Background: Adrenocortical hormones, particularly 11-oxygenated androgens, are pivotal in female reproductive health and fertility. Standardized detection kits and population-specific reference intervals are lacking in China, hindering related clinical applications. Methods: A HPLC-tandem mass spectrometry (HPLC-MS/MS) pipeline was developed, rigorously validated, and applied to simultaneously quantify corticosterone, cortisone, cortisol, 18-OH cortisol, androstenedione (A4), 11β-hydroxyandrostenedione (11-OH A4), dehydroepiandrosterone, and dehydroepiandrosterone sulfate in serum samples from 455 reproductive-aged women (18–45 yrs) in Guangxi, China. Age-dependent concentration trends were analyzed, and reference intervals stratified by age (2.5th to 97.5th percentiles) were established. Correlations with body-composition metrics, ethnicity, and the menstrual cycle were investigated. Results: The HPLC-MS/MS method demonstrated high precision (intra- and inter-assay CVs < 15%), accuracy, and sensitivity. All eight hormones exhibited significant age-related declines (P < 0.001 for seven hormones; P = 0.001 for 11-OH A4). Notably, 11-OH A4 levels were significantly lower in the 35–45-yr (3.05 nmol/L) and 25–34-yr (3.09 nmol/L) age groups than in the 18–24-yr (3.57 nmol/L) age group, whereas no significant difference was observed between the 35–45-yr and 25–34-yr age groups. Weak negative correlations were observed between the body mass index and corticosterone and cortisone levels, whereas ethnicity and the menstrual cycle showed no significant associations with hormone levels. Conclusions We developed an HPLC-MS/MS-based method for simultaneously quantifying eight adrenocortical hormones, including 11-OH A4, and defined age-specific reference intervals for reproductive-aged Chinese women. These findings advance the clinical utility of adrenocortical hormones in diagnosing and managing reproductive disorders.

ObjectiveTo investigate the mechanism by which Luoshi Neiyi prescription treats endometriosis （EMs） through the Toll-like receptor 4 （TLR4）/nuclear factor-κB （NF-κB） signaling pathway. MethodsAnimal experiments were conducted with 50 female SD rats， which were randomized into a sham operation group （10 rats） and a modeling group （40 rats）. An autologous endometrial transplantation method was used for the modeling of EMs. The 36 successfully modeled rats were randomly allocated into four groups （n=9 each）： The model group， the low-dose （7.87 g·kg^-1） Luoshi Neiyi prescription group， the high-dose （15.74 g·kg^-1） Luoshi Neiyi prescription group， and the dienogest （0.20 mg·kg^-1） group. The physiological status and ectopic lesion volumes of rats in each group were observed. Hematoxylin-eosin （HE） staining was used to observe the morphology of the eutopic endometrial tissue. Enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of inflammatory factors including interleukin-1β （IL-1β） and prostaglandin E₂ （PGE₂） in the serum of EMs rats. Immunohistochemistry was used to detect the expression of matrix metalloproteinase-9 （MMP-9） and vascular endothelial growth factor A （VEGFA） in the eutopic endometrial tissue. Western blot was adopted to determine the protein levels of TLR4， myeloid differentiation factor 88 （MyD88）， phosphorylated nuclear factor-κB （p-NF-κB）/NF-κB， MMP-9， and VEGFA in the eutopic endometrial tissue. In the cell experiments， the cell-counting kit-8 （CCK-8） assay was employed to screen the optimal concentration of Luoshi Neiyi prescription-containing serum， and a scratch assay was performed to assess the migration ability of iheESCs cells. Interventions with Luoshi Neiyi prescription-containing serum， resatorvid （TAK-242， a TLR4 inhibitor）， and lipopolysaccharides （LPS， a TLR4 agonist） were conducted， and Western blot was used to detect the expression of proteins related to the TLR4/NF-κB signaling pathway. ResultsThis experiment successfully replicated 36 EMs rat models. Compared with the sham operation group， the model group exhibited visible ectopic lesions on the abdominal wall and an increase in the writhing response score （P<0.01）. Furthermore， HE staining revealed the model group exhibited a thickened eutopic epithelium， stromal cell disarrangement， and evident infiltration of inflammatory cells. In addition， the model group showed elevated serum levels of IL-1β and PGE₂ （P<0.05， P<0.01）， increased positive expression of MMP-9 and VEGFA in the ectopic endometrial tissue （P<0.01）， and up-regulated protein levels of TLR4， MyD88， p-NF-κB/NF-κB， MMP-9， and VEGFA in the ectopic endometrial tissue （P<0.05， P<0.01）. Compared with the model group， all treatment groups exhibited a reduction in the ectopic lesion volume （P<0.01）. Furthermore， the writhing response scores were decreased in the high-dose Luoshi Neiyi prescription group and the dienogest group （P<0.05， P<0.01）. The pathological state of the ectopic endometrial tissue was alleviated to varying degrees in the treatment groups. Low-dose Luoshi Neiyi prescription reduced serum PGE₂ levels， and high-dose Luoshi Neiyi prescription and dienogest decreased serum IL-1β and PGE₂ levels in EMs rats （P<0.05， P<0.01）. The treatment groups showed decreased positive expression of MMP-9 and VEGFA in the ectopic endometrial tissue （P<0.05， P<0.01） and down-regulated protein levels of TLR4， MyD88， p-NF-κB/NF-κB， MMP-9， and VEGFA in the ectopic endometrial tissue （P<0.05， P<0.01）. The cell experiments showed that 5%， 10%， and 20% Luoshi Neiyi prescription-containing sera significantly reduced the viability and inhibited the migration of iheESCs. Compared with the control group， 5%， 10%， and 20% Luoshi Neiyi prescription-containing serum groups and the TAK-242 group showed reduced protein levels of TLR4， MyD88， p-NF-κB/NF-κB， MMP-9， and VEGFA （P<0.01）. Compared with the control group， the LPS group showed increased expression of the above proteins （P<0.01）. Compared with the LPS group， the LPS+5%， 10%， and 20% Luoshi Neiyi prescription-containing serum groups showed reduced expression of the above proteins （P<0.05， P<0.01）. ConclusionLuoshi Neiyi prescription may modulate the TLR4/NF-κB signaling pathway to reduce the inflammatory response and histopathological damage in the eutopic endometrium and suppress the adhesive， invasive， and angiogenic biological processes in the ectopic endometrial tissue， thereby exerting its therapeutic effect on EMs.

Temporal lobe epilepsy(TLE)is the most common form of focal epilepsy in adults,characterized by spontaneous recurrent seizures,with most patients experiencing drug resistance and cognitive dysfunction.MicroRNAs(miRNAs)play a critical role in the pathological process of TLE through their regulation of post-transcriptional gene expression.The pathogenesis of TLE has not been fully elucidated,lacking effective clinical therapeutic targets and prognostic markers.This review sum-marized the expression changes of miRNAs in TLE and their research progress as potential biomarkers,aiming to provide new insights into the early diagnosis,prognosis evaluation,and pathogenic mecha-nisms of TLE.

Objective:To establish a clinical predictive model for poor clinical outcomes in patients with secondary hemophagocytic lymphohistiocytosis (sHLH) and to evaluate its clinical application value.Methods:Patients diagnosed with sHLH who met the study criteria and were initially admitted to the Emergency Department of Peking University People’s Hospital between September 2017 and December 2024 were enrolled. Clinical data were collected, and patients were categorized into a death group or a survival group based on clinical outcomes as the observational endpoint. Differences in clinical data between the two groups were compared. Univariate and multivariate logistic regression analyses were conducted to screen significant variables, and a predictive model nomogram was developed using the R programming language. The discriminative ability, calibration, and clinical utility of the predictive model were assessed using the receiver operating characteristic curve, net reclassification improvement index, calibration curve, and decision curve analysis. K-fold cross-validation was employed to evaluate the model's performance. The model was compared with the Acute Physiology and Chronic Health Evaluation Ⅱ (APACHE Ⅱ) score and the Sequential Organ Failure Assessment (SOFA) score.Results:A total of 116 cases were enrolled in the study, comprising 36 cases in the mortality group and 80 cases in the survival group. Multivariate logistic analysis identified age, platelet count, prothrombin time, total bilirubin, altered mental status, and cardiac involvement as factors significantly associated with clinical outcomes. Based on these factors, an early warning model for adverse clinical prognosis was established, and a corresponding nomogram was developed. The model demonstrated excellent discriminative ability, calibration, and clinical utility (AUC=0.950; Hosmer-Lemeshow test: χ2=2.5476, P=0.980; calibration curve: R 2=0.649, P=0.906), outperforming both the APACHE Ⅱ and SOFA scores in predicting adverse outcomes (both P<0.01). Conclusions:This study established an early warning model for adverse clinical prognosis in sHLH based on objective clinical data. The model aids in the clinical assessment of sHLH patients, facilitates early warning, and supports clinical decision-making for treatment.

Feline herpesvirus type Ⅰ(FHV-1)was used as the vector.The gI and gE genes of FHV-1 were replaced with the feline calicivirus(FCV)VP1 gene and the red fluorescent protein(mCherry)gene by CRISPR/Cas9 systems and homologous recombination technology,and the re-combinant virus strain FHV △gI&gE/VP1-mCherry+was successfully rescued.The recombinant virus strain was purified by plaque assay.The biological characteristics and genetic stability of the recombinant virus were analyzed by indirect immunofluorescence assay,plaque morphological anal-ysis,and PCR.The results of the indirect immunofluorescence identification showed that the re-combinant virus FHV △gI&gE/VP1-mCherry+could express the VP1 protein in F81 cells,and the growth characteristics of the recombinant virus were not significantly different from those of the parent virus FHV-1.The plaque morphology and staining results indicated that the area of the plaque formed by the recombinant virus was smaller than that of the parent virus,suggesting that the spread ability of the recombinant virus between cells was reduced after the deletion of the gI and gE genes.The result of PCR showed that the VP1 gene could still be detected after 15 succes-sive passages of the recombinant virus,indicating that the recombinant virus had good genetic stability.In this study,the recombinant virus strain expressing the FCV VP1 protein was successfully prepared,which will lay a foundation for the development of engineered FCV and FHV-1 vaccine.

Moloney leukemia virus 10(MOV10)gene knockout(MKO)mouse neuro 2a(N2a)cell lines was constructed by CRISPR/Cas9(clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9)gene editing technology.First,a recombinant plasmid pMD18T-U6-sgRNA expressing MOV10 gene-specific guide RNA(sgRNA)was constructed,and then pMD18T-U6-sgRNA and pMJ920-Cas9-eGFP were co-transfected into N2a.The results showed that the MKO N2a cell line had normal cell activity and cell proliferation ability.The infection test of the MKO N2a cell line was carried out using the rabies virus(RABV)and vesicular stomatitis virus(VSV)of the Rhabdoviridae family.The results showed that the replication level of the Rhabdoviridae virus in the MKO N2a cell line was significantly enhanced.The results showed that a MKO N2a cell line was successfully constructed in this study,which provided a preliminary basis for the exploration of the biological function and antiviral mechanism of MOV10 and the develop-ment of a recombinant viral vector vaccine with RABV/VSV as the vector.

This study aims to establish an indirect ELISA method for detecting RVFV antibodies u-sing recombinant proteins of Rift Valley fever virus(RVFV)Gn protein Ⅱ-Ⅲ structural domains as the encapsulated antigen which was expressed by the Escherichia coli(E.coli)expression sys-tem.The gene sequences encoding the Ⅱ and Ⅲ subdomains of RVFV Gn protein were inserted in-to pET-30a(+)to construct the recombinant plasmid pET-RVFV Gn-D Ⅱ-Ⅲ.After transforma-tion of the recombinant plasmid into DE3(BL21)competent cells,the recombinant Gn-D Ⅱ-Ⅲ protein was induced with IPTG and purified using affinity chromatography.An indirect ELISA method for the detection of RVFV antibodies was developed using purified recombinant protein as coating antigen and SPA-HRP as the enzyme-labelled secondary antibody.Western blot analysis confirmed that the RVFV Gn-D Ⅱ-Ⅲ protein was successfully expressed.The optimal expression conditions for RVFV Gn-D Ⅱ-Ⅲ protein were induced with 0.8 mmol/L IPTG at 37 ℃ for 5 h.The Gn-D Ⅱ-Ⅲ protein was purified using affinity chromatography with a purity of 91.9％,and the purified protein was used as the encapsulated antigen to develop an ELISA assay for RVFV anti-bodies.The specificity evaluation showed that the method specifically detected RVFV-positive sera and did not cross-react with sera positive for West Nile virus(WNV),Ebola virus(EBOV),Mar-burg virus(MARV)and tick-borne encephalitis virus(TBEV).When the RVFV Gn-D Ⅲ-Ⅲ posi-tive serum was diluted to 6 400 times,the test result still showed positive results,demonstrating the method had good sensitivity.The repeatability evaluation results indicated that the variation co-efficients for both intra-and inter-batch responses was less than 10％,indicating that the method had good repeatability.In conclusion,the RVFV Gn-D Ⅱ-Ⅲ protein was successfully expressed u-sing the E.coli expression system.The purified recombinant Gn-D Ⅱ-Ⅲ protein was used as the encapsulated antigen to develop an indirect ELISA assay for RVFV antibodies,which provides a preliminary basis for the diagnosis of RVF and the research and development of RVF vaccines.

To establish an indirect enzyme linked immunosorbent assay(ELISA)method for the detection of Zaire Ebola virus(ZEBOV)specific antibodies,the full-length of ZEBOV GP1 gene was amplified by PCR and cloned into pET-30a(+)vector to generate the pET-30a(+)-GP1 plasmid.After expressed in the E.coli expression system,the purified GP1 protein was used as coating antigen to establish the indirect ELISA method for detection of ZEBOV antibody.The con-ditions including concentration of coating antigen and serum dilution were determined by chess-board titration.Specificity,sensitivity,and reproducibility of the established ELISA detection meth-od were evaluated.GP1 protein was successfully prepared by prokaryotic expression,and was used as the coatingantigen for indirect ELISA.By optimizing the reaction conditions,the optimal concen-tration of the coating antigen was determined to be 0.5 g/L;the optimal dilution of serum was cal-culated to be 1∶3 200;the optimal dilution of enzyme-labeled secondary antibody was measured to be 1∶20 000.The established method exhibited excellent specificity,sensitivity,and reproducibili-ty.In the present study,the GP1 protein was successfully expressed in the E.coli expression sys-tem and the high purity GP1 protein was used as the coating protein to establish an indirect ELISA assay for ZEBOV antibody.This method is highly specific,sensitive,and reproducible,which provides technical support for the fur-ther study of the biological function of GP1 and the detection of ZEBOV antibody in serum.