[DNA assembly by multi-fragment digestion/ligation and homologous recombination].

To develop an accurate and efficient protocol for multi-fragment assembly and multi-site mutagenesis, we integrated and optimized the common multi-fragment assembly methods and validated the established method by using fructose-1,6-diphosphatase 1 (FBP1) with 4 mutant sites. The fragments containing mutations were assembled by introducing mutant sites and Bsa I recognition sequences. After digestion/ligation, the ligated fragment was amplified with the primers containing overlap region to the linearized vector. The amplified fragment was ligated to the linearized vector and the ligation product was transformed into Escherichia coli. After screening and sequencing, the recombinant plasmid with 4 mutant sites was obtained. This protocol overcame the major defects of Gibson assembly and Golden Gate assembly, serving as an efficient solution for multi-fragment assembly and multi-site mutagenesis.

Semaglutide alters gut microbiota and improves NAFLD in db/db mice.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease associated with type 2 diabetes mellitus (T2D). NAFLD can progress to nonalcoholic steatohepatitis (NASH), cirrhosis, and even cancer, all of which have a very poor prognosis. Semaglutide, a novel glucagon-like peptide-1 (GLP-1) receptor agonist, has been recognized as a specific drug for the treatment of diabetes. In this study, we used a gene mutation mouse model (db/db mice) to investigate the potential liver-improving effects of semaglutide. The results showed that semaglutide improved lipid levels and glucose metabolism in db/db mice. HE staining and oil red staining showed alleviation of liver damage and reduction of hepatic lipid deposition after injection of semaglutide. In addition, semaglutide also improved the integrity of gut barrier and altered gut microbiota, especially Alloprevotella, Alistpes, Ligilactobacillus and Lactobacillus. In summary, our findings validate that semaglutide induces modifications in the composition of the gut microbiota and ameliorates NAFLD, positioning it as a promising therapeutic candidate for addressing hepatic steatosis and associated inflammation.

Muscone attenuates susceptibility to ventricular arrhythmia by inhibiting NLRP3 inflammasome activation in rats after myocardial infarction.

Fibrosis and abnormal expression of connexin 43 (Cx43) in the ventricle play vital roles in ventricular arrhythmias (VAs) after myocardial infarction (MI). Muscone, an active monomer of heart-protecting musk pill, has various biological activities, but its effect on susceptibility to VAs in rats with MI has not been determined. In the present study, we investigated the effects of muscone on ventricular inflammation, fibrosis, Cx43 expression, and the occurrence of VAs after MI. An MI model was established by ligating the proximal left anterior descending coronary artery. Then, the MI model rats were administered muscone (2 mg/kg/day) or vehicle (saline)via intragastric injection for 14 days. Cardiac function was evaluated by echocardiography, and an in vivo electrophysiological study was performed on Day 14. Cardiac inflammation, fibrosis, and Cx43 expression were determined by histochemical analysis and western blot analysis. Our results indicated that muscone treatment significantly improved cardiac function and inhibited ventricular inflammation, fibrosis, and nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 3 (NLRP3) inflammasome activation. Electrocardiogrphy and electrophysiology studies showed that muscone shortened the QRS interval, QT interval, QTc interval, and action potential duration; prolonged the effective refractory period; and reduced susceptibility to VAs in rats after MI. Furthermore, Cx43 expression in the BZ was increased by muscone treatment, and this change was coupled by inhibition of the NLRP3/IL-1ß/p38 MAPK pathway. Taken together, our results demonstrated that muscone reduces susceptibility to VA, mainly by decreasing ventricular inflammation and fibrosis, and attenuates abnormal Cx43 expression by inhibiting NLRP3 inflammasome activation after myocardial infarction in rats.

Multivariate response surface methodology assisted modified QuEChERS method for the rapid determination of 39 pesticides and metabolites in medlar.

A modified QuEChERS method coupled with high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was established for residue analysis of 39 pollutants (34 commonly used multi-class pesticides and 5 metabolites) in medlar matrices (fresh, dried, and medlar juice). Samples were extracted using water with 0.1 % formic acid: acetonitrile (5: 10, v/v). The phase-out salts and five different cleanup sorbents (including N-propyl ethylenediamine (PSA), octadecyl silane bonded silica gel (C18), graphitized carbon black (GCB), Carbon nanofiber (C-Fiber) and MWCNTs) were investigated to improve the purification efficiency. The Box-Behnken Design (BBD) study was employed for an optimal solution of the volume of extraction solvent, phase-out salt, and the purification sorbents for the analytical method. The average recoveries of the target analytes in the three medlar matrices ranged from 70 % to 119 % with relative standard deviations (RSDs) of 1.0 %-19.9 %. Screening of market samples (fresh and dried medlars) collected from the major producing regions in China showed that 15 pesticides and metabolites were detected in the samples at concentrations of 0.01-2.22 mg/kg, and none of which exceeded the maximum residue limits (MRLs) set in China. The results showed that the risk of food safety by consumption of medlar products caused by the use of pesticides was low. The validated method could be used for rapid and accurate screening of multi-class multi-pesticide residues in Medlar for food safety.

Experience with successful treatment of severe recurrent vulvar adhesions: A case report.

Vulvar adhesions are defined as partial or complete adherence of the labia minora and/or labia majora. Vulvar adhesions are rare, especially in postmenopausal women.This article describes a case of postmenopausal recurrent vulvar adhesions successfully treated with surgery. The patient was a 52-year-old woman who had undergone manual separation and surgical adhesion release due to vulvar adhesions, which recurred soon after treatment. The patient then came to our hospital for treatment because of complete dense adhesions to the vulva and laboured urination. The patient received surgical treatment, the anatomical structure of the vulva recovered well, and the symptoms affecting the urinary system disappeared. There was no readhesion during the 3-month follow-up.

Key metabolites and mechanistic insights in forchlorfenuron controlling kiwifruit development.

Forchlorfenuron (CPPU) is a plant growth regulator widely applied on kiwifruit to improve yield, however, there are rarely reports on its effects on the nutrients of kiwifruits. Based on UHPLC-Q-TOF-MS, the effects of CPPU on metabolism profile and nutrient substances of two kiwifruit varieties during development were investigated by non-targeted metabolomics. A total of 115 metabolites were identified, and 29 differential metabolites were confirmed and quantified using certified reference standards. Metabolic profile indicated that CPPU promoted kiwifruit development during the main expansion stages at the molecular level, and the effects varied slightly for different varieties. In the early and middle stages of kiwifruit development, the anthocyanin, flavone and flavonol biosynthesis were down-regulated in both varieties, and flavanols biosynthesis was down-regulated only in Hayward variety. Arginine biosynthesis was down-regulated at all stages till the harvest. Although the synthesis of these nutrient substances in kiwifruits was mostly down-regulated by CPPU, the negative effects became mild at harvest time, and positively, the significant increase of sucrose and decrease of organic acids at harvest time could help to improve the taste of kiwifruits.

Prediction of the immunological and prognostic value of five signatures related to fatty acid metabolism in patients with cervical cancer.

A growing attention has been attached to the role of fatty acid metabolism (FAM) in the development of cancer, and cervical cancer (CC) is still the primary cause of cancer-associated death in women worldwide. Therefore, it is imperative to explore the possible prognostic significance of FAM in CC. In this study, CC samples and corresponding normal samples were acquired from the Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx). Single sample gene set enrichment analysis (ssGSEA) was conducted for calculating FAM-related scores (FAMRs) to screen FAM-related genes (FAMRGs). Two subtypes related to FAM were identified by consistent clustering. Among them, subtype C2 had a poor prognosis, and C1 had a high level of immune cell infiltration, while C2 had a high possibility of immune escape and was insensitive to chemotherapy drugs. Based on the differentially expressed genes (DEGs) in the two subtypes, a 5-gene signature (PLCB4, FBLN5, TSPAN8, CST6, and SERPINB7) was generated by the least absolute shrinkage and selection operator (LASSO) and Akaike information criterion (AIC). The model demonstrated a high prognostic accuracy (area under the curve (AUC)>0.7) in multiple cohorts and was one independent prognostic factor for CC patients. Accordingly, FAMRGs can be adopted as a biomarker for the prediction of CC patients' prognosis and help guide the immunotherapy of CC.

Metal formate framework-assisted solid fluorescent material based on carbonized nanoparticles for the detection of latent fingerprints.

Latent fingerprints (LFPs) powders play a crucial part in the detection of LFPs by powder-dusting methods in forensic sciences, due to the facile, onsite, and easy-to-operate properties. However, conventional LFPs powders suffer from some drawbacks, such as high toxicity, low contrast, sensitivity and selectivity. Herein, a novel fluorescent composite powder was synthesized and used for the detection of LFPs. The resultant composite powder shows an enhanced fluorescence due to the protection of the emission of fluorescent carbonized nanoparticles (FCNPs) from being quenched in the solid state. Subsequently, the fluorescent composite powder was successfully applied to detect LFPs on various substrates. This work provides a promising strategy for developing FCNPs-based fluorescent powders for the detection of LFPs in forensic science.

Noise exposure and its relationship with postinfarction cardiac remodeling: implications for NLRP3 inflammasome activation.

In recent years, high-decibel noise has emerged as a causative risk factor for ischemic heart disease. Massive noise overdose is associated with increased endocrine, neural, and immune stress responses. The NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 3) inflammasome, the most characterized supramolecular complex and a potent mediator of inflammatory signaling, has been reported to be a marker of increased ischemic heart disease vulnerability. Our study evaluated the association of noise exposure with postinfarction cardiac remodeling and its effect on NLRP3 inflammasome activation. Rats were exposed to a noisy environment (14 days, 24 h/per day, 70 ± 5 dB), and speck formation by the NLRP3 inflammasome scaffold protein ASC (apoptosis-associated speck-like protein) was assessed by confocal immunofluorescence. Echocardiography, pathological analysis, and in vivo electrophysiology were performed. Our results revealed the improved postinfarction cardiac function, mitigated fibrosis, and decreased arrhythmia vulnerability and sympathetic sprouting in low-environment noise groups. Moreover, western blotting of NLRP3, caspase-1, ASC, IL-1ß, and IL-18 and confocal microscopy of ASC speck showed that the priming and activation of NLRP3 inflammasome were higher in the NE group than in the NI group. In conclusion, our findings reveal a previously unidentified association between NLRP3 inflammasome activation and noise exposure, underscoring the significance of effective noise prevention in improving postinfarction prognosis.

Residue Behavior and Risk Assessment of Pyraclostrobin and Thifluzamide in Cowpea.

A rapid and sensitive analytical method for determination of pyraclostrobin and thifluzamide in cowpea was established based on QuEChERS sample preparation and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Average recoveries of pyraclostrobin and thifluzamide on cowpea were 100%-105% and 99%-105% with RSDs of 1%-5% and 2%-6%, respectively. The storage stability tests showed degradation rates of < 20% for samples stored at - 18℃ within 12 weeks. The field trials at eight locations in China showed that the residues of pyraclostrobin in cowpea at 3 and 5 days after spraying were 0.081-0.49 mg/kg and 0.029-0.48 mg/kg, and the residues of thifluzamide were 0.12-0.46 mg/kg and 0.047-0.50 mg/kg, respectively, which were all lower than the corresponding maximum residue limits in China. The dissipation of both pyraclostrobin and thifluzamide in cowpea were fast with half-lives (T1/2) of 1.5-2.3 days and 1.7-2.4 days. This study provided risk assessment data for establishment of good agricultural practice in cowpea plant.

Method validation, residue analysis and dietary risk assessment of trifloxystrobin and trifloxystrobin acid in milk, eggs and pork.

Trifloxystrobin (TFS) is a widely used strobilurin fungicide and its residues accumulating in animal-derived food could result in potential harm to consumers. By optimization of extraction solvents and cleanup sorbents, a residue analysis method for TFS and its metabolite trifloxystrobin acid (TFSA) was established in milk, eggs and pork based on QuEChERS sample preparation and LC-MS/MS. The calibration curves exhibited good linearity with determination coefficients (R2 ) >0.9930 over the range of 0.5-250 ng/ml for both TFS and TFSA. The recoveries of the two analytes were 81-100% with RSD 3-10% and 76-96% with RSD 2-13%, respectively. The limit of quantification (LOQ) was 1 ng/g for both analytes. The milk, egg and pork samples, 30 each, were collected from the 30 main producing regions in China, and residues of TFS and TFSA were analyzed. The concentrations of both analytes were lower than the corresponding LOQs and maximum residue limits. Long-term dietary risk assessment showed that the hazard quotients were 0.001-0.003%, indicating an absence of unacceptable risks in milk, eggs and pork to the health of common consumers in China.

Method validation and residue analysis of methoxyfenozide and metaflumizone in Chinese broccoli under field conditions by liquid chromatography with tandem mass spectrometry.

Methoxyfenozide and metaflumizone are insecticides used on Chinese broccoli to prevent insects and increase yield. However, the residues are potentially harmful to the environment and consumers. In this study, the quick, easy, cheap, effective, rugged, safe method with high-performance liquid chromatography with tandem mass spectrometry was modified and validated for determination of methoxyfenozide and metaflumizone in Chinese broccoli. The clean-up efficiency of different sorbents including C18 , primary secondary amine, graphitized carbon black, and carbon nanofiber was compared. Recoveries of the validated method were 71.8-94.6% with relative standard deviations of 1.5-3.2% and the limits of quantification were 0.01 and 0.005 mg/kg for methoxyfenozide and metaflumizone, respectively. A storage stability test showed almost no degradation of methoxyfenozide in Chinese broccoli, however, the degradation rate of metaflumizone was 22.9% after 10-wk storage at -20°C. In field trials in four producing regions, the dissipation of both methoxyfenozide and metaflumizone in Chinese broccoli was fast, with half-lives of only 1.0-5.1 and 0.7-2.5 days, respectively. Terminal residues after application of the two pesticides were all below 1.0 mg/kg after 5 days.

Hydrolysis Kinetics of a Novel 3,4-Dichloroisothiazole Plant-Activator Candidate LY5-24-2 by UPLC-Q-TOF.

Hydrolysis characteristics of a novel 3,4-dichloroisothiazole based fungicide with activating plant defense responses as a candidate plant-activator LY5-24-2 were investigated under different conditions (pH and temperature) using ultra-performance liquid chromatography (UPLC) and quadrupole Time-of-Flight (Q-TOF). The hydrolysis case complied with the first-order kinetic model, with half-lives ranging from 4.8 h to 3.2 days at pH 4, 7, 9 and temperature at 25 and 50℃. One of the hydrolysis metabolite 3,4-dichloroisothiazole-5-carboxylic acid (metabolite 1, M1) was determined and quantified using authentic standard. The other hydrolysate 3-chloro-5-(trifluoromethyl) pyridin-2-amine (metabolite 2, M2) was determined and identified according to accurate mass information, fragmentation patterns and principle component analysis (PCA). By utilizing high-resolution mass spectrometry and multivariate statistical analysis, hydrolysis dynamic of the metabolites was characterized and figured out. This research provided a non-target screening method to analyze hydrolysis metabolites of a new plant-activator and to find its degradation products in aqueous solution.

Identification of Potential Hub Genes and miRNA-mRNA Pairs Related to the Progression and Prognosis of Cervical Cancer Through Integrated Bioinformatics Analysis.

In recent years, the incidence and mortality of cervical cancer have increased worldwide. At the same time, increasing data have confirmed that miRNA-mRNA plays a positive or negative regulatory role in many cancers. This study attempted to screen effective miRNA-mRNA in the progression of cervical cancer, and to study the mechanism of miRNA-mRNA in the progression of cervical cancer. The expression profile data of GSE7410, GSE 63514, GSE 86100 and TCGA-CESC were downloaded, and 34 overlapping differentially expressed genes (22 up-regulated and 12 down-regulated) and 166 miRNAs (74 down-regulated and 92 up-regulated) were screened through limma package. Then, miR-197-3p/TYMS pairs were obtained by PPI, functional enrichment, Kaplan-Meier plotter analysis, Cox univariate and multivariate analysis, risk modeling, WGCNA, qPCR and dual-luciferase experiments. The results showed that TYMS was an independent prognostic factor of cervical cancer, and its expression level was negatively correlated with cervical cancer tissue grade (TMN), tumor grade, age, microsatellite stability and tumor mutation load, and positively correlated with methyl expression in DNMT1, DNMT2, DNMT3A and DNMT3B. Functional experiments showed that TYMS knockout could promote the proliferation, migration and invasion of HeLa cells and reduce apoptosis. Overexpression of TYMS showed the opposite trend, miR-197-3p was negatively correlated with the expression of TYMS. MiR-197-3p inhibitor reversed the effect of si-TYMS on the proliferation of HeLa cells. In conclusion, these results reveal that TYMS plays a very important role in the prognosis and progression of cervical cancer, and has the potential to be thought of as cervical cancer biomarkers. At the same time, miR-197-3p/TYMS axis can regulate the deterioration of cervical cancer cells, which lays a foundation for the molecular diagnosis and treatment of cervical cancer.

Method validation, storage stability and field trial for residues of florasulam and pyroxsulam in cereal by liquid chromatography with tandem mass spectrometry.

A QuEChERS method with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was modified and validated for the determination of florasulam and pyroxsulam residues in wheat grain and straw. The validated method was applied to cereals including oat, millet, corn and rice. Average recoveries were 76-113% with RSDs 2-15%. The limits of quantitation (LOQ) were 0.005 mg/kg for wheat grain and 0.01 mg/kg for wheat straw and four cereals. Ion suppression for florasulam (-28% to -76%) was observed in all the matrices except corn, whereas ion enhancement were shown for pyroxsulam (44% to 83%). Degradation rates of florasulam and pyroxsulam were 6% and 23%, respectively, in wheat grain and straw after eight-week storage at -20°C. The ultimate residues in field trials in ten regions were all ≤0.05 mg/kg, and long term dietary risk assessment indicated that hazard quotients were 0.02% and 0.001% for florasulam and pyroxsulam, respectively, which shows that it is safe to spray the two herbicides on wheat.

Efficient CO2 capture by a novel deep eutectic solvent through facile, one-pot synthesis with low energy consumption and feasible regeneration.

With steady growth on CO2 emission over the years, removal of CO2 from the environment is a critical issue in preventing global warming and climate change. Due to negligible volatility and remarkable thermal stability, ionic liquids have become potential media for CO2 capture. However, their drawbacks such as high price, toxicity and inefficient energy consumption have severely limited their applications in CO2 capture. Here, a promising deep eutectic solvent was synthesized by a one-pot method, simply mixing NH4Cl with monoethanolamine (MEA) at 30 °C for 30 min to form [MEAHCl][MEA] confirmed by 1H NMR. [MEAHCl][MEA] with EG exhibits a rapid initial kinetics of CO2 absorption, around 22.5 wt% within 10 min. It also has good thermal-stability, high tolerance to water contents, economically feasibility of regeneration and robust performance in recycle. This novel capture system which can be prepared on-site is feasible for industrialization and offers an efficient, inexpensive and safe system for CO2 capture.