Investigation of electrocatalysis for tiered-tower micro-electro-mechanical-system-based biosensors: application in the early detection of the thrombosis factor trimethylamine N-oxide.

Trimethylamine N-oxide (TMAO) has been recognized as a biomarker for the early detection of thrombosis. However, testing for TMAO typically requires expensive laboratory equipment and skilled technicians, making it unsuitable for home care pre-screening. To enable its widespread use in home applications, it is crucial to develop a scalable and sensitive device capable of catalyzing TMAO metabolism with a specific enzyme that is tailored for point-of-care use. This study presents an investigation of a MEMS-based two-tiered-tower biosensor array with a detection limit of 0.1 µM for TMAO, aiming to diagnose chronic metabolic diseases using urine or serum samples. Based on the augmented Cole-Cole model, the proposed parameters R_catalyzed, C_catalyzed, and Rp_catalyzed can predict the catalytic impedance of enzymatic activities such as the redox effects of analytes and characterize the small-signal current caused by catalysis. The proposed MEMS biosensor, integrated with a readout circuitry, demonstrates a high sensitivity of 41 ADC counts per µM TMAO (or 4.5 mV µM-1 TMAO), a response time of 1 second, a repetition rate of 98.9%, and a drift over time of 0.5 mV. The sensor effectively distinguishes TMAO based on minute capacitance changes induced by the TorA enzyme, resulting in a discernible distinction of 10.6%. These measurements were successfully compared to conventional cyclic voltammetry (CV) results, showing a variance of only 0.024%. The proposed biosensor is well-suited for pre-screening thrombosis factors for the early detection and prevention of thrombosis in point-of-care applications. The device is cost-effective, lightweight, and demonstrates excellent performance, with a conversion rate of 88% of TMAO and a selectivity rate of 97% for the by-product TMA, allowing for the prediction of cardiovascular risks.

Native Mass Spectrometry of Membrane Protein-Lipid Interactions in Different Detergent Environments.

Native mass spectrometry (MS) reveals the role of specific lipids in modulating membrane protein structure and function. Membrane proteins solubilized in detergents are often introduced into the mass spectrometer. However, detergents commonly used for structural studies, such as dodecylmaltoside, tend to generate highly charged ions, leading to protein unfolding, thereby diminishing their utility in characterizing protein-lipid interactions. Thus, there is a critical need to develop approaches to investigate protein-lipid interactions in different detergents. Here, we demonstrate how charge-reducing molecules, such as spermine and trimethylamine-N-oxide, enable the opportunity to characterize lipid binding to the bacterial water channel (AqpZ) and ammonia channel (AmtB) in complex with regulatory protein GlnK in different detergent environments. We find that protein-lipid interactions not only are protein-dependent but also can be influenced by the detergent and type of charge-reducing molecule. AqpZ-lipid interactions are enhanced in LDAO (n-dodecyl-N,N-dimethylamine-N-oxide), whereas the interaction of AmtB-GlnK with lipids is comparable among different detergents. A fluorescent lipid binding assay also shows detergent dependence for AqpZ-lipid interactions, consistent with results from native MS. Taken together, native MS will play a pivotal role in establishing optimal experimental parameters that will be invaluable for various applications, such as drug discovery as well as biochemical and structural investigations.

Trimethylamine N-oxide detection by electrochemical sensor based on screen printed electrode modified with molecularly imprinted polypyrrole-molybdenum(III) sulfide nanosheets.

Trimethylamine N-oxide (TMAO) is a gut metabolite produced by dietary L-carnitine and choline metabolism. Its altered level in the serum has been implicated in human health and diseases such as colorectal cancer, chronic kidney diseases, cardiovascular diseases, etc. Early detection of TMAO in body fluids has been presumed to be significant in understanding the pathogenesis and treatment of many diseases. Hence, developing reliable and rapid technologies for its detection may augment our understanding of pathogenesis and diagnosis of diseases. Hence, in the present work, polypyrrole (Ppy)@molybdenum(III)sulfide (Mo2S3) nanosheets (NS) composite molecularly imprinted polymer (MIP) (Ppy@Mo2S3-MIP) based electrochemical sensor has been fabricated for the detection of TMAO. Polypyrrole (Ppy) and Mo2S3 NS have been synthesized by chemical oxidative polymerization and hydrothermal techniques, respectively. The synthesized nanocomposite has been validated using different techniques such as X-ray diffraction (XRD), Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The fabricated Ppy@Mo2S3-MIP sensor showed a linear detection range from 30 µM to 210 µM, a sensitivity of 1.21 µA µM-1 cm-2 and a limit of detection as 1.4 µM for the detection of TMAO and found more robust and improved when compared with Ppy-MIP using identical parameters. The fabricated sensor is also highly selective towards TMAO. It can be further used to detect TMAO in human samples such as urine quickly.

Do the biogenic amines ethylamine, ethanolamine and methylamine reach toxic concentrations in foods?

Ethylamine, ethanolamine and methylamine are biogenic amines (BA) - active metabolites that, despite having important biological functions, may accumulate at toxic concentrations in certain foods. Very little information exists on the toxicity of these BA in this context. This study provides new insights into their cytotoxicity with respect to a human intestinal epithelial cell line, as assessed using real-time cell analyzer technology. A preliminary evaluation of the cytotoxic mode of action was also performed. The present results show that only ethylamine was cytotoxic for these cells at food concentrations. These new data should help establish legal limits for these BA in foods.

Destabilization of the D2 domain of Thermotoga maritima arginine binding protein induced by guanidinium thiocyanate and its counteraction by stabilizing agents.

D2 is a structural and cooperative domain of Thermotoga maritima Arginine Binding Protein, that possesses a remarkable conformational stability, with a denaturation temperature of 102.6°C, at pH 7.4. The addition of potassium thiocyanate causes a significant decrease in the D2 denaturation temperature. The interactions of thiocyanate ions with D2 have been studied by means of isothermal titration calorimetry measurements and molecular dynamics simulations. It emerged that: (a) 20-30 thiocyanate ions interact with the D2 surface and are present in its first solvation shell; (b) each of them makes several contacts with protein groups, both polar and nonpolar ones. The addition of guanidinium thiocyanate causes a marked destabilization of the D2 native state, because both the ions are denaturing agents. However, on adding to the solution containing D2 and guanidinium thiocyanate a stabilizing agent, such as TMAO, sucrose or sodium sulfate, a significant increase in denaturation temperature occurs. The present results confirm that counteraction is a general phenomenon for globular proteins.

Trimethylamine N-oxide (TMAO) doubly locks the hydrophobic core and surfaces of protein against desiccation stress.

Interactions between proteins and osmolytes are ubiquitous within cells, assisting in response to environmental stresses. However, our understanding of protein-osmolyte interactions underlying desiccation tolerance is limited. Here, we employ solid-state NMR (ssNMR) to derive information about protein conformation and site-specific interactions between the model protein, SH3, and the osmolyte trimethylamine N-oxide (TMAO). The data show that SH3-TMAO interactions maintain key structured regions during desiccation and facilitate reversion to the protein's native state once desiccation stress is even slightly relieved. We identify 10 types of residues at 28 sites involved in the SH3-TMAO interactions. These sites comprise hydrophobic, positively charged, and aromatic amino acids located in SH3's hydrophobic core and surface clusters. TMAO locks both the hydrophobic core and surface clusters through its zwitterionic and trimethyl ends. This double locking is responsible for desiccation tolerance and differs from ideas based on exclusion, vitrification, and water replacement. ssNMR is a powerful tool for deepening our understanding of extremely weak protein-osmolyte interactions and providing insight into the evolutionary mechanism of environmental tolerance.

Modulation of Biomolecular Liquid-Liquid Phase Separation by Preferential Hydration and Interaction of Small Osmolytes with Proteins.

We examined the effects of trimethylamine N-oxide (TMAO) and urea (known osmolytes) on the liquid-liquid phase separation (LLPS) of fused in sarcoma (FUS) and three FUS-LLPS states: LLPS states at atmospheric pressure with low- and high-salt concentrations and a re-entrant LLPS state above 2 kbar. Temperature- and pressure-scan turbidity measurements revealed that TMAO and urea contributed to stabilizing and destabilizing LLPS, respectively. These results can be attributed to the excluded volume effect of TMAO (preferential hydration) and preferential interaction of urea with proteins. Additionally, TMAO counteracted the effects of equimolar urea on LLPS, a phenomenon not previously reported. The concept of the m-value for osmolyte-induced protein folding and unfolding can be applied to the osmolyte's effects on LLPS. In conclusion, biomolecular LLPS can be modulated by preferential hydration and the interaction of small osmolytes with proteins, thereby facilitating LLPS formation, even in extreme environments characterized by high-salt, high-urea, and high-pressure conditions.

Michael addition-activated alkylation of G-quadruplex DNA with methylamine-protected vinyl-quinazolinone derivatives.

The role of G-quadruplex (G4) in cellular processes can be investigated by the covalent modification of G4-DNA using alkylating reagents. Controllable alkylating reagents activated by external stimuli can react elegantly and selectively. Herein, we report a chemical activation system that can significantly boost the reaction rate of methylamine-protected vinyl-quinazolinone (VQ) derivative for the alkylation of G4-DNA. The two screened activators can transform low-reactive VQ-NHR' to highly reactive intermediates following the Michael addition mechanism. This approach expands the toolbox of activable G4 alkylating reagents.

Insights into the dynamic interactions of RNase a and osmolytes through computational approaches.

Osmolytes are small organic molecules that are known to stabilize proteins and other biological macromolecules under various stressful conditions. They belong to various categories such as amino acids, methylamines, and polyols. These substances are commonly known as 'compatible solutes' because they do not disrupt cellular processes and help regulate the osmotic balance within cells. In the case of ribonuclease A (RNase A), which is prone to aggregation, the presence of osmolytes can help to maintain its structural stability and prevent unwanted interactions leading to protein aggregation. In this study, we investigated the interaction between RNase A and several osmolytes using molecular docking and molecular dynamics (MD) simulations. We performed molecular docking to predict the binding mode and binding affinity of each osmolyte with RNase A. MD simulations were then carried out to investigate the dynamics and stability of the RNase A-osmolyte complexes. Our results show that two osmolytes, glucosylglycerol and sucrose have favorable binding affinities with RNase A. The possible role of these osmolytes in stabilizing the RNase A and prevention of aggregation is also explored. By providing computational insights into the interaction between RNase A and osmolytes, the study offers valuable information that could aid in comprehending the mechanisms by which osmolytes protect proteins and help in designing therapeutics for protein-related disorders based on osmolytes. These findings may have significant implications for the development of novel strategies aimed at preventing protein misfolding and aggregation in diverse disease conditions.Communicated by Ramaswamy H. Sarma.

Tracing Immunological Interaction in Trimethylamine N-Oxide Hydrogel-Derived Zwitterionic Microenvironment During Promoted Diabetic Wound Regeneration.

The diabetic wound healing is challenging due to the sabotaged delicate balance of immune regulation via an undetermined pathophysiological mechanism, so it is crucial to decipher multicellular signatures underlying diabetic wound healing and seek therapeutic strategies. Here, this work develops a strategy using novel trimethylamine N-oxide (TMAO)-derived zwitterionic hydrogel to promote diabetic wound healing, and explore the multi-cellular ecosystem around zwitterionic hydrogel, mapping out an overview of different cells in the zwitterionic microenvironment by single-cell RNA sequencing. The diverse cellular heterogeneity is revealed, highlighting the critical role of macrophage and neutrophils in managing diabetic wound healing. It is found that polyzwitterionic hydrogel can upregulate Ccl3+ macrophages and downregulate S100a9+ neutrophils and facilitate their interactions compared with polyanionic and polycationic hydrogels, validating the underlying effect of zwitterionic microenvironment on the activation of adaptive immune system. Moreover, zwitterionic hydrogel inhibits the formation of neutrophil extracellular traps (NETs) and promotes angiogenesis, thus improving diabetic wound healing. These findings expand the horizons of the sophisticated orchestration of immune systems in zwitterion-directed diabetic wound repair and uncover new strategies of novel immunoregulatory biomaterials.

Mo(IV) Ion-Modulated BSA-Protected Gold Nanocluster Probe for Fluorescence Turn-On Detection of Trimethylamine N-Oxide (TMAO).

Trimethylamine N-oxide (TMAO), a molecule produced by the microbiota, has been associated with human health and illness. Its early discovery in body fluids may affect our understanding of the pathophysiology and treatment of many illnesses. Therefore, our knowledge of the pathophysiology and diagnostics of disorders associated with TMAO might be enhanced by the creation of dependable and fast methods for TMAO detection. Therefore, we developed a fluorescent probe for detecting TMAO utilizing an on-off-on strategy. Bovine serum albumin (BSA)@AuNCs luminescence is effectively quenched by Mo4+ because BSA@AuNCs and Mo4+ have a strong binding relationship. Mo4+ ions can substantially decrease the emission intensity of gold nanoclusters by establishing a BSA@AuNCs-Mo system. Then, the luminescence of BSA@AuNCs was restored due to the interaction between Mo4+ and TMAO. A significant linear relationship was seen between the emission intensity and TMAO concentration within the 0-201 µM range, with a detection limit of 1.532 µM. Additionally, the method can measure TMAO in blood and urine samples.

High-Performance Trimethylamine Sensor Based on an Imine Covalent Organic Framework.

As trimethylamine (TMA) is widely used in agriculture and industry, inhalation of TMA can cause very serious negative effects on human health. However, most of the current gas sensors for detecting TMA are commonly performed at high temperatures and cannot meet market needs. Inspired by this, we prepared imine covalent organic frameworks (TB-COF) synthesized from two monomers, 1,3,5-tris(4-aminophenyl)benzene (TAPB) and 1,3,5-benzotricarboxaldehyde (BTCA), using acetic acid as a catalyst at room temperature. Based on this, three sensors were prepared for gas sensitivity testing, namely, TA, BT, and TB-COF sensors. The three sensors were tested for 15 different gases at room temperature. From the whole gas sensitivity data, the TB-COF sensor made by compositing TA and BT has a higher sensitivity (6845.9%) to TMA at 500 ppm, which is 6.1 and 5.4 times higher than the response of TA and BT sensors, respectively. The TB-COF sensor adsorbs and desorbs TMA in a controlled 23 s cycle with a low detection limit of 28.6 ppb. This result indicates that TB-COF prepared at room temperature can be used as a gas-sensitive sensing material for real-time monitoring of TMA. The gas sensing results demonstrate the great potential of COFs for sensor development and application and provide ideas for further development of COFs-based gas sensors.

Fast real-time monitoring of meat freshness based on fluorescent sensing array and deep learning: From development to deployment.

A fluorescent sensor array (FSA) combined with deep learning (DL) techniques was developed for meat freshness real-time monitoring from development to deployment. The array was made up of copper metal nanoclusters (CuNCs) and fluorescent dyes, having a good ability in the quantitative and qualitative detection of ammonia, dimethylamine, and trimethylamine gases with a low limit of detection (as low as 131.56 ppb) in range of 5 âˆ¼ 1000 ppm and visually monitoring the freshness of various meats stored at 4 °C. Moreover, SqueezeNet was applied to automatically identify the fresh level of meat based on FSA images with high accuracy (98.17 %) and further deployed in various production environments such as personal computers, mobile devices, and websites by using open neural network exchange (ONNX) technique. The entire meat freshness recognition process only takes 5 âˆ¼ 7 s. Furthermore, gradient-weighted class activation mapping (Grad-CAM) and uniform manifold approximation and projection (UMAP) explanatory algorithms were used to improve the interpretability and transparency of SqueezeNet. Thus, this study shows a new idea for FSA assisted with DL in meat freshness intelligent monitoring from development to deployment.

Effect of trimethylamine-N-oxide on the phase separation of aqueous polyethylene glycol-600-Dextran-75 two-phase systems.

The emergence of phase separation in both intracellular biomolecular condensates (membrane-less organelles) and in vitro aqueous two-phase systems (ATPS) relies on the formation of immiscible water-based phases/domains. The solvent properties and arrangement of hydrogen bonds within these domains have been shown to differ and can be modulated with the addition of various inorganic salts and osmolytes. The naturally occuring osmolyte, trimethylamine-N-oxide (TMAO), is well established as a biological condensate stabilizer whose presence results in enhanced phase separation of intracellular membrane-less compartments. Here, we show the unique effect of TMAO on the mechanism of phase separation in model PEG-600-Dextran-75 ATPS using dynamic and static light scattering in conjunction with ATR-FTIR and solvatochromic analysis. We observe that the presence of TMAO may enhance or destabilize phase separation depending on the concentration of phase forming components. Additionally, the behavior and density of mesoscopic polymer agglomerates, which arise prior to macroscopic phase separation, are altered by the presence and concentration of TMAO.

Trimethylamine-N-oxide depletes urea in a peptide solvation shell.

Trimethylamine-N-oxide (TMAO) and urea are metabolites that are used by some marine animals to maintain their cell volume in a saline environment. Urea is a well-known denaturant, and TMAO is a protective osmolyte that counteracts urea-induced protein denaturation. TMAO also has a general protein-protective effect, for example, it counters pressure-induced protein denaturation in deep-sea fish. These opposing effects on protein stability have been linked to the spatial relationship of TMAO, urea, and protein molecules. It is generally accepted that urea-induced denaturation proceeds through the accumulation of urea at the protein surface and their subsequent interaction. In contrast, it has been suggested that TMAO's protein-stabilizing effects stem from its exclusion from the protein surface, and its ability to deplete urea from protein surfaces; however, these spatial relationships are uncertain. We used neutron diffraction, coupled with structural refinement modeling, to study the spatial associations of TMAO and urea with the tripeptide derivative glycine-proline-glycinamide in aqueous urea, aqueous TMAO, and aqueous urea-TMAO (in the mole ratio 1:2 TMAO:urea). We found that TMAO depleted urea from the peptide's surface and that while TMAO was not excluded from the tripeptide's surface, strong atomic interactions between the peptide and TMAO were limited to hydrogen bond donating peptide groups. We found that the repartition of urea, by TMAO, was associated with preferential TMAO-urea bonding and enhanced urea-water hydrogen bonding, thereby anchoring urea in the bulk solution and depleting urea from the peptide surface.

Structurally different chemical chaperones show similar mechanical roles with independent molecular mechanisms.

Osmolytes are well known to protect the protein structure against different chemical and physical denaturants. Since their actions with protein surfaces are mechanistically complicated and context dependent, the underlying molecular mechanism is not fully understood. Here, we combined single-molecule magnetic tweezers and molecular dynamics (MD) simulation to explore the mechanical role of osmolytes from two different classes, trimethylamine N-oxide (TMAO) and trehalose, as mechanical stabilizers of protein structure. We observed that these osmolytes increase the protein L mechanical stability by decreasing unfolding kinetics while accelerating the refolding kinetics under force, eventually shifting the energy landscape toward the folded state. These osmolytes mechanically stabilize the protein L and plausibly guide them to more thermodynamically robust states. Finally, we observed that osmolyte-modulated protein folding increases mechanical work output up to twofold, allowing the protein to fold under a higher force regime and providing a significant implication for folding-induced structural stability in proteins.

Modulation of protein-saccharide interactions by deep-sea osmolytes under high pressure stress.

Deep-sea organisms must cope with high hydrostatic pressures (HHP) up to the kbar regime to control their biomolecular processes. To alleviate the adverse effects of HHP on protein stability most organisms use high amounts of osmolytes. Little is known about the effects of these high concentrations on ligand binding. We studied the effect of the deep-sea osmolytes trimethylamine-N-oxide, glycine, and glycine betaine on the binding between lysozyme and the tri-saccharide NAG3, employing experimental and theoretical tools to reveal the combined effect of osmolytes and HHP on the conformational dynamics, hydration changes, and thermodynamics of the binding process. Due to their different chemical makeup, these cosolutes modulate the protein-sugar interaction in different ways, leading to significant changes in the binding constant and its pressure dependence. These findings suggest that deep-sea organisms may down- and up-regulate reactions in response to HHP stress by altering the concentration and type of the intracellular osmolyte.

Molecular Aggregation Behavior and Microscopic Heterogeneity in Binary Osmolyte-Water Solutions.

Osmolytes, small organic compounds, play a key role in modulating the protein stability in aqueous solutions, but the operating mechanism of the osmolyte remains inconclusive. Here, we attempt to clarify the mode of osmolyte action by quantitatively estimating the microheterogeneity of osmolyte-water mixtures with the aid of molecular dynamics simulation, graph theoretical analysis, and spatial distribution measurement in the four osmolyte solutions of trimethylamine-N-oxide (TMAO), tetramethylurea (TMU), dimethyl sulfoxide, and urea. TMAO, acting as a protecting osmolyte, tends to remain isolated with no formation of osmolyte aggregates while preferentially interacting with water, but there is a strong aggregation propensity in the denaturant TMU solution, characterized by favored hydrophobic interactions between TMU molecules. Taken together, the mechanism of osmolyte action on protein stability is proposed as a comprehensive one that encompasses the direct interactions between osmolytes and proteins and indirect interactions through the regulation of water properties in the osmolyte-water mixtures.

Identification of selective 5-LOX and FLAP inhibitors as novel anti-inflammatory agents by ligand-based virtual screening.

Inflammation is a multifaceted biological process in which the conversion of arachidonic acid to eicosanoids, including prostaglandins and leukotrienes (LTs), plays a crucial role. 5-Lipoxygenase (5-LOX) is a key enzyme in cellular LT biosynthesis, and it is supported by the accessory protein 5-lipoxygenase-activating protein (FLAP). Pharmacological interventions to modulate LTs aim at either decreasing their biosynthesis or at mitigating their biological effects. Therefore, inhibiting 5-LOX or FLAP represents a useful strategy to reduce inflammation. Herein we present the identification and pharmacological evaluation of novel inhibitors targeting 5-LOX or FLAP. By means of a ligand-based virtual screening approach, we selected 38 compounds for in vitro assays. Among them, ALR-38 exhibits direct 5-LOX inhibition, while ALR-6 and ALR-27 showed potential as FLAP inhibitors. These latter not only reduced LT production but also promoted the generation of specialized pro-resolving mediators in specific human macrophage phenotypes. Interestingly, the identified compounds turned out to be selective for their respective targets, as none of them displayed activity towards microsomal prostaglandin E2 synthase-1 and soluble epoxide hydrolase, which are other proteins involved in eicosanoid biosynthesis. Thus, these compounds are endowed with potential therapeutic utility in mitigating inflammatory responses and might offer a venue for tackling inflammation-based disorders.

TMAO: Protecting proteins from feeling the heat.

Osmolytes are ubiquitous in the cell and play an important role in controlling protein stability under stress. The natural osmolyte trimethylamine N-oxide (TMAO) is used by marine animals to counteract the effect of pressure denaturation at large depths. The molecular mechanism of TMAO stabilization against pressure and urea denaturation has been extensively studied, but unlike the case of other osmolytes, the ability of TMAO to protect proteins from high temperature has not been quantified. To reveal the effect of TMAO on folded and unfolded protein ensembles and the hydration shell at different temperatures, we study a mutant of the well-characterized, fast-folding model protein B (PRB). We carried out, in total, >190 µs all-atom simulations of thermal folding/unfolding of PRB at multiple temperatures and concentrations of TMAO. The simulations show increased thermal stability of PRB in the presence of TMAO. Partly structured, compact ensembles are favored over the unfolded state. TMAO forms two shells near the protein: an outer shell away from the protein surface has altered H-bond lifetimes of water molecules and increases hydration of the protein to help stabilize it; a less-populated inner shell with an opposite TMAO orientation closer to the protein surface binds exclusively to basic side chains. The cooperative cosolute effect of the inner and outer shell TMAO has a small number of TMAO molecules "herding" water molecules into two hydration shells at or near the protein surface. The stabilizing effect of TMAO on our protein saturates at 1 M despite higher TMAO solubility, so there may be little evolutionary pressure for extremophiles to produce higher intracellular TMAO concentrations, if true in general.