PHD2 safeguards modest mesendoderm development.

PHD2 is essential in modulating HIF-1α levels upon oxygen fluctuations. Hypoxia, a hallmark of uterus, and HIF-1α have recently emerged as opposing regulators of mesendoderm specification, suggesting a role for PHD2 therein. We found that PHD2 expression initially covered the epiblast and gradually receded from the primitive streak, which was identical to hypoxia and exclusive to HIF-1α. The investigations performed in mESCs, embryoids, and mouse embryos together demonstrated that PHD2 negatively regulated mesendoderm specification. Single-cell RNA sequencing revealed that PHD2 governed the transition from epiblast to mesendoderm. The downstream effect of PHD2 relied on the HIF-1α regulated Wnt/ß-catenin pathway, while it was regulated upstream by miR-429. In summary, our research highlights PHD2's essential role in mesendoderm specification and its interactions with hypoxia and HIF-1α.

Full bio-based flame retardant towards multifunctional polylactic acid: Crystallization, flame retardant, antibacterial and enhanced mechanical properties.

The preparation of flame-retardant high-performance polylactic acid (PLA) composites by adding green flame retardants have always been a challenge. In this work, an intumescent flame-retardant PCR based on phytic acid, chitosan, and resveratrol was successfully designed. Adding 4 wt% of PCR, the PLA-PCR composite was classified as UL-94 V-0 grade, with a LOI value increasing from 19.7 % (pure PLA) to 26.0 %, a peak heat release rate decreasing from 433 to 344 kW/m2. Owing to excellent compatibility of PCR, the mechanical strength and toughness of PLA-PCR composites have been improved, as reflected by a ~ 16 % increase in tensile strength, a ~ 73 % increase in impact strength and a ~ 57 % increase in toughness. In addition, PCR also presented plasticization effect on PLA, making it easier to process. This work provided a highly efficient and environmental-friendly modification approach for the development of multifunctional polymers.

The Discovery and Characterization of a Potent DPP-IV Inhibitory Peptide from Oysters for the Treatment of Type 2 Diabetes Based on Computational and Experimental Studies.

The inhibition of dipeptidyl peptidase-IV (DPP-IV) is a promising approach for regulating the blood glucose levels in patients with type 2 diabetes (T2D). Oysters, rich in functional peptides, contain peptides capable of inhibiting DPP-IV activity. This study aims to identify the hypoglycemic peptides from oysters and investigate their potential anti-T2D targets and mechanisms. This research utilized virtual screening for the peptide selection, followed by in vitro DPP-IV activity assays to validate the chosen peptide. Network pharmacology was employed to identify the potential targets, GO terms, and KEGG pathways. Molecular docking and molecular dynamics simulations were used to provide virtual confirmation. The virtual screening identified LRGFGNPPT as the most promising peptide among the screened oyster peptides. The in vitro studies confirmed its inhibitory effect on DPP-IV activity. Network pharmacology revealed that LRGFGNPPT exerts an anti-T2D effect through multiple targets and signaling pathways. The key hub targets are AKT1, ACE, and REN. Additionally, the molecular docking results showed that LRGFGNPPT exhibited a strong binding affinity with targets like AKT1, ACE, and REN, which was further confirmed by the molecular dynamics simulations showcasing a stable peptide-target interaction. This study highlights the potential of LRGFGNPPT as a natural anti-T2D peptide, providing valuable insights for potential future pharmaceutical or dietary interventions in T2D management.

Compositional characteristics of the gut microbiome in patients with uremia.

ABSTRACT: During acute or chronic uremia, the cumulative harmful effects of uremic toxins result in numerous health problems and, ultimately, mortality. Previous research has identified that uremic retention solutes originate from the gut microbiome, indicating that uremia may be closely associated with gut microbiome dysbiosis. To deepen our understanding of the compositional characteristics of the gut microbiome in patients with uremia and thereby promote precision medicine in the treatment of uremia, we conducted a study of the compositional characteristics of the gut microbiome in 20 patients with uremia. The gut microbiome diversity of uremic patients and the control group showed certain differences. Nonmetric multidimensional scaling analysis showed that the beta diversity of the gut microbiome of uremic patients was significantly different from that of the healthy control individuals, with a distinct clustering effect in the uremic patient group, and it also showed a similarly distinct clustering effect in the healthy control group. The Chao1 index and Sobs index were significantly lower in the uremic patient group than in the healthy control group ( P < 0.05). By analyzing the composition and abundance distribution of the gut microbiome in the uremic patient group and healthy control group, we found that the relative abundance of the gut microbiome constituents Fusobacteriota , Enterobacteriaceae, Oscillospirales, Ruminococcaceae, and Lachnospiraceae was significantly increased in the intestines of uremic patients. We also detected the rare taxa Erysipelotrichaceae, which was present only in the uremic patient group. Predictive functional analysis suggested that an increased abundance of Ruminococcaceae and Lachnospirales, which are associated with indoxyl sulfate and phenylacetyl glutamine, and an increased abundance of Oscillospirales, which is associated with pyruvate metabolism, in uremic patients may strongly influence the gut environment according to renal function, resulting in dysbiosis associated with uremic toxin production. Rare taxa such as Erysipelotrichaceae have been suggested to be detrimental to intestinal disease. Further research into these gut microbiomes may provide new ideas for the prevention and treatment of uremia with the gut microbiome.

Impact of earthworms on antibiotic resistance genes removal in ampicillin-contaminated soil through bacterial community alteration.

The emergence of antibiotic resistance genes (ARGs) as contaminants in soil poses a significant threat to public health. Earthworms (Eisenia foetida), which are common inhabitants of soil, have been extensively studied for their influence on ARGs. However, the specific impact of earthworms on penicillin-related ARGs remains unclear. In this study, we investigate the role of earthworms in mitigating ARGs, specifically penicillin-related ARGs, in ampicillin-contaminated soil. Utilizing high-throughput quantitative PCR (HT-qPCR), we quantified a significant reduction in the relative abundance of penicillin-related ARGs in soil treated with earthworms, showing a decrease with a p-value of <0.01. Furthermore, high-throughput 16S rRNA gene sequencing revealed that earthworm intervention markedly alters the microbial community structure, notably enhancing the prevalence of specific bacterial phyla such as Proteobacteria, Firmicutes, Chloroflexi, and Tenericutes. Our findings not only demonstrate the effectiveness of earthworms in reducing the environmental load of penicillin-related ARGs but also provide insight into the alteration of microbial communities as a potential mechanism. This research contributes to our understanding of the role of earthworms in mitigating the spread of antibiotic resistance and provides valuable insights for the development of strategies to combat this global health issue.

Bioactive Compounds from Vicia sativa L. and Vicia monantha Retz. with Unveiling Antiviral Potentials in Newly Green Synthesized CdO Nanoparticles.

BACKGROUND: in the current study, a comparative phytochemical analysis was carried out to explore the phenolic and flavonoid contents in the aerial parts of Vicia sativa L and Vicia monantha Retz growing in cultivated, reclaimed, and desert habitats. METHODS: High-performance liquid chromatography (HPLC) was used to detect Vicia methanolic extracts' individual phenolic and flavonoid constituents. The first-time synthesis of cadmium oxide nanoparticles (CdO NPs) using the aqueous extract of V. monantha has been developed using a green approach. Also, the cytotoxicity of V. monantha extract and CdO NPs was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for unveiling them as anti-HAV and anti-AdV. RESULTS: Our results indicated that in the case of desert habitat, the contents of total phenolics (76.37 mg/g) and total flavonoids (65.23 mg/g) of V. monantha were higher than those of V. sativa (67.35 mg/g and 47.34 mg/g, respectively) and the contents of these secondary metabolites were even increased in V. monantha collected from reclaimed land (phenolics: 119.77 mg/g, flavonoids: 88.61 mg/g). Also, V. monantha surpassed V. sativa in the contents of some individual HPLC constituents, and hence, V. monantha was used to synthesize the green CdO NPs and subsequent antiviral tests. The average size of CdO NPs was determined to be 24.28 nm, and the transmission electron microscopy (TEM) images of CdO NPs clearly showed their spherical form and varying particle sizes, with different diameters in the range of 19-29 nm. MTT assay was positive to the exposure of CdO NPs in the normal cell line, proposing that CdO NPs can reduce cell viability. V. monantha extract showed promising antiviral activity against Hepatitis A virus (HAV) and Adenovirus (AdV) with SI of 16.40 and 10.54. On the other hand, CdONPs had poor antiviral activity against HAV with an SI of 4.74 and moderate antiviral activity against AdV with an SI of 10.54. CONCLUSION: V. monantha is now considered a new, valuable natural resource for phenolics and flavonoids, especially when grown in reclaimed soil. The green CdO NPs based on V. monantha extract showed a promising antiviral effect against HAV and AdV.

Effect of baking on the structure and bioavailability of protein-binding zinc from oyster (Crassoetrea hongkongensis).

To compare the bioavailability of protein-binding zinc, we investigated the impact of baking on the structure of zinc-binding proteins. The results showed that zinc-binding proteins enriched in zinc with relative molecular weights distributed at 6 kDa and 3 kDa. Protein-binding zinc is predisposed to separate from proteins' interiors and converge on proteins' surface after being baked, and its structure tends to be crystalline. Especially -COO, -C-O, and -C-N played vital roles in the sites of zinc-binding proteins. However, baking did not affect protein-binding zinc's bioavailability which was superior to that of ZnSO4 and C12H22O14Zn. They were digested in the intestine, zinc-binding complexes that were easily transported and uptaken by Caco-2 cells, with transport and uptake rates as high as 62.15% and 15.85%. Consequently, baking can alter the conformation of zinc-binding proteins without any impact on protein-binding zinc's bioavailability which is superior to that of ZnSO4 and C12H22O14Zn.

Effects of elevated levels of intracellular nitric oxide on Pseudomonas aeruginosa biofilm in chronic skin wound and slow-killing infection models

Nitric oxide (NO), produced through the denitrification pathway, regulates biofilm dynamics through the quorum sensing system in Pseudomonas aeruginosa. NO stimulates P. aeruginosa biofilm dispersal by enhancing phosphodiesterase activity to decrease cyclic di-GMP levels. In a chronic skin wound model containing a mature biofilm, the gene expression of nirS, encoding nitrite reductase to produce NO, was low, leading to reduced intracellular NO levels. Although low-dose NO induces biofilm dispersion, it is unknown whether it influences the formation of P. aeruginosa biofilms in chronic skin wounds. In this study, a P. aeruginosa PAO1 strain with overexpressed nirS was established to investigate NO effects on P. aeruginosa biofilm formation in an ex vivo chronic skin wound model and unravel the underlying molecular mechanisms. Elevated intracellular NO levels altered the biofilm structure in the wound model by inhibiting the expression of quorum sensing–related genes, which was different from an in vitro model. In Caenorhabditis elegans as a slow-killing infection model, elevated intracellular NO levels increased worms’ lifespan by 18%. Worms that fed on the nirS-overexpressed PAO1 strain for 4 h had complete tissue, whereas worms that fed on empty plasmid–containing PAO1 had biofilms on their body, causing severe damage to the head and tail. Thus, elevated intracellular NO levels can inhibit P. aeruginosa biofilm growth in chronic skin wounds and reduce pathogenicity to the host. Targeting NO is a potential approach to control biofilm growth in chronic skin wounds wherein P. aeruginosa biofilms are a persistent problem. (AU)

Identification and Characterization of Nigrospora Species and a Novel Species, Nigrospora anhuiensis, Causing Black Leaf Spot on Rice and Wild Rice in the Anhui Province of China.

Rice production in the Anhui province is threatened by fungal diseases. We obtained twenty-five fungal isolates from rice and wild rice leaves showing leaf spot disease collected along the Yangtze River. A phylogenetic analysis based on internal transcribed spacer (ITS), translation elongation factor 1 alpha (TEF1-α), and beta tubulin (TUB2) sequences revealed one isolate (SS-2-JB-1B) grouped with Nigrospora sphaerica, one (QY) with Nigrospora chinensis, twenty-two with Nigrospora oryzae, and one isolate (QY-2) grouped in its own clade, which are related to but clearly different from N. oryzae. Nineteen tested isolates, including sixteen strains from the N. oryzae clade and the three isolates of the other three clades, caused disease on detached rice leaves. The three isolates that did not belong to N. oryzae were also able to cause disease in rice seedlings, suggesting that they were rice pathogens. Isolate QY-2 differed from the other isolates in terms of colony morphology, cell size, and susceptibility to fungicides, indicating that this isolate represents a new species that we named Nigrospora anhuiensis. Our analysis showed that N. sphaerica, N. chinensis, and the new species, N. anhuiensis, can cause rice leaf spot disease in the field. This research provides new knowledge for understanding rice leaf spot disease.

m6A-Mediated Upregulation of lncRNA CHASERR Promotes the Progression of Glioma by Modulating the miR-6893-3p/TRIM14 Axis.

Long noncoding RNAs (lncRNAs) play crucial roles in tumor progression and are dysregulated in glioma. However, the functional roles of lncRNAs in glioma remain largely unknown. In this study, we utilized the TCGA (the Cancer Genome Atlas database) and GEPIA2 (Gene Expression Profiling Interactive Analysis 2) databases and observed the overexpression of lncRNA CHASERR in glioma tissues. We subsequently investigated this phenomenon in glioma cell lines. The effects of lncRNA CHASERR on glioma proliferation, migration, and invasion were analyzed using in vitro and in vivo experiments. Additionally, the regulatory mechanisms among PTEN/p-Akt/mTOR and Wnt/ß-catenin, lncRNA CHASERR, Micro-RNA-6893-3p(miR-6893-3p), and tripartite motif containing14 (TRIM14) were investigated via bioinformatics analyses, quantitative real-time PCR (qRT-PCR), western blot (WB), RNA immunoprecipitation (RIP), dual luciferase reporter assay, fluorescence in situ hybridization (FISH), and RNA sequencing assays. RIP and RT-qRCR were used to analyze the regulatory effect of N6-methyladenosine(m6A) on the aberrantly expressed lncRNA CHASERR. High lncRNA CHASERR expression was observed in glioma tissues and was associated with unfavorable prognosis in glioma patients. Further functional assays showed that lncRNA CHASERR regulates glioma growth and metastasis in vitro and in vivo. Mechanistically, lncRNA CHASERR sponged miR-6893-3p to upregulate TRIM14 expression, thereby facilitating glioma progression. Additionally, the activation of PTEN/p-Akt/mTOR and Wnt/ß-catenin pathways by lncRNA CHASERR, miR-6893-3p, and TRIM14 was found to regulate glioma progression. Moreover, the upregulation of lncRNA CHASERR was observed in response to N6-methyladenosine modification, which was facilitated by METTL3/YTHDF1-mediated RNA transcripts. This study elucidates the m6A/lncRNACHASERR/miR-6893-3p/TRIM14 pathway that contributes to glioma progression and underscores the potential of lncRNA CHASERR as a novel prognostic indicator and therapeutic target for glioma.

TMEM64 aggravates the malignant phenotype of glioma by activating the Wnt/ß-catenin signaling pathway.

Transmembrane protein 64 (TMEM64), a member of the family of transmembrane protein, is an α-helical membrane protein. Its precise role in various types of tumors, including glioma, is unclear. This study used immunohistochemical (IHC) staining, western blotting, and quantitative reverse transcription polymerase chain reaction (qRT-PCR) techniques to show that TMEM64 expression was significantly higher in glioma cells and tissues compared to normal cells and tissues, respectively. Additionally, a correlation between high TMEM64 expression and higher grade as well as a worse prognosis was found. TMEM64 enhanced cell proliferation and tumorigenicity while inhibiting glioma cell apoptosis in vitro and in vivo, according to loss- and gain-of-function studies. Mechanistically, it was discovered that TMEM64 increased the malignant phenotype of gliomas by accelerating the translocation of ß-catenin from the cytoplasm to the nucleus, thereby activating the Wnt/ß-catenin signaling pathway. Stimulation with the Wnt/ß-catenin signaling pathway activator CHIR-99021 successfully reversed the malignant phenotype of glioma; however, these effects were inhibited upon TMEM64 silencing. Stimulation with the Wnt/ß-catenin signaling pathway inhibitor XAV-939 successfully rescued the malignant phenotype of glioma, which was promoted upon TMEM64 overexpression. Our results provide that TMEM64 as a novel prognostic biomarker and a potential treatment target for glioma.

Carboxymethyl cellulose and carboxymethyl chitosan-based composite nanogel as a stable delivery vehicle for oyster peptides: Characterization, absorption and transport mechanism.

An oyster peptide (OPs)-loaded composite nanogel based on carboxymethyl cellulose and carboxymethyl chitosan (CMC@CMCS@OPs) was prepared, and the characterization, absorption and transport mechanism were further investigated. CMC@CMCS@OPs, a dense spherical microstructure with a diameter of ∼64 nm, which enhanced the thermal and digestive stabilities of individual OPs and improved its retention rate of hypoglycemic activity in vitro. The swelling response and in-vitro release profiles showed that CMC@CMCS@OPs could help OPs achieve targeted and controlled release in the intestine. In addition, CMC@CMCS@OPs had no cytotoxicity on Caco-2 cells, and its apparent permeability coefficients increased 4.70-7.45 times compared with OPs, with the absorption rate increased by 129.38 %. Moreover, the transcytosis of CMC@CMCS@OPs nanogel occurred primarily through the macropinocytosis pathway, endocytosis pathway and intestinal efflux transporter-mediated efflux. Altogether, these results suggested that CMC@CMCS@OPs nanogel could be as an effective OPs delivery device for enhancing its stability and absorption.

Effects of elevated levels of intracellular nitric oxide on Pseudomonas aeruginosa biofilm in chronic skin wound and slow-killing infection models.

Nitric oxide (NO), produced through the denitrification pathway, regulates biofilm dynamics through the quorum sensing system in Pseudomonas aeruginosa. NO stimulates P. aeruginosa biofilm dispersal by enhancing phosphodiesterase activity to decrease cyclic di-GMP levels. In a chronic skin wound model containing a mature biofilm, the gene expression of nirS, encoding nitrite reductase to produce NO, was low, leading to reduced intracellular NO levels. Although low-dose NO induces biofilm dispersion, it is unknown whether it influences the formation of P. aeruginosa biofilms in chronic skin wounds. In this study, a P. aeruginosa PAO1 strain with overexpressed nirS was established to investigate NO effects on P. aeruginosa biofilm formation in an ex vivo chronic skin wound model and unravel the underlying molecular mechanisms. Elevated intracellular NO levels altered the biofilm structure in the wound model by inhibiting the expression of quorum sensing-related genes, which was different from an in vitro model. In Caenorhabditis elegans as a slow-killing infection model, elevated intracellular NO levels increased worms' lifespan by 18%. Worms that fed on the nirS-overexpressed PAO1 strain for 4 h had complete tissue, whereas worms that fed on empty plasmid-containing PAO1 had biofilms on their body, causing severe damage to the head and tail. Thus, elevated intracellular NO levels can inhibit P. aeruginosa biofilm growth in chronic skin wounds and reduce pathogenicity to the host. Targeting NO is a potential approach to control biofilm growth in chronic skin wounds wherein P. aeruginosa biofilms are a persistent problem.

Using teacher-student neural networks based on knowledge distillation to detect anomalous samples in the otolith images.

Otoliths are small calcium carbonate structures found in the inner ear of fish and they, as one of important information carriers, are applied in diverse ecological fields. Otoliths are usually photographed and used to explore many unsolved biological and ecological questions. However, many anomalies may occur in the large volume of otolith image data due to natural or artificial consequences, which brings a huge bias to the aimed study and even misleading results. In this study, we first propose a specific definition of otolith anomalies and provide a dataset of otolith anomalies with Electrona carlsbergi, one of the most abundant species of lanternfishes, as the study subject. We modify a multiresolution knowledge distillation neural network model, the state-of-the-art anomaly detection model to a multiresolution knowledge distillation network model with asymmetric inputs, which uses grayscale maps to align the features of color maps in the feature space, to help improve otolith anomalies detection. Our fine-tuned anomaly detection network obtains a better anomaly identification performance with a Receiving Operating Characteristic Area Under the Curve value of 0.9843. Our result shown that multiresolution knowledge distillation networks can efficiently identify abnormal otolith image sample, which is of great importance for conducting otolith-based science.

Antioxidant, anti-inflammatory, and anti-apoptotic effects of genkwanin against aflatoxin B1-induced testicular toxicity.

Aflatoxin B1 (AFB1) is the most hazardous aflatoxin that causes significant damage to the male reproductive system. Genkwanin (GNK) is a bioactive flavonoid that shows antioxidant and anti-inflammatory potential. Therefore, the current study was planned to evaluate the effects of GNK against AFB1-induced testicular toxicity. Forty-eight male rats were distributed into four groups (n = 12 rats). AFB1 (50 µg/kg) and GNK (20 mg/kg) were administered to the rats for eight weeks. Results of the current study revealed that AFB1 exposure induced adverse effects on the Nrf2/Keap1 pathway and reduced the expressions and activities of antioxidant enzymes. Additionally, it increased the levels of oxidative stress markers. Furthermore, expressions of steroidogenic enzymes were down-regulated by AFB1 intoxication. Besides, AFB1 exposure reduced the levels of gonadotropins and plasma testosterone, which subsequently reduced the epididymal sperm count, motility, and hypo-osmotic swelled (HOS) sperms, while increasing the number of dead sperms and causing morphological anomalies of the head, midpiece, and tail of the sperms. In addition, AFB1 decreased the activities of testicular function marker enzymes and the levels of inflammatory markers. Moreover, it severely affected the apoptotic profile by up-regulating the expressions of Bax and Casp3, while down-regulating the Bcl2 expression. Besides, AFB1 significantly damaged the histoarchitecture of testicular tissues. However, GNK treatment reversed all the AFB1-induced damages in the rats. Taken together, the current study reports the potential use of GNK as a therapeutic agent to prevent AFB1-induced testicular toxicity due to its antioxidant, anti-inflammatory, and anti-apoptotic properties.

Recent advances in taste transduction mechanism, analysis methods and strategies employed to improve the taste of taste peptides.

Taste peptides are oligopeptides that enhance both aroma and taste of food, and they are classified into five categories based on their taste characteristics: salty, sour, umami, sweet, bitter, and kokumi peptide. Recently, taste peptides have attracted the attention of several fields of research in food science and commercial applications. However, research on taste receptors of taste peptides and their taste transduction mechanisms are not clearly understood and we present a comprehensive review about these topics here. This review covers the aspects of taste peptides perceived by their receptors in taste cells, the proposed transduction pathway, as well as structural features of taste peptides. Apart from traditional methods, molecular docking, peptidomic analysis, cell and animal models and taste bud biosensors can be used to explore the taste mechanism of taste peptides. Furthermore, synergistic effect, Maillard reaction, structural modifications and changing external environment are employed to improve the taste of taste peptides. Consequently, we discussed the current challenges and future trends in taste peptide research. Based on the summarized developments, taste peptides derived from food proteins potentially appear to be important taste substances. Their applications meet the principles of "safe, nutritious and sustainable" in food development.

Alkaline phosphatase LapA regulates quorum sensing-mediated virulence and biofilm formation in Pseudomonas aeruginosa PAO1 under phosphate depletion stress.

IMPORTANCE: Our previous study demonstrated that the expression of lapA was induced under phosphate depletion conditions, but its roles in virulence and biofilm formation by Pseudomonas aeruginosa remain largely unknown. This study presents a systematic investigation of the roles of lapA in virulence induction and biofilm formation by constructing a lapA-deficient strain with P. aeruginosa PAO1. The results showed that deletion of the lapA gene evidently reduced elastase activity, swimming motility, C4-HSL, and 3-oxo-C12-HSL production, and increased rhamnolipid production under phosphate depletion stress. Moreover, lapA gene deletion inhibited PAO1 biofilm formation in porcine skin explants by reducing the expression levels of las and rhl quorum sensing systems and extracellular polymeric substance synthesis. Finally, lapA gene deletion also reduced the virulence of PAO1 in Caenorhabditis elegans in fast-kill and slow-kill infection assays. This study provides insights into the roles of lapA in modulating P. aeruginosa virulence and biofilm formation under phosphate depletion stress.

Two Different Isocitrate Dehydrogenases from Pseudomonas aeruginosa: Enzymology and Coenzyme-Evolutionary Implications.

Pseudomonas aeruginosa PAO1, as an experimental model for Gram-negative bacteria, harbors two NADP+-dependent isocitrate dehydrogenases (NADP-IDHs) that were evolved from its ancient counterpart NAD-IDHs. For a better understanding of PaIDH1 and PaIDH2, we cloned the genes, overexpressed them in Escherichia coli and purified them to homogeneity. PaIDH1 displayed higher affinity to NADP+ and isocitrate, with lower Km values when compared to PaIDH2. Moreover, PaIDH1 possessed higher temperature tolerance (50 °C) and wider pH range tolerance (7.2-8.5) and could be phosphorylated. After treatment with the bifunctional PaIDH kinase/phosphatase (PaIDH K/P), PaIDH1 lost 80% of its enzymatic activity in one hour due to the phosphorylation of Ser115. Small-molecule compounds like glyoxylic acid and oxaloacetate can effectively inhibit the activity of PaIDHs. The mutant PaIDH1-D346I347A353K393 exhibited enhanced affinity for NAD+ while it lost activity towards NADP+, and the Km value (7770.67 µM) of the mutant PaIDH2-L589 I600 for NADP+ was higher than that observed for NAD+ (5824.33 µM), indicating a shift in coenzyme specificity from NADP+ to NAD+ for both PaIDHs. The experiments demonstrated that the mutation did not alter the oligomeric state of either protein. This study provides a foundation for the elucidation of the evolution and function of two NADP-IDHs in the pathogenic bacterium P. aeruginosa.

Enzymatic Characterization of the Isocitrate Dehydrogenase with Dual Coenzyme Specificity from the Marine Bacterium Umbonibacter marinipuiceus.

Isocitrate dehydrogenase (IDH) can be divided into NAD+-dependent and NADP+-dependent types based on the coenzyme specificity. It is worth noting that some IDHs exhibit dual coenzyme specificity characteristics. Herein, a dual coenzyme-dependent IDH from Umbonibacter Marinipuiceus (UmIDH) was expressed, purified, and identified in detail for the first time. SDS-PAGE and Gel filtration chromatography analyses showed that UmIDH is an 84.7 kDa homodimer in solution. The Km values for NAD+ and NADP+ are 1800.0 ± 64.4 µM and 1167.7 ± 113.0 µM in the presence of Mn2+, respectively. Meanwhile, the catalytic efficiency (kcat/Km) of UmIDH is only 2.3-fold greater for NADP+ than NAD+. The maximal activity for UmIDH occurred at pH 8.5 (with Mn2+) or pH 8.7 (with Mg2+) and at 35 °C (with Mn2+ or Mg2+). Heat inactivation assay revealed that UmIDH sustained 50% of maximal activity after incubation at 57 °C for 20 min with either Mn2+ or Mg2+. Moreover, three putative core coenzyme binding residues (R345, L346, and V352) of UmIDH were evaluated by site-directed mutagenesis. This recent work identified a unique dual coenzyme-dependent IDH and achieved the groundbreaking bidirectional modification of this specific IDH's coenzyme dependence for the first time. This provides not only a reference for the study of dual coenzyme-dependent IDH, but also a basis for the investigation of the coenzyme-specific evolutionary mechanisms of IDH.