Development of a vitamin B5 hyperproducer in Escherichia coli by multiple metabolic engineering.

Vitamin B5 [D-pantothenic acid (D-PA)] is an essential water-soluble vitamin that is widely used in the food and feed industries. Currently, the relatively low fermentation efficiency limits the industrial application of D-PA. Here, a plasmid-free D-PA hyperproducer was constructed using systematic metabolic engineering strategies. First, pyruvate was enriched by deleting the non-phosphotransferase system, inhibiting pyruvate competitive branches, and dynamically controlling the TCA cycle. Next, the (R)-pantoate pathway was enhanced by screening the rate-limiting enzyme PanBC and regulating the other enzymes of this pathway one by one. Then, to enhance NADPH sustainability, NADPH regeneration was achieved through the novel "PEACES" system by (1) expressing the NAD + kinase gene ppnk from Clostridium glutamicum and the NADP + -dependent gapCcae from Clostridium acetobutyricum and (2) knocking-out the endogenous sthA gene, which interacts with ilvC and panE in the D-PA biosynthesis pathway. Combined with transcriptome analysis, it was found that the membrane proteins OmpC and TolR promoted D-PA efflux by increasing membrane fluidity. Strain PA132 produced a D-PA titer of 83.26 g/L by two-stage fed-batch fermentation, which is the highest D-PA titer reported so far. This work established competitive producers for the industrial production of D-PA and provided an effective strategy for the production of related products.

Spautin-1 Ameliorates Acute Pancreatitis via Inhibiting Impaired Autophagy and Alleviating Calcium Overload.

Acute pancreatitis is characterized by zymogen pre-activation. Severe inflammation caused by zymogen activation can eventually lead to multiple organ dysfunctions, which contributes to the high mortality rate of severe acute pancreatitis. However, there is no specific treatment available for acute pancreatitis therapy. Here, we show that spautin-1, which effectively inhibits autophagy flux, ameliorated the pathogenesis of acute pancreatitis induced by cerulein or L-Arginine. CaMKII phosphorylation due to cytosolic calcium oeverload was revealed in this paper. It was also demonstrated that autophagic protein aggregates degradation blockade accompanying with impaired autophagy correlated positively to intra acinar cells digestive aymogen activation sitimulated by cerulein or L-Arginine. The role of spautin-1 in ameliorating acute pancreatitis was shown here to be associated with impaired autophagy inhibition and Ca2+ overload alleviation. We provided a promising therapy for acute pancreatitis here through targeting both impaired autophagy and increased cytosolic calcium.

Mechanism of the protective effect of Yulangsan flavonoid on myocardial ischemia/reperfusion injury in rats.

AIMS: Effect and mechanism of Yulangsan flavonoid (YLSF) on rat myocardial ischemia/reperfusion injury (MI/RI) has been investigated. METHODS: Sprague-Dawley (SD) rats were randomly divided into seven groups (sham group, model group and NS group: 2 mL of normal saline/kg body weight was administered; diltiazem group: 5 mg of diltiazem hydrochloride/kg body weight was administered; YLSFL, YLSFM and YLSFH groups: 20, 40 and 80 mg of YLSF/kg body weight was administered) and the MI/RI model was established. Myocardial infarct area, levels of myocardial enzymes and nitric oxide synthase (NOS) were measured. Caspase-3 and adenine nucleotide translocator-1 (ANT1) mRNA expression were evaluated by reverse transcription polymerase chain reaction (RT-PCR). Pathological structure and cardiocyte ultrastructure were also analysed. RESULTS: Compared with the MI/RI group, pretreatment with YLSF or diltiazem hydrochloride decreased the infarct area, levels of inducible nitric oxide synthase (iNOS), caspase-3 as well as the leakage of myocardial enzyme and increased activities of total nitric oxide synthase (tNOS) as well as constitutive nitric oxide synthase (cNOS). Cellular edema and the infiltration of inflammatory cells were alleviated. CONCLUSIONS: The experiment showed that YLSF protected the heart against MI/RI, possibly by reducing lipid peroxidation damage, regulating NOS activity and modulating the apoptosis genes expression.

Evaluation of the therapeutic efficiency and efficacy of blood purification in the treatment of severe acute pancreatitis.

This study aimed to evaluate the therapeutic efficacy and effect of blood purification (BP) therapy on severe acute pancreatitis (SAP). Information on 305 patients (BP group 68, control group 237) diagnosed with SAP was retrieved from the Medical Information Mart for Intensive Care IV (MIMIC IV) database. Firstly, the influence of BP treatment was preliminarily evaluated by comparing the outcome indicators of the two groups. Secondly, multiple regression analysis was used to screen the mortality risk factors to verify the impact of BP on the survival outcome of patients. Then, the effect of BP treatment was re-validated with baseline data. Finally, cox regression was used to make the survival curve after matching to confirm whether BP could affect the death outcome. The results indicated that the BP group had a lower incidence of shock (p = 0.012), but a higher incidence of acute kidney injury (AKI) (p < 0.001), with no differences observed in other outcome indicators when compared to the control group. It was also found that the 28-day survival curve of patients between the two groups was significantly overlapped (p = 0.133), indicating that BP treatment had no significant effect on the survival outcome of patients with SAP. Although BP is beneficial in stabilizing hemodynamics, it has no effect on short- and long-term mortality of patients. The application of this technology in the treatment of SAP should be done with caution until appropriate BP treatment methods are developed, particularly for patients who are not able to adapt to renal replacement therapy.

Decreased expression of TXNIP is associated with poor prognosis and immune infiltration in kidney renal clear cell carcinoma.

The most prevalent and insidious type of kidney cancer is kidney clear cell carcinoma (KIRC). Thioredoxin-interacting protein (TXNIP) encodes a thioredoxin-binding protein involved in cellular energy metabolism, redox homeostasis, apoptosis induction and inflammatory responses. However, the relationship between TXNIP, immune infiltration and its prognostic value in KIRC remains unclear. Thus, the present study evaluated the potential for TXNIP as a prognostic marker in patients with KIRC. Data from The Cancer Genome Atlas were used to assess relative mRNA expression levels of TXNIP in different types of cancer. The protein expression levels of TXNIP were evaluated using the Human Protein Atlas. Enrichment analysis of genes co-expressed with TXNIP was performed to assess relevant biological processes that TXNIP may be involved in. CIBERSORT was used to predict the infiltration of 21 tumor-infiltrating immune cells (TIICs). Univariate and multivariate Cox regression analyses were used to assess the relationship between TXNIP expression and prognosis. Single-cell RNA-sequencing datasets were used to evaluate the mRNA expression levels of TXNIP in certain immune cells in KIRC. The CellMiner database was used to analyze the relationship between TXNIP mRNA expression and drug sensitivity in KIRC. The results from the present study demonstrated that TXNIP expression was significantly decreased in KIRC tissue compared with that in normal tissue, as confirmed by western blotting and reverse transcription-quantitative PCR. In addition, downregulated TXNIP expression was significantly associated with poor prognosis, a high histological grade and an advanced stage. The Cell Counting Kit-8 assay demonstrated that TXNIP overexpression significantly suppressed tumor cell proliferation. Univariate and multivariate Cox regression analyses indicated that TXNIP served as a separate prognostic factor in KIRC. Moreover, TXNIP expression was significantly correlated with the accumulation of several TIICs and its overexpression significantly downregulated the mRNA expression levels of CD25 and cytotoxic T-lymphocyte-associated protein 4, immune cell surface markers in CD4+ T lymphocytes. In conclusion, TXNIP may be used as a possible biomarker to assess unfavorable prognostic outcomes and identify immunotherapy targets in KIRC.

Construction and validation of a bioinformatics­based screen for cuproptosis­related genes and risk model for Alzheimer's disease.

The present study aimed to validate the association between core cuproptosis genes (CRGs) and Alzheimer's disease (AD) from both bioinformatics and experimental perspectives and also to develop a risk prediction model. To this end, 78 human­derived temporal back samples were analyzed from GSE109887, and the biological functions of the resulting CRGs were explored by cluster analysis, weighted gene co­expression network analysis and similar methods to identify the best machine model. Moreover, an external dataset GSE33000 and a nomogram were used to validate the model. The mRNA and protein expression of CRGs were validated using the SH­SY5Y cell model and the Sprague­Dawley rat animal model. The RT­qPCR and western blotting results showed that the mRNA and protein expression content of dihydrolipoamide dehydrogenase, ferredoxin 1, glutaminase and pyruvate dehydrogenase E1 subunit ß decreased, and the expression of dihydrolipoamide branched chain transacylase E2 increased in AD, which supported the bioinformatic analysis results. The CRG expression alterations affected the aggregation and infiltration of certain immune cells. The present study also confirmed the accuracy and validity of AD diagnostic models and nomograms, and validated the association between five CRGs and AD, indicating a significant difference between patients with AD and healthy individuals. Therefore, CRGs are expected to serve as relevant biomarkers for the diagnosis and prognostic monitoring of AD.

Identification and immunological characterization of genes associated with ferroptosis in Alzheimer's disease and experimental demonstration.

The incidence of Alzheimer's disease (AD) is rising globally, yet its treatment and prediction of this condition remain challenging due to the complex pathophysiological mechanisms associated with it. Consequently, the objective of the present study was to analyze and characterize the molecular mechanisms underlying ferroptosis­related genes (FEGs) in the pathogenesis of AD, as well as to construct a prognostic model. The findings will provide new insights for the future diagnosis and treatment of AD. First, the AD dataset GSE33000 from the Gene Expression Omnibus database and the FEGs from FerrDB were obtained. Next, unsupervised cluster analysis was used to obtain the FEGs that were most relevant to AD. Subsequently, enrichment analyses were performed on the FEGs to explore biological functions. Subsequently, the role of these genes in the immune microenvironment was elucidated through CIBERSORT. Then, the optimal machine learning was selected by comparing the performance of different machine learning models. To validate the prediction efficiency, the models were validated using nomograms, calibration curves, decision curve analysis and external datasets. Furthermore, the expression of FEGs between different groups was verified using reverse transcription quantitative PCR and western blot analysis. In AD, alterations in the expression of FEGs affect the aggregation and infiltration of certain immune cells. This indicated that the occurrence of AD is strongly associated with immune infiltration. Finally, the most appropriate machine learning models were selected, and AD diagnostic models and nomograms were built. The present study provided novel insights that enhance understanding with regard to the molecular mechanism of action of FEGs in AD. Moreover, the present study provided biomarkers that may facilitate the diagnosis of AD.

Systematic engineering of Escherichia coli for efficient production of nicotinamide riboside from nicotinamide and 3-cyanopyridine.

Nicotinamide riboside (NR), a key biosynthetic precursor of NAD+, is receiving increasing attention because of its role. In this study, a whole-cell catalysis method to efficiently synthesize NR was established. First, the performance of 5'-nucleotidase (UshA) from Escherichia coli was confirmed to have high catalytic activity to synthesize NR. Then, the endogenous NR degradation pathway was detected, and the genes (rihA, rihB, and rihC) involved in NR degradation were knocked out, which enabled NR biosynthesis. In addition, the important role of the signal peptide of UshA in NR transport had been confirmed. Subsequently, nitrile hydratase was introduced to achieve the conversion of 3-cyanopyridine to NR. Finally, the NR titer reached 25.6 and 29.8 g/L with nicotinamide and 3-cyanopyridine, respectively, as substrates in a 5-L bioreactor, the efficient biosynthesis of NR in E. coli by using nicotinamide and 3-cyanopyridine.

Systematic Engineering of Escherichia coli for Efficient Production of Nicotinamide Mononucleotide From Nicotinamide.

Research studies on NAD+ have proven its crucial role in aging and disease. Nicotinamide mononucleotide (NMN), as the key intermediate of NAD+, plays a significant role in supplying and maintaining NAD+ levels. In the present study, a biocatalytic method for the efficient synthesis of NMN was established. First, Escherichia coli was systematically modified to make it more conducive to the biosynthesis and accumulation of NMN. Next, the performance of nicotinamide phosphoribosyltransferase from Vibrio bacteriophage KVP40 (VpNadV) was determined, which has the best catalytic activity to produce NMN from nicotinamide. The accumulation of extracellular NMN was further increased after the introduction of an NMN transporter. Fine-tuning of gene expression and copy number led to the synthesis of NMN at the yield of 2.6 g/L at the shake flask level. The introduction of a nicotinamide transporter, BcniaP, could not obviously increase the production of NMN at the shake flask level, but it decreased the production of NMN at the bioreactor level. Finally, the titer of NMN reached 16.2 g/L with a conversion ratio of 97.0% from nicotinamide, both of which are highest according to currently available reports. The fed-batch fermentation with direct supplementation of nicotinamide could facilitate the industrial-scale production of NMN compared to that achieved by the whole-cell catalysis process. These results also represent the highest reported yield of NMN synthesized from nicotinamide in E. coli.

Transcriptome Comparison of Brain and Kidney Endothelial Cells in Homeostasis.

Endothelial cells are heterogeneous, stemming from multiple organs, but there is still little known about the connection between the brain and kidney endothelial cells, especially in homeostasis. In this study, scRNA-seq results were obtained to compare genetic profiles and biological features of tissue-specific endothelial cells. On this basis, seven endothelial cell subpopulations were identified, two of which were upregulated genes in pathways related to stroke and/or depression, as characterized by neuroinflammation. This study revealed the similarities and distinctions between brain and kidney endothelial cells, providing baseline information needed to fully understand the relationship between renal diseases and neuroinflammation, such as stroke and depression.

Effect of Zhuang Jing Decoction on Learning and Memory Ability in Aging Rats.

With the average life span of humans on the rise, aging in the world has drawn considerable attentions. The monoamine neurotransmitters and neurotrophic factors in brain areas are involved in learning and memory processes and are an essential part of normal synaptic neurotransmission and plasticity. In the present study, the effect of Zhuang Jing Decoction (ZJD) on the learning and memory ability in aging rats was examined in vivo using Morris water maze. Furthermore, the levels of monoamine neurotransmitters and neurotrophic factors in brain were detected by high-performance liquid chromatography with a fluorescence detector and enzyme-linked immunosorbent assay, respectively. These data showed that oral administration with ZJD at the dose of 30 g·kg(-1) exerted an improved effect on learning and memory ability in aging rats. The results revealed that ZJD could effectively adjust the monoamine neurotransmitters and neurotrophic factors, restore the balance of the level of monoamine neurotransmitters and neurotrophic factors in brain, and finally attenuate the degeneration of learning and memory ability. These findings suggested that ZJD might be a potential agent as cognitive-enhancing drug in improving learning and memory ability. It may exert through regulating the levels of monoamine neurotransmitters and neurotrophic factors in brain, which demonstrated that ZJD had certain antiaging effects.

Protective effect of Millettia pulchra polysaccharide on cognitive impairment induced by D-galactose in mice.

A polysaccharide (PMP) was isolated from Millettia pulchra and purified by DEAE-cellulose and Sephadex G-75 chromatography. The results showed that PMP was composed of d-glucose and d-arabinose in a molar ratio of 90.79% and 9.21%, with an average molecular weight of about 14,301 Da. Furthermore, the effect of PMP on cognitive impairment induced by d-galactose in mice was evaluated. Treatment with PMP significantly reversed d-galactose-induced learning and memory impairments, as measured by behavioral tests. One of the potential mechanisms of this action was to reduce oxidative stress and suppress inflammatory responses. Furthermore, our results also showed that PMP markedly reduced the content and deposition of ß-amyloid peptide, improved the dysfunction of synaptic plasticity, increased the levels of acetylcholine, but decreased cholinesterase activity. These results suggest that PMP exerts an effective protection against d-galactose-induced cognitive impairment, and PMP may be a major bioactive ingredient in M. pulchra.

Effects of Yulangsan polysaccharide on monoamine neurotransmitters, adenylate cyclase activity and brain-derived neurotrophic factor expression in a mouse model of depression induced by unpredictable chronic mild stress.

The present study established a mouse model of depression induced by unpredictable chronic mild stress. The model mice were treated with Yulangsan polysaccharide (YLSPS; 150, 300 and 600 mg/kg) for 21 days, and compared with fluoxetine-treated and normal control groups. Enzyme-linked immunosorbent assay, radioimmunity and immunohistochemical staining showed that following treatment with YLSPS (300 and 600 mg/kg), monoamine neurotransmitter levels, prefrontal cortex adenylate cyclase activity and hippocampal brain-derived neurotrophic factor expression were significantly elevated, and depression-like behaviors were improved. Open-field and novelty-suppressed feeding tests showed that mouse activity levels were increased and feeding latency was shortened following treatment. Our results indicate that YLSPS inhibits depression by upregulating monoamine neurotransmitters, prefrontal cortex adenylate cyclase activity and hippocampal brain-derived neurotrophic factor expression.