conditions, having a molecular mass of 24.1667kDa. KSCP was found to contain 30.3% helix turns and 69.7% random coils without a beta-pleated sheet. KSCP digested casein and fibrin, and its activity was inhibited by iodoacetic acid (IAA). KSCP was optimally active at pH 6.0 at the temperature of 40°C. KSCP exhibited anticoagulant properties by interfering in the intrinsic pathway of the blood coagulation cascade. Furthermore, KSCP dissolved both whole blood and plasma clots and platelet aggregation., Conclusion: KSCP purified from kenaf seed extract showed antithrombotic potential. Hence, it could be a better candidate for the management of thrombotic complications., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)