Research progress on metabolites of nitrofurazone in aquatic products.

The carcinogenic and teratogenic risks of nitrofurazone (NFZ) led to its restriction in aquatic products. Semicarbazide (SEM), one of its metabolites, is a primary focus of modern monitoring techniques. However, the SEM residue in aquatic products is believed to be formed through endogenous mechanisms, especially for aquatic crustaceans. In this article, we will discuss the source of SEM, including its usage as an antibiotic in aquatic products (nitrofurazone), its production during food processing (azodicarbonamide and hypochlorite treatment), its occurrence naturally in the body, and its intake from the environment. SEM detection techniques were divided into three groups: derivatization, extraction/purification, and analytical methods. Applications based on liquid chromatography and its tandem mass spectrometry, immunoassay, and electrochemical methods were outlined, as were the use of various derivatives and their assisted derivatization, as well as extraction and purification techniques based on liquid-liquid extraction and solid-phase extraction. The difficulties of implementing SEM for nitrofurazone monitoring in aquatic products from crustaceans are also discussed. Possible new markers and methods for detecting them are discussed. Finally, the present research on monitoring illicit nitrofurazone usage through its metabolites is summarised, and potential problems that need to be overcome by continuing research are proposed with an eye toward giving references for future studies.

[Exposure Level and Risk Impact Assessment of Pesticides and Veterinary Drugs in Aquaculture Environment].

To explore the exposure level of pesticides and veterinary drugs in an aquaculture environment and its impact on the ecological environment, this study took the aquaculture environment in Shanghai as an example, and samples of water, sediment, and inputs from 40 major aquaculture farms were collected from July to September 2022. The types and contents of pesticides and veterinary drugs were screened using high-performance liquid chromatography-electrostatic field orbital ion trap mass spectrometry, and the risk quotient (RQ) method was used to assess the ecological risk of pesticide contamination in water and sediment. The results showed that 13 drugs were screened out from 204 samples (72 samples of water, 72 samples of mud, and 60 samples of input), namely, chlorpromazine, carbendazim, thiophanate, diazepam, florfenicol, simazine, amantidine, diazepam, trimethoprim, ciprofloxacin, ofloxacin, mebendazole, and enrofloxacin. Among them, 12 species were found in water samples with concentrations ranging from 0.016 µg·L-1 to 2.084 µg·L-1. The concentrations of seven species in the mud samples ranged from 0.018 µg·kg-1 to 23.101 µg·kg-1. The results showed that there were four types of inputs, ranging from 1.979 µg·kg-1 to 101.940 µg·kg-1. Seven drugs were found in both water and sediment. The risk quotient (RQ) results showed that there were some high and middle risks in both water and sediment samples of aquaculture farms, and the ecological risks of carbendazim were the highest in both water and sediment samples of aquaculture farms; the RQ values were 3.848 and 1.580, respectively, indicating high risk. It is suggested to strengthen the control and management of exogenous pesticides and veterinary drugs in aquaculture environments to protect the ecosystem health of the aquaculture environment.

One step cleanup of 160 pesticides and veterinary drugs in aquatic products using melamine-based automatic pressure filtration purification method combined with HPLC-MS/MS.

A 15-channel pressure filtration purification method was presented for high throughput sample preparation of aquatic products. A cost-effective device was constructed and melamine sponge was selected as the cleanup sorbent. Upon interfacing with HPLC-MS/MS, the analytical procedure demonstrated its suitability for quantifying 160 pesticides and veterinary drug residues in aquatic products such as fish, shrimp, and crab. The method achieved sample recoveries ranging from 61.3 to 124.9 %. The detection limits were established between 0.5 and 1.0 µg/kg, while the quantitation limits were confirmed to be within the range of 1.0-2.0 µg/kg. The method was applied to quantify the pesticide and veterinary drug residues in mostly consumed aquatic products from five coastal provinces in China. The results showed significant differences between different aquatic products in the concentrations of pesticide and veterinary drug residues, implying the necessity of supervision for the accurate determination of pesticides and veterinary drugs.

Dietary methionine supplementation during the estrous cycle improves follicular development and estrogen synthesis in rats.

The follicle is an important unit for the synthesis of steroid hormones and the oocyte development and maturation in mammals. However, the effect of methionine supply on follicle development and its regulatory mechanism are still unclear. In the present study, we found that dietary methionine supplementation during the estrous cycle significantly increased the number of embryo implantation sites, as well as serum contents of a variety of amino acids and methionine metabolic enzymes in rats. Additionally, methionine supplementation markedly enhanced the expression of rat ovarian neutral amino acid transporters, DNA methyltransferases (DNMTs), and cystathionine gamma-lyase (CSE); meanwhile, it significantly increased the ovarian concentrations of the metabolite S-adenosylmethionine (SAM) and glutathione (GSH). In vitro data showed that methionine supply promotes rat follicle development through enhancing the expression of critical gene growth differentiation factor 9 and bone morphogenetic protein 15. Furthermore, methionine enhanced the relative protein and mRNA expression of critical genes related to estrogen synthesis, ultimately increasing estrogen synthesis in primary ovarian granulosa cells. Taken together, our results suggested that methionine promoted follicular growth and estrogen synthesis in rats during the estrus cycle, which improved embryo implantation during early pregnancy. These findings provided a potential nutritional strategy to improve the reproductive performance of animals.

Novel filter-press single-step cleanup approach facilitated rapid screening and accurate quantification of 112 veterinary drugs in aquatic products.

In this study, a novel filter-press cleanup column was developed as a single-step cleanup approach for the rapid screening and quantification of 112 veterinary drugs in fish samples. Fish muscle samples were extracted with acetonitrile and ethyl acetate, sequentially. After concentration and reconstitution, N-propylethylenediamine (PSA) sorbent, packed in filter-press column, allows rapid single-step cleanup operation, while UHPLC-Q-Orbitrap-HRMS provides high-precision mass information in multi-residue screening. Under optimum settings, the detection and quantification limits were validated at 0.5 and 2.0 µg·kg-1, for all analytes, respectively. The ranges of recoveries were from 35.3 to 138.4%. Most of these target analytes (82%) could be measured with recoveries between 60 and 130%, and intra-day RSDs ranging from 1.9 to 26.1%. This method was further applied to evaluate the residual of veterinary drugs in fish samples from four cities in China, and results demonstrated its practicability for multi-residue monitoring veterinary residues for food safety administration.

CD34+ cell-derived fibroblast-macrophage cross-talk drives limb ischemia recovery through the OSM-ANGPTL signaling axis.

CD34+ cells improve the perfusion and function of ischemic limbs in humans and mice. However, there is no direct evidence of the differentiation potential and functional role of these cells in the ischemic muscle microenvironment. Here, we combined the single-cell RNA sequencing and genetic lineage tracing technology, then provided exact single-cell atlases of normal and ischemic limb tissues in human and mouse, and consequently found that bone marrow (BM)-derived macrophages with antigen-presenting function migrated to the ischemic site, while resident macrophages underwent apoptosis. The macrophage oncostatin M (OSM) regulatory pathway was specifically turned on by ischemia. Simultaneously, BM CD34+-derived proregenerative fibroblasts were recruited to the ischemia niche, where they received macrophage-released OSM and promoted angiopoietin-like protein-associated angiogenesis. These findings provided mechanisms on the cellular events and cell-cell communications during tissue ischemia and regeneration and provided evidence that CD34+ cells serve as fibroblast progenitors promoting tissue regeneration.

Multi-Residue Screening of Pesticides in Aquatic Products Using High-Performance Liquid Chromatography-Tandem High-Resolution Mass Spectrometry.

Pesticide residues in aquatic products are of great concern due to the risk of environmental transmission and their extensive use in aquaculture. In our work, a quick screening approach was developed for the qualitative and semi-quantitative screening of 87 pesticide residues in aquatic products. The sample preparation was investigated, including extract solvent, extract methods, buffer salts, lipid removal, cleanup materials and filter membranes for aquatic products. Samples were extracted using a modified QuEChERS procedure, and two clean-up procedures were developed for UHPLC-Q/Orbitrap MS analysis based on the fat content of the aquatic products. The screening detection limits for all studied pesticides were distributed between 1 and 500 µg/kg in the three representative matrices. Seventy-one pesticides could be analyzed with a screening limit between 1 and 25 µg/kg in grass carp and crayfish, sixty-one pesticides could be screened for limits between 1 and 50 µg/kg in crab. The accuracy results showed that recoveries ranged from 50 to 120% for 60, 56 and 52 pesticides at medium-level for grass carp, crayfish and crab, respectively. At high spiking levels, 74, 65 and 59 pesticides were recovered within the range of 50-120% for the three matrices, respectively. The relative standard deviations of most compounds in different matrices were less than 20%. With this method, the local farmed aquatic products were tested for pesticide residues. In these samples, ethoxyquinoline, prometryn and phoxim were frequently detected. The majority of these confirmed compounds did not exceed 2.00 µg/kg. A grass carp with trichlorfon at 4.87 µg/kg and two carps with ethoxyquinoline at 200 µg/kg were detected, indicating the potential dietary risk.

Exogenous Ca2+ promotes transcription factor phosphorylation to suppress ethylene biosynthesis in apple.

Ethylene biosynthesis in apple (Malus domestica) fruit can be suppressed by calcium ions (Ca2+) during storage; however, the underlying mechanisms are unclear. In this study, we identified the apple transcription factor MCM1-AGAMOUS-DEFICIENS-SRF5 (MdMADS5), which functions as a transcriptional activator of the ethylene biosynthesis-related gene 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID SYNTHASE1 (MdACS1), a partner of the calcium sensor CALCIUM-DEPENDENT PROTEIN KINASES7 (MdCDPK7). Ca2+ promoted the MdCDPK7-mediated phosphorylation of MdMADS5, which resulted in the degradation of MdMADS5 via the 26S proteasome pathway. MdCDPK7 also phosphorylated 1-AMINOCYCLOPROPANE-1-CARBOXYLIC ACID OXIDASE1 (MdACO1), the key enzyme in ethylene biosynthesis, leading to MdACO1 degradation and inhibition of ethylene biosynthesis. Our results reveal that Ca2+/MdCDPK7-MdMADS5 and Ca2+/MdCDPK7-MdACO1 are involved in Ca2+-suppressed ethylene biosynthesis, which delays apple fruit ripening. These findings provide insights into fruit ripening, which may lead to the development of strategies for extending the shelf life of fruit.

DDIT3 regulates key enzymes in the methionine cycle and flux during embryonic development.

One-carbon metabolism is a key metabolic network that integrates nutritional signals with embryonic development. However, the response of one-carbon metabolism to methionine status and the regulatory mechanisms are poorly understood. Herein, we found that methionine supplementation during pregnancy significantly increased fetal number and average fetal weight. In addition, methionine modulated one-carbon metabolism primarily through 2 metabolic enzymes, cystathionine ß-synthase (CBS) and methionine adenosyltransferase 2A (MAT2A), which were significantly increased in fetal liver tissues and porcine trophoblast (pTr) cells in response to proper methionine supplementation. CBS and MAT2A overexpression enhanced the DNA synthesis in pTr cells. More importantly, we identified a transcription factor, DNA damage-inducible transcript 3 (DDIT3), that was the primary regulator of CBS and MAT2A, which bound directly to promoters and negatively regulated the expression of CBS and MAT2A. Taken together, our findings identified that DDIT3 targeting CBS and MAT2A was a novel regulatory pathway that mediated cellular one-carbon metabolism in response to methionine signal and provided promising targets to improve pregnancy health.

T lymphocyte-derived extracellular vesicles aggravate abdominal aortic aneurysm by promoting macrophage lipid peroxidation and migration via pyruvate kinase muscle isozyme 2.

T lymphocyte and macrophage infiltration in the aortic wall is critical for abdominal aortic aneurysm (AAA). However, how T lymphocytes interact with macrophages in the pathogenesis of AAA remains largely uncharacterized. In an elastase-induced murine AAA model, we first found that the expression of pyruvate kinase muscle isozyme 2 (PKM2), the last rate-limiting enzyme in glycolysis, was increased in infiltrated T lymphocytes of vascular lesions. T lymphocyte-specific PKM2 deficiency in mice (LckCrePKM2fl/fl) or intraperitoneal administration of the sphingomyelinase inhibitor GW4869 caused a significant attenuation of the elastase-increased aortic diameter, AAA incidence, elastic fiber disruption, matrix metalloproteinases (MMPs) expression, and macrophage infiltration in the vascular adventitia compared with those in PKM2fl/fl mice. Mechanistically, extracellular vesicles (EVs) derived from PKM2-activated T lymphocytes elevated macrophage iron accumulation, lipid peroxidation, and migration in vitro, while macrophages treated with EVs from PKM2-null T lymphocytes or pretreated with the lipid peroxidation inhibitors ferrostatin-1 (Fer-1), liproxstatin-1 (Lip-1), or the iron chelating agent deferoxamine mesylate (DFOM) reversed these effects. In vascular lesions of elastase-induced LckCrePKM2fl/fl mice with AAA, the oxidant system weakened, with downregulated 4-hydroxynonenal (4-HNE) levels and strengthened antioxidant defense systems with upregulated glutathione peroxidase 4 (GPX4) and cystine/glutamate antiporter solute carrier family 7 member 11 (Slc7a11) expressions in macrophages. High-throughput metabolomics showed that EVs derived from PKM2-activated T lymphocytes contained increased levels of polyunsaturated fatty acid (PUFA)-containing phospholipids, which may provide abundant substrates for lipid peroxidation in target macrophages. More importantly, upregulated T lymphocyte PKM2 expression was also found in clinical AAA subjects, and EVs isolated from AAA patient plasma enhanced macrophage iron accumulation, lipid peroxidation, and migration ex vivo. Therefore, from cell-cell crosstalk and metabolic perspectives, the present study shows that PKM2-activated T lymphocyte-derived EVs may drive AAA progression by promoting macrophage redox imbalance and migration, and targeting the T lymphocyte-EV-macrophage axis may be a potential strategy for early warning and treating AAA.

Ethylene-activated MdPUB24 mediates ubiquitination of MdBEL7 to promote chlorophyll degradation in apple fruit.

Chlorophyll (Chl) degradation is a natural phenomenon that occurs during ripening in many fleshy fruit species, and also during fruit storage. The plant hormone ethylene is a key factor in promoting Chl degradation during fruit storage, but the mechanisms involved in this induction are largely unknown. In this study, an apple (Malus domestica) BEL1-LIKE HOMEODOMAIN transcription factor 7 (MdBEL7), potentially functioning as a transcriptional repressor of the Chl catabolic genes (CCGs), including MdCLH, MdPPH2 and MdRCCR2, was identified as a partner of the ethylene-activated U-box type E3 ubiquitin ligase MdPUB24 in a yeast library screen. Yeast-two-hybrid, co-immunoprecipitation and luciferase complementation imaging assays were then used to verify the interaction between MdBEL7 and MdPUB24. In vitro and in vivo ubiquitination experiments revealed that MdPUB24 functions as an E3 ubiquitin ligase to ubiquitinate MdBEL7, thereby causing its degradation through the 26S proteasome pathway. Transient overexpression of MdPUB24 in apple fruit led to a decrease in MdBEL7 abundance and increased expression of CCG genes, including MdCLH, MdPPH2 and MdRCCR2, as well as greater Chl degradation. Taken together, the data indicated that an ethylene-activated U-box type E3 ubiquitin ligase MdPUB24 directly interacts with and ubiquitinates MdBEL7. Consequent degradation of MdBEL7 results in enhanced expression of MdCLH, MdPPH2 and MdRCCR2, and thus Chl degradation during apple fruit storage. Our results reveal that an ethylene-MdPUB24-MdBEL7 module regulates Chl degradation by post-translational modification during apple fruit storage.

Butyrate drives the acetylation of histone H3K9 to activate steroidogenesis through PPARγ and PGC1α pathways in ovarian granulosa cells.

Maintaining ovarian steroidogenesis is of critical importance, considering that steroid hormones are required for successful establishment and maintenance of pregnancy and proper development of embryos and fetuses. Investigating the mechanism that butyrate modulates the ovarian steroidogenesis is beneficial for understanding the impact of lipid nutrition on steroidogenesis. Herein, we identified that butyrate improved estradiol and progesterone synthesis in rat primary ovarian granulosa cells and human granulosa KGN cells and discovered the related mechanism. Our data indicated that butyrate was sensed by GPR41 and GPR43 in ovarian granulosa cells. Butyrate primarily upregulated the acetylation of histone H3K9 (H3K9ac). Chromatin immune-precipitation and sequencing (ChIP-seq) data of H3K9ac revealed the influenced pathways involving in the mitochondrial function (including cellular metabolism and steroidogenesis) and cellular antioxidant capacity. Additionally, increasing H3K9ac by butyrate further stimulated the PPARγ/CD36/StAR pathways to increase ovarian steroidogenesis and activated PGC1α to enhance mitochondrial dynamics and alleviate oxidative damage. The improvement in antioxidant capacity and mitochondrial dynamics by butyrate enhanced ovarian steroidogenesis. Collectively, butyrate triggers histone H3K9ac to activate steroidogenesis through PPARγ and PGC1α pathways in ovarian granulosa cells.

CBS and MAT2A improve methionine-mediated DNA synthesis through SAMTOR/mTORC1/S6K1/CAD pathway during embryo implantation.

OBJECTIVES: Early pregnancy loss is a major clinical concern in animal and human reproduction, which is largely influenced by embryo implantation. The importance of methionine for embryo implantation is widely neglected. MATERIALS AND METHODS: We performed a series of experiments with primiparous rats fed diets containing different levels of methionine during early pregnancy to investigate the role of methionine in embryonic implantation and pregnancy outcomes, and used them to perform in vivo metabolic assessments and in vitro uterine explant culture. In addition, through transcriptome analysis and silencing the expression of cystathionine ß-synthase (CBS, the key enzyme in transsulfuration pathway) and cell adhesion assay, we measured signalling within Ishikawa, pTr and JAR cells. RESULTS: We determined the relevance and underlying mechanism of methionine on embryo implantation. We showed that methionine deprivation sharply decreased embryo implantation sites, expression of CBS and transsulfuration pathway end products, which were reversed by maternal methionine supplementation during early pregnancy. Moreover, we found CBS improved methionine-mediated cell proliferation and DNA synthesis by CBS inhibition or interference. In addition, transcriptome analysis also revealed that CBS influenced the signalling pathway-associated cell proliferation and DNA synthesis, as well as a correlation between CBS and methionine adenosyltransferase 2A (MAT2A), implying that MAT2A was possibly involved in cell proliferation and DNA synthesis. Further analysis revealed that MAT2A influenced S-adenosylmethionine receptor SAMTOR expression, and SAMTOR activated mTORC1 and its downstream S6K1 and CAD, ultimately enhancing DNA synthesis in the embryo and uterus. CONCLUSIONS: Taken together, these studies demonstrate that CBS and MAT2A improve methionine-mediated DNA synthesis through SAMTOR/mTORC1/S6K1/CAD pathway during embryo implantation.

Peroxisome Proliferator-Activated Receptor-γ Coactivator-1α (PGC-1α) Regulates the Expression of B-Cell Lymphoma/Leukemia-2 (Bcl-2) and Promotes the Survival of Mesenchymal Stem Cells (MSCs) via PGC-1α/ERRα Interaction in the Absence of Serum, Hypoxia, and High Glucose Conditions.

BACKGROUND To study the effect of estrogen-related receptor α (ERRα) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) on mesenchymal stem cells (MSCs) apoptosis, and further investigated its detailed molecular mechanisms in the absence of serum, hypoxia, and high glucose conditions. MATERIAL AND METHODS In our study, we first evaluated the expression rates of CD14, CD34, CD45, CD44, CD29, and Sca-1 surface markers on MSCs by flow cytometry. Then, the ability of osteogenic and fatty differentiation of MSCs was determined by osteogenic differentiation and adipogenesis reagent kit. Next, Annexin V-APC/7-AAD apoptosis kit was used for detecting the apoptosis rate of MSCs. RT-PCR and Western blotting were used for detection of mRNA expression and proteins expression, respectively. RESULTS Our data showed that the MSCs used in our study were capable of self-renewal and differentiating into many cell lineages, such as osteogenic differentiation and adipogenesis. Our results further showed that over-expression of PGC-1α could protect MSCs from apoptosis induced by rotenone. We also found that PGC-1α over-expression could enhance the expression of anti-apoptotic gene Bcl-2, and inhibit the expression of pro-apoptotic gene Bax in MSCs. In addition, our data demonstrated that PGC-1α could induce upregulation of Bcl-2 and further promote the survival of MSCs by interacting with ERRα. CONCLUSIONS In the absence of serum, hypoxia and high glucose conditions, PGC-1α can regulate the expression of Bcl-2 and promote the survival of MSCs via PGC-1α/ERRα interaction.

Effects of diabetes mellitus on prognosis of the patients with colorectal cancer undergoing resection: a cohort study with 945 patients.

BACKGROUND: Diabetes mellitus plays an important role in cancer prevalence and outcomes. The aim of this study was to evaluate the influence of DM on stages and outcomes among patients with colorectal cancer. METHODS: The study enrolled 945 patients who were diagnosed as having colorectal carcinoma from August 1994 to December 2002. In the cohort, 26 patients were diagnosed as having DM. With a median follow-up of 45.8 months, differences in overall survival and disease-free survival between the diabetes and non-diabetes groups were analyzed. RESULTS: Kaplan and Meier analysis showed that there were no significant differences between the two groups in overall survival rates at 3 years or 5 years. At 5 years, patients with DM, compared with patients without diabetes, experienced a significantly lower disease-free survival rate (34.2% diabetics vs. 55.1% non-diabetics; P = 0.025). CONCLUSIONS: DM was associated with an increased risk of recurrence in patients with colorectal cancer.