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.

Engineering Escherichia coli for high-yielding 2,5-Dimethylpyrazine synthesis from L-Threonine by reconstructing metabolic pathways and enhancing cofactors regeneration.

2,5-Dimethylpyrazine (2,5-DMP) is important pharmaceutical raw material and food flavoring agent. Recently, engineering microbes to produce 2,5-DMP has become an attractive alternative to chemical synthesis approach. In this study, metabolic engineering strategies were used to optimize the modified Escherichia coli BL21 (DE3) strain for efficient synthesis of 2,5-DMP using L-threonine dehydrogenase (EcTDH) from Escherichia coli BL21, NADH oxidase (EhNOX) from Enterococcus hirae, aminoacetone oxidase (ScAAO) from Streptococcus cristatus and L-threonine transporter protein (EcSstT) from Escherichia coli BL21, respectively. We further optimized the reaction conditions for synthesizing 2,5-DMP. In optimized conditions, the modified strain can convert L-threonine to obtain 2,5-DMP with a yield of 2897.30 mg/L. Therefore, the strategies used in this study contribute to the development of high-level cell factories for 2,5-DMP.

Bioinformatic mapping of a more precise Aspergillus niger degradome.

Aspergillus niger has the ability to produce a large variety of proteases, which are of particular importance for protein digestion, intracellular protein turnover, cell signaling, flavour development, extracellular matrix remodeling and microbial defense. However, the A. niger degradome (the full repertoire of peptidases encoded by the A. niger genome) available is not accurate and comprehensive. Herein, we have utilized annotations of A. niger proteases in AspGD, JGI, and version 12.2 MEROPS database to compile an index of at least 232 putative proteases that are distributed into the 71 families/subfamilies and 26 clans of the 6 known catalytic classes, which represents ~ 1.64% of the 14,165 putative A. niger protein content. The composition of the A. niger degradome comprises ~ 7.3% aspartic, ~ 2.2% glutamic, ~ 6.0% threonine, ~ 17.7% cysteine, ~ 31.0% serine, and ~ 35.8% metallopeptidases. One hundred and two proteases have been reassigned into the above six classes, while the active sites and/or metal-binding residues of 110 proteases were recharacterized. The probable physiological functions and active site architectures of these peptidases were also investigated. This work provides a more precise overview of the complete degradome of A. niger, which will no doubt constitute a valuable resource and starting point for further experimental studies on the biochemical characterization and physiological roles of these proteases.

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.

Association of meteorological factors with pediatric acute appendicitis in China: A 7-year retrospective analysis.

Acute appendicitis (AA) affects between 7% and 8% of the world population and is one of the most common general surgical emergencies. The concept of seasonal patterns in the incidence of AA remains controversial. Thus, this study aimed to investigate whether meteorological factors are related to variations in the rate of pediatric AA cases at the Children's Hospital in Chongqing, China.In total, in this retrospective survey, 3436 children younger than 18 years who had been hospitalized with AA from January 1, 2008 to December 31, 2013 were enrolled, and the meteorological factors during this period were collected.Patients with AA showed a male/female ratio of 1.81:1; the highest incidence age ranged from 6 to 12 years old (P < .0001). The highest incidences of pediatric AA occurred in summer and autumn, with a peak in September and a trough in February. Pearson correlation analysis showed that the monthly mean temperature (r = 0.357, P = .001), monthly mean relative humidity (r = -0.357, P = .001), and monthly mean sunshine duration (r = 0.235, P = -0.031) were relatively weak correlated with pediatric AA. Multiple linear regression analysis indicated that pediatric AA occurrence was positively affected by monthly mean temperature (P < .0001) and negatively affected by monthly mean humidity (P < .0001) and monthly sum of sunshine (P < .0001), while monthly mean air pressure (P = .092), monthly wind speed (P = .143) and monthly precipitation (P = .297) were marginally associated with pediatric AA.Pediatric AA is associated with climatic factors. Specifically, pediatric AA is more likely related to the following meteorological conditions of: high temperature (20 °C-30 °C), low humidity, and less sunshine.

Bile salt hydrolases: Structure and function, substrate preference, and inhibitor development.

The worldwide trend of limiting the use of antibiotic growth promoters (AGPs) in animal production creates challenges for the animal feed industry, thus necessitating the development of effective non-antibiotic alternatives to improve animal performance. Increasing evidence has shown that the growth-promoting effect of AGPs is highly correlated with the reduced activity of bile salt hydrolase (BSH, EC 3.5.1.24), an intestinal bacteria-producing enzyme that has a negative impact on host fat digestion and energy harvest. Therefore, BSH inhibitors may become novel, attractive alternatives to AGPs. Detailed knowledge of BSH substrate preferences and the wealth of structural data on BSHs provide a solid foundation for rationally tailored BSH inhibitor design. This review focuses on the relationship between structure and function of BSHs based on the crystal structure, kinetic data, molecular docking and comparative structural analyses. The molecular basis for BSH substrate recognition is also discussed. Finally, recent advances and future prospectives in the development of potent, safe, and cost-effective BSH inhibitors are described.

Improved ethanol productivity from lignocellulosic hydrolysates by Escherichia coli with regulated glucose utilization.

BACKGROUND: Lignocellulosic ethanol could offer a sustainable source to meet the increasing worldwide demand for fuel. However, efficient and simultaneous metabolism of all types of sugars in lignocellulosic hydrolysates by ethanol-producing strains is still a challenge. RESULTS: An engineered strain Escherichia coli B0013-2021HPA with regulated glucose utilization, which could use all monosaccharides in lignocellulosic hydrolysates except glucose for cell growth and glucose for ethanol production, was constructed. In E. coli B0013-2021HPA, pta-ackA, ldhA and pflB were deleted to block the formation of acetate, lactate and formate and additional three mutations at glk, ptsG and manZ generated to block the glucose uptake and catabolism, followed by the replacement of the wild-type frdA locus with the ptsG expression cassette under the control of the temperature-inducible λ pR and pL promoters, and the final introduction of pEtac-PA carrying Zymomonas mobilis pdc and adhB for the ethanol pathway. B0013-2021HPA was able to utilize almost all xylose, galactose and arabinose but not glucose for cell propagation at 34 °C and converted all sugars to ethanol at 42 °C under oxygen-limited fermentation conditions. CONCLUSIONS: Engineered E. coli strain with regulated glucose utilization showed efficient metabolism of mixed sugars in lignocellulosic hydrolysates and thus higher productivity of ethanol production.

Exploring the Metabolomic Responses of Bacillus licheniformis to Temperature Stress by Gas Chromatography/Mass Spectrometry.

Owing to its high protein secretion capacity, simple nutritional requirements, and GRAS (generally regarded as safe) status, Bacillus licheniformis is widely used as a host for the industrial production of enzymes, antibiotics, and peptides. However, as compared with its close relative Bacillus subtilis, little is known about the physiology and stress responses of B. licheniformis. To explore its temperature-stress metabolome, B. licheniformis strains ATCC 14580 and B186, with respective optimal growth temperatures of 42°C and 50°C, were cultured at 42°C, 50°C, and 60°C and their corresponding metabolic profiles were determined by gas chromatography/mass spectrometry and multivariate statistical analyses. It was found that with increased growth temperatures, the two B. licheniformis strains displayed elevated cellular levels of proline, glutamate, lysine, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, and octadecanoic acid, and decreased levels of glutamine and octadecenoic acid. Regulation of amino acid and fatty acid metabolism is likely to be associated with the evolution of protective biochemical mechanisms of B. licheniformis. Our results will help to optimize the industrial use of B. licheniformis and other important Bacillus species.

A novel strategy for production of ethanol and recovery of xylose from simulated corncob hydrolysate.

OBJECTIVES: To develop a xylose-nonutilizing Escherichia coli strain for ethanol production and xylose recovery. RESULTS: Xylose-nonutilizing E. coli CICIM B0013-2012 was successfully constructed from E. coli B0013-1030 (pta-ack, ldhA, pflB, xylH) by deletion of frdA, xylA and xylE. It exhibited robust growth on plates containing glucose, arabinose or galactose, but failed to grow on xylose. The ethanol synthesis pathway was then introduced into B0013-2012 to create an ethanologenic strain B0013-2012PA. In shaking flask fermentation, B0013-2012PA fermented glucose to ethanol with the yield of 48.4 g/100 g sugar while xylose remained in the broth. In a 7-l bioreactor, B0013-2012PA fermented glucose, galactose and arabinose in the simulated corncob hydrolysate to 53.4 g/l ethanol with the yield of 48.9 g/100 g sugars and left 69.6 g/l xylose in the broth, representing 98.6% of the total xylose in the simulated corncob hydrolysate. CONCLUSIONS: By using newly constructed strain B0013-2012PA, we successfully developed an efficient bioprocess for ethanol production and xylose recovery from the simulated corncob hydrolysate.

Periplasmic Export of Bile Salt Hydrolase in Escherichia coli by the Twin-Arginine Signal Peptides.

Bile salt hydrolase (BSH, EC 3.5.1.24) is considered as an ideal way with lower cost and less side effects to release the risk of coronary heart disease caused by hypercholesterolemia. As bile salt hydrolase from Lactobacillus plantarum BBE7 could not be efficiently exported by PelB signal peptide of the general secretory (Sec) pathway, three twin-arginine signal peptides from twin-arginine translocation (Tat) pathway were synthesized, fused with bsh gene, inserted into expression vectors pET-20b(+) and pET-22b(+), and transformed into four different Escherichia coli hosts, respectively. Among the 24 recombinant bacteria obtained, E. coli BL21 (DE3) pLysS (pET-20b(+)-dmsA-bsh) showed the highest BSH activity in periplasmic fraction, which was further increased to 1.21 ± 0.03 U/mL by orthogonal experimental design. And, signal peptide dimethyl sulfoxide reductase subunit DmsA (DMSA) had the best activity of exported BSH. More importantly, the presence of BSH in the periplasm had proven to be caused by the export rather than cell leakage. For the first time, we report the periplasmic expression of BSH by signal peptides from the Tat pathway. This will lay a solid foundation for the purification and biochemical characterization of BSH from the supernatant, and strategies adopted here could be used for the periplasmic expression of other proteins in E. coli.

Codon and Propeptide Optimizations to Improve the Food-grade Expression of Bile Salt Hydrolase in Lactococcus lactis.

To achieve the food-grade expression of bile salt hydrolase (BSH, EC 3.5.1.24) from Lactobacillus plantarum BBE7, the nisin controlled gene expression system (NICE), food-grade selection maker and signal peptide of Lactococcus lactis were used in this study. The open reading frame of BSH was optimized based on the codon bias of L. lactis, resulting in 12-fold and 9.5% increases in the intracellular and extracellular BSH activities, respectively. Three synthetic propeptides, LEISSTCDA (acidic), LGISSTCNA (neutral) and LKISSTCHA (basic) were also fused with signal peptide SPusp45 of vector pNZ8112 and introduced into the food-grade expression vector pNZ8149, respectively. Among these propeptides, acidic propeptide was effective in increasing the secretion efficiency and yield of BSH in recombinant bacteria, while neutral propeptide had no significant effect on the secretion of BSH. In contrast, basic propeptide strongly reduced the extracellular expression of BSH. By using codon optimization and the acidic propeptide together, the extracellular BSH activity was increased by 11.3%, reaching its maximum of 3.56 U/mg. To the best of our knowledge, this is the first report on the intracellular and extracellular expression of BSH using food-grade expression system, which would lay a solid foundation for large-scale production of BSH and other heterologous proteins in L. lactis.