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Biological macromolecules can condense into liquid domains. In cells, these condensates form membraneless organelles that can organize chemical reactions. However, little is known about the physical consequences of chemical activity in and around condensates. Working with model bovine serum albumin (BSA) condensates, we show that droplets swim along chemical gradients. Active BSA droplets loaded with urease swim toward each other. Passive BSA droplets show diverse responses to externally applied gradients of the enzyme’s substrate and products. In all these cases, droplets swim toward solvent conditions that favor their dissolution. We call this behavior “dialytaxis”, and expect it to be generic, as conditions which favor dissolution typically reduce interfacial tension, whose gradients are well-known to drive droplet motion through the Marangoni effect. These results could potentially suggest alternative physical mechanisms for active transport in living cells, and may enable the design of fluid micro-robots. Here the authors identify a generic coupling in phase-separated liquids between motility and phase equilibria perturbations: phase-separated droplets swim to their dissolution. This suggests alternative transport mechanism for biomolecular condensates.

Bio-catalytic micro- and nanomotors self-propel by the enzymatic conversion of substrates into products. Despite the advances in the field, the fundamental aspects underlying enzyme-powered self-propulsion have rarely been studied. In this work, we select four enzymes (urease, acetylcholinesterase, glucose oxidase, and aldolase) to be attached on silica microcapsules and study how their turnover number and conformational dynamics affect the self-propulsion, combining both an experimental and molecular dynamics simulations approach. Urease and acetylcholinesterase, the enzymes with higher catalytic rates, are the only enzymes capable of producing active motion. Molecular dynamics simulations reveal that urease and acetylcholinesterase display the highest degree of flexibility near the active site, which could play a role on the catalytic process. We experimentally assess this hypothesis for urease micromotors through competitive inhibition (acetohydroxamic acid) and increasing enzyme rigidity (β-mercaptoethanol). We conclude that the conformational changes are a precondition of urease catalysis, which is essential to generate self-propulsion. Self-propulsion of biocatalytic micro- and nanomotors is facilitated by enzymes converting substrates into products. Here, the authors show that intrinsic enzymatic properties such as conformational changes are crucial for the self-propulsion of silica microcapsules modified with urease.

Living cells harvest energy from their environments to drive the chemical processes that enable life. We introduce a minimal system that operates at similar protein concentrations, metabolic densities, and length scales as living cells. This approach takes advantage of the tendency of phase-separated protein droplets to strongly partition enzymes, while presenting minimal barriers to transport of small molecules across their interface. By dispersing these microreactors in a reservoir of substrate-loaded buffer, we achieve steady states at metabolic densities that match those of the hungriest microorganisms. We further demonstrate the formation of steady pH gradients, capable of driving microscopic flows. Our approach enables the investigation of the function of diverse enzymes in environments that mimic cytoplasm, and provides a flexible platform for studying the collective behavior of matter driven far from equilibrium. Living cells can harvest environmental energy to drive chemical processes. Here the authors design a minimal artificial system that achieves steady states at similar metabolic densities to microorganisms.

Examining enzyme kinetics is critical for understanding cellular systems and for using enzymes in industry. The Michaelis-Menten equation has been widely used for over a century to estimate the enzyme kinetic parameters from reaction progress curves of substrates, which is known as the progress curve assay. However, this canonical approach works in limited conditions, such as when there is a large excess of substrate over enzyme. Even when this condition is satisfied, the identifiability of parameters is not always guaranteed, and often not verifiable in practice. To overcome such limitations of the canonical approach for the progress curve assay, here we propose a Bayesian approach based on an equation derived with the total quasi-steady-state approximation. In contrast to the canonical approach, estimates obtained with this proposed approach exhibit little bias for any combination of enzyme and substrate concentrations. Importantly, unlike the canonical approach, an optimal experiment to identify parameters with certainty can be easily designed without any prior information. Indeed, with this proposed design, the kinetic parameters of diverse enzymes with disparate catalytic efficiencies, such as chymotrypsin, fumarase, and urease, can be accurately and precisely estimated from a minimal amount of timecourse data. A publicly accessible computational package performing such accurate and efficient Bayesian inference for enzyme kinetics is provided.

Clinically significant problems such as kidney stones and stomach ulcers are linked to the activation of the urease enzyme. At low pH, this enzyme gives an ideal environment to Helicobacter pylori in the stomach which is the cause of gastric ulcers and peptic ulcers. In recent work, we have developed a library of 4-fluorocinnamaldehyde base thiosemicarbazones and assessed them for their potential against urease enzyme. The synthesized compounds displayed significant to moderate inhibition potential with IC 50 values ranging from 2.7 ± 0.5 µM to 29.0 ± 0.5 µM. compound 3c displayed the highest inhibition potential followed by 3a and 3b . Two compounds of the series 3f and 3 g remained inactive against urease. The kinetic study of compound 3c exhibited a competitive type of inhibition with a K i value of 3.26 ± 0.0048 µM. SAR analysis was also thoroughly done. Molecular docking was used to analyze the interaction pattern of each derivative, and the outcomes demonstrated that the compounds had excellent binding interactions with the active site.

This study examines the chemical composition, antioxidant properties, and urease inhibitory effects of Hyoscyamus muticus L. subsp. falezlez (Coss.) Maire. Using LC-ESI-MS/MS, 19 distinct phenolic compounds were identified, with chlorogenic acid being the most abundant. The ethanol extract demonstrated notable antioxidant activity, highlighting its potential for therapeutic use. Urease inhibition assays revealed a remarkable 91.35% inhibition by the H. muticus extract, with an IC50 value of 5.6 ± 1.20 μg/mL, indicating its promising role in addressing conditions linked to urease activity. Molecular docking studies further investigated the interaction between H. muticus phenolic compounds and urease, identifying hyperoside as a leading candidate, with a binding energy of −7.9 kcal/mol. Other compounds, such as rutin, luteolin, apigenin, kaempferol, hesperetin, chlorogenic acid, and rosmarinic acid, also demonstrated significant binding affinities, suggesting their potential to disrupt urease function. These findings highlight the therapeutic potential of H. muticus as a source of natural bioactive compounds, offering promising avenues for the development of novel treatments for urease-related disorders and oxidative stress.

The development of new bioactive compounds is important for progress in therapeutic research. In the present study, we describe the multistep synthetic approach to develop a library of novel benzimidazole analogs incorporating piperazine rings in order to increase their biological activity. In order to synthesize the desired benzimidazole analogs, the synthesis started with the easily accessible precursors between aniline and chloroacetyl chloride. It proceeded via a series of reactions, such as condensation, cyclization, and N -alkylation. TLC optimized each step, and spectroscopic methods such as CHN, IR, EIMS, 1 H-NMR, and 13 C-NMR were used to characterize the final products. The urease inhibitory activity of the synthesized compounds was evaluated. It was discovered that almost all compounds were quite effective, even more potent (IC 50  = 0.15–12.17 µ M) than the standard thiourea (IC 50  = 23.11 ± 0.21 µ M). The structure-activity relationship (SAR) is also established, which displayed that compound 9 L (IC 50  = 0.15 ± 0.09 µ M) with -NO 2 substitutions at meta position play a major role in urease inhibition and figure out as the most potent analog of the library. These results were further verified by molecular docking analysis, which indicated favorable binding energies and interactions of the compounds with the urease active site. This study not only depicts the importance of multistep synthesis but also the structure-based modification approach to produce new pharmacophores for therapeutic applications.

Non-mechanical nano- and microscale pumps that function without the aid of an external power source and provide precise control over the flow rate in response to specific signals are needed for the development of new autonomous nano- and microscale systems. Here we show that surface-immobilized enzymes that are independent of adenosine triphosphate function as self-powered micropumps in the presence of their respective substrates. In the four cases studied (catalase, lipase, urease and glucose oxidase), the flow is driven by a gradient in fluid density generated by the enzymatic reaction. The pumping velocity increases with increasing substrate concentration and reaction rate. These rechargeable pumps can be triggered by the presence of specific analytes, which enables the design of enzyme-based devices that act both as sensor and pump. Finally, we show proof-of-concept enzyme-powered devices that autonomously deliver small molecules and proteins in response to specific chemical stimuli, including the release of insulin in response to glucose. Self-powered micropumps that are turned on by the presence of their respective substrates are formed from surface-immobilized, ATP-independent enzymes. Coupling substrate-sensing with transport enables the design of devices that deliver cargo in response to specific stimuli. Demonstrated here is the release of insulin at a rate proportional to ambient glucose concentration.

This study reports the plant extract-assisted synthesis of iron oxide (Fe 3 O 4 ) using the aqueous extract of Thevetia peruviana . The synthesized IONPs were confirmed via UV–Vis spectroscopy (295 nm) and characterized using FTIR and SEM. Density Functional Theory (DFT) calculations indicated a thermodynamically and mechanically stable system with semimetallic behavior and visible light absorption. The biological activities of the IONPs were evaluated, including enzyme inhibition assays for urease, α-glucosidase, carbonic anhydrase-II, and xanthine oxidase, as well as anticancer activity. The Fe₃O₄ NPs exhibited potent enzyme inhibition, including urease (94.78%, IC₅₀ = 24.98 µg/mL), α-glucosidase (86.09%), and carbonic anhydrase-II (82.98%, IC₅₀ = 24.78 µg/mL). Additionally, molecular docking was performed to evaluate the interaction of Fe₃O₄ NPs with target enzymes, supporting their inhibitory potential. The NPs also demonstrated notable anticancer activity, particularly against MDR 2780AD (IC₅₀ = 0.39 µg/mL). These results showed significant enzyme inhibition and anticancer properties, indicating the potential of these green-synthesized IONPs in biomedical applications.

A novel and sensitive fluorescence ratiometric method is developed for urea detection based  on the pH-sensitive response of two fluorescent carbon dot (CD) systems: R-CDs/methyl red (MR) and NIR-CDs/Cu 2+ . The sensing mechanism involves breaking down urea using the enzyme urease, releasing ammonia and increasing pH. At higher pH, the fluorescence of NIR-CDs is quenched due to the enhanced interaction with Cu 2+ , while the fluorescence of R-CDs is restored as the acidic MR converts to its basic form, removing the inner filter effect. The ratiometric signal (F 608 /F 750 ) of the R-CDs/MR and NIR-CDs/Cu 2+ intensities changed in response to the pH induced by urea hydrolysis, enabling selective and sensitive urea detection. Detailed spectroscopic and morphological investigations confirmed the fluorescence probe design and elucidated the sensing mechanism. The method exhibited excellent sensitivity (0.00028 mM LOD) and linearity range (0.001 – 8.0 mM) for urea detection, with successful application in milk samples for monitoring adulteration, demonstrating negligible interference and high recovery levels (96.5% to 101.0%). This ratiometric fluorescence approach offers a robust strategy for selective urea sensing in complicated matrices. Graphical Abstract