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Abstract Background Ethacrynic acid (EA) is a loop diuretic that is approved orally and parenterally to manage edema-associated diseases. Nevertheless, it was earlier reported that it is also associated with bleeding upon its parenteral administration. In this report, we investigated the effects of EA on human factor XIIIa (FXIIIa) of the coagulation process using a variety of techniques. Methods A series of biochemical and computational methods have been used in this study. The potency and efficacy of human FXIIIa inhibition by EA was evaluated using a bisubstrate-based fluorescence trans-glutamination assay under near physiological conditions. To establish the physiological relevance of FXIIIa inhibition by EA, the effect on FXIIIa-mediated polymerization of fibrin(ogen) as well as the formation of fibrin(ogen) – α2-antiplasmin complex was evaluated using SDS-PAGE experiments. The selectivity profile of EA against other coagulation proteins was assessed by evaluating EA’s effect on human clotting times in the activated partial thromboplastin time (APTT) and the prothrombin time (PT) assays. We also used molecular modeling studies to put forward a putative binding mode for EA in the active site of FXIIIa. Results involving EA were the average of at least three experiments and the standard error ± 1 was provided. In determining the inhibition parameters, we used non-linear regression analysis. Results FXIIIa is a transglutaminase that works at the end of the coagulation process to form an insoluble, rigid, and cross-linked fibrin rich blood clot. In fact, inhibition of FXIIIa-mediated biological processes has been reported to result in a bleeding diathesis. Inhibition of FXIIIa by EA was investigated given the nucleophilic nature of the thiol-containing active site of the enzyme and the Michael acceptor-based electrophilicity of EA. In a bisubstrate-based fluorescence trans-glutamination assay, EA inhibited FXIIIa with a moderate potency (IC 50 ~ 105 µM) and efficacy (∆Y ~ 66%). In SDS-PAGE experiments, EA appears to significantly inhibit the FXIIIa-mediated polymerization of fibrin(ogen) as well as the formation of fibrin(ogen) – α2-antiplasmin complex which indicates that EA affects the physiological functions of FXIIIa. Interestingly, EA did not affect the clotting times of human plasma in the APTT and the PT assays at the highest concentration tested of 2.5 mM suggesting the lack of effects on the coagulation serine proteases and potentially the functional selectivity of EA with respect to the clotting process. Molecular modeling studies demonstrated that the Michael acceptor of EA forms a covalent bond with catalytic residue of Cys314 in the active site of FXIIIa. Conclusions Overall, our studies indicate that EA inhibits the physiological function of human FXIIIa in vitro which may potentially contribute to the bleeding complications that were reported with the association of the parenteral administration of EA.

Abstract The inhibition of factor XIa (FXIa) is a trending paradigm for the development of new generations of anticoagulants without a substantial risk of bleeding. In this report, we present the discovery of a benzyl tetra‐phosphonate derivative as a potent and selective inhibitor of human FXIa. Biochemical screening of four phosphonate/phosphate derivatives has led to the identification of the molecule that inhibited human FXIa with an IC50 value of ∼7.4 μM and a submaximal efficacy of ∼68 %. The inhibitor was at least 14‐fold more selective to FXIa over thrombin, factor IXa, factor Xa, and factor XIIIa. It also inhibited FXIa‐mediated activation of factor IX and prolonged the activated partial thromboplastin time of human plasma. In Michaelis‐Menten kinetics experiment, inhibitor 1 reduced the VMAX of FXIa hydrolysis of a chromogenic substrate without significantly affecting its KM suggesting an allosteric mechanism of inhibition. The inhibitor also disrupted the formation of FXIa – antithrombin complex and inhibited thrombin‐mediated and factor XIIa‐mediated formation of FXIa from its zymogen factor XI. Inhibitor 1 has been proposed to bind to or near the heparin/polyphosphate‐binding site in the catalytic domain of FXIa. Overall, inhibitor 1 is the first benzyl tetraphosphonate small molecule that allosterically inhibits human FXIa, blocks its physiological function, and prevents its zymogen activation by other clotting factors under in vitro conditions. Thus, we put forward benzyl tetra‐phosphonate 1 as a novel lead inhibitor of human FXIa to guide future efforts in the development of allosteric anticoagulants.

Highly efficient intracellular delivery strategies are essential for developing therapeutic, diagnostic, biological, and various biomedical applications. The recent advancement of micro/nanotechnology has focused numerous researches towards developing microfluidic device-based strategies due to the associated high throughput delivery, cost-effectiveness, robustness, and biocompatible nature. The delivery strategies can be carrier-mediated or membrane disruption-based, where membrane disruption methods find popularity due to reduced toxicity, enhanced delivery efficiency, and cell viability. Among all of the membrane disruption techniques, the mechanoporation strategies are advantageous because of no external energy source required for membrane deformation, thereby achieving high delivery efficiencies and increased cell viability into different cell types with negligible toxicity. The past two decades have consequently seen a tremendous boost in mechanoporation-based research for intracellular delivery and cellular analysis. This article provides a brief review of the most recent developments on microfluidic-based mechanoporation strategies such as microinjection, nanoneedle arrays, cell-squeezing, and hydroporation techniques with their working principle, device fabrication, cellular delivery, and analysis. Moreover, a brief discussion of the different mechanoporation strategies integrated with other delivery methods has also been provided. Finally, the advantages, limitations, and future prospects of this technique are discussed compared to other intracellular delivery techniques.

Single-neuron actions are the basis of brain function, as clinical sequelae, neuronal dysfunction or failure for most of the central nervous system (CNS) diseases and injuries can be identified via tracing single-neurons. The bulk analysis methods tend to miscue critical information by assessing the population-averaged outcomes. However, its primary requisite in neuroscience to analyze single-neurons and to understand dynamic interplay of neurons and their environment. Microfluidic systems enable precise control over nano-to femto-liter volumes via adjusting device geometry, surface characteristics, and flow-dynamics, thus facilitating a well-defined micro-environment with spatio-temporal control for single-neuron analysis. The microfluidic platform not only offers a comprehensive landscape to study brain cell diversity at the level of transcriptome, genome, and/or epigenome of individual cells but also has a substantial role in deciphering complex dynamics of brain development and brain-related disorders. In this review, we highlight recent advances of microfluidic devices for single-neuron analysis, i.e., single-neuron trapping, single-neuron dynamics, single-neuron proteomics, single-neuron transcriptomics, drug delivery at the single-neuron level, single axon guidance, and single-neuron differentiation. Moreover, we also emphasize limitations and future challenges of single-neuron analysis by focusing on key performances of throughput and multiparametric activity analysis on microfluidic platforms.

The anticoagulant activity of lignosulfonic acid sodium (LSAS), a non-saccharide heparin mimetic, was investigated in this study. LSAS is a relatively safe industrial byproduct with similar polyanionic characteristics to that of heparin. Human plasma clotting assays, fibrin polymerization testing, and enzyme inhibition assays were exploited to investigate the anticoagulant activity of LSAS. In normal human plasma, LSAS selectively doubled the activated partial thromboplastin time (APTT) at ~308 µg/mL. Equally, LSAS doubled APTT at ~275 µg/mL in antithrombin-deficient plasma. Yet, LSAS doubled APTT at a higher concentration of 429 µg/mL using factor XI-deficient plasma. LSAS did not affect FXIIIa-mediated fibrin polymerization at 1000 µg/mL. Enzyme assays revealed that LSAS inhibits factor XIa (FXIa) with an IC50 value of ~8 μg/mL. LSAS did not inhibit thrombin, factor IXa, factor Xa, factor XIIIa, chymotrypsin, or trypsin at the highest concentrations tested and demonstrated significant selectivity against factor XIIa and plasmin. In Michaelis–Menten kinetics, LSAS decreased the VMAX of FXIa hydrolysis of a tripeptide chromogenic substrate without significantly changing its KM indicating an allosteric inhibition mechanism. The inhibitor also disrupted the generation of FXIa–antithrombin complex, inhibited factor XIIa-mediated and thrombin-mediated activation of the zymogen factor XI to FXIa, and competed with heparin for binding to FXIa. Its action appears to be reversed by protamine sulfate. Structure–activity relationship studies demonstrated the advantageous selectivity and allosteric behavior of LSAS over the acetylated and desulfonated derivatives of LSAS. LSAS is a sulfonated heparin mimetic that demonstrates significant anticoagulant activity in human plasma. Overall, it appears that LSAS is a potent, selective, and allosteric inhibitor of FXIa with significant anticoagulant activity in human plasma. Altogether, this study introduces LSAS as a promising lead for further development as an anticoagulant.

The coronavirus disease-2019 (COVID-19) pandemic continues to challenge health care systems around the world. Scientists and pharmaceutical companies have promptly responded by advancing potential therapeutics into clinical trials at an exponential rate. Initial encouraging results have been realized using remdesivir and dexamethasone. Yet, the research continues so as to identify better clinically relevant therapeutics that act either as prophylactics to prevent the infection or as treatments to limit the severity of COVID-19 and substantially decrease the mortality rate. Previously, we reviewed the potential therapeutics in clinical trials that block the early stage of the viral life cycle. In this review, we summarize potential anti-COVID-19 therapeutics that block/inhibit the post-entry stages of the viral life cycle. The review presents not only the chemical structures and mechanisms of the potential therapeutics under clinical investigation, i.e., listed in clinicaltrials.gov, but it also describes the relevant results of clinical trials. Their anti-inflammatory/immune-modulatory effects are also described. The reviewed therapeutics include small molecules, polypeptides, and monoclonal antibodies. At the molecular level, the therapeutics target viral proteins or processes that facilitate the post-entry stages of the viral infection. Frequent targets are the viral RNA-dependent RNA polymerase (RdRp) and the viral proteases such as papain-like protease (PLpro) and main protease (Mpro). Overall, we aim at presenting up-to-date details of anti-COVID-19 therapeutics so as to catalyze their potential effective use in fighting the pandemic.

Background: Medicinal plants are a source of phytochemicals and they are used for the treatment of several oxidative stress-related or other diseases for their effectiveness, low toxicity and easy availability. Five traditionally used and less characterized herbaceous weeds of West Bengal, India, namely, Heliotropium indicum, Tridax procumbens, Cleome rutidosperma, Commelina benghalensis and Euphorbia hirta, were investigated for the current research study. Methods: Aqueous and 70% ethanolic extracts of the leaves were analyzed for estimation of essential phytochemicals and to evaluate their in vitro antioxidant status, medicinal properties and cytotoxic effects. To the best of our knowledge, several assays and comparative evaluations using these herbs are reported for the first time. For quantitative study, UV-vis spectrophotometry and high-performance liquid chromatography with diode array detector HPLC-DAD techniques were used. Antibacterial properties were investigated using the Kirby-Bauer disc diffusion method. For in vitro anti-lithiatic study, a titration method was used. The cell viability assay was done using peripheral blood mononuclear cells. Results: The aqueous extract exhibits higher content of polyphenols, flavonoids, tannins and inhibition percentage values for free radical scavenging assays, whereas the 70% ethanolic extract exhibits higher content of alkaloids and cardiac glycosides. HPLC-DAD analysis of 70% ethanolic extracts led us to identify 10 predominant phenolic constituents. Euphorbia hirta extracts showed minimum cytotoxicity (cell death ~2.5% and 4% in water and 70% ethanolic extract, respectively), whereas Cleome rutidosperma and Tridax procumbens’ 70% ethanolic extracts showed higher cell death (~13% and 28%, respectively), compared with the control (cell death ~10-12%). Conclusions: The study concluded that of all the medicinal weeds selected for the current study, Euphorbia hirta possesses the highest amount of bioactive compounds and hence exhibits the highest in vitro antioxidant activity and promising in vitro medicinal properties.

Background: Proteases cleave proteins, thereby providing essential amino acids for protein synthesis, and degrade misfolded and damaged proteins to maintain homeostasis. Proteases also serve as signaling molecules, therapeutic agents and find wide applications in biotechnology and pharmaceutical industry. Plant-derived proteases are suitable for many biomedical applications due to their easy availability and activity over a wide range of pH, temperature, and substrates. Moringa oleifera Lam (Moringaceae) is a very common food plant with medicinal property and geographically distributed in tropical countries. Here, we isolate proteases from the leaves of Moringa oleifera and characterize its enzymatic activity. Methods: Proteases were isolated from the aqueous leaf extract of Moringa oleifera by ammonium sulfate precipitation and purified by ion exchange chromatography. Subsequently, the enzyme kinetics was determined using casein as a substrate and calibrated over different pH and temperature range for maximal activity. Results: We obtained purified fraction of the protease having a molecular weight of 51 kDa. We observed that for the maximal caseinolytic activity of the protease, a pH of 8 and temperature of 37ºC was found to be most effective. Conclusion: The plant-derived proteolytic enzymes are finding increasing clinical and industrial applications. We could extract, purify and characterize the enzymatic activity of proteases from the leaves of Moringa oleifera. Further molecular characterization, substrate specificity and activity of the extracted protease are required for determining its suitability as a proteolytic enzyme for various applications.

The ability to deliver foreign molecules into a single living cell with high transfection efficiency and high cell viability is of great interest in cell biology for applications in therapeutic development, diagnostics and drug delivery towards personalized medicine. Many chemical and physical methods have been developed for cellular delivery, however most of these techniques are bulk approach, which are cell-specific and have low throughput delivery. On the other hand, electroporation is an efficient and fast method to deliver exogenous biomolecules such as DNA, RNA and oligonucleotides into target living cells with the advantages of easy operation, controllable electrical parameters and avoidance of toxicity. The rapid development of micro/nanofluidic technologies in the last two decades, enables us to focus an intense electric field on the targeted cell membrane to perform single cell micro-nano-electroporation with high throughput intracellular delivery, high transfection efficiency and cell viability. This review article will emphasize the basic concept and working mechanism associated with electroporation, single cell electroporation and biomolecular delivery using micro/nanoscale electroporation devices, their fabrication, working principles and cellular analysis with their advantages, limitations, potential applications and future prospects. [ABSTRACT FROM AUTHOR]