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 To investigate the status of population dynamics and distribution changes of Aedes aegypti in Guangdong Province.Methods Continuous monitoring was conducted from May 2018 to July 2024 in Wushi Town and Qishui Town,Leizhou City,Zhanjiang City,Guangdong Province.Additionally,a survey of the distribution of Ae.aegypti along the Leizhou Peninsula coast was carried out.Results The density of Ae.aegypti in Zhanjiang showed a gradual decline from 2018 to 2024.The last detection of adult Ae.aegypti in Wushi Town was in September 2021,and the last larva was found in October 2023.No Ae.aegypti was detected in Qishui Town during surveys from 2021 to 2024.A survey of 18 coastal villages in the Leizhou Peninsula revealed no detections of Ae.aegypti.Conclusions This study provides a basis for understanding the distribution and population density fluctuations of Ae.aegypti,assessing its invasion risk,and scientifically conducting relevant prevention and control efforts.

ObjectiveBased on functional near-infrared spectroscopy (fNIRS), we investigated the brain activity characteristics of gross motor tasks in children with autism spectrum disorder (ASD) and motor dysfunctions (MDs) to provide a theoretical basis for further understanding the mechanism of MDs in children with ASD and designing targeted intervention programs from a central perspective. MethodsAccording to the inclusion and exclusion criteria, 48 children with ASD accompanied by MDs were recruited into the ASD group and 40 children with typically developing (TD) into the TD group. The fNIRS device was used to collect the information of blood oxygen changes in the cortical motor-related brain regions during single-handed bag throwing and tiptoe walking, and the differences in brain activation and functional connectivity between the two groups of children were analyzed from the perspective of brain activation and functional connectivity. ResultsCompared to the TD group, in the object manipulative motor task (one-handed bag throwing), the ASD group showed significantly reduced activation in both left sensorimotor cortex (SMC) and right secondary visual cortex (V2) (P<0.05), whereas the right pre-motor and supplementary motor cortex (PMC&SMA) had significantly higher activation (P<0.01) and showed bilateral brain region activity; in terms of brain functional integration, there was a significant decrease in the strength of brain functional connectivity (P<0.05) and was mainly associated with dorsolateral prefrontal cortex (DLPFC) and V2. In the body stability motor task (tiptoe walking), the ASD group had significantly higher activation in motor-related brain regions such as the DLPFC, SMC, and PMC&SMA (P<0.05) and showed bilateral brain region activity; in terms of brain functional integration, the ASD group had lower strength of brain functional connectivity (P<0.05) and was mainly associated with PMC&SMA and V2. ConclusionChildren with ASD exhibit abnormal brain functional activity characteristics specific to different gross motor tasks in object manipulative and body stability, reflecting insufficient or excessive compensatory activation of local brain regions and impaired cross-regions integration, which may be a potential reason for the poorer gross motor performance of children with ASD, and meanwhile provides data support for further unraveling the mechanisms underlying the occurrence of MDs in the context of ASD and designing targeted intervention programs from a central perspective.

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 To investigate the therapeutic effects and mechanism of enalapril maleate tablet on doxorubicin-induced heart failure rats based on mitogen-activated protein kinase(MAPK)signaling pathway.Methods Eleven of the 40 male SD rats were randomly selected as the normal group(equivalent to 0.9％NaCl),and the remaining 29 were prepared with intraperitoneal injection of 3 mg·kg-1·w-1 doxorubicin to prepare heart failure model.After successful modeling,they were randomly divided into model group(n=15 cases)and experimental group(n=14 cases).Experimental group was given 1.8 mg·kg-1·d-1 enalapril maleate suspension for gavage;normal and model groups were given the same amount of 0.9％NaCl by gavage.After 8 weeks,the rats were subjected to cardiac ultrasound,the left ventricular ejection fractions(LVEF)of each group were recorded,the serum myocardial injury index level was detected by enzyme-linked immunosorbent assay,and the expression levels of mRNA and protein related to the MAPK signaling pathway were detected by real-time quantitative polymerase chain reaction and Western blot.Results The LVEF values of control,model and experimental groups were(77.85±3.34％)％,(41.39±2.87％)％and(60.10±6.53％)％;serum brain natriuretic peptide contents were(219.30±10.59),(333.90±61.19)and(260.00±16.10)pg·mL-1;the relative expression levels of Mapk8ip2 were 1.00±0.01,2.60±0.12 and 2.00±0.08;the relative expression levels of Mapk8ip3 were 1.00±0.00,6.77±1.04 and 3.66±0.54;the relative expression levels of Mapk1 were 1.00±0.00,4.40±0.14 and 2.71±0.24;the relative expression levels of Mapk3 were 1.00±0.01,7.83±0.34 and 2.71±0.24;the relative expression levels of P38-MAPK were 1.00±0.05,1.14±0.02 and 1.02±0.03;the relative expression levels of extracellular regulated protein kinase 1/2 protein were 1.00±0.07,1.49±0.03 and 1.16±0.10;the relative expression levels of c-Jun N-terminal kinase 1/2 protein were 1.00±0.03,1.65±0.19 and 1.14±0.01,respectively.Compared with the model group,the differences of above indexes in the normal and experimental groups were statistically significant(all P＜0.01).Conclusion Enalapril maleate tablets have therapeutic effects on rats with heart failure,and the mechanism may be achieved by regulating the MAPK signaling pathway.

BACKGROUND:Indolepropionic acid has been shown to reduce diabetes-induced central nervous system inflammation.However,there is a lack of research on whether to inhibit microglia M1 polarization for the treatment of spinal cord injury. OBJECTIVE:To investigate the mechanism of indolepropionic acid inhibition of microglial cell M1 polarization for the treatment of spinal cord injury through cell and animal experiments. METHODS:(1)In vitro experiments:BV2 cell viability was assessed using the CCK-8 assay to determine optimal concentrations of indolepropionic acid.Subsequently,BV2 cells were categorized into control group,administration group(50 μmol/L indolepropionic acid),lipopolysaccharide group(100 ng/mL lipopolysaccharide),and treatment group(100 ng/mL lipopolysaccharide + 50 μmol/L indolepropionic acid).Nitric oxide content was quantified using the Griess method.Real-time quantitative PCR and western blot assay were employed to measure mRNA and protein levels of pro-inflammatory factors.Cell immunofluorescence staining was conducted to assess inducible nitric oxide synthase expression.The Seahorse assay was employed to assess glycolytic stress levels in BV2 cells.(2)In vivo experiments:30 SD rats were randomly divided into three groups:sham surgery group,spinal cord injury group,and indolepropionic acid group.Motor function recovery in rats after spinal cord injury was assessed using BBB scoring and the inclined plane test.Immunofluorescence staining of spinal cord tissue was conducted to evaluate the expression of inducible nitric oxide synthase in microglial cells.ELISA was employed to measure protein expression levels of the pro-inflammatory cytokines interleukin-1β and tumor necrosis factor-α in spinal cord tissue. RESULTS AND CONCLUSION:(1)In vitro experiments:Indolepropionic acid exhibited significant suppression of BV2 cell viability when its concentration exceeded 50 μmol/L.Indolepropionic acid achieved this by inhibiting the activation of the nuclear factor κB signaling pathway,thereby suppressing the mRNA and protein expression levels of pro-inflammatory cytokines(interleukin-1β and tumor necrosis factor-α),as well as the M1 polarization marker,inducible nitric oxide synthase,in BV2 cells.Additionally,indolepropionic acid notably reduced the glycolytic level in BV2 cells induced by lipopolysaccharides.(2)In vivo experiments:Following indolepropionic acid intervention in spinal cord injury rats,there was a noticeable increase in BBB scores and the inclined plane test angle.There was also a significant decrease in the number of M1-polarized microglial cells in spinal cord tissue,accompanied by a marked reduction in the protein expression levels of pro-inflammatory cytokines(interleukin-1β and tumor necrosis factor-α).(3)These results conclude that indolepropionic acid promotes functional recovery after spinal cord injury by improving the inflammatory microenvironment through inhibition of microglia M1 polarization.

ObjectiveTo investigate the genetic polymorphism and structure of 47 autosomal microhaplotypes in the Han population in Changshu City, Jiangsu Province, and to evaluate the forensic efficiencies and forensic parameters. MethodsThe DNA library of unrelated individual samples was prepared according to MHSeqTyper47 kit manual and sequenced on the MiSeq FGx platform. Microhaplotype genotyping and sequencing depth statistics were processed using MHTyper. The genetic information of samples was then evaluated. The fixation index and genetic distance between the Jiangsu Changshu population and the reference populations in the 1000 Genomes Project phase 3 (1KG) were calculated, and forensic parameters were evaluated. ResultsThe fixation index and genetic distance between the Han population in Changshu, Jiangsu, and the CHB (Han Chinese in Beijing, China) reference population in 1KG were the lowest. The effective allele number (A_e) of each locus is also the closest between the two populations. The combined matching probability (CMP) of the Changshu Han population is close to the 5 populations of the East Asian reference super-population in 1KG, which is 1.25×10^-36, and the combined probability of exclusion reached 0.999 999 999 964 1. ConclusionThis study reported the genetic polymorphism and allele frequency of 47 microhaplotypes in a Han population in Changshu City, Jiangsu Province. This information provides a data basis for 47 microhaplotypes in forensic applications. In addition, the polymorphism differences between the 1KG reference population and the Han population in Changshu, Jiangsu were compared, and the genetic structure of 47 microhaplotypes in the Han population in Changshu, Jiangsu was revealed. In general, the reference data of the East Asian super-population in 1KG is more in line with the genetic characteristics of Han population in Changshu, Jiangsu.

Objective To explore medium and long term efficacy of oblique lateral interbody fusion(OLIF)in treating lumbar specific infection.Methods From October 2017 to January 2021,24 patients with lumbar specific infection were treated by OLIF combined with vertebral screw internal fixation,including 15 males and 9 females,aged from 27 to 61 years old with an average of(43.0±15.0)years old;the courses of disease ranged from 6 to 24 months with an average of(14.0±7.0)months;7 patients with L2-L3,12 patients with L3-L4 and 5 patients with L4-L5;19 patients with tuberculosis infection and 5 patients with brucella infection.The amount of intraoperative blood loss,operative time and complications were recorded,and erythro-cyte sedimentation rate(ESR),C-reactive protein(CRP),visual analogue scale(VAS),Japanese Orthopaedic Association(JOA)score and American Spinal Injury Association(ASIA)rating were compared before and one month after opertaion.Re-sults All patients were followed up from 9 to 24 months with an average of(13.0±6.0)months.Operative time was(132.5±21.4)min,and intraoperative blood loss was(227.3±43.1)ml.ESR and CRP were decreased from(82.34±18.62)mmol·h-1 and(53.08±21.84)mg·L-1 before operation to(33.52±17.31)mmol·h-1 and(15.48±8.36)mg·L-1 at one month after opera-tion,respectively(P＜0.05).VAS was decreased from(7.52±1.36)before opertaion to(1.74±0.87)at one month after opera-tion(P＜0.05).JOA was increased from(17.86±3.95)before operation to(24.72±3.19)at one month after operation(P＜0.05).Four patients had neurological symptoms before operation,and were classified to grade D according to ASIA classifica-tion,who were recovered to grade E at 1 month after operation.One patient was suffered from psoas major muscle injury after operation,and returned to normal at 3 weeks.One patient was suffered from abdominal distension and difficulty in defecation,and relieved after gastrointestinal decompression and enema.No complications such as abdominal organ injury and poor wound healing occurred in all patients.Conclusion OLIF combined with vertebral screw internal fixation is a new minimally invasive surgical method for the treatment of lumbar specific infection,especially the lesion located on the middle lumbar vertebra.It has advantages of less trauma,short operation time,less blood loss,convenient operation,complete removal of the lesion,safety and effectiveness,and has good medium-and long-term efficacy for lumbar specific infection.

AIM To analyze the constituents absorbed into blood and brain from Zhishe Tongluo Capsules.METHODS Sixteen rats were randomly assigned into four groups and given intragastric administration(3.1 g/kg),after which the cerebral ischemia-reperfusion injury(MACO)model was established,the blood and brain tissues were collected,and UHPLC-Q Exactive Focus MS/MS was adopted in the identification of prototype constituents.RESULTS Total 70 constituents were identified,20 of which were found in the blood,mainly including flavonoids,tanshinones and Ligusticum chuanxiong phthalides,and 7 of them could enter the brain through blood-brain barrier.Compared with the normal administration group,the MACO administration group demonstrated added constituents absorbed into blood containing 3-hydroxybenzoic acid,calycosin-7-glucoside,curcumenol,senkyunolide B,dihydrotanshinone I and cryptotanshinone;removed constituents absorbed into brain containing puerarin,elemicin,sedanolide,and added those containing salvianolic acid A,senkyunolide I,dihydrotanshinone I in the left brain tissues(infarcted side).CONCLUSION The constituents absorbed into blood and brain from Zhishe Tongluo Capsules,along with the enhanced absorptions of phthalides,quinones and phenols in MACO rats in vivo may be the active substances for treating cerebral infarction.