Objective To gain insights into the biological characteristics of different populations of Culex tritaeniorhynchus within and around the Yellow Sea wetland from the perspective of the occurrence of resistance, we investigated the levels of resistance to deltamethrin and kdr gene mutation in the wetland and its peripheral areas. Methods Specimens were collected from Cx. tritaeniorhynchus populations at two monitoring sites in the Rare Bird National Nature Reserve and Tiaozi Ni Wetland Scenic Area, and also from two populations in Yancheng City and the Liuhe District of Nanjing, and the resistance of these mosquitoes to deltamethrin was determined using the CDC biotest bottle method. For each concentration of deltamethrin assessed, a random subset of exposed specimens was selected for amplification of the kdr gene fragment, followed by Sanger sequencing to identify and analyze resistance-associated mutations. Results The LC50 levels of deltamethrin among mosquitoes from the four populations in Luhe, Yancheng, the Rare Bird National Nature Reserve and the Tiaozi Ni Wetland Scenic Area were 2.048 5, 7.798 2, 3.473 3, and 17.695 5 mg/mL, respectively, with corresponding concentrations of deltamethrin ranging from 0.005 to 5.000,0.050 to 50.000,0.050 to 25.000 and 0.050 to 50.000 mg/mL, respectively. Furthermore, the ranges of the KT50 values were 11.76-107.43, 67.05-216.30,29.77-107.43 and 28.40-329.51 min; the 1-h knockdown rates were 34.58%-99.15%, 9.52%-43.80%, 55.09%-73.01%, and 10.09%-68.07%; and the 24-h mortality rates were 12.15%-67.52%,9.52%-79.56%,13.17%-82.21%, and 11.01%-78.99%, respectively. With respect to kdr gene mutation, we assayed a total of 63,70,59, and 57 mosquitoes for the four populations, for which we detected L1014F mutation frequencies of 14.29%, 35.00%, 20.34%, and 31.58%, respectively, with a majority of these mutations being heterozygous for resistance. In addition, five adult mosquitoes were identified has having synonymous mutations at site 1011[i. e. , AAT(asparagine)mutation to AAC(asparagine)]. Conclusions Our findings revealed the clear resistance of Cx. tritaeniorhynchus to deltamethrin in the Yancheng region of the Yellow Sea wetland, and the resistance phenotype and kdr frequency of Cx. tritaeniorhynchus in the wetland environment were comparable to those of Cx. tritaeniorhynchus in the wetland environment, thereby indicating that the resistance of different populations of Cx. tritaeniorhynchus was homogeneous under the pressure of different insecticide selection within and around the wetland. However, the underlying mechanisms need to be further studied.

Objective To investigate the characteristics of immune exhaustion in sepsis and analyze its association with gut microbiota translocation.Methods A total of 130 mice were randomly divided into a cecal ligation and puncture(CLP)group(n=100)and a Sham group(n=30)Mouse model of sepsis was established with CLP procedure.Flow cytometry was used to analyze the proportions of peripheral blood CD4+T and CD8+T cells and programmed cell death protein 1(PD-1)positive T cell subsets in mice.Bacterial colonization in organs such as the heart,liver and kidneys was quantified by plating homogenates of the organs.Pathological changes in immune organs were observed with HE staining.The expression and localization of CD4?,CD8?,and PD-1?cells in immune organs were detected with immunohistochemical staining,and Image J software was employed for subsequent quantification of the number of the positive cells.Results HE staining demonstrated that immune organs exhibited varying degrees of pathological damages with disease progression.Compared with the Sham mice,the CLP mice exhibited significantly increased bacterial colonization in parenchymal organs and peripheral blood(P<0.05),notably in the liver,which showed the most severe infection.In the CLP group,the proportion of CD4+T lymphocytes in peripheral blood at days 1,3,and 5 postoperatively was decreased by 56%,70.57%,and 87.42%,respectively,when compared with the Sham group(P<0.001).The proportion of CD8+T lymphocytes was decreased by 48.33%relative to the Sham group only at day 5(P<0.001).In contrast,the proportion of CD4+T cell subsets expressing PD-1 was increased to 673.08,423.08,and 600 times that of the Sham group,respectively,at the same postoperative time points(P<0.001).Immunohistochemical results showed that,in the CLP group,the proportion of CD4+T cells in the thymus,spleen,and mesenteric lymph nodes was increased to 7.65,2.66,and 3.7 times that of the Sham group,respectively,at the early-stage peak(P<0.001),and then these proportions were decreased by 82.8%(P<0.001),41.9%(P<0.01),and 60.15%(P<0.001),respectively,at the late-stage trough when compared with the early-stage peak in the corresponding organs.The proportion of CD8+positive cells was increased in the early stage and then decreased insignificantly,while the proportion of PD-1+positive cells was increased continuously,and reached 6.24,13.9,and 20.96 times that of the Sham group at the peak in the thymus,spleen,and mesenteric lymph nodes respectively(P<0.001),with their expression regions showing a rough overlap with those of CD4+cells.Conclusion During sepsis,the inflammatory response can cause severe damage to immune organs and persistent exhaustion of CD4?T lymphocytes,leading to declined defenses against infection,which may be the main causes for exacerbated gut microbiota translocation and then systemic infection.

As a cheap and effective antibiotic,sulfonamides are often used in animal husbandry.However,their residues in animal-derived foodstuffs will threaten human health.Consequently,a high-performance liquid chromatography(HPLC)method integrated with supramolecular solvent microextraction was successfully established for simultaneous quantification of sulfonamide residues sulfachlorpyridazine,sulfamethoxazole,sulfamethoxypyridazine and sulfadimethoxine in milk matrices.This approach exhibited prominent characteristics of operational simplicity,environmental sustainability,and high extraction efficiency.The supramolecular solvents prepared by tributyl octylphosphine tetrafluoroborate and tetrahydrofuran were employed as extraction solvents.The analytes underwent isolation and concentration via dispersive liquid-liquid microextraction(DLLME)prior to quantitative determination using high-performance liquid chromatography(HPLC).The composition and microscopic morphology of the supramolecular solvent were characterized through a series of analytical techniques,including phase diagram,Fourier transform infrared spectroscopy,scanning electron microscopy,and inverted fluorescence microscopy and so on.The density and pH value of supramolecular solvents were determined.The extraction conditions were optimized through the one-factor experiments.The experimental results demonstrated that under the optimal extraction conditions,the four kinds of sulfonamide antibiotics exhibited excellent linearity within respective detection range(R2 ≥ 0.9998)and the limits of detection(LOD)were 0.67-1.45 μg/L.Compared with literature methods,this approach offered some advantages such as simplicity of operation and less reagent consumption,and could be used for analysis and detection of sulfonamide antibiotic residues in milk samples.The present method provided technical support for food safety regulation and paved a new way for the application of supramolecular solvents in the field of extraction and separation.

A method for determination and targeted screening of diflorasone components in health products using ultra performance liquid chromatography-quadrupole time of flight mass spectrometry(UPLC-Q-TOF/MS)was established.Four representative diflorasone and esters(diflorasone,diflorasone diacetate,diflorasone-17-propionate,and diflorasone-21-propionate)were selected to optimize the pretreatment conditions,and 10 mL of extraction solvent dosage,15 min of extraction time and 5 g of salting-out agent as the optimal conditions were selected by response surface methodology.The results showed that the four analytes exhibited good linearity within the concentration range of 2.0?100 μg/L with the chromatographic peak area,and the correlation coefficients(R2)were all greater than 0.9990,while the results of recovery and relative standard deviation could satisfy the requirements of determination.The common characteristic ions of diflorasone and esters werem/z121 andm/z335,and their specific structures were obtained by analyzing the cleavage pathway based on the optimized determination conditions.A targeted screening method for other esters of diflorasone based on characteristic ions guidance strategy was established.This method had many advantages such as high efficiency,high sensitivity and good reproducibility,and could be used for targeted screening of diflorasone and esters in health products.The developed characteristic ion guided strategy could be employed to construct mass spectral databases for various glucocorticoids,enabling comprehensive targeted screening across a broad range of compounds.

Objective To verify the efficacy of a domestic human DNA quantification kit based on real-time fluorescence quantitative PCR in detecting the total human DNA concentration,male DNA concen-tration in mixed male/female DNA samples,the degree of DNA degradation and inhibitor tolerance.Methods Samples with different concentrations,different male/female ratios,different concentrations of inhibitors,and different degradation degrees were tested using the domestic human DNA quantification kit based on real-time fluorescence quantitative PCR.This kit was compared with a similar product on the market and was applied to the detection of DNA from real cases.Results This human DNA quan-tification kit can effectively detect human DNA as low as 0.001 65 ng/μL,and 6.25 pg/μL of male DNA in mixed samples with a male-to-female ratio of 1∶15 000.Even when the sample contains as high as 400 ng/μL of humic acid or 1 000 μmol/L of hemin alone,the DNA concentration can still be accurately detected.The degradation index can effectively characterize the degradation degree of the sample.This kit has been successfully applied in forensic practice.Conclusion This human DNA quan-tification kit is accurate and reliable in detection.It can accurately reflect the degradation of DNA and inhibitor tolerance.It has good performance in quantitative accuracy,determination of the male/female ratio in mixed samples,and inhibitor tolerance.It has application potential in forensic case examination.

Aim To investigate the effect of Huangqi injection(HI)and Huangqi oral solution(HO)on cisplatin-induced nephrotoxicity(CIN)based on un-targeted metabolomics technology and the underlying mechanisms.Methods Sprague Dawley(SD)rats were randomly divided into the blank group,cisplatin(CDDP)model group,HI treatment group,and HO treatment group,then the CIN model was built with low dose multiple intraperitoneal injections of CDDP.Pre-liminary evaluation of the renal protective efficacy of HI and HO was performed by measuring serum creatinine(Scr),blood urea nitrogen(BUN),and organ indi-ces.Further screening and identification of potential biomarkers(PBs)related to CIN and HI/HO pharma-cological effects were attained through metabolomics studies of renal tissues,and pathway enrichment analy-sis was conducted.Results HI and HO significantly restored the abnormal increase in renal function indica-tors and abnormal decrease in organ indices caused by CDDP,as well as significantly improved the abnormal renal metabolic profile induced by CDDP,indicating that both HI and HO had good alleviating effects on CIN.HI significantly reversed 47 out of 54 CIN related PBs,mainly involving metabolic pathways such as glycerophospholipid metabolism,tryptophan metabo-lism,pantothenate and CoA biosynthesis;HO signifi-cantly reversed 18 out of 54 CIN related PBs,mainly involving metabolic pathways such as taurine and hypo-taurine metabolism,ascorbate and aldarate metabo-lism,pentose and glucuronate interconversions.Con-clusions Both HI and HO have significant alleviating effects on CIN.In the short term,HI salleviating effect is superior to that of HO.Overall,the mechanisms by which both alleviate CIN are mainly related to regula-ting lipid metabolism,amino acid metabolism.

The ocean plays a critical role in the global carbon cycle,and base on the"dual carbon"goals,ocean carbon sinks have received widespread attention.Shellfish aquaculture is one of the most important sources of carbon sinks in fisheries,which has an important impact on the offshore carbon cy-cle.As global temperature rises and ocean acidification intensifies,the capacity of the ocean to absorb CO2 will change.However,the effects of high temperature on the physiology and transcriptome related to carbon metabolism in Mytilus coruscus are not clear enough.This study investigated the effects of high temperatures on the total carbon content,carbon metabolism,antioxidant-related enzyme activities,and the transcriptome of Mytilus coruscus.The results showed that high temperature significantly inhibited the activities of hexokinase and pyruvate kinase,increased carbonic anhydrase activity(P＜0.05),de-creased the ATP content of digestive glands(P＜0.05),and affected glycolysis and the tricarboxylic acid cycle,leading to a significant decrease in the mussel's ability to sequester carbon.High temperature re-sulted in significant(P＜0.05)increases in the levels of reactive oxygen species and malondialdehyde,and enhanced the activities of superoxide dismutase and catalase.Observations by transmission electron microscopy showed that high temperatures damaged the subcellular structure of the digestive gland in Mytilus coruscus,resulting in the shrinkage of the nucleolus,swelling of the endoplasmic reticulum,and a significant reduction in the mitochondrial cristae.Comparative transcriptomic analysis showed that the upregulated DEGs were mainly enriched in protein processing in the endoplasmic reticulum,antigen pro-cessing and presentation,and MAPK signaling pathway.The downregulated DEGs were mainly enriched in necroptosis,DNA replication,and the NF-kappa B signaling pathway.In antioxidant-related DEGs,the upregulated DEGs include vitamin K epoxide reductase,peroxidases,heat shock protein 105 kD,heat shock protein 70 kD,and superoxide dismutase;The downregulated DEGs mainly included NADPH oxidase,glutathione reductase,cytochrome b-245,cytochrome P450,and quinone reductase.The up-regulated genes enriched in the carbon metabolism pathway included chitinase,phosphatidylinositol 4,5-bisphosphate 3-kinase,phosphoenolpyruvate carboxykinase,galactokinase,and inositol trisphosphate 3-kinase.The downregulated genes included aldose-1-epimerase,carbonic anhydrase,galactose mutaro-tase,acyl-CoA synthetase,alcohol dehydrogenase,and hexokinase.In conclusion,high temperature has an inhibitory effect on the activities of enzymes and the expression of genes related to carbon metabolism in Mytilus coruscus.The results of this study are intended to provide a scientific basis for the healthy de-velopment of mussel aquaculture and the assessment of carbon sinks.

Aim To explore the mechanism of the syn-ergistic effect of the ferroptosis inducer erastin com-bined with shikonin in promoting ferroptosis in human hypertrophic scar fibroblasts(HSFBs).Methods Hypertrophic scar tissues provided by the General Hos-pital of Ningxia Medical University were collected,and HSFBs were extracted.HSFBs were identified by HE staining and immunofluorescence.The inhibitory rates of Era and SHK on HSFBs at different concentrations were detected by CCK-8 assay,and the IC50 value was calculated.CompuSyn software was used to calculate the co-use index(CI).Control group,Erastin(Era)group,shikonin(SHK)group and Era+SHK group were set up,and the number and morphological chan-ges of cells were observed after 24 hours of interven-tion.The ability of cell migration and invasion was de-tected by scratch test and Transwell test.The changes of malondialdehyde(MDA),total iron ion and reactive oxygen species(ROS)were detected by corresponding biochemical kits.The expressions of collagen I,α-SMA and GOT1,SLC7A11,GPX4 and FTH1 were detected by Western blot.Results The IC50 value of Era and SHK of primary HSFBs was 2.22 μmol·L-1 and 3.94μmol·L-1 respectively,which was used as the single drug concentration for subsequent experiments.The CompuSyn software was employed to calculate the CI value when the two drugs were used in combination,and the concentrations corresponding to CI=0.39597(Era:1.2 μmol·L-1+SHK:1.5 μmol·L-1)were selected as subsequent combination concentrations(Because when CI was equal to 0.395 97,the concen-tration of each drug was lower than the concentration of single drug,and the inhibition rate of combined drug was greater than 50％).Compared with the monother-apy group,the number of HSFBs in the SHK+Era group was significantly reduced,cell membrane showed breakage and vesiculation,cell wrinkling became smal-ler,and cytoplasm was concentrated.The migration and invasion ability of HSFBs in the SHK+Era group were obviously weakened(P＜0.05),and the expres-sion of fibrosis-related proteins collagen Ⅰ and α-SMA was reduced(P＜0.05);the contents of MDA,total i-ron ions,and ROS in HSFBs of the SHK+Era group increased(P＜0.05),and the protein expression lev-els of SLC7A11,GOT1,GPX4,and FTH1 further de-creased(P＜0.05).Conclusions Erastin in combi-nation with shikonin can synergistically inhibit the pro-liferation,migration and fibrosis levels of HSFBs.The mechanism may be that erastin enhances the inhibition of shikotin on GOT1,increases the levels of cellular i-ron ions,ROS,and lipid peroxides,thereby promoting ferroptosis in HSFBs.

Objective·To investigate the molecular mechanisms and biological functions of the E3 ubiquitin ligase membrane-associated RING-CH 9(MARCH9)in regulating the ubiquitination of receptor protein tyrosine phosphatase alpha(RPTPα).Methods·Western blotting was employed to identify the ubiquitination type of RPTPα and to evaluate the regulatory effect of MARCH9 on its ubiquitination level;Comparative analysis of RPTPα protein stability was performed among wild-type MARCH9,catalytically inactive MARCH9 mutants(MARCH9 S198A or MARCH9-HC/CC),and endogenous MARCH9 knockdown via shRNA.Proteasome inhibitor MG132,autophagy inhibitor 3-MA,and lysosomal inhibitor chloroquine(CQ)were used to determine the degradation pathway of MARCH9-mediated RPTPα ubiquitination.The mechanism underlying 43℃heat shock-induced RPTPα degradation was explored.Stable lung cancer cell lines with MARCH9 single-knockdown(H1299-sh MARCH9)and MARCH9/RPTPα double-knockdown(H1299-sh MARCH9-sh RPTPα)were established using lentiviral vectors.CCK-8 proliferation assay,colony formation assay,and soft agar assay were conducted to evaluate the effects of MARCH9 or RPTPα on lung cancer cell proliferation and clonogenicity.Vasculogenic mimicry formation assay and scratch wound healing assay were performed to assess the impacts on tumor cell invasion and migration.Subcutaneous xenograft models in nude mice were established to examine in vivo tumorigenicity.Bioinformatics analysis was used to compare the expression differences and prognostic correlations of MARCH9 and RPTPα in lung cancer patients.Results·RPTPα predominantly underwent K63-linked poly-ubiquitination,which was significantly enhanced by MARCH9 overexpression.Wild-type MARCH9,but not its catalytic mutants,markedly reduced RPTPα protein stability,while endogenous MARCH9 knockdown increased RPTPα levels.CQ,not MG132 or 3-MA,restored RPTPα stability,indicating that MARCH9 mediated lysosomal degradation of RPTPα through ubiquitination.Heat shock at 43℃specifically enhanced MARCH9-RPTPα interaction,promoting RPTPα degradation.Functional assays revealed that,compared to control H1299 cells,MARCH9-knockdown cells exhibited elevated RPTPα levels,accelerated proliferation,enhanced clonogenicity and invasive capacity,and increased tumorigenicity in nude mice.These phenotypes could be reversed by double knockdown of MARCH9/RPTPα.Bioinformatics analysis demonstrated that high RPTPα expression correlated with poor prognosis and tumor metastasis in lung cancer patients,while MARCH9 showed inverse correlations.Conclusion·MARCH9 mediates K63-linked ubiquitination-dependent lysosomal degradation of phosphatase RPTPα,providing new insights into developing RPTPα-targeted cancer therapeutic strategies.

Objective:To evaluate the clinical efficacy of Tianma Xionglin Zhixuan Tablets in treating hypertension patients with liver yang hyperactivity or comorbid phlegm-stasis syndrome and explore its therapeutic mechanisms through plasma metabolomics.Methods:Thirty-six hypertension patients(4 dropouts)diagnosed with liver yang hyperactivity or phlegm-stasis syndrome were enrolled as the treatment group from June 2022 to September 2023 at the First Affiliated Hospital of Hunan University of Chinese Medicine,while 30 healthy volunteers with balanced constitutions were recruited as the blank group.Plasma samples were collected from patients pre-and post-treatment and from healthy volunteers.Clinical outcomes,including syndrome scores,office blood pressure(BP),and 24-hour ambulatory BP,were recorded.Plasma metabolomic profiling was performed using liquid chromatography-mass spectrometry(LC-MS).Results:Compared with baseline,Tianma Xionglin Zhixuan Tablets significantly reduced traditional Chinese medicine syndrome scores(P＜0.01),office systolic/diastolic BP(P＜0.01),and 24-hour ambulatory BP parameters(24-hour mean BP,daytime/nighttime mean BP;all P＜0.01).Metabolomic analysis identified 45 differential metabolites between the blank group and pretreatment patients,and 64 metabolites altered post-treatment(VIP＞1,P＜0.05).Enrichment analysis of 16 overlapping endogenous metabolites revealed that Tianma Xionglin Zhixuan Tablets primarily modulated arachidonic acid metabolism and sphingolipid metabolism pathways.Conclusion:Tianma Xionglin Zhixuan Tablets demonstrates significant clinical efficacy in hypertension patients with liver yang hyperactivity or phlegm-stasis syndrome,potentially mediated through regulation of arachidonic acid and sphingolipid metabolism.