Inhibitory effect of compounds extracted from Monochoria hastata (L.) Solms on SARS-CoV-2 main protease: An insight from molecular docking and MD-simulation studies.

Using molecular docking and other studies, 20 compounds extracted from Monochoria hastata (L.) Solms were screened, and their inhibitory efficiency examined against main protease (3CLpro) of SARS CoV-2. All the compounds were found to binding with 3CLpro through van der Waals and electrostatic forces of attractions. Among them, Azelaic dihydrazide (ADZ) was found to have the highest docking score. 3CLpro-ADZ complex was studied by MD simulation. ADZ was found to disrupt the structure of 3CLpro after 2 ns. RMSD and RMSF analysis along with sequence and binding energy analysis suggest that ADZ can be a potential drug against SARS CoV-2.

Phenolate-thiazole based reversible "turn-on" chemosensor for the selective detection of carbonate anion: X-ray crystallography, DFT/TDFT, and cell study.

A new phenolate-thiazole derivative (L) has been synthesized and structurally characterized.The chemo-sensing activity of L is detected by the naked eye for the aqueous carbonate anion in the pH range of 4 to 8. The selective 'turn-on' fluorescence occurs through the formation of a stable intermediate L∙CO32-(1) following the PET mechanism. The limit of detection (LOD) is found 0.18 µM based on the absorbance-based assay.The quinonoid form of bromophenol unit binds strongly with CO32- through thiazole nitrogen and hydrazinic nitrogen. Further, the selective holding of CO32- anion over other planar tetranuclear anions (e.g., SO32-, NO3-) happens with several intra and intermolecular hydrogen bonds as envisaged by the DFT/TDFT study. The formation mechanism of L∙CO32- is proposed based on experimental and theoretical studies. The biological experiments (MTT and cell imaging)reveal the non-cytotoxicity nature of L and the biocompatible uptake of L mostly in the cytoplasm at physiological pH.

A multi-type branching process model for epidemics with application to COVID-19.

In this paper we model an infectious disease epidemic using Multi-type Branching Process where the number of offsprings of different types follow non-identical Poisson distributions whose parameters may vary over time. We allow for variation in parameters due to the behavior of citizens, government interventions in the form of lockdown, testing and contact tracing and the infectiousness of the variant of the virus in circulation at a time-point in a location. The model can be used to estimate several unknown quantities of interest in an epidemic such as the number of undetected cases and number of people quarantined following contact tracing. The model is fitted to the publicly available COVID-19 caseload data of India, South Korea, UK and US and is seen to provide good fit. It also provides good short-term forecast of the caseload for these countries. This model can be useful for health policy planners in assessing the impact of various intervention strategies such as testing, contact tracing, quarantine etc.

Large-Scale Purification and Characterization of Recombinant Receptor-Binding Domain (RBD) of SARS-CoV-2 Spike Protein Expressed in Yeast.

SARS-CoV-2 spike protein is an essential component of numerous protein-based vaccines for COVID-19. The receptor-binding domain of this spike protein is a promising antigen with ease of expression in microbial hosts and scalability at comparatively low production costs. This study describes the production, purification, and characterization of RBD of SARS-CoV-2 protein, which is currently in clinical trials, from a commercialization perspective. The protein was expressed in Pichia pastoris in a large-scale bioreactor of 1200 L capacity. Protein capture and purification are conducted through mixed-mode chromatography followed by hydrophobic interaction chromatography. This two-step purification process produced RBD with an overall productivity of ~21 mg/L at >99% purity. The protein's primary, secondary, and tertiary structures were also verified using LCMS-based peptide mapping, circular dichroism, and fluorescence spectroscopy, respectively. The glycoprotein was further characterized for quality attributes such as glycosylation, molecular weight, purity, di-sulfide bonding, etc. Through structural analysis, it was confirmed that the product maintained a consistent quality across different batches during the large-scale production process. The binding capacity of RBD of spike protein was also assessed using human angiotensin-converting enzyme 2 receptor. A low binding constant range of KD values, ranging between 3.63 × 10-8 to 6.67 × 10-8, demonstrated a high affinity for the ACE2 receptor, revealing this protein as a promising candidate to prevent the entry of COVID-19 virus.

TURBOMOLE: Today and Tomorrow.

TURBOMOLE is a highly optimized software suite for large-scale quantum-chemical and materials science simulations of molecules, clusters, extended systems, and periodic solids. TURBOMOLE uses Gaussian basis sets and has been designed with robust and fast quantum-chemical applications in mind, ranging from homogeneous and heterogeneous catalysis to inorganic and organic chemistry and various types of spectroscopy, light-matter interactions, and biochemistry. This Perspective briefly surveys TURBOMOLE's functionality and highlights recent developments that have taken place between 2020 and 2023, comprising new electronic structure methods for molecules and solids, previously unavailable molecular properties, embedding, and molecular dynamics approaches. Select features under development are reviewed to illustrate the continuous growth of the program suite, including nuclear electronic orbital methods, Hartree-Fock-based adiabatic connection models, simplified time-dependent density functional theory, relativistic effects and magnetic properties, and multiscale modeling of optical properties.

A comparative finite element analysis of artificial intervertebral disc replacement and pedicle screw fixation of the lumbar spine.

Titanium alloy-based Pedicle screw-rod fusion is a very common technique to provide higher fusion regularity than other methods. In recent times, Carbon-fibre-reinforced (CFR)-PEEK rod is used to better reduce the fusion rate. Alternatively, total disc replacement (TDR) is also very common for the non-fusion treatment method for degenerative disc disease (DDD). This study aims to investigate flexibility (ROM), stability, stress condition in implant, implant adjacent bone of the implanted lumbar spine during different physiological movements and loading environments. The finite element (FE) intact model of the lumbar spine (L2-L5) with two-level pedicle screw-rod fusion at L3-L4-L5 and two-level artificial disc replacement was developed. CFR-PEEK was taken for rod for semi-rigid fusion. UHMWPE was taken as core part of the artificial disc. The FE models were simulated under 6, 8 and 10 Nm moments in left right lateral bending, flexion and extension movements. The total ROM was reduced for two-level pedicle screw fixation and increased for the artificial disc replacement model during flexion extension compared to the intact spine. The total ROM was reduced by around 54% and 25% for two-level fixation and increased by 30% and 19.5% for artificial disc replacement spine, under flexion-extension and left-right lateral bending respectively. For screw fixation, the ROM increased by 15% and 18% reduced by 4.5% and 14% for disc replacement at the adjacent segments for flexion-extension and left-right lateral bending.

2.2.2-Cryptand complexes of neptunium(III) and plutonium(III).

New coordination environments are reported for Np(III) and Pu(III) based on pilot studies of U(III) in 2.2.2-cryptand (crypt). The U(III)-in-crypt complex, [U(crypt)I2][I], obtained from the reaction between UI3 and crypt, is treated with Me3SiOTf (OTf = O3SCF3) in benzene to form the [U(crypt)(OTf)2][OTf] complex. Similarly, the isomorphous Np(III) and Pu(III) complexes were obtained similarly starting from [AnI3(THF)4]. All three complexes (1-An; An = U, Np, Pu) contain an encapsulated actinide in a THF-soluble complex. Absorption spectroscopy and DFT calculations are consistent with 5f3 U(III), 5f4 Np(III), and 5f5 Pu(III) electron configurations.

In Vivo Anticoagulant and Thrombolytic Activities of a Fibrinolytic Serine Protease (Brevithrombolase) With the k-Carrageenan-Induced Rat Tail Thrombosis Model.

In the present study, in vivo thrombolysis efficiency of Brevithrombolase, a nontoxic fibrinolytic enzyme purified from Brevibacillus brevis strain FF02B, was affirmed by significant inhibition of thrombus formation in the k-carrageenan-induced rat tail, in a dose-dependent manner. Brevithrombolase at a dose of 600 µg/kg showed an efficacy that was comparable to streptokinase and plasmin, in dissolving in vivo thrombus of k-carrageenan-treated rats under identical conditions. The in vivo anticoagulant property of Brevithrombolase was demonstrated by its prolongation of activated partial thromboplastin time, prothrombin time, and thrombin time in Wistar rats. However, the Brevithrombolase-treated rats demonstrated an insignificant decrease in fibrinogen (Fg) level of plasma compared with Fg level of control group of rats corroborating in vivo as well as in vitro anticoagulant activity of Brevithrombolase is due to its hydrolytic action on thrombin. These findings unequivocally suggest that Brevithrombolase may serve a promising alternative to the commercial thrombolytic drugs.

Antiplatelet and antithrombotic activity of a fibrin(ogen)olytic protease from Bacillus cereus strain FF01.

Fibrin(ogen)olytic enzymes offer great promise for the treatment of thrombosis associated disorders. The present study describes the characterization of an extracellular fibrin(ogen)olytic serine protease (named Bacethrombase) purified from the Bacillus cereus strain FF01. The molecular mass of the Bacethrombase was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix assisted laser desorption/ionization-time-of-flight-mass spectroscopy analyses at 39.5 kDa and 38,450.51 Da, respectively. The peptide mass fingerprinting and analyses of the composition of the amino acids revealed the similarity of the Bacethrombase to the bacterial serine proteases. The secondary structure of the Bacethrombase was composed of 14% helix, 6.6% beta-sheet, and 79.4% random coil. Bacethrombase was found to contain 48% sialic acid and it preferentially degraded the Aα-chain of fibrinogen, as well as fibrin. The anticoagulant potency of the Bacethrombase was comparable with that of warfarin and heparin, and was corroborated by its fibrinogenolytic activity rather than the inhibition of thrombin, prothrombin or FXa. Bacethrombase demonstrated antiplatelet activity, and dose-dependently inhibited the ADP-induced platelet aggregation. Bacethrombase (10 mg/kg) did not show toxicity after i.v. administration in Wistar rats; however, it revealed an in vivo anticoagulant effect and significantly inhibited the carrageenan-induced in vivo thrombus formation in rats.

Characterization, mechanism of anticoagulant action, and assessment of therapeutic potential of a fibrinolytic serine protease (Brevithrombolase) purified from Brevibacillus brevis strain FF02B.

In this study, biochemical and pharmacological characterization of Brevithrombolase, a fibrinolytic serine protease purified from Brevibacillus brevis strain FF02B has been reported. An assessment of its thrombolytic potency has also been made. The molecular mass of this monomeric protease was determined as 55 kDa, and 56043 Da, respectively, by SDS-PAGE and MALDI-TOF-MS. In the analytical studies, the N-terminal sequence of Brevithrombolase was found to be blocked; however, the peptide mass fingerprinting and amino acid composition analyses demonstrated the similarity of Brevithrombolase with endopeptidases in possessing serine in their catalytic triad. This finding was confirmed by the observation that the serine protease inhibitors decrease the catalytic (fibrinolytic) activity of Brevithrombolase. The secondary structure of Brevithrombolase was composed of 30.6% alpha helix and 69.4% random coil. Brevithrombolase showed the Km and Vmax values towards the chromogenic substrate for plasmin at 0.39 mM and 14.3 µmol/min, respectively. Brevithrombolase demonstrated optimum fibrinolytic activity at pH 7.4 and 37 °C, and showed marginal hydrolytic activity towards globulin, casein and fibrinogen. The anticoagulant potency of Brevithrombolase was comparable to the low molecular mass heparin/antithrombin-III and warfarin. Among the three enzymes-Brevithrombolase, plasmin and streptokinase-the fibrinolytic activity and in vitro thrombolytic potency of Brevithrombolase was found to be superior. The RP-HPLC and SDS-PAGE analyses suggested a similar pattern of fibrin degradation by Brevithrombolase and plasmin, indicating that former enzyme is a plasmin-like fibrinolytic serine protease. Brevithrombolase did not show in vitro cytotoxicity on HT29 and HeLa cells or hemolytic activity. At a dose of 10 mg/kg, Brevithrombolase did not exhibit lethality or toxicity on Wistar strain albino rats. Brevithrombolase did not inhibit factor Xa, and its mechanism of anticoagulant action was associated with the enzymatic cleavage of thrombin. The combined properties of Brevithrombolase indicate its therapeutic potential in peptide-based cardiovascular drug development.

Mechanism of in vivo anticoagulant and haemolytic activity by a neutral phospholipase A(2) purified from Daboia russelii russelii venom: correlation with clinical manifestations in Russell's Viper envenomed patients.

A 13.0 kDa neutral phospholipase A2 (NEUPHOLIPASE) purified from venom of Daboia russelii russelii from eastern India was identified by peptide mass fingerprinting analysis. It exerted dose-dependent PLA2, anticoagulant and indirect haemolytic activities. NEUPHOLIPASE showed preferential binding followed by hydrolysis of phosphatidylserine > phosphatidylcholine >> phosphatidylethanolamine. Circular dichroism analysis of NEUPHOLIPASE showed a high content of alpha helix (54.6%) followed by beta-turn (29.7%) in its secondary structure. Gas-chromatographic analysis of plasma from PLA2-treated mice suggested preferential hydrolysis of pro-coagulant phospholipid PS was the primary mechanism to account for in vivo anticoagulant effect of NEUPHOLIPASE. The NEUPHOLIPASE-treated mice blood showed a significant decrease (p < 0.01) in WBC as well as RBC counts with a corresponding decline in Hb content due to indirect damage to erythrocyte membranes by plasma phospholipids hydrolysis products rather than the direct haemolytic activity of PLA2 under study. NEUPHOLIPASE was non-lethal to BALB/c mice, however; it was detrimental to liver of treated-mice. Pathological symptoms observed in NEUPHOLIPASE-treated mice were correlated with the actual clinical manifestations in Russell's Viper envenomed patients from eastern India indicating some contribution of NEUPHOLIPASE in Russell's Viper venom induced toxicity and pathogenesis.