Microgravity mutagenesis in E. coli: A molecular mechanism for high-yield cadaverine production

Transglutaminase (TGase) has been widely applied in the food industry. However, achieving high-yield TGase production remains a challenge, limiting its broader industrial application. In this study, a high-yield strain with stable genetic traits was obtained through UV-ARTP combined mutagenesis, achieving a maximum TGase activity of 13.77 U/mL, representing a 92.43% increase. Using this strain as a forward mutation gene pool, comparative genomic research identified 95 mutated genes, which were mostly due to base substitutions that led to changes in codon usage preference. Transcriptomic analysis revealed significant expression changes in 470 genes, with 232 upregulated and 238 downregulated genes. By investigating potential key regulatory factors, comprehensive analysis indicated that changes in codon usage preference, amino acid metabolism, carbon metabolism, protein export processes, TGase activation, and spore production pathways collectively contributed to the enhancement of TGase activity. Subsequently, the in vitro activation efficiency of TGase was further improved using co-cultivation techniques with neutral proteases secreted by Bacillus amyloliquefaciens CICC10888, and a TGase activity of 16.91 U/mL was achieved, accounting for a 22.71% increase. This study provides a comprehensive understanding of the mechanisms underlying high-yield TGase production and valuable insights and data references for future research.

Background: Peroxidation of renal cells is a critical event in the nucleation and formation of calcium oxalate crystals under hyperoxaluric conditions. We previously demonstrated that oxalate-induced peroxidative injury is one of the major mechanisms in promoting crystal attachment to renal epithelial cells. Methods: In this study we have demonstrated that the mechanism of oxalate-induced peroxidative injury is through the induction of TGF-β1 and glutathione (GSH) redox imbalance in LLC-PK1 cells. Results: LLC-PK1, renal epithelial cells exposed to oxalate had significantly higher reactive oxygen species (ROS) production; higher TGF-β1 levels, as measured by ELISA (1.89 ± 0.035 fold increase) or Western blot (1.65 ± 0.01 fold increase); increased malondialdehyde formation; increased LDH release, and loss of cell viability. In addition, oxalate exposure significantly decreased GSH content, glutathione reductase, glucose-6-phosphate dehydrogenase activities, and increased oxidized GSH content. Treatment with vitamin E, neutralizing anti-TGF-β antibody, or diphenylene iodium, an inhibitor of NAD(P)H oxidase, significantly inhibited oxalate-induced ROS production and prevented peroxidative injury and cytolysis. Vitamin E, catalase, or desferoxamine treatment also significantly restored the oxalate-induced cellular GSH redox status toward the control level, and vitamin E treatment significantly attenuated the oxalate-mediated increase in TGF-β1 protein in cultured LLC-PK1 cells. Conclusions: This is the first study to demonstrate that the mechanism of oxalate-induced free radical production in renal tubular epithelial cells is through the activation of NAD(P)H oxidase via cytokine TGF-β1 induction. These results also provide direct evidence that antioxidant therapy might prevent calcium oxalate nucleation and kidney stone formation by preventing oxalate-mediated peroxidative injury and GSH redox imbalance.

The hydroxyl radical ((center dot)OH) has been considered to be one of the most reactive oxygen species that are produced in biological systems. Frequently, (center dot)OH formation is ascribed to the transition metal-catalyzed Fenton reaction. During the study of the molecular mechanism for the genotoxicity of the wood preservative pentachlorophenol (PCP), we found that (center dot)OH could be produced by H(2)O(2) and PCP metabolite tetrachloro-1,4-benzoquinone (TCBQ) independent of transition metal ions. Further studies showed that TCBQ, but not its corresponding semiquinone radical, is essential for (center dot)OH production. Metal-independent production of (center dot)OH could also be observed with other halogenated quinones and H(2)O(2). Based on these data, we propose that (center dot)OH production by TCBQ and H(2)O(2) is not through a semiquinone-dependent organic Fenton reaction, but rather through a novel nucleophilic substitution and homolytical decomposition mechanism. This represents a novel mechanism for (center dot)OH production not requiring the catalysis of redox-active transition metal ions, and may partly explain the potential carcinogenicity of the widely used biocides such as PCP and other polyhalogenated aromatic compounds.

Thermoregulation is an essential homeostatic process in which critical mechanisms of heat production and dissipation are controlled centrally in large part by the hypothalamus and peripherally by activation of the sympathetic nervous system. Drugs that disrupt the components of this highly orchestrated multi-organ process can lead to life-threatening hyperthermia. In most cases, hyperthermic agents raise body temperature by increasing the central and peripheral release of thermoregulatory neurotransmitters that ultimately lead to heat production in thermogenic effector organs skeletal muscle (SKM) and brown adipose tissue (BAT). In many cases hyperthermic drugs also decrease heat dissipation through peripheral changes in blood flow. Drug-induced heat production is driven by the stimulation of mechanisms that normally regulate the adaptive thermogenic responses including both shivering and non-shivering thermogenesis (NST) mechanisms. Modulation of the mitochondrial electrochemical proton/pH gradient by uncoupling protein 1 (UCP1) in BAT is the most well characterized mechanism of NST in response to cold, and may contribute to thermogenesis induced by sympathomimetic agents, but this is far from established. However, the UCP1 homologue, UCP3, and the ryanodine receptor (RYR1) are established mediators of toxicant-induced hyperthermia in SKM. Defining the molecular mechanisms that orchestrate drug-induced hyperthermia will be essential in developing treatment modalities for thermogenic illnesses. This review will briefly summarize mechanisms of thermoregulation and provide a survey of pharmacologic agents that can lead to hyperthermia. We will also provide an overview of the established and candidate molecular mechanisms that regulate the actual thermogenic processes in heat effector organs BAT and SKM.

Monascus species are capable of producing various active metabolites, including monacolin K (MK) and pigments. Studies have shown that the overexpression of the mok I gene from the MK synthesis gene cluster in Monascus species can significantly increase MK production; however, the molecular mechanism has not yet been fully elucidated. Therefore, this study focused on the mok I gene of Monascus pilosus to construct overexpression strains of the mok I gene, resulting in high-yield MK production. Sixteen positive transformants were obtained, seven of which produced 9.63% to 41.39% more MK than the original strain, with no citrinin detected in any of the transformants. The qRT-PCR results revealed that the expression levels of mok I in the transformed strains TI-13, TI-24, and TI-25 increased by more than 50% compared to the original strain at various fermentation times, with the highest increase being 10.9-fold. Furthermore, multi-omics techniques were used to analyze the molecular mechanisms underlying enhanced MK production in transformed strains. The results indicated that mok I overexpression may enhance MK synthesis in M. pilosus by regulating the expression of key genes (such as MAO, HPD, ACX, and PLC) and the synthesis levels of key metabolites (such as delta-tocopherol and alpha-linolenic acid) in pathways linked to the biosynthesis of cofactors, the biosynthesis of unsaturated fatty acids, tyrosine metabolism, ubiquinone and other terpenoid-quinone biosynthesis, alpha-linolenic acid metabolism, and glycerophospholipid metabolism. These findings provide a theoretical basis for further study of the metabolic regulation of MK in Monascus species and for effectively enhancing their MK production.

Exploring the Vital Role of Microbiota Metabolites in Early-Life Health.

Since the beginning of the century, our knowledge of inherited thrombocytopenias greatly advanced, and we presently know 30 forms with well-defined genetic defects. This great advancement changed our view of these disorders, as we realized that most patients have only mild thrombocytopenia with inconspicuous bleeding or no bleeding tendency at all. However, better knowledge of inherited thrombocytopenias also revealed that some of the most prevalent forms expose to the risk of acquiring during infancy or adulthood additional disorders that endanger the life of patients much more than hemorrhages. Thus, inherited thrombocytopenias are complex disorders with quite different clinical features and prognosis. Identification of novel genes whose mutations result in low platelet count greatly advanced also our knowledge of the megakaryocyte biology and proved beyond any doubt that the defective proteins play an essential role in platelet biogenesis or survival in humans. Based on the study of inherited thrombocytopenias, we better understood the sequence of molecular events regulating megakaryocyte differentiation, maturation, and platelet release. Since nearly 50% of patients have as yet unidentified genetic or molecular mechanisms underlying their inherited thrombocytopenia, further studies are expected to reveal new clinical entities and new molecular mechanisms of platelet production.

Simple SummaryTranscriptome is the study of gene expression at the RNA level. In this study, high-throughput transcriptome sequencing technology was used to compare and analyze the gene expression differences in the hypothalamus, pituitary, and ovary tissues of Xinjiang Yili geese with high and low egg production. We preliminarily screened 30 candidate genes related to egg production regulation of Xinjiang Yili geese, including the calcium signaling pathway. Therefore, this study laid a theoretical foundation for further elucidating the molecular mechanism of egg production of Yili geese in Xinjiang.The study was conducted to investigate the transcriptomic differences of the hypothalamic-pituitary-gonadal axis between Xinjiang Yili geese with high and low egg production and to find candidate genes regulating the egg production of Xinjiang Yili geese. The 8 selected Xinjiang Yili Geese with high or low egg production (4 for each group) were 3 years old, with good health, and under the same feeding condition. High-throughput sequencing technology was used to sequence cDNA libraries of the hypothalami, pituitary glands, and ovaries. The sequencing data were compared and analyzed, and the transcripts with significant differences were identified and analyzed with bioinformatics. The study showed that the transcriptome sequencing data of the 24 samples contained a total of 1,176,496,146 valid reads and 176.47 gigabase data. Differential expression analyses identified 135, 56, and 331 genes in the hypothalami, pituitary glands, and ovaries of Xinjiang Yili geese with high and low egg production. Further annotation of these differentially expressed genes in the non-redundant protein sequence database (Nr) revealed that 98, 52, and 309 genes were annotated, respectively. Through the annotations of GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases, 30 candidate genes related to the egg production of Xinjiang Yili geese were preliminarily selected. The gap junction, focal adhesion, and ECM-receptor interaction signaling pathways were enriched with the hypothalamic, pituitary, and ovarian differentially expressed genes, and the calcium signaling pathway was enriched with the pituitary and ovarian differentially expressed genes. Thus, these pathways in the hypothalamic-pituitary-gonadal axis may play an important role in regulating egg production of Xinjiang Yili geese. The results provided the transcriptomic information of the hypothalamic-pituitary-gonadal axis of Xinjiang Yili geese and laid the theoretical basis for revealing the molecular mechanisms regulating the egg-laying traits of Xinjiang Yili geese.

It is well-known that highly reactive hydroxyl radicals (HO•) can be produced by the classic Fenton system and our recently discovered haloquinone/H2O2 system, but rarely from thiol-derivatives. Here, we found, unexpectedly, that HO• can be generated from H2O2 and thiourea dioxide (TUO2), a widely used and environmentally friendly bleaching agent. A carbon-centered radical and sulfite were detected and identified as the transient intermediates, and urea and sulfate as the final products, with the complementary application of electron spin-trapping, oxygen-18 isotope labeling coupled with HPLC/MS analysis. Density functional theory calculations were conducted to further elucidate the detailed pathways for HO• production. Taken together, we proposed that the molecular mechanism for HO• generation by TUO2/H2O2: TUO2 tautomerizes from sulfinic acid into ketone isomer (TUO2-K) through proton transfer, then a nucleophilic addition of H2O2 on the S atom of TUO2-K, forming a S-hydroperoxide intermediate TUO2-OOH, which dissociates homolytically to produce HO•. Our findings represent the first experimental and computational study on an unprecedented new molecular mechanism of HO• production from simple thiol-derived sulfinic acids, which may have broad chemical, environmental, and biomedical significance for future research on the application of the well-known bleaching agent and its analogs.

Glutamate production is a typical success in industrial fermentation. Annual production of glutamate by Corynebacterium glutamicum is over 1.5 million tons per year worldwide. It is well known that there are some triggers of glutamate overproduction by C. glutamicum: depletion of biotin, which is required for cell growth; addition of detergent; addition of β-lactam antibiotics such as penicillin; and addition of ethambutol or cerulenin. A marked change in metabolic pathways occurs after glutamate overproduction is triggered. In this chapter, recent studies on the molecular mechanisms of glutamate production are described with a particular focus on triggering mechanisms, changes in key enzyme activities, and secretion of glutamate. Recent advances in genome-wide studies, including genomics, proteomics, metabolomics, and on metabolic flux analysis of flux redistribution during glutamate overproduction are discussed as well. The biosynthesis of the related amino acids glutamine and proline and strategies for their overproduction are also described.

Plants produce specialized metabolites in various organs which serve important functions in defense and development. However, the molecular regulatory mechanisms of oleoresin production in stems from broadleaved tree species are not fully understood. To determine whether endogenous developmental cues play a role in the regulation of oleoresin biosynthesis in tree stems, anatomy, multi-omics and molecular experiments were utilized to investigate the change of secretory structures, chemical profiles and gene expression in different ontogenetic stages of Sindora glabra tree, which accumulates copious amount of sesquiterpene-rich oleoresin in stems. The size of secretory canals and the concentration of five sesquiterpenes in Sindora stems exhibited obvious increase with plant age, from 0.5- to 20-year-old plants. Moreover, α-copaene and β-copaene were found to be stem-specific sesquiterpenes. Metabolomic analysis revealed that salicylic acid highly accumulated in mature stems, but the content of triterpenes was greatly decreased. The expression of three repressors AUX/IAA, DELLA and JAZ involved in hormone signaling transduction pathways was significantly downregulated in stems of 10- and 20-year-old plants. Two key genes SgTPS3 and SgTPS5 were identified, whose expression was highly correlated with the accumulation patterns of specific sesquiterpenes and their enzymatic products were consistent with the chemical profiles in the stem. The promoters of three SgTPSs exhibiting high activity were isolated. Furthermore, we demonstrated that SgSPL15 directly interacts with SgTPS3 and SgTPS5 promoters and activates SgTPS5 expression but SgSPL15 inhibits SgTPS3 expression. In addition, SgSPL15 enhanced sesquiterpene levels by upregulating AtTPSs expression in Arabidopsis. These results suggested that sesquiterpene biosynthesis in S. glabra stem was dependent on the regulation of endogenous hormones as well as plant age, and SgSPL15 might act as a buffering factor to regulate sesquiterpene biosynthesis by targeting SgTPS genes.

The importance of this study lies in its unveiling of a molecular regulatory mechanism associated with the most important food poisoning toxin and the evolution of Staphylococcal food poisoning (SFP)-associated clone. SFP is primarily caused by Staphylococcus aureus, with Staphylococcal enterotoxin A (SEA) being commonly involved in many cases. Thus, SEA has been recognized as a major toxin type. However, despite almost a century since its discovery, the complete mechanism of SEA production is as yet unknown. In this study, we analyzed an SEA-producing SFP clone isolated in East Asia and discovered that this strain, besides acquiring the high SEA-producing phage, exhibits remarkably high SEA production due to the low activity of SarS, an intrinsic regulatory factor. This is the first report documenting the evolution of the SFP clone through the coordinated action of exogenous mobile genetic factors and endogenous regulators on this notorious toxin.

Molecular Biology of Orchid Flowers: With Emphasis on Phalaenopsis

The MDCK cell line is the primary cell line used for influenza vaccine production. Using genetic engineering technology to change the expression and activity of genes that regulate virus proliferation to obtain high-yield vaccine cell lines has attracted increasing attention. A comprehensive understanding of the key genes, targets, and molecular mechanisms of viral regulation in cells is critical to achieving this goal, yet the post-transcriptional regulation mechanism involved in virus proliferation-particularly the effect of lncRNA on influenza virus proliferation-is still poorly understood. Therefore, this study used high-throughput RNA-seq technology to identify H1N1 infection-induced lncRNA and mRNA expression changes in MDCK cells and explore the regulatory relationship between these crucial lncRNAs and their target genes. In response to H1N1 infection in MDCK cells 16 h post-infection (hpi) relative to uninfected controls, we used multiple gene function annotation databases and initially identified 31,501 significantly differentially expressed (DE) genes and 39,920 DE lncRNAs (|log2FC| > 1, p < 0.05). Among these, 102 lncRNAs and 577 mRNAs exhibited predicted correlations with viral response mechanisms. Based on the magnitude of significant expression differences, related research, and RT-qPCR expression validation at the transcriptional level, we further focused on 18 DE mRNAs and 32 DE lncRNAs. Among these, the differential expression of the genes RSAD2, CLDN1, HCLS1, and IFIT5 in response to influenza virus infection was further verified at the protein level using Western blot technology, which showed results consistent with the RNA-seq and RT-qPCR findings. We then developed a potential molecular regulatory network between these four genes and their six predicted lncRNAs. The results of this study will contribute to a more comprehensive understanding of the molecular mechanism of host cell non-coding RNA-mediated regulation of influenza virus replication. These results may also identify methods for screening target genes in the development of genetically engineered cell lines capable of high-yield artificial vaccine production.

Nitrogen is an essential macronutrient for plant growth and development, taken up mainly as ammonium (NH₄⁺) and nitrate (NO₃⁻). To compare growth, physiological traits, and osmotic responses of licorice (Glycyrrhiza glabra) to different N sources across culture systems, a completely randomized design with three replicates tested four cultivation methods, aeroponics, nutrient-film technique (NFT), substrate culture, and soil culture, and three nitrogen regimes: NH₄⁺, NO₃⁻, and an equimolar NH₄⁺ + NO₃⁻ mixture. Sole NH₄⁺ nutrition markedly increased proline, total soluble carbohydrates, sucrose, total phenolic compounds, and total flavonoids in both leaves and roots, and elevated activities of phenylalanine ammonia-lyase, polyphenol oxidase, guaiacol peroxidase, glutamine synthetase, and overall free-radical-scavenging capacity. Plants fed with ammonium consistently exhibited the highest glycyrrhizic acid levels across all soilless culture systems. The soil cultivation system showed the lowest glycyrrhizic acid concentrations and no significant differences among nitrogen sources. However, NH₄⁺ decreased biomass, chlorophyll pigments, maximal quantum yield of PS II (Fv/Fm), performance index (PI), total soluble protein, starch, nitrate-reductase activity, and tissue concentrations of N, Ca²⁺, K⁺, Mg²⁺, and P. All parameters were significantly influenced by the culture system; aeroponics produced the highest growth and chlorophyll levels, followed by NFT, substrate culture, and soil culture. Licorice thus benefits most from a mixed NH₄⁺ + NO₃⁻ supply in advanced soilless systems, with aeroponics providing the remarkable overall performance. In conclusion, licorice cultivation benefits from the synergistic effects of combined nitrogen forms and advanced soilless culture systems. Future studies should aim to elucidate the molecular mechanisms underlying the observed physiological responses to different nitrogen sources and further optimize nutrient ratios for sustainable, high-yield licorice production in aeroponic systems. Additionally, the long-term effects of these cultivation strategies on the quality and bioactivity of medicinal compounds in licorice should be investigated.