Expression Levels Of Enzymes Research Articles

Mitochondrial dysfunction, iron accumulation, lipid peroxidation, and inflammasome activation in cellular models derived from patients with multiple sclerosis.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS). Despite advancements in managing relapsing active illness, effective treatments for the irreversible progressive decline in MS remain limited. Research employing skin fibroblasts obtained from patients with neurological disorders revealed modifications in cellular stress pathways and bioenergetics. However, research using MS patient-derived cellular models is scarce. In this study, we collected fibroblasts from two MS patients to investigate cellular pathological alterations. We observed that MS fibroblasts showed a senescent morphology associated with iron/lipofuscin accumulation and altered expression of iron metabolism proteins. In addition, we found increased lipid peroxidation and downregulation of antioxidant enzymes expression levels in MS fibroblasts. When challenged against erastin, a ferroptosis inducer, MS fibroblasts showed decreased viability, suggesting increased sensitivity to ferroptosis. Furthermore, MS fibroblasts presented alterations in the expression levels of autophagy-related proteins. Interestingly, these alterations were associated with mitochondrial dysfunction and inflammasome activation. These findings were validated in 7 additional patient-derived cell lines. Our findings suggest that the underlying stress phenotype of MS fibroblasts may be disease-specific and recapitulate the main cellular pathological alterations found in the disease such as mitochondrial dysfunction, iron accumulation, lipid peroxidation, inflammasome activation, and pro-inflammatory cytokine production.

PbMADS49 Regulates Lignification During Stone Cell Development in 'Dangshansuli' (Pyrus bretschneideri) Fruit.

Lignified stone cell content is one of the critical factors affecting 'Dangshansuli' fruit quality. The function of MADS-box transcription factors in regulating lignin biosynthesis in pear fruit is still less. In this study, PbMADS49 gene silencing inhibited the lignin biosynthesis and stone cell secondary wall development of pear fruit mainly through reducing the expression levels of lignin monomer polymerisation key enzymes (PbPRX33 and PbPRX45). PbMADS49 was a transcriptional repressor inhibiting its transcription by binding to the CArG element in the target gene promoter. Combined with the co-expression network and promoter cis-acting element analysis, we hypothesised that PbMADS49 positively regulates the transcription of PbPRX33 through PbWRKY63. The gene silencing effect of homologous genes PbPRX33-1 and PbPRX33-2 was consistent with PbMADS49, and PbPRX33-2 was more significant than PbPRX33-1. This study shows that PbMADS49 is a positive regulator of stone cell lignification, providing new insights into the development mechanism of pear stone cells.

Air-liquid interface culture alters the characteristics and functions of monolayers generated from human iPS cell‑derived enterocyte‑like cell organoids.

To evaluate the intestinal absorption and metabolism of orally administered drugs, human induced pluripotent stem (iPS) cell‑derived enterocyte‑like cells (ELCs) are expected to be useful. In a previous report, we succeeded in developing a highly functional monolayer platform (ELC-org-mono) from human iPS cell-derived ELCs through an organoid culture and demonstrated its suitability for pharmacokinetic studies. In recent years, the air-liquid interface (ALI) culture model was developed, allowing for the culture of epithelial tissue under conditions that mimic the in vivo environment. In the present study, we applied ALI culture to ELC-org-mono for further improvement of intestinal functions. ALI culture of ELC-org-mono greatly developed goblet cells and enhanced the gene expression levels of many drug-metabolizing enzymes, drug transporters and intestinal differentiation markers. However, their activities were not enhanced. RNA-seq analysis suggested that ALI culture increased the expression of genes related to metabolic processes but decreased glycolytic processes. Analysis of glycolytic capacity confirmed that ALI culture decreased glycolytic activities. Thus, there is room for some adjustment in the ALI culture model to optimize its applicability to pharmacokinetic studies using ELC-org-mono.

Sexual spores in mushrooms: bioactive compounds, factors and molecular mechanisms of spore formation.

Throughout the life cycle of mushrooms, countless spores are released from the fruiting bodies. The spores have significant implications in the food and medicine industries due to pharmacological effects attributed to their bioactive ingredients. Moreover, high concentration of mushroom spores can induce extrinsic allergic reactions in mushroom cultivation workers. Therefore, it is important to study the bioactive ingredients of medicinal mushroom spores and molecular mechanisms of spore formation to develop healthcare products utilizing medicinal mushroom spores and breed sporeless/low- or high-spore-producing strains. This review summarizes the bioactive compounds of mushroom spores, the influence factors and molecular mechanisms of spore formation. Many bioactive compounds extracted from mushroom spores have a wide range of pharmacological activities. Several exogenous factors such as temperature, humidity, light, nutrients, and culture matrix, and endogenous factors such as metabolism-related enzymes activities and expression levels of genes related to sporulation individually or in combination affect the formation, size, and discharge of spores. The future research directions are also discussed for supplying references to analyze the bioactive compounds of spores and the molecular mechanisms of spore formation in mushrooms.

Machine learning reveals novel compound for the improved production of chitooligosaccharides in Escherichia coli.

In order to improve predictability of outcome and reduce costly rounds of trial-and-error, machine learning models have been of increasing importance in the field of synthetic biology. Besides applications in predicting genome annotation, process parameters and transcription initiation frequency, such models have also been of help for pathway optimization. The latter is a common strategy in metabolic engineering and improves the production of a desirable compound by optimizing enzyme expression levels of the production pathway. However, engineering steps might not lead to sufficient improvement, and bottlenecks may remain hidden among the hundreds of metabolic reactions occurring in a living cell, especially if the production pathway is highly interconnected with other parts of the cell's metabolism. Here, we use the synthesis of chitooligosaccharides (COS) to show that the production from such complex pathways can be improved by using machine learning models and feature importance analysis to find new compounds with an impact on COS production. We screened Escherichia coli libraries of engineered transcription regulators with an expected broad range of metabolic diversity and trained several machine learning models to predict COS production titers. Subsequent feature analysis led to the finding of iron, whose addition we could show improved COS production in vivo up to 2-fold. Additionally, the analysis revealed important clues for future engineering steps.

Methyl Jasmonate Was Involved in Hydrogen Sulfide-Alleviated Cadmium Stress in Cucumber Plants Through ROS Homeostasis and Chlorophyll Metabolism.

Cadmium (Cd), as one of the most toxic nonessential elements, severely prohibits plant growth and development. Hydrogen sulfide (H2S) and methyl jasmonate (MeJA) play essential roles in plant response to abiotic stress. However, the potential mechanism of H2S and MeJA in alleviating Cd stress in plants remains unclear. In the current study, the importance and crosstalk of H2S and MeJA in the Cd tolerance of cucumber seedlings have been investigated. Our results revealed that Cd stress obviously prohibited the growth of cucumber seedlings. Optimal concentrations of H2S donor sodium hydrosulfide (NaHS) or MeJA treatment, respectively, or in combination, significantly enhanced seedling growth under Cd stress. However, the positive effects of H2S during seedling growth were obviously reversed by the application of MeJA biosynthesis inhibitors, which implied that MeJA might be involved in the H2S-improved growth of cucumber seedlings under Cd stress. Moreover, Cd stress resulted in the increase in hydrogen peroxide (H2O2), superoxide radical (O2·-) accumulation, and impaired the functioning of the ascorbate-glutathione cycle. Both H2S and MeJA decreased the reactive oxygen species (ROS) level and ameliorated the negative effects of Cd stress through significantly increasing the ratio of ascorbate (AsA)/dehydroascorbic acid (DHA) and reduced glutathione (GSH)/oxidized glutathione (GSSG). Besides that, the expression level of ROS scavenge genes was significantly upregulated by the application of exogenous H2S or MeJA treatment. Moreover, H2S and MeJA significantly enhanced the chlorophyll concentration and inhibited chlorophyll degradation through decreasing the expression levels of chlorophyll catabolic enzymes. Additionally, exogenous H2S and MeJA obviously enhanced the chlorophyll fluorescence. However, MeJA biosynthesis inhibitors significantly suppressed the positive role of H2S. The above results suggested MeJA is involved in H2S-induced Cd stress alleviation in cucumber seedlings through enhancing ROS-scavenge capacity and improving the photosynthesis system.

Split Application of Potassium Reduces Rice Chalkiness by Regulating Starch Accumulation Process Under High Temperatures

Chalkiness in rice is adversely affected by high temperatures during the flowering and grain-filling stages. Potassium (K) is essential for improving grain quality and heat resilience. The effects of split application K fertilizer on rice chalkiness under high temperatures during the flowering and grain-filling stages were investigated in this study. Four treatments, including ambient temperatures with basal K fertilizer (AT-K1), high temperatures with basal K fertilizer (HT-K1), high temperatures with 70% K pre-transplanting and 30% K at the heading stage (HT-K2), and high temperatures with 30% K pre-transplanting and 70% K at the heading stage (HT-K3), were conducted. The results revealed that the chalky grain rate and chalkiness degree were reduced by 9.2–13.72% and 12.16–19.91%, respectively, by the split application of K fertilizer through effectively modulating the sucrose-to-starch conversion process in the rice grains, relative to the single basal application of K fertilizer under high temperatures. Specifically, the split application of K fertilizer reduced the enzymatic activities of SuSy, ADPGase, and SBE by 3.17–34.20% at 5–10 DAA, and GBSS and SSS by 6.48–13.50% at 5 DAA, but enhanced them by 5.50–54.90% from 15 DAA and 2.07–97.10% from 10 DAA. Similarly, the gene expression levels of enzymes involved in this process were decreased by 3.52–24.12% at 5 DAA but increased by 8.61–30.00% at 20 DAA by the split application of K fertilizer. This modulation led to a retardation in the excessive accumulation of starch during the early grain-filling stage but a higher starch accumulation rate during the middle and later stages, combined with a longer duration of starch accumulation, ultimately resulting in higher starch accumulation and reduced rice chalkiness. These results suggest that the application of K fertilizer during the heading stage is effective in compensating the deterioration of rice chalkiness by high temperatures.

CHI-KAT8i5 suppresses ESCC tumor growth by inhibiting KAT8-mediated c-Myc stability.

The integrated analysis of histone modifier enzymes in solid tumors, especially in esophageal squamous cell carcinoma (ESCC), is still inadequate. Here, we investigate the expression levels of histone modifier enzymes in ESCC tissues. Notably, KAT8 (lysine acetyltransferase 8) is identified as a prognostic and therapeutic biomarker in ESCC. Esophageal-tissue-specific deletion of KAT8 in mice led to less tumor burden after induction of tumorigenesis via 4-nitroquinoline N-oxide (4NQO) treatment compared with wild-type mice. Meanwhile, silencing KAT8 significantly suppresses tumor growth in cell-line-derived xenograft (CDX) and patient-derived xenograft (PDX) models. Mechanically, we confirm that KAT8 regulates c-Myc protein stability by directly binding it. Furthermore, we design and screen a specific KAT8 inhibitor (CHI-KAT8i5) that significantly attenuates tumor growth invitro and invivo, providing promising potential for clinical application. Thus, our work identifies that KAT8 could serve as a potential clinically relevant biomarker and therapeutic target in patients with ESCC and that KAT8 inhibitor is a promising lead candidate for ESCC therapy.

Impact of copper stress in the intestinal barriers and gut microbiota of Chinese stripe-necked turtle (Mauremys sinensis).

Copper is used to treat algal blooms, macrophyte infestations and other environmental issues, but its rising ambient levels harm aquatic animals, especially their intestines. However, its impact on turtles' digestive health is not well understood, and the risks are unclear. This study investigates the effects of copper on the intestinal health of Chinese stripe-necked turtle, focusing on histomorphology, mucosal barrier function, gene expression, and gut microbiota. Copper stress caused intestinal damage, characterized by shortened villi, inflammatory cell infiltration, and reduced epithelial layer thickness, as well as decreased acidic mucins, increased villi edema and inflammation. The mRNA expression level of bacteriostatic enzymes significantly reduced. Furthermore, This study found that copper exposure increases gut permeability by suppressing tight junction genes and triggers an inflammatory response in the gut, as indicated by elevated inflammatory cytokines. At the phylum level, Firmicutes exhibited a significant decrease, whereas Bacteroidota displayed a notable increase, and Fusobacteriota showed a substantial reduction in relative abundance in copper-treated groups. Similarly, at genus level Romboutsia, Cetobacterium decreased, while Turicibacter and Sarcina significantly increases in copper-treated groups compared to the control. This indicating the unique properties of copper including its essentiality, reactivity, and accumulation enables it to profoundly impact gut bacteria, altering both their composition and function. Copper's dual role as a nutrient and toxicant uniquely impacts gut microbes. Our findings suggest that copper stress compromises the intestinal physical, immune, chemical, and microbial barrier in M. sinensis, all of which contribute to the turtle's poor health.

Personalized statin therapy: Targeting metabolic processes to modulate the therapeutic and adverse effects of statins.

Statins are widely used for treating lipid disorders and cardiovascular diseases. However, the therapeutic efficiency and adverse effects of statins vary among different patients, which numerous clinical and epidemiological studies have attributed to genetic polymorphisms in statin-metabolizing enzymes and transport proteins. The metabolic processes of statins are relatively complex, involving spontaneous or enzyme-catalyzed interconversion between more toxic lactone metabolites and active acid forms in the liver and bloodstream, influenced by multiple factors, including the expression levels of many metabolic enzymes and transporters. Addressing the variable statin therapeutic outcomes is a pressing clinical challenge. Transcription factors and epigenetic modifications regulate the metabolic enzymes and transporters involved in statin metabolism and disposition and, therefore, hold promise as 'personalized' targets for achieving optimized statin therapy. In this review, we explore the potential for customizing therapy by targeting the metabolism of statin medications. The biochemical bases of adverse reactions to statin drugs and their correlation with polymorphisms in metabolic enzymes and transporters are summarized. Next, we mainly focus on the regulatory roles of transcription factors and epigenetic modifications in regulating the gene expression of statin biochemical machinery. The recommendations for future therapies are finally proposed by targeting the central regulatory factors of statin metabolism.

Resilience to Alzheimer's disease associates with alterations in perineuronal nets.

Some individuals show intact cognition despite the presence of neuropathological hallmarks of Alzheimer's disease (AD). The plasticity of parvalbumin (PV)-containing interneurons might contribute to resilience. Perineuronal nets (PNNs), that is, extracellular matrix structures around neurons, modulate PV neuron function. We hypothesize that PNNs play a role in resilience to AD. PNN amount and morphology were determined in immunolabelled sections of the frontal cortex of control, AD and resilient subjects. Expression levels of genes related to PNNs and microglia signatures were evaluated by bulk RNA sequencing. The expression of the PNN-component aggrecan around PV neurons is decreased in resilient and AD subjects, whereas PNN-sugar chains are reduced only in resilient subjects. In AD, fewer presynaptic terminals on PV neurons are detected and genes related to PNN degradation are upregulated. These data show distinct PNN changes in individuals resilient to AD, which may contribute to preserved cognition despite the neuropathology. Aggrecan levels are decreased in the frontal cortex of AD and resilient subjects. In resilient subjects, WFA+ PNNs are reduced around neuronal somata. In AD patients, PV neurons show disrupted WFA peridendritic staining and synaptic loss. Expression levels of PNN-degrading enzymes are higher in AD. Excitatory neurons bearing a PNN show low amounts of ptau.

Effect of the SGLT2 inhibitor ipragliflozin on the expression of genes that regulate skin function.

Skin disorders occur more frequently with sodium-dependent glucose cotransporter type 2 (SGLT2) inhibitors than with other antidiabetic drugs. We conducted basic research using ipragliflozin, with the aim of identifying new measures to prevent skin disorders caused by SGLT2 inhibitors. db/db type 2 diabetes model mice were orally administered ipragliflozin (10 mg/kg or 30 mg/kg) once a day for 28 days and skin function genes were analysed by real-time RT-PCR or Western blotting. No difference in the expression level of collagen (Col1a1 and Col1a2) in the skin was detected between the ipragliflozin treatment group and the control group. On the other hand, the expression levels of enzymes involved in the synthesis and decomposition of hyaluronic acid (Has2 and Hayl1) and enzymes involved in the synthesis and decomposition of ceramide (Sptlc1, Sptlc2, Asah1, and Acer1) were significantly decreased by the administration of ipragliflozin. Furthermore, the expression levels of filaggrin (Flg), loricrin (Lor), elastin (Eln), and aquaporin-3 (Aqp3) in the skin were lower in the ipragliflozin treatment group than in the control group. It was revealed that ipragliflozin reduces the expression of genes involved in skin barrier and moisturizing functions, which this may be one of the mechanisms through which this drug causes skin disorders.

Excessive glutathione intake contributes to chemotherapy resistance in breast cancer: a propensity score matching analysis

BackgroundWe aim to explore the impact of excessive glutathione (GSH) intake on chemotherapy sensitivity in breast cancer.MethodsClinicopathological data were collected from 460 breast cancer patients who underwent adjuvant chemotherapy from January 2016 to December 2019 from Zhengzhou University People’s Hospital. The clinicopathological characteristics following GSH treatment were collected and compared with those in Non-GSH group after 1:2 propensity score matching (PSM). Intracellular GSH levels and the expression of antioxidant enzymes (NRF2, GPX4 and SOD1) were evaluated in tumor tissues in 51 patients receiving neoadjuvant chemotherapy.ResultsThe recurrence rate after adjuvant chemotherapy was significantly higher in the GSH group (n = 28, 31.8%) than that in the Non-GSH group (n = 39, 22.2%; P = 0.010). Additionally, patients in the HGSH group (high GSH intake, ≥ 16 days) exhibited an elevated recurrence rate compared to that in the LGSH group (low GSH intake, < 16 days) (n = 15 (46.8%) vs. n = 52 (22.4%); P = 0.003). Cox regression revealed that High GSH intake, Ki67 ≥ 30%, Triple negative and Lymphovascular invasion were independent risk factors of progression after adjuvant chemotherapy. Among patients receiving neoadjuvant chemotherapy, intracellular GSH levels and the expression levels of antioxidant enzymes (NRF2, GPX4 and SOD1) in the resistant patients were substantially higher (P < 0.001).ConclusionsExcessive GSH intake may contribute to chemotherapy resistance in breast cancer, and the levels of intracellular GSH and antioxidant enzymes are elevated in resistant patients after neoadjuvant chemotherapy, indicating that the standardization of GSH intake may assist in reducing chemotherapy resistance.

Engineered β1-3-N-Acetylglucosaminyltransferase Facilitating the One-Pot Multienzyme Synthesis of Human Milk Oligosaccharides.

β1-3-linked N-acetylglucosaminide is a prevalent carbohydrate motif found in oligosaccharides, polysaccharides, glycoproteins, and glycolipids. It is a crucial component of human milk oligosaccharides (HMOs). Neisseria meningitidis β1-3-N-acetylglucosaminyltransferase (NmLgtA) catalyzes the formation of a glycosidic bond and has the potential for use in synthesizing HMOs. However, this application is hindered by challenges such as low levels of enzyme expression, poor stability, and significant aggregation. Since there is no available crystal structure for NmLgtA, we used its AlphaFold 2 predicted structure to identify potential unfavorable factors. We then modified the enzyme by removing the 17 N-terminal amino acids and substituting nine specific residues. The engineered NmLgtA-Opti exhibited improved thermal stability, increased soluble protein expression, complete relief from aggregation, and enhanced catalysis while maintaining its catalytic specificity and substrate promiscuity. Furthermore, NmLgtA-Opti maximizes substrate utilization and can be employed in a sequential one-pot multienzyme platform for high-yield production of HMOs.

Optimization of Ultraviolet-B Treatment for Enrichment of Total Flavonoids in Buckwheat Sprouts Using Response Surface Methodology and Study on Its Metabolic Mechanism.

Buckwheat possesses significant nutritional content and contains different bioactive compounds, such as total flavonoids, which enhance its appeal to consumers. This study employed single-factor experiments and the response surface methodology to identify the optimal germination conditions for enhancing the total flavonoid content in buckwheat sprouts through ultraviolet-B treatment. The research showed that buckwheat sprouts germinated for 3 days at a temperature of 28.7 °C while being exposed to ultraviolet-B radiation at an intensity of 30.0 μmol·m-2·s-1 for 7.6 h per day during the germination period resulted in the highest total flavonoid content of 1872.84 μg/g fresh weight. Under these specified conditions, ultraviolet-B treatment significantly elevated the activity and gene expression levels of enzymes related to the phenylpropanoid metabolic pathway, including phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumarate coenzyme A ligase, and chalcone isomerase. Ultraviolet-B treatment caused oxidative damage to buckwheat sprouts and inhibited their growth, but ultraviolet-B treatment also enhanced the activity of key enzymes in the antioxidant system, such as catalase, peroxidase, superoxide dismutase, and ascorbate peroxidase. This research provided a technical reference and theoretical support for enhancing the isoflavone content in buckwheat sprouts through ultraviolet-B treatment.

Versican Proteolysis by ADAMTS: Understanding Versikine Expression in Canine Spontaneous Mammary Carcinomas.

Background: The present study investigates VKINE, a bioactive proteolytic fragment of the proteoglycan VCAN, as a novel and significant element in the tumor extracellular matrix (ECM). Although VKINE has been recognized for its immunomodulatory potential in certain tumor types, its impact on ECM degradation and prognostic implications remains poorly understood. Objectives: This study aimed to evaluate VCAN proteolysis and its association with ADAMTS enzymes involved in extracellular matrix remodeling in spontaneous canine mammary gland cancer. Methods: The expression levels of VKINE, ADAMTS enzymes, and collagen fibers were comparatively analyzed in situ and in invasive areas of carcinoma in mixed tumor (CMT) and carcinosarcoma (CSS) with different prognoses. Results: VKINE was notably expressed in the stroma adjacent to the invasion areas in CMT, whereas ADAMTS-15 was identified as the enzyme associated with VCAN proteolysis. Inverse correlations were observed between type III collagen and VCAN expression in in situ areas. Conclusions: Our findings suggest that VKINE and ADAMTS-15 play crucial roles in the tumor microenvironment, influencing invasiveness and type III collagen deposition. This study contributes to a better understanding of the dynamics within the ECM, paving the way for potential new tools in diagnosing and treating human and canine mammary tumors.

Activation of the kynurenine pathway identified in individuals with covert hepatic encephalopathy.

HE is a neuropsychiatric complication of liver disease characterized by systemic elevation in ammonia and proinflammatory cytokines. These neurotoxins cross the blood-brain barrier and cause neuroinflammation, which can activate the kynurenine pathway (KP). This results in dysregulated production of neuroactive KP metabolites, such as quinolinic acid, which is known to cause astrocyte and neuronal death. Our aim was to compare KP activity between patients with covert HE (CHE), patients without encephalopathic cirrhosis (NHE), and healthy controls (HCs). This was a single-center prospective cohort study conducted between 2018 and 2021 at St Vincent's Hospital, Sydney. Overall, 13 patients with CHE, 10 patients with NHE, and 12 with HC were recruited. Patients with cirrhosis were diagnosed with CHE if they scored ≤-4 on the Psychometric Hepatic Encephalopathy Score. KP metabolite levels were quantified on plasma samples via HPLC and gas chromatography/mass spectrometry. One-way Kruskal-Wallis test was used to compare the expression levels of KP enzymes. KP was highly activated in patients with cirrhosis, demonstrated by higher levels of activity in the rate-limiting enzymes, indoleamine 2,3-dioxygenase, and tryptophan-2,3-dioxygenase in both CHE (65.04±20.72, p=0.003) and patients with NHE (64.85±22.10, p=0.015) compared to HC (40.95±7.301). Higher quinolinic acid concentrations were demonstrated in CHE (3726nM±3385, p<0.001) and patients with NHE (1788nM±632.3, p=0.032) compared to HC (624nM±457). KP activation was positively correlated with inflammatory marker C-reactive protein in patients with CHE (Rs=0.721, p≤0.01). KP is highly activated in patients with CHE, resulting in heightened production of neurotoxic metabolites. Dysregulation of the pathway is demonstrable in patients who do not yet show clinical signs of neurocognitive impairment. Therapeutic agents that modulate KP activity may be able to alleviate symptoms of patients with CHE.

Multienzyme Cascade Synthesis of Rare Sugars From Glycerol in Bacillus subtilis.

Rare sugars are valuable and unique monosaccharides extensively utilized in the food, cosmetics, and pharmaceutical industries. Considering the high purification costs and the complex processes of enzymatic synthesis, whole-cell conversion has emerged as a significantly important alternative. The Escherichia coli strain was initially used in whole-cell synthesis of rare sugars. However, its pathogenic nature poses limitations to its widespread applications. Consequently, there is an urgent need to explore biologically safe strains for the efficient production of rare sugars. In this study, the generally regarded as safe (GRAS) strain Bacillus subtilis was employed as the chassis cells to produce rare sugars via whole-cell conversion. Three genes encoding alditol oxidase (AldO), L-rhamnulose-1-phosphate aldolase (RhaD), and fructose-1-phosphatase (YqaB) involved in rare sugars biosynthesis were heterogeneously expressed in B. subtilis to convert the only substrate glycerol into rare sugars. To enhance the expression levels of the relevant enzymes in B. subtilis, different promoters for aldO, rhaD, and yqaB were investigated and optimized in this system. Under the optimized reaction conditions, the maximum total production titer was 16.96g/L of D-allulose and D-sorbose with a conversion yield of 33.9% from glycerol. Furthermore, the engineered strain produced 26.68g/L of D-allulose and D-sorbose through fed-batch for the whole-cell conversion, representing the highest titer from glycerol reported to date. This study demonstrated an efficient and cost-effective method for thesynthesis of rare sugars, providing a food-grade platform with the potential to meet the growing demand for rare sugars in industries.

Effect of Elderberry (Sambucus nigra L.) Extract Intake on Normalizing Testosterone Concentration in Testosterone Deficiency Syndrome Rat Model Through Regulation of 17β-HSD, 5α-Reductase, and CYP19A1 Expression.

Background/Objectives. Men experience Leydig cell and mitochondrial dysfunction due to the accumulation of reactive oxygen species during aging, leading to hormonal imbalances in the body. This results in symptoms of testosterone deficiency syndrome (TDS) as testosterone levels decline. Consequently, there is a growing need for alternative therapies, such as phytotherapy, to regulate testosterone secretion. Methods. In this study, we evaluated the potential of elderberry extract powder (KSB191) as a functional ingredient for improving TDS by analyzing its mechanism in regulating testosterone imbalance. The major compounds of KSB191 were rutin and fructose-leucine, and the efficacy of KSB191 was confirmed by observing increases in total testosterone, free testosterone, and sperm motility in an aged rat model with decreased testosterone levels. Additionally, we assessed safety by analyzing levels of prostate-specific antigen, alanine aminotransferase, aspartate aminotransferase, and creatinine. Results. To confirm the effectiveness of KSB191 in increasing testosterone synthesis and inhibiting its breakdown, we analyzed the expression levels of genes related to testosterone synthesis and degradation in the testis tissue. KSB191 not only increases the expression levels of enzymes (3β-HSD, CYP17A1, and 17β-HSD) that catalyze testosterone synthesis in Leydig cells, but also reduces the expression of enzymes (5α-reductase and CYP19A1) that degrade testosterone, thereby enhancing testosterone production in the body. Conclusions. KSB191 is predicted to be a novel functional ingredient that acts on Leydig cells and increases testosterone synthesis (particularly, the increase in free testosterone), ultimately alleviating the symptoms of TDS.

Physiology and Metabolism Alterations in Flavonoid Accumulation During Buckwheat (Fagopyrum esculentum Moench.) Sprouting.

In this research, we investigated the physiological modifications, flavonoid metabolism, and antioxidant systems of two buckwheat (Fagopyrum esculentum Moench.) cultivars, Pintian and Suqiao, during germination. The results demonstrated an initial increase followed by a subsequent decline in the flavonoid content of the buckwheat sprouts throughout germination. On the third day of germination, the highest flavonoid concentrations were observed, with the Pintian and Suqiao varieties reaching 996.75 and 833.98 μg/g fresh weight, respectively. Both the activity and relative gene expression level of the flavonoid metabolizing enzyme showed a significant rise in 3-day-old buckwheat sprouts, which was strongly correlated with the flavonoid content. The correlation analysis revealed that the buckwheat sprouts accumulated flavonoids by enhancing the activities and gene expression levels of flavonoid synthases. The antioxidant capacity and the activities and gene expression profiles of the antioxidant enzymes in both buckwheat cultivars notably increased after three days of germination. The correlation analysis indicated a significant positive link between antioxidant capacity and the activity and gene expression levels of the antioxidant enzymes, flavonoid content, and total phenol content. This research demonstrated that germination treatment can significantly boost the accumulation of flavonoids and total phenols, thereby enhancing the antioxidant properties of buckwheat sprouts, despite variations among different buckwheat varieties.