Combining Flexible Region Design and Automatic Design to Enhance the Thermal Stability and Catalytic Efficiency of Leucine Dehydrogenase.

Leucine dehydrogenase (LeuDH, EC 1.4.1.9) can reversibly catalyze the oxidative deamination of l-leucine and some other specific α-amino acids to form the corresponding α-ketoacids. This reaction has great significance in the field of food additives and the pharmaceutical industry. The LeuDH from Exiguobacterium sibiricum (EsLeuDH) has high catalytic efficiency but limited thermal stability, hindering its widespread industrial application. In this study, a mutant N5F/I12L/A352Y of EsLeuDH (referred to as M2) was developed with enhanced thermal stability and catalytic activity through rational modification. The M2 mutant exhibits a half-life at 60 °C (t1/2(60 °C)) of 975.7 min and a specific activity of 69.6 U mg-1, which is 5.4 and 2.1 times higher than those of EsLeuDH, respectively. This research may facilitate the utilization of EsLeuDH at elevated temperatures, enhancing its potential for industrial applications. The findings offer a practical and efficient approach for optimizing LeuDH and other industrial enzymes.

Exploring the Pathogenesis and Treatment of PSD from the Perspective of Gut Microbiota.

Post-stroke depression (PSD) is a psychological disease that can occur following a stroke and is associated with serious consequences. Research on the pathogenesis and treatment of PSD is still in the infancy stage. Patients with PSD often exhibit gastrointestinal symptoms; therefore the role of gut microbiota in the pathophysiology and potential treatment effects of PSD has become a hot topic of research. In this review, describe the research on the pathogenesis and therapy of PSD. We also describe how the gut microbiota influences neurotransmitters, the endocrine system, energy metabolism, and the immune system. It was proposed that the gut microbiota is involved in the pathogenesis and treatment of PSD through the regulation of neurotransmitter levels, vagal signaling, hypothalamic-pituitary-adrenal axis activation and inhibition, hormone secretion and release, in addition to immunity and inflammation.

The role of cellular senescence-related genes in Asthma: Insights from bioinformatics and animal experiments.

BACKGROUND: Asthma is a heterogeneous chronic respiratory disease, affecting about 10% of the global population. Cellular senescence is a multifaceted phenomenon defined as the irreversible halt of the cell cycle, commonly referred to as the senescence-associated secretory phenotype. Recent studies suggest that cellular senescence may play a role in asthma. This study aims to dissect the role and biological mechanisms of CSRGs in asthma, enhancing our understanding of the progression of asthma. METHODS: The study utilized the GSE147878 dataset, employing methods like WGCNA, Differential analysis, Cibersort, GO, KEGG, unsupervised clustering, and GSVA to explore CSRGs functions and immune cell patterns in asthma. Machine learning identified key diagnostic genes, validated externally with the GSE165934 dataset and through qRT-PCR and WB experiments in animal models. RESULT: From the GSE147878 dataset, 24 CSRGs were identified, highlighting their role in immune and inflammatory processes in asthma. Differences in CD4 naive T cells and activated dendritic cells between asthma and control groups underscored CSRGs' role in immune regulation. Cluster analysis revealed two distinct asthma patient groups with unique immune microenvironments. Machine learning identified five genes, leading to a TF-miRNA-mRNA network and singling out RHOA and RBM39 as key diagnostic genes, which were experimentally validated. Finally, a nomogram was created based on these genes. CONCLUSION: This study, utilizing bioinformatics and animal experiments, identified RHOA and RBM39 as key diagnostic genes for asthma, providing new insights into the potential role and biological mechanisms of CSRGs in asthma.

Exploration of the Binding Mechanism of Cyclic Dinucleotide Analogs to Stimulating Factor Proteins and the Implications for Subsequent Analog Drug Design.

Cyclic dinucleotides (CDNs) are cyclic molecules consisting of two nucleoside monophosphates linked by two phosphodiester bonds, which act as a second messenger and bind to the interferon gene stimulating factor (STING) to activate the downstream signaling pathway and ultimately induce interferon secretion, initiating an anti-infective immune response. Cyclic dinucleotides and their analogs are lead compounds in the immunotherapy of infectious diseases and tumors, as well as immune adjuvants with promising applications. Many agonists of pathogen recognition receptors have been developed as effective adjuvants to optimize vaccine immunogenicity and efficacy. In this work, the binding mechanism of human-derived interferon gene-stimulating protein and its isoforms with cyclic dinucleotides and their analogs was theoretically investigated using computer simulations and combined with experimental results in the hope of providing guidance for the subsequent synthesis of cyclic dinucleotide analogs.

Nanomaterial-Based Drug Delivery Systems for Ischemic Stroke.

Ischemic stroke is a leading cause of death and disability in the world. At present, reperfusion therapy and neuroprotective therapy, as guidelines for identifying effective and adjuvant treatment methods, are limited by treatment time windows, drug bioavailability, and side effects. Nanomaterial-based drug delivery systems have the characteristics of extending half-life, increasing bioavailability, targeting drug delivery, controllable drug release, and low toxicity, thus being used in the treatment of ischemic stroke to increase the therapeutic effects of drugs. Therefore, this review provides a comprehensive overview of nanomaterial-based drug delivery systems from nanocarriers, targeting ligands and stimulus factors of drug release, aiming to find the best combination of nanomaterial-based drug delivery systems for ischemic stroke. Finally, future research areas on nanomaterial-based drug delivery systems in ischemic stroke and the implications of the current knowledge for the development of novel treatment for ischemic stroke were identified.

Mycobacterium tuberculosis Fatty Acyl-CoA Synthetase fadD33 Promotes Bacillus Calmette-Guérin Survival in Hostile Extracellular and Intracellular Microenvironments in the Host.

Tuberculosis, caused by Mycobacterium tuberculosis (M. tb), remains a significant global health challenge. The survival of M. tb in hostile extracellular and intracellular microenvironments is crucial for its pathogenicity. In this study, we discovered a Bacillus Calmette-Guérin (BCG) mutant B1033 that potentially affected mycobacterium pathogenicity. This mutant contained an insertion mutation gene, fadD33, which is involved in lipid metabolism; however, its direct role in regulating M. tb infection is not well understood. Here, we found that the absence of fadD33 reduced BCG adhesion and invasion into human pulmonary alveolar epithelial cells and increased the permeability of the mycobacterial cell wall, allowing M. tb to survive in the low pH and membrane pressure extracellular microenvironment of the host cells. The absence of fadD33 also inhibited the survival of BCG in macrophages by promoting the release of proinflammatory cytokines, such as interleukin (IL)-1ß, IL-6, and tumors necrosis factor-α, through the mitogen-activated protein kinase p38 signaling pathway. Overall, these findings provide new insights into M. tb mechanisms to evade host defenses and might contribute to identifying potential therapeutic and vaccine targets for tuberculosis prevention.

Engineering of formate dehydrogenase for improving conversion potential of carbon dioxide to formate.

Formate dehydrogenase (FDH) is a D-2-hydroxy acid dehydrogenase, which can reversibly reduce CO2 to formate and thus act as non-photosynthetic CO2 reductase. In order to increase catalytic efficiency of formate dehydrogenase for CO2 reduction, two mutants V328I/F285W and V354G/F285W were obtained of which reduction activity was about two times more than the parent CbFDHM2, and the formate production from CO2 catalyzed by mutants were 2.9 and 2.7-fold higher than that of the parent CbFDHM2. The mutants had greater potential in CO2 reduction. The optimal temperature for V328I/F285W and V354G/F285W was 55 °C, and they showed increasement of relative activity under 45 °C to 55 °C compared with parent. The optimal pH for the mutants was 9.0, and they showed excellent stability in pH 4.0-11.5. The kcat/Km values of mutants were 1.75 times higher than that of the parent. Then the molecular basis for its improvement of biochemical characteristics were preliminarily elucidated by computer-aided methods. All of these results further established a solid foundation for molecular modification of formate dehydrogenase and CO2 reduction.

Regulatory Effects of Quercetin on Bone Homeostasis: Research Updates and Future Perspectives.

The imbalance of bone homeostasis has become a major public medical problem amid the background of an aging population, which is closely related to the occurrence of osteoporosis, osteoarthritis, and fractures. Presently, most drugs used in the clinical treatment of bone homeostasis imbalance are bisphosphonates, calcitonin, estrogen receptor modulators, and biological agents that inhibit bone resorption or parathyroid hormone analogs that promote bone formation. However, there are many adverse reactions. Therefore, it is necessary to explore potential drugs. Quercetin, as a flavonol compound with various biological activities, is widely distributed in plants. Studies have found that quercetin can regulate bone homeostasis through multiple pathways and targets. An in-depth exploration of the pharmacological mechanism of quercetin is of great significance for the development of new drugs. This review discusses the therapeutic mechanisms of quercetin on bone homeostasis, such as regulating the expression of long non-coding RNA, signaling pathways of bone metabolism, various types of programmed cell death, bone nutrients supply pathways, anti-oxidative stress, anti-inflammation, and activation of Sirtuins. We also summarize recent progress in improving quercetin bioavailability and propose some issues worth paying attention to, which may help guide future research efforts.

Machine learning in the prediction of post-stroke cognitive impairment: a systematic review and meta-analysis.

Objective: Cognitive impairment is a detrimental complication of stroke that compromises the quality of life of the patients and poses a huge burden on society. Due to the lack of effective early prediction tools in clinical practice, many researchers have introduced machine learning (ML) into the prediction of post-stroke cognitive impairment (PSCI). However, the mathematical models for ML are diverse, and their accuracy remains highly contentious. Therefore, this study aimed to examine the efficiency of ML in the prediction of PSCI. Methods: Relevant articles were retrieved from Cochrane, Embase, PubMed, and Web of Science from the inception of each database to 5 December 2022. Study quality was evaluated by PROBAST, and c-index, sensitivity, specificity, and overall accuracy of the prediction models were meta-analyzed. Results: A total of 21 articles involving 7,822 stroke patients (2,876 with PSCI) were included. The main modeling variables comprised age, gender, education level, stroke history, stroke severity, lesion volume, lesion site, stroke subtype, white matter hyperintensity (WMH), and vascular risk factors. The prediction models used were prediction nomograms constructed based on logistic regression. The pooled c-index, sensitivity, and specificity were 0.82 (95% CI 0.77-0.87), 0.77 (95% CI 0.72-0.80), and 0.80 (95% CI 0.71-0.86) in the training set, and 0.82 (95% CI 0.77-0.87), 0.82 (95% CI 0.70-0.90), and 0.80 (95% CI 0.68-0.82) in the validation set, respectively. Conclusion: ML is a potential tool for predicting PSCI and may be used to develop simple clinical scoring scales for subsequent clinical use. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=383476.

Trends and Challenges in AIoT/IIoT/IoT Implementation.

For the next coming years, metaverse, digital twin and autonomous vehicle applications are the leading technologies for many complex applications hitherto inaccessible such as health and life sciences, smart home, smart agriculture, smart city, smart car and logistics, Industry 4.0, entertainment (video game) and social media applications, due to recent tremendous developments in process modeling, supercomputing, cloud data analytics (deep learning, etc.), communication network and AIoT/IIoT/IoT technologies. AIoT/IIoT/IoT is a crucial research field because it provides the essential data to fuel metaverse, digital twin, real-time Industry 4.0 and autonomous vehicle applications. However, the science of AIoT is inherently multidisciplinary, and therefore, it is difficult for readers to understand its evolution and impacts. Our main contribution in this article is to analyze and highlight the trends and challenges of the AIoT technology ecosystem including core hardware (MCU, MEMS/NEMS sensors and wireless access medium), core software (operating system and protocol communication stack) and middleware (deep learning on a microcontroller: TinyML). Two low-powered AI technologies emerge: TinyML and neuromorphic computing, but only one AIoT/IIoT/IoT device implementation using TinyML dedicated to strawberry disease detection as a case study. So far, despite the very rapid progress of AIoT/IIoT/IoT technologies, several challenges remain to be overcome such as safety, security, latency, interoperability and reliability of sensor data, which are essential characteristics to meet the requirements of metaverse, digital twin, autonomous vehicle and Industry 4.0. applications.

High level expression of nicotinamide nucleoside kinase from Saccharomyces cerevisiae and its purification and immobilization by one-step method.

Nicotinamide riboside kinase (NRK) plays an important role in the synthesis of ß -nicotinamide nucleotide (NMN). NMN is a key intermediate of NAD+ synthesis, and it actually contribute to the well-being of our health. In this study, gene mining technology was used to clone nicotinamide nucleoside kinase gene fragments from S. cerevisiae, and the ScNRK1 was achieved a high level of soluble expression in E. coli BL21. Then, the reScNRK1 was immobilized by metal affinity label to optimize the enzyme performance. The results showed that the enzyme activity in the fermentation broth was 14.75 IU/mL, and the specific enzyme activity after purification was 2252.59 IU/mg. After immobilization, the optimum temperature of the immobilized enzyme was increased by 10°C compared with the free enzyme, and the temperature stability was improved with little change in pH. Moreover, the activity of the immobilized enzyme remained above 80% after four cycles of immobilized reScNRK1, which makes the enzyme more advantageous in the enzymatic synthesis of NMN.

Falls caused by balance disorders in the elderly with multiple systems involved: Pathogenic mechanisms and treatment strategies.

Falls are the main contributor to both fatal and nonfatal injuries in elderly individuals as well as significant sources of morbidity and mortality, which are mostly induced by impaired balance control. The ability to keep balance is a remarkably complex process that allows for rapid and precise changes to prevent falls with multiple systems involved, such as musculoskeletal system, the central nervous system and sensory system. However, the exact pathogenesis of falls caused by balance disorders in the elderly has eluded researchers to date. In consideration of aging phenomenon aggravation and fall risks in the elderly, there is an urgent need to explore the pathogenesis and treatments of falls caused by balance disorders in the elderly. The present review discusses the epidemiology of falls in the elderly, potential pathogenic mechanisms underlying multiple systems involved in falls caused by balance disorders, including musculoskeletal system, the central nervous system and sensory system. Meanwhile, some common treatment strategies, such as physical exercise, new equipment based on artificial intelligence, pharmacologic treatments and fall prevention education are also reviewed. To fully understand the pathogenesis and treatment of falls caused by balance disorders, a need remains for future large-scale multi-center randomized controlled trials and in-depth mechanism studies.

Research progress in the risk factors and screening assessment of dysphagia in the elderly.

With the aging of the population, the incidence of dysphagia has gradually increased and become a major clinical and public health issue. Early screening of dysphagia in high-risk populations is crucial to identify the risk factors of dysphagia and carry out effective interventions and health management in advance. In this study, the current epidemiology, hazards, risk factors, preventive, and therapeutic measures of dysphagia were comprehensively reviewed, and a literature review of screening instruments commonly used globally was conducted, focusing on their intended populations, main indicators, descriptions, and characteristics. According to analysis and research in the current study, previous studies of dysphagia were predominantly conducted in inpatients, and there are few investigations and screenings on the incidence and influencing factors of dysphagia in the community-dwelling elderly and of dysphagia developing in the natural aging process. Moreover, there are no unified, simple, economical, practical, safe, and easy-to-administer screening tools and evaluation standards for dysphagia in the elderly. It is imperative to focus on dysphagia in the community-dwelling elderly, develop unified screening and assessment tools, and establish an early warning model of risks and a dietary structure model for dysphagia in the community-dwelling elderly.

Research progress on transcranial magnetic stimulation for post-stroke dysphagia.

Dysphagia is one of the most common manifestations of stroke, which can affect as many as 50-81% of acute stroke patients. Despite the development of diverse treatment approaches, the precise mechanisms underlying therapeutic efficacy remain controversial. Earlier studies have revealed that the onset of dysphagia is associated with neurological damage. Neuroplasticity-based transcranial magnetic stimulation (TMS), a recently introduced technique, is widely used in the treatment of post-stroke dysphagia (PSD) by increasing changes in neurological pathways through synaptogenesis, reorganization, network strengthening, and inhibition. The main objective of this review is to discuss the effectiveness, mechanisms, potential limitations, and prospects of TMS for clinical application in PSD rehabilitation, with a view to provide a reference for future research and clinical practice.

Gene cloning of a highly active phytase from Lactobacillus plantarum and further improving its catalytic activity and thermostability through protein engineering.

Phytase belongs to orthophosphate monoester hydrolase, which can catalyze the gradual hydrolysis of phytic acid to inositol phosphate. It can be added to animal feed to reduce the anti-nutritional factor of phytic acid in feed. The thermostability and specific activity of phytases are two key factors determining their potential applications. In this study, a highly active 233-aa phytase gene (LpPHY233) from Lactobacillus plantarum was cloned and expressed in Escherichia coli (E. coli), achieving 800 times higher activity than that expressed in L. plantarum. Next, the temperature characteristic and catalytic performance of LpPHY233 was improved by disulfide bond engineering and C-terminal truncation, respectively. Surprisingly, the specific activity of the C-terminal truncated mutant LpPHY200 was about 5.6 times higher than that of LpPHY233, and the optimal temperature for the mutant LpPHY233S58C/K61C introduced disulfide bond was 15 °C higher than that of LpPHY233. Moreover, these phytase mutants displayed excellent pH property and kinetic parameters, and have great application prospect in feed additives field. The molecular basis for its catalytic performance was preliminarily explained by in silico design methods. Our results provided a solid theoretical foundation for further molecular modification and industrial application of phytases.

Gene editing in monogenic autism spectrum disorder: animal models and gene therapies.

Autism spectrum disorder (ASD) is a lifelong neurodevelopmental disease, and its diagnosis is dependent on behavioral manifestation, such as impaired reciprocal social interactions, stereotyped repetitive behaviors, as well as restricted interests. However, ASD etiology has eluded researchers to date. In the past decades, based on strong genetic evidence including mutations in a single gene, gene editing technology has become an essential tool for exploring the pathogenetic mechanisms of ASD via constructing genetically modified animal models which validates the casual relationship between genetic risk factors and the development of ASD, thus contributing to developing ideal candidates for gene therapies. The present review discusses the progress in gene editing techniques and genetic research, animal models established by gene editing, as well as gene therapies in ASD. Future research should focus on improving the validity of animal models, and reliable DNA diagnostics and accurate prediction of the functional effects of the mutation will likely be equally crucial for the safe application of gene therapies.

Expression of Novel L-Leucine Dehydrogenase and High-Level Production of L-Tert-Leucine Catalyzed by Engineered Escherichia coli.

Leucine dehydrogenase (LDH) is a NAD+-dependent oxidoreductase, which can selectively catalyze α-keto acids to obtain α-amino acids and their derivatives. It plays a key role in the biosynthesis of L-tert-leucine (L-Tle). As a non-naturally chiral amino acid, L-Tle can be used as an animal feed additive, nutrition fortifier, which is a perspective and important building block in the pharmaceutical, cosmetic, and food additive industry. In this study, four hypothetical leucine dehydrogenases were discovered by using genome mining technology, using the highly active leucine dehydrogenase LsLeuDH as a probe. These four leucine dehydrogenases were expressed in Escherichia coli BL21(DE3), respectively, and purified to homogeneity and characterized. Compared with the other enzymes, the specific activity of PfLeuDH also shows stronger advantage. In addition, the highly selective biosynthesis of L-Tle from trimethylpyruvic acid (TMP) was successfully carried out by whole-cell catalysis using engineered E. coli cells as biocatalyst, which can efficiently coexpress leucine dehydrogenase and formate dehydrogenase. One hundred-millimolar TMP was catalyzed for 25 h, and the yield and space-time yield of L-Tle reached 87.38% (e.e. >99.99%) and 10.90 g L-1 day-1. In short, this research has initially achieved the biosynthesis of L-Tle, laying a solid foundation for the realization of low-cost and large-scale biosynthesis of L-Tle.

Prevalence of Dysphagia in China: An Epidemiological Survey of 5943 Participants.

OBJECTIVE: To determine the prevalence of dysphagia among an older population and patients with stroke, head and neck cancers (HNCs) or neurodegenerative diseases (NDDs) in China, to identify the factors associated with this condition, and to explore the relationship between dysphagia and nutritional status. METHODS: This study included participants 65 years and older living in the community or in nursing homes and patients who had sustained a stroke, HNC, or NDD also recruited in hospitals from 14 provinces of China. The presence of dysphagia was determined by use of a questionnaire, water swallowing test, and/or a videofluoroscopic swallowing study. Logistic regression analysis was used to assess the possible associated risk factors. Body mass index was assessed as an indicator of malnutrition. RESULTS: A total of 5943 persons met the inclusion criteria and 2341 (39.4%) were identified with dysphagia, including the following: 51.14% of patients with stroke, 34.4% in HNCs, 48.3% in NDDs, and 19.2% of otherwise healthy older adults. The elderly with comorbidity (OR = 2.90, p < 0.01) and stroke patients (OR = 2.27, p < 0.01) were significantly more likely to exhibit signs of dysphagia. Dysphagic participants were at significantly greater risk of malnutrition (OR = 1.91, p < 0.01) compared to those without dysphagia. CONCLUSION: Dysphagia is prevalent in China among older individuals and people who have suffered a stroke, HNCs, or NDDs. The prevalence of dysphagia increases steadily with increasing age and presence of comorbid disease. People with dysphagia are more likely to suffer from malnutrition.

Efficient expression of novel glutamate decarboxylases and high level production of γ-aminobutyric acid catalyzed by engineered Escherichia coli.

Glutamate decarboxylase (GAD) has the potential of converting L-glutamate to gamma-aminobutyric acid (GABA), which is an important non-proteinogenic amino acid that has a potential use as food additive or dietary supplement for its physiological functions. A novel pyridoxal 5'-phosphate (PLP)-dependent glutamate decarboxylase (LsGAD) was cloned from GRAS (generally recognized as safe) Lactobacillus senmaizukei by genome mining and efficiently expressed in Escherichia coli BL21. The LsGAD displayed excellent temperature property, pH property and kinetic parameters compared with the probe LbGAD and the other GADs. By increasing the copy number of the LsGAD encoding gene, the expression level of LsGAD and the biosynthesis yield of GABA were increased, which was near to 2 times of that was expressed in single copy. These results established a solid foundation for increasing the added value of L-glutamate and the biosynthesis of GABA.

High level and enantioselective production of L-phenylglycine from racemic mandelic acid by engineered Escherichia coli using response surface methodology.

L-Phenylglycine (L-PHG) is a member of unnatural amino acids, and becoming more and more important as intermediate for pharmaceuticals, food additives and agrochemicals. However, the existing synthetic methods for L-PHG mainly rely on toxic cyanide chemistry and multistep processes. To provide green, safe and high enantioselective alternatives, we envisaged cascade biocatalysis for the one-pot synthesis of L-PHG from racemic mandelic acid. A engineered E. coli strain was established to co-express mandelate racemase, D-mandelate dehydrogenase and L-leucine dehydrogenase and catalyze a 3-step reaction in one pot, enantioselectively transforming racemic mandelic acid to give L-PHG (e.e. >99 %). After the conditions for biosynthesis of L-PHG optimized by response surface methodology, the yield and space-time yield of L-PHG can reach 87.89 % and 79.70 g·L-1·d-1, which was obviously improved. The high-yielding and enantioselective synthetic methods use cheap and green reagents, and E. coli whole-cell catalysts, thus providing green and useful alternative methods for manufacturing L-PHG.