Search Results (20)

Long-range intercellular communication is essential for multicellular biological systems to regulate multiscale cell–cell interactions and maintain life. Growing evidence suggests that intercellular calcium waves (ICWs) act as a class of long-range signals that influence a broad spectrum of cellular functions and behaviors. Importantly, mechanical signals, ranging from single-molecule-scale to tissue-scale in vivo, can initiate and modulate ICWs in addition to relatively well-appreciated biochemical and bioelectrical signals. Despite these recent conceptual and experimental advances, the full nature of underpinning mechanotransduction mechanisms by which cells convert mechanical signals into ICW dynamics remains poorly understood. This review provides a systematic analysis of quantitative ICW dynamics around three main stages: initiation, propagation, and regeneration/relay. We highlight the landscape of upstream molecules and organelles that sense and respond to mechanical stimuli, including mechanosensitive membrane proteins and cytoskeletal machinery. We clarify the roles of downstream molecular networks that mediate signal release, spread, and amplification, including adenosine triphosphate (ATP) release, purinergic receptor activation, and gap junction (GJ) communication. Furthermore, we discuss the broad pathophysiological implications of ICWs, covering pathophysiological processes such as cancer metastasis, tissue repair, and developmental patterning. Finally, we summarize recent advances in optical imaging and artificial intelligence (AI)/machine learning (ML) technologies that reveal the precise spatial-temporal-functional dynamics of ICWs and ATP waves. By synthesizing these insights, we offer a comprehensive framework of ICW mechanobiology and propose new directions for mechano-therapeutic strategies in disease diagnosis, cancer immunotherapies, and drug discovery. Full article

Enzyme catalysis represents a promising approach for sustainable chemical synthesis, yet its industrial applications face limitations due to the inefficient regeneration and high cost of essential cofactors, such as adenosine-5′-triphosphate (ATP) and nicotinamide adenine dinucleotide phosphate (NADPH). While natural metabolic systems efficiently recycle cofactors through spatially organized enzymes, replicating this efficiency in vitro remains challenging. Here, we prepare a five-enzyme condensate system using liquid–liquid phase separation (LLPS) mediated by intrinsically disordered proteins (IDPs). By colocalizing a carboxylic acid reductase from Norcadia iowensis (NiCAR) with a reductive aminase from Aspergillus oryzae (AspRedAm) and three cofactor-regenerating enzymes, we generated a phase-separated catalytic condensate that enhanced ATP and NADPH recycling efficiency by 4.7-fold and 1.9-fold relative to free enzymes, respectively. Catalytic performance was correlated with the extent of phase separation, as confirmed by fluorescence microscopy, which revealed clear enrichment of ATP and NADPH within the condensates. This proximity effect enabled efficient cofactor turnover in the one-step reaction, achieving substrate conversion above 90% within 6 h and enhancing the space–time yield (STY) of the chiral imines 1.6-fold, with only one-fifth of the standard cofactor load. This approach creates a scalable and economic tool for performing multienzyme cascade reactions in vitro that are driven by the efficient recycling of multiple cofactors. Full article

Nucleoside-5′-triphosphates (5′-NTPs) are essential building blocks of nucleic acids in nature and play an important role in molecular biology, diagnostics, and mRNA therapeutic synthesis. Chemical synthesis has long been the standard method for producing modified 5′-NTPs. However, chemical routes face limitations, including low regio- and stereoselectivity, along with the need for protection/deprotection cycles, resulting in low yields, high costs, and lengthy processes. In contrast, enzymatic synthesis methods offer significant advantages, such as improved regio- and stereoselectivity and the use of mild reaction conditions, which often leads to higher product yields in “one-pot” reactions. Despite the extensive review of chemical synthesis routes for 5′-NTPs, there has not yet been any comprehensive analysis of enzymatic approaches. Initially, this review provides a brief overview of the enzymes involved in nucleotide metabolism, introducing valuable biocatalysts for 5’-NTP synthesis. Furthermore, the available enzymatic methods for efficient 5′-NTP synthesis using purified enzymes and starting from either nucleobases or nucleosides are examined, highlighting their respective advantages and disadvantages. Special attention is also given to the importance of ATP regeneration systems for 5′-NTP synthesis. We aim to demonstrate the remarkable potential of enzymatic in vitro cascade reactions, promoting their broader application in both basic research and industry. Full article

Long-lasting brain fatigue is a consequence of stroke or traumatic brain injury associated with emotional, psychological, and physical overload, distress in hypertension, atherosclerosis, viral infection, and aging-related chronic low-grade inflammatory disorders. The pathogenesis of brain fatigue is linked to disrupted neurotransmission, the glutamate-glutamine cycle imbalance, glucose metabolism, and ATP energy supply, which are associated with multiple molecular targets and signaling pathways in neuroendocrine-immune and blood circulation systems. Regeneration of damaged brain tissue is a long-lasting multistage process, including spontaneously regulating hypothalamus-pituitary (HPA) axis-controlled anabolic–catabolic homeostasis to recover harmonized sympathoadrenal system (SAS)-mediated function, brain energy supply, and deregulated gene expression in rehabilitation. The driving mechanism of spontaneous recovery and regeneration of brain tissue is a cross-talk of mediators of neuronal, microglia, immunocompetent, and endothelial cells collectively involved in neurogenesis and angiogenesis, which plant adaptogens can target. Adaptogens are small molecules of plant origin that increase the adaptability of cells and organisms to stress by interaction with the HPA axis and SAS of the stress system (neuroendocrine-immune and cardiovascular complex), targeting multiple mediators of adaptive GPCR signaling pathways. Two major groups of adaptogens comprise (i) phenolic phenethyl and phenylpropanoid derivatives and (ii) tetracyclic and pentacyclic glycosides, whose chemical structure can be distinguished as related correspondingly to (i) monoamine neurotransmitters of SAS (epinephrine, norepinephrine, and dopamine) and (ii) steroid hormones (cortisol, testosterone, and estradiol). In this narrative review, we discuss (i) the multitarget mechanism of integrated pharmacological activity of botanical adaptogens in stress overload, ischemic stroke, and long-lasting brain fatigue; (ii) the time-dependent dual response of physiological regulatory systems to adaptogens to support homeostasis in chronic stress and overload; and (iii) the dual dose-dependent reversal (hormetic) effect of botanical adaptogens. This narrative review shows that the adaptogenic concept cannot be reduced and rectified to the various effects of adaptogens on selected molecular targets or specific modes of action without estimating their interactions within the networks of mediators of the neuroendocrine-immune complex that, in turn, regulates other pharmacological systems (cardiovascular, gastrointestinal, reproductive systems) due to numerous intra- and extracellular communications and feedback regulations. These interactions result in polyvalent action and the pleiotropic pharmacological activity of adaptogens, which is essential for characterizing adaptogens as distinct types of botanicals. They trigger the defense adaptive stress response that leads to the extension of the limits of resilience to overload, inducing brain fatigue and mental disorders. For the first time, this review justifies the neurogenesis potential of adaptogens, particularly the botanical hybrid preparation (BHP) of Arctic Root and Ashwagandha, providing a rationale for potential use in individuals experiencing long-lasting brain fatigue. The review provided insight into future research on the network pharmacology of adaptogens in preventing and rehabilitating long-lasting brain fatigue following stroke, trauma, and viral infections. Full article

d-Tagatose is a rare sugar with low calories, and is extensively used in food, beverage, and drug additives. In this study, an in vitro multienzyme cascade route for d-tagatose synthesis from sucrose (MCTS) was designed, which contains five enzymes (sucrose phosphorylase, fructokinase, d-fructose 6-phosphate 4-epimerase, d-tagatose 6-phosphate phosphatase, and polyphosphate kinase). The whole MCTS route comprised a sucrose phosphorylation reaction, and a phosphorylation–dephosphorylation reaction coupled with an ATP regeneration system. After optimization, the conversion of d-tagatose from 10 mM sucrose reached 82.3%. At an elevated sucrose concentration of 50 mM, 72.4% of d-tagatose conversion and 0.27 g·L^–1·h⁻¹ of space–time yield were obtained. Furthermore, ADP consumption decreased to 1% of the sucrose concentration after introducing the ATP regeneration system. The MCTS strategy is an efficient and cost-effective approach for d-tagatose production. Full article

The pyrimidine nucleoside uridine and its phosphorylated derivates have been shown to be involved in the systemic regulation of energy and redox balance and promote the regeneration of many tissues, including the myocardium, although the underlying mechanisms are not fully understood. Moreover, rearrangements in mitochondrial structure and function within cardiomyocytes are the predominant signs of myocardial injury. Accordingly, this study aimed to investigate whether uridine could alleviate acute myocardial injury induced by isoprenaline (ISO) exposure, a rat model of stress-induced cardiomyopathy, and to elucidate the mechanisms of its action related to mitochondrial dysfunction. For this purpose, a biochemical analysis of the relevant serum biomarkers and ECG monitoring were performed in combination with transmission electron microscopy and a comprehensive study of cardiac mitochondrial functions. The administration of ISO (150 mg/kg, twice with an interval of 24 h, s.c.) to rats caused myocardial degenerative changes, a sharp increase in the serum cardiospecific markers troponin I and the AST/ALT ratio, and a decline in the ATP level in the left ventricular myocardium. In parallel, alterations in the organization of sarcomeres with focal disorganization of myofibrils, and ultrastructural and morphological defects in mitochondria, including disturbances in the orientation and packing density of crista membranes, were detected. These malfunctions were improved by pretreatment with uridine (30 mg/kg, twice with an interval of 24 h, i.p.). Uridine also led to the normalization of the QT interval. Moreover, uridine effectively inhibited ISO-induced ROS overproduction and lipid peroxidation in rat heart mitochondria. The administration of uridine partially recovered the protein level of the respiratory chain complex V, along with the rates of ATP synthesis and mitochondrial potassium transport, suggesting the activation of the potassium cycle through the mitoK_ATP channel. Taken together, these results indicate that uridine ameliorates acute ISO-induced myocardial injury and mitochondrial malfunction, which may be due to the activation of mitochondrial potassium recycling and a mild uncoupling leading to decreased ROS generation and oxidative damage. Full article

Theanine is a non-protein amino acid that is highly represented in tea plants and is one of the delicious ingredients in tea. In recent years, the global market demand for theanine has continued to rise, and the industry has developed rapidly. Here, we designed and constructed a promising pathway in Escherichia coli to produce L-theanine. This biosynthesis pathway employs four enzymes to achieve the production of L-theanine. This route involves the co-expression of four functional enzymes: γ-glutamylmethylamide synthetase (GMAS) from Methyloversatilis universalis, polyphosphate kinase (PPK) from E. coli, alanine transaminase from Bacillus subtilis (BsAld), and alanine decarboxylase from Camellia sinensis (CsAlaDC). Polyphosphate kinase from Escherichia coli was overexpressed in E. coli FD02, constructing an ATP regeneration system that increased the titer of L-theanine by 13.4% compared to E. coli FD01. A titer of 334 mg/L of L-theanine was produced via engineering strain FD03 in shake flasks. Moreover, glutamine permease from Saccharomyces cereviside (GNP1) was overexpressed in E. coli FD04, and the L-theanine titer increased by 14.7%. Finally, 2.9 g/L of L-theanine was obtained via FD04 in a 1 L bioreactor. In addition, the molecular docking results indicated that L-glutamate could bind to the hydrophobic cavity of GMAS due to the formation of hydrogen bonds and hydrophobic interactions with the surrounding amino acid residues. Full article

Deoxyadenosine triphosphate (dATP) is an important biochemical molecule. In this paper, the synthesis of dATP from deoxyadenosine monophosphate (dAMP), catalyzed by Saccharomyces cerevisiae, was studied. By adding chemical effectors, an efficient ATP regeneration and coupling system was constructed to achieve efficient synthesis of dATP. Factorial and response surface designs were used to optimize process conditions. Optimal reaction conditions were as follows: dAMP 1.40 g/L, glucose 40.97 g/L, MgCl₂·6H₂O 4.00 g/L, KCl 2.00 g/L, NaH₂PO₄ 31.20 g/L, yeast 300.00 g/L, ammonium chloride 0.67 g/L, acetaldehyde 11.64 mL/L, pH 7.0, temperature 29.6 °C. Under these conditions, the substrate conversion was 93.80% and the concentration of dATP in the reaction system was 2.10 g/L, which was 63.10% higher than before optimization, and the concentration of product was 4 times higher than before optimization. The effects of glucose, acetaldehyde, and temperature on the accumulation of dATP were analyzed. Full article

Soil salinization is spread in the dryland of NW China due to the dry or extreme dry climate. Increased salinization damages the health and function of soil systems and influences the microbial community structure and function. Some studies have been conducted to reveal the microbial community structure and isolate the microorganisms of saline soil or salt-lake sediments in this region. However, the functions of microorganisms and their response to salinization, i.e., their adaptation strategy to a wide salinization range in arid environments, are less understood. Here, we applied metagenomics technology to investigate the microbial community structure, function, and their relationship with salinization, and discussed the adaptative strategy of microorganisms to different saline environments. A total of 42 samples were sequenced on the Illumina PE500 platform. The archaea and bacteria constituted the dominant kingdoms; Actinobacteria, Proteobacteria, Bacteroidetes, and Firmicutes were the dominant bacterial phyla; and Euryarchaeota were the dominant archaeal phylum. The microbial communities showed significant structure divergence according to the salt concentration (saline (mean EC 22 mS/cm) and hypersaline (mean EC 70 mS/cm)), wherein the communities were dominated by bacteria in saline soils and archaea in hypersaline soils. Most of the dominant bacterial representation decreased with salinity, while the archaea increased with salinity. KEGG functional annotation showed that at level 2, the cell motility, environmental adaptation, signal transduction, signaling molecules and interaction, glycan biosynthesis and metabolism, and metabolism of other amino acids were reduced from saline to hypersaline, whereas the metabolism of cofactors and vitamins, folding sorting and degradation, replication and repair, transcription and translation, amino acid biosynthesis, glycolysis/gluconeogenesis, and carbon fixation increased with salinity. The increased salt content decreased the carbohydrate activities of microorganisms. The osmolyte regulation substance synthesis and absorption-related genes were more abundant in saline soils than in hypersaline soils, whereas the Na⁺/H⁺ antiporter genes (mnhB-E) and H⁺/Na⁺-transporting ATPase genes (atpA-F, I, K) were significantly higher in hypersaline soils. This indicated that in saline soils, microorganisms primarily synthesize and/or uptake compatible solutes to cope with osmotic stress, whereas in the hypersaline habitat, the high-salt-in strategy was predicated to be adopted by the halophilic/extremely halophilic microorganisms, coupled with a high abundance of replication and repair, cofactors and vitamin metabolism, nucleotide metabolism, and carbon fixation to provide energy and ensure cell regeneration. In conclusion, increases in salinity influence the microbial communities’ structure and function, as well as the adaptation of microorganisms. Full article

Chilling injury (CI) caused by exposure to low temperatures is a serious problem in the postharvest cold storage of pepper fruit. Melatonin (MT) has been reported to minimize CI in several plants. To evaluate the effectiveness of MT to minimize CI in green horn pepper and the possible mechanism involved, freshly picked green horn peppers were treated with MT solution at 100 μmol L⁻¹ or water and then stored at 4 °C for 25 d. Results showed that MT treatment reduced CI in green horn pepper fruit, as evidenced by lower CI rate and CI index. MT treatment maintained lower postharvest metabolism rate and higher fruit quality of green horn peppers, as shown by reduced weight loss and respiratory rate, maintened fruit firmness and higher contents of chlorophyll, total phenols, flavonoids, total soluble solids and ATP. Additionally, the contents of hydrogen peroxide, superoxide radical, and malondialdehyde were kept low in the MT-treated fruit, and the activities of the enzymes peroxidase, superoxide dismutase, and catalase were significantly elevated. Similarly, the ascorbate–glutathione cycle was enhanced by elevating the activities of ascorbate peroxidase, glutathione reductase, dehydroascorbate reductase, and monodehydroascorbate reductase, to increase the regeneration of ascorbic acid and glutathione. Our results show that MT treatment protected green horn pepper fruit from CI and maintained high fruit quality during cold storage by triggering the antioxidant system Full article

An extensive research field in regenerative medicine is electrical stimulation (ES) and its impact on tissue and cells. The mechanism of action of ES, particularly the role of electrical parameters like intensity, frequency, and duration of the electric field, is not yet fully understood. Human MG-63 osteoblasts were electrically stimulated for 10 min with a commercially available multi-channel system (IonOptix). We generated alternating current (AC) electrical fields with a voltage of 1 or 5 V and frequencies of 7.9 or 20 Hz, respectively. To exclude liquid-mediated effects, we characterized the AC-stimulated culture medium. AC stimulation did not change the medium’s pH, temperature, and oxygen content. The H₂O₂ level was comparable with the unstimulated samples except at 5 V_7.9 Hz, where a significant increase in H₂O₂ was found within the first 30 min. Pulsed electrical stimulation was beneficial for the process of attachment and initial adhesion of suspended osteoblasts. At the same time, the intracellular Ca²⁺ level was enhanced and highest for 20 Hz stimulated cells with 1 and 5 V, respectively. In addition, increased Ca²⁺ mobilization after an additional trigger (ATP) was detected at these parameters. New knowledge was provided on why electrical stimulation contributes to cell activation in bone tissue regeneration. Full article

The biocatalytic system comprised of RizA and acetate kinase (AckA) combines the specific synthesis of bioactive arginyl dipeptides with efficient ATP regeneration. Immobilization of this coupled enzyme system was performed and characterized in terms of activity, specificity and reusability of the immobilisates. Co-immobilization of RizA and AckA into a single immobilisate conferred no disadvantage in comparison to immobilization of only RizA, and a small addition of AckA (20:1) was sufficient for ATP regeneration. New variants of RizA were constructed by combining mutations to yield variants with increased biocatalytic activity and specificity. A selection of RizA variants were co-immobilized with AckA and used for the production of the salt-taste enhancers Arg-Ser and Arg-Ala and the antihypertensive Arg-Phe. The best variants yielded final dipeptide concentrations of 11.3 mM Arg-Ser (T81F_A158S) and 11.8 mM Arg-Phe (K83F_S156A), the latter of which represents a five-fold increase in comparison to the wild-type enzyme. T81F_A158S retained more than 50% activity for over 96 h and K83F_S156A for over 72 h. This study provides the first example of the successful co-immobilization of an l-amino acid ligase with an ATP-regenerating enzyme and paves the way towards a bioprocess for the production of bioactive dipeptides. Full article

Adhesion molecules regulate cell proliferation, migration, survival, neuritogenesis, synapse formation and synaptic plasticity during the nervous system’s development and in the adult. Among such molecules, the neural cell adhesion molecule L1 contributes to these functions during development, and in synapse formation, synaptic plasticity and regeneration after trauma. Proteolytic cleavage of L1 by different proteases is essential for these functions. A proteolytic fragment of 70 kDa (abbreviated L1-70) comprising part of the extracellular domain and the transmembrane and intracellular domains was shown to interact with mitochondrial proteins and is suggested to be involved in mitochondrial functions. To further determine the role of L1-70 in mitochondria, we generated two lines of gene-edited mice expressing full-length L1, but no or only low levels of L1-70. We showed that in the absence of L1-70, mitochondria in cultured cerebellar neurons move more retrogradely and exhibit reduced mitochondrial membrane potential, impaired Complex I activity and lower ATP levels compared to wild-type littermates. Neither neuronal migration, neuronal survival nor neuritogenesis in these mutants were stimulated with a function-triggering L1 antibody or with small agonistic L1 mimetics. These results suggest that L1-70 is important for mitochondrial homeostasis and that its absence contributes to the L1 syndrome phenotypes. Full article

Adenosine triphosphate (ATP), as a universal energy currency, takes a central role in many biochemical reactions with potential for the synthesis of numerous high-value products. However, the high cost of ATP limits industrial ATP-dependent enzyme-catalyzed reactions. Here, we investigated the effect of cell-surface display of phosphotransferase on ATP regeneration in recombinant Escherichia coli. By N-terminal fusion of the super-folder green fluorescent protein (sfGFP), we successfully displayed the phosphotransferase of Pseudomonas brassicacearum (PAP-Pb) on the surface of E. coli cells. The catalytic activity of sfGFP-PAP-Pb intact cells was 2.12 and 1.47 times higher than that of PAP-Pb intact cells, when the substrate was AMP and ADP, respectively. The conversion of ATP from AMP or ADP were up to 97.5% and 80.1% respectively when catalyzed by the surface-displayed enzyme at 37 °C for only 20 min. The whole-cell catalyst was very stable, and the enzyme activity of the whole cell was maintained above 40% after 40 rounds of recovery. Under this condition, 49.01 mg/mL (96.66 mM) ATP was accumulated for multi-rounds reaction. This ATP regeneration system has the characteristics of low cost, long lifetime, flexible compatibility, and great robustness. Full article

Arginyl dipeptides like Arg-Ser, Arg-Ala, and Arg-Gly are salt-taste enhancers and can potentially be used to reduce the salt content of food. The l-amino acid ligase RizA from B. subtilis selectively synthesizes arginyl dipeptides. However, industrial application is prevented by the high cost of the cofactor adenosine triphosphate (ATP). Thus, a coupled reaction system was created consisting of RizA and acetate kinase (AckA) from E. coli providing ATP regeneration from acetyl phosphate. Both enzymes were recombinantly produced in E. coli and purified by affinity chromatography. Biocatalytic reactions were varied and analyzed by RP-HPLC with fluorescence detection. Under optimal conditions the system produced up to 5.9 g/L Arg-Ser corresponding to an ATP efficiency of 23 g Arg-Ser per gram ATP. Using similar conditions with alanine or glycine as second amino acid, 2.6 g/L Arg-Ala or 2.4 g/L Arg Gly were produced. The RizA/AckA system selectively produced substantial amounts of arginyl dipeptides while minimizing the usage of the expensive ATP. Full article