This study was aimed at developing an in vitro fluorescent substrate assay for the activity of leucyl aminopeptid-ase(LAP)from Echinococcus multilocularis and comparing it with the chemical chromogenic substrate enzyme activity assay.Through the establishment of reaction conditions for the fluorescent substrate-based in vitro enzyme activity assay,we com-pared the differences between the fluorescent substrate L-Leucine-7-amido-4-methylocoumarin(Leu-AMC)and the chemical chromogenic substrate L-Leucine-4-nitroanilide(Leu-pNA)through molecular docking,inhibition rates,and precision measures.Molecular docking revealed that the fluorescent substrate Leu-AMC had higher affinity for the protein than the chemical chromogenic substrate Leu-pNA.Through analysis of the effects of varying reaction conditions on fluorescence intensi-ty,we optimized the fluorescent substrate enzyme activity assay to demonstrate favorable performance at a reaction temperature of 37℃,a pH of 9.0,a protein concentration of 800 nmol/L,and a reaction duration of 60 minutes.Leu-AMC exhibited significant and distinct responses at a 5 μmol/L substrate concentration,under varying substrate conditions.The fluo-rescent substrate assay demonstrated more significant intergroup differences than the chemical chromogenic substrate assay when various inhibitors were added.This study established a fluorescence-based enzyme activity assay for leucyl aminopeptidase from Echinococcus multilocularis by using Leu-AMC as the substrate;this method demonstrated a more significant intergroup difference and sensitivity than the chemical chromogenic substrate assay.

This study was aimed at developing an in vitro fluorescent substrate assay for the activity of leucyl aminopeptid-ase(LAP)from Echinococcus multilocularis and comparing it with the chemical chromogenic substrate enzyme activity assay.Through the establishment of reaction conditions for the fluorescent substrate-based in vitro enzyme activity assay,we com-pared the differences between the fluorescent substrate L-Leucine-7-amido-4-methylocoumarin(Leu-AMC)and the chemical chromogenic substrate L-Leucine-4-nitroanilide(Leu-pNA)through molecular docking,inhibition rates,and precision measures.Molecular docking revealed that the fluorescent substrate Leu-AMC had higher affinity for the protein than the chemical chromogenic substrate Leu-pNA.Through analysis of the effects of varying reaction conditions on fluorescence intensi-ty,we optimized the fluorescent substrate enzyme activity assay to demonstrate favorable performance at a reaction temperature of 37℃,a pH of 9.0,a protein concentration of 800 nmol/L,and a reaction duration of 60 minutes.Leu-AMC exhibited significant and distinct responses at a 5 μmol/L substrate concentration,under varying substrate conditions.The fluo-rescent substrate assay demonstrated more significant intergroup differences than the chemical chromogenic substrate assay when various inhibitors were added.This study established a fluorescence-based enzyme activity assay for leucyl aminopeptidase from Echinococcus multilocularis by using Leu-AMC as the substrate;this method demonstrated a more significant intergroup difference and sensitivity than the chemical chromogenic substrate assay.

OBJECTIVE: This study was aimed at investigating the carrier rate of, and molecular variation in, α- and β-globin gene mutations in Hunan Province. METHODS: We recruited 25,946 individuals attending premarital screening from 42 districts and counties in all 14 cities of Hunan Province. Hematological screening was performed, and molecular parameters were assessed. RESULTS: The overall carrier rate of thalassemia was 7.1%, including 4.83% for α-thalassemia, 2.15% for β-thalassemia, and 0.12% for both α- and β-thalassemia. The highest carrier rate of thalassemia was in Yongzhou (14.57%). The most abundant genotype of α-thalassemia and β-thalassemia was -α ^3.7/αα (50.23%) and β ^IVS-II-654/β ^N (28.23%), respectively. Four α-globin mutations [CD108 (ACC>AAC), CAP +29 (G>C), Hb Agrinio and Hb Cervantes] and six β-globin mutations [CAP +8 (C>T), IVS-II-848 (C>T), -56 (G>C), beta nt-77 (G>C), codon 20/21 (-TGGA) and Hb Knossos] had not previously been identified in China. Furthermore, this study provides the first report of the carrier rates of abnormal hemoglobin variants and α-globin triplication in Hunan Province, which were 0.49% and 1.99%, respectively. CONCLUSION Our study demonstrates the high complexity and diversity of thalassemia gene mutations in the Hunan population. The results should facilitate genetic counselling and the prevention of severe thalassemia in this region.
Humans ; beta-Thalassemia/genetics* ; alpha-Thalassemia/genetics* ; Hemoglobinopathies/genetics* ; China/epidemiology* ; High-Throughput Nucleotide Sequencing

ObjectiveLower urinary tract symptoms （LUTS） caused by benign prostatic enlargement （BPE） are influenced by prostate calcification （PC）. The aim of this study is to explore the related factors of BPE with PC in men receiving physical examinations， and to provide some references for the prevention and treatment of PC in BPE patients.MethodsThe clinical data of 863 cases of BPE among 3433 men who underwent physical examination in the Fourth Affiliated Hospital of Anhui Medical University from October 2018 to June 2021 were analyzed retrospectively The blood and urine examination indexes between group A （BPE with PC） and group B （BPE without PC） were compared by univariate analysis. The related factors of BPE with PC were analyzed by logistic regression.ResultsThe incidence of BPE （defined as prostate volume ≥20 mL） with PC in BPE patients was 37.5% （324 / 863）. Univariate analysis showed that age， blood urea nitrogen， and urinary pH value in group A were significantly higher than those in group B （P<0.05）， and height， weight and blood uric acid of group A were lower than those of group B group （P<0.05）. The proportion of patients with inhomogeneous prostatic echo in group A was higher than that in group B （P<0.05）. There was no significant difference in the proportion of patients with metabolic syndrome and components between the two groups （P>0.05）. Multivariate logistic regression analysis showed that inhomogeneous echo of the prostate， urinary pH and age （especially 80~89 years old） were the related factors of BPE with PC in BPE patients undergoing checkups ［OR 95%CI 2.082 （1.111， 3.900）； OR 95%CI 1.419 （1.152， 1.747） and OR 95%CI 17.829 （3.224， 98.594）］.ConclusionsThe incidence of BPE with PC in BPE patients undergoing checkups is higher. In addition to age， inhomogeneous echo of the prostate and urine pH are related factors for BPE with PC in BPE patients undergoing physical examinations.

Objective: A method for the determination of acetochlor and its metabolites in urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was established. Methods: After cleaned-up by a HLB extraction cartridges, the urine was eluted with 1% acetic acid acetonitrile solution. The target compounds were separated by ACQUITY UPLC®HSS T3 Column (2.1 mm×100 mm×1.8 μm) by using 1% formic acid solution and acetonitrile as mobile phase with gradient elution program, and analyzed in positive electrospray ionization mode by liquid chromatography tandem mass spectrometry. Results: All the target compounds showed good linear relationships in the range of 1-50 μg/L, and the correlation coefficients (r) were higher than 0.997. The recoveries rates at three different spiked levels for all target compounds in blank matrices were 107.6%-129.1%, and the relative standard deviations (RSD) were 1.5%-9.9% (n=6) . The limits of detection and quantitation of the method were 0.04-0.11 μg/L and 0.15-0.42 μg/L, respectively, and target substances were detected in all urine samples from occupational exposure workers to acetochlor. Conclusion: This method is suitable for rapid screening and analysis of acetochlor and metabolites in urine with the advantages of accuracy, rapidity, simplicity, high sensitivity and good specificity.
Acetonitriles ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Humans ; Solid Phase Extraction ; Tandem Mass Spectrometry ; Toluidines

MicroRNAs (miRNAs) are considered to be involved in the pathogenic initiation and progression of chronic nonbacterial prostatitis (CNP); however, the comprehensive expression profile of dysregulated miRNAs, relevant signaling pathways, and core machineries in CNP have not been fully elucidated. In the current research, CNP rat models were established through the intraprostatic injection of carrageenan into the prostate. Then, next-generation sequencing was performed to explore the miRNA expression profile in CNP. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) bioinformatical analyses were conducted to reveal the enriched biological processes, molecular functions, and cellular components and signaling pathways. As a result, 1224, 1039, and 1029 known miRNAs were annotated in prostate tissues from the blank control (BC), normal saline injection (NS), and carrageenan injection (CAR) groups (n = 3 for each group), respectively. Among them, 84 miRNAs (CAR vs BC) and 70 miRNAs (CAR vs NS) with significantly different expression levels were identified. Compared with previously reported miRNAs with altered expression in various inflammatory diseases, the majority of deregulated miRNAs in CNP, such as miR-146b-5p, miR-155-5p, miR-150-5p, and miR-139-5p, showed similar expression patterns. Moreover, bioinformatics analyses have enriched mitogen-activated protein kinase (MAPK), cyclic adenosine monophosphate (cAMP), endocytosis, mammalian target of rapamycin (mTOR), and forkhead box O (FoxO) signaling pathways. These pathways were all involved in immune response, which indicates the critical regulatory role of the immune system in CNP initiation and progression. Our investigation has presented a global view of the differentially expressed miRNAs and potential regulatory networks containing their target genes, which may be helpful for identifying the novel mechanisms of miRNAs in immune regulation and effective target-specific theragnosis for CNP.
Animals ; Computational Biology ; Databases, Genetic ; Gene Expression Profiling ; High-Throughput Nucleotide Sequencing ; Male ; MicroRNAs/metabolism* ; Prostate/metabolism* ; Prostatitis/metabolism* ; Rats ; Rats, Wistar

This study aims to validate our hypothesis that acid-sensing ion channels (ASICs) may contribute to the symptom of pain in patients with chronic prostatitis (CP). We first established a CP rat model, then isolated the L5-S2 spinal dorsal horn neurons for further studies. ASIC1a was knocked down and its effects on the expression of neurogenic inflammation-related factors in the dorsal horn neurons of rat spinal cord were evaluated. The effect of ASIC1a on the Ca²⁺ ion concentration in the dorsal horn neurons of rat spinal cord was measured by the intracellular calcium ([Ca²⁺]i) intensity. The effect of ASIC1a on the p38/mitogen-activated protein kinase (MAPK) signaling pathway was also determined. ASIC1a was significantly upregulated in the CP rat model as compared with control rats. Acid-induced ASIC1a expression increased [Ca²⁺]i intensity in the dorsal horn neurons of rat spinal cord. ASIC1a also increased the levels of neurogenic inflammation-related factors and p-p38 expression in the acid-treated dorsal horn neurons. Notably, ASIC1a knockdown significantly decreased the expression of pro-inflammatory cytokines. Furthermore, the levels of p-p38 and pro-inflammatory cytokines in acid-treated dorsal horn neurons were significantly decreased in the presence of PcTx-1, BAPTA-AM, or SB203580. Our results showed that ASIC1a may contribute to the symptom of pain in patients with CP, at least partially, by regulating the p38/MAPK signaling pathway.
Acid Sensing Ion Channel Blockers/pharmacology* ; Acid Sensing Ion Channels/genetics* ; Animals ; Calcium/metabolism* ; Chelating Agents/pharmacology* ; Chronic Disease ; Cytokines/metabolism* ; Disease Models, Animal ; Egtazic Acid/pharmacology* ; Gene Knockdown Techniques ; Imidazoles/pharmacology* ; Inflammation/metabolism* ; MAP Kinase Signaling System/genetics* ; Male ; Pain/genetics* ; Peptides/pharmacology* ; Phosphorylation/drug effects* ; Posterior Horn Cells/metabolism* ; Prostatitis/complications* ; Protein Kinase Inhibitors/pharmacology* ; Pyridines/pharmacology* ; Rats ; Spider Venoms/pharmacology* ; Up-Regulation ; p38 Mitogen-Activated Protein Kinases/metabolism*

Objective: The proposal of the present study was to investigate whether the TP53 rs1042522 polymorphism confers susceptibility to prostate cancer (PCa), by performing an updated meta-analysis. Methods: Eligible publications investigating the association between the TP53 rs1042522 polymorphism and PCa susceptibility were selected from PubMed, Google Scholar, and Web of Science. We used STATA 12.0 software to conduct the analyses. Odds ratio (OR) with 95%confidence interval (CI) was calculated. Results: A total of 17 case-control studies were retrieved reporting a total of 2683 cases and 2981 controls. However, no sig-nificant association was uncovered between the TP53 rs1042522 polymorphism and PCa susceptibility in the overall population under the five genetic models. In the stratification analysis by source of control, an increased susceptibility to PCa was identified in the population-based (P-B) group (CG vs. GG:OR=1.48, 95%CI:1.24-1.77, P<0.01;CC/CG vs. GG:OR=1.32, 95%CI:1.12-1.57, P<0.01), whereas a decreased susceptibility was uncovered in the hospital-based (H-B) group (CG vs. GG:OR=0.67, 95%CI:0.46-0.96, P=0.03;CC/CG vs. GG:OR=0.67, 95%CI:0.46-0.99, P=0.04) under heterozygous and dominant model. Conclusion: This study did not find an association between the TP53 rs1042522 polymorphism and PCa susceptibility in the overall population and corresponding subgroup analyses except in the stratification analysis by source of control. The results suggest that the TP53 rs1042522 polymorphism is not a risk factor for PCa.

Objective: The proposal of the present study was to investigate whether the TP53 rs1042522 polymorphism confers susceptibility to prostate cancer (PCa), by performing an updated meta-analysis. Methods: Eligible publications investigating the association between the TP53 rs1042522 polymorphism and PCa susceptibility were selected from PubMed, Google Scholar, and Web of Science. We used STATA 12.0 software to conduct the analyses. Odds ratio (OR) with 95%confidence interval (CI) was calculated. Results: A total of 17 case-control studies were retrieved reporting a total of 2683 cases and 2981 controls. However, no sig-nificant association was uncovered between the TP53 rs1042522 polymorphism and PCa susceptibility in the overall population under the five genetic models. In the stratification analysis by source of control, an increased susceptibility to PCa was identified in the population-based (P-B) group (CG vs. GG:OR=1.48, 95%CI:1.24-1.77, P<0.01;CC/CG vs. GG:OR=1.32, 95%CI:1.12-1.57, P<0.01), whereas a decreased susceptibility was uncovered in the hospital-based (H-B) group (CG vs. GG:OR=0.67, 95%CI:0.46-0.96, P=0.03;CC/CG vs. GG:OR=0.67, 95%CI:0.46-0.99, P=0.04) under heterozygous and dominant model. Conclusion: This study did not find an association between the TP53 rs1042522 polymorphism and PCa susceptibility in the overall population and corresponding subgroup analyses except in the stratification analysis by source of control. The results suggest that the TP53 rs1042522 polymorphism is not a risk factor for PCa.

This study was to investigate the mechanism of gingerols antagonizing the inflammatory effect of toxic raphides from Pinella pedatisecta. Mice peritonitis models induced by toxic raphides from P. pedatisecta were applied to observe the effect of gingerols on inflammatory mediators PGE2 in the exudates of abdominal inflammation in mice; rats peritoneal macrophage in vitro culture models were adopted to study the anti-inflammatory effects of gingerol against toxic raphides, with TNF-α and IL-1β in supernatant as indexes. Scanning electron microscopy was used to observe the changes in surface morphology of macrophages treated by raphides and gingerols. Macrophages-neutrophils co-cultured models were used to study the antagonism of gingerols against the effect of toxic raphides' stimulation on neutrophils migration. Results showed that gingerols could significantly inhibit the production of PGE2 in the exudates of abdominal inflammation induced by toxic raphides from P. pedatisecta in mice. Gingerols could significantly inhibit the toxic raphides from P. pedatisecta to induce the release of inflammatory factors, with certain dose dependence. Scanning electron microscopy showed that gingerols could significantly inhibit phagocytosis of macrophages, cytomembrane injury, and neutrophils migration induced by toxic raphides from P. pedatisecta. The results showed that the antagonism mechanism of gingerols against the toxic raphides from P. pedatisecta may be associated with inhibiting the pro-inflammatory toxicity including macrophage activation, inflammatory factors release, and neutrophils migration.