This study was aimed at establishing a method of ultra-fast quantitative PCR for Brucella detection.We used an exogenous recombinant plasmid as the internal reference and targeted the T4SS secretion system,an important Brucella viru-lence factor,to design specific primers and probes.The sensitivity,specificity,and repeatability of this method were evaluated,and a standard curve was constructed.The coincidence rate of detection findings with this method versus quantitative PCR was determined.This method markedly decreased the detection time to only 10 minutes.The standard curve demonstrated a good linear relationship(Y=-3.410 7x+38.357,R2=0.998 5)with a low minimum detection limit of 10 copies/μL.The method exhibited good specificity and did not specifically amplify several common clinical bacteria other than Brucella.The de-tection of three concentrations of positive plasmids yielded coefficients of variation(CVs)of 0.20％to 0.91％,thus demonstra-ting the method's excellent repeatability.Furthermore,140 clinical samples were analyzed concurrently with the fluorescence PCR method,which yielded a 100％compliance rate and consistent results.Our findings indicated that the Brucella ultra-fast quantitative PCR was ultrafast;had high sensitivity,high specificity,and good specificity;and can be used for the clinical de-tection of Brucella and emergency investigation of epidemics.Therefore,this method is valuable for the early diagnosis of Bru-cella.

Objective To investigate the feasibility of breast-conserving surgery(BCS)after neoadjuvant chemotherapy(NC)for triple-negative breast cancer(TNBC).Methods One hundred and thirty-two TNBC patients who were admitted to The Second Affiliated Hospital of Nanchang University from Jun.2014 to Jun.2017 were divided into NC group(62 patients who received NC followed by BCS)and control group(70 patients who received only conventional standard BCS).The response rate was calculated for patients in the NC group.The general clinicopathological data of the 2 groups were compared.The disease-free survival(DFS)of the 2 groups was analyzed using the Kaplan-Meier method,and the effect of NC followed by BCS on the DFS was analyzed using Cox proportional hazards regression method.Results The response rate of patients in the NC group was 96.8%(60/62).There were no differences in age,histological grade,axillary lymph node status or Ki67 index between the 2 groups(all P＞0.05),and the maximum tumor diameter after NC in the NC group was similar to that of the control group(P＞0.05).The ratio of intraventricular cancerous thrombus invasion cases was higher in the NC group than in the control group(P＜0.01).The 1-,3-,and 5-year DFS rates in the NC group were 100%(62 cases),93.5%(58 cases),and 69.4%(43 cases),respectively,with a mean of 55.5 months;the 1-,3-,and 5-year DFS rates in the control group were 100%(70 cases),95.7%(67 cases),and 72.9%(51 cases),respectively,with a mean of 55.6 months;and there was no difference in DFS between the 2 groups(P＞0.05).Cox proportional hazards regression analysis showed that BCS after NC was not a risk factor for DFS in patients with TNBC(hazard ratio=1.133,95%confidence interval 0.600-2.139,P=0.701).Conclusion The response rate to NC is high in TNBC patients,and the treatment strategy of BCS after NC is feasible.

AIM To investigate the ameliorative effects of Compound Fufangteng Mixture(CFM)on cyclophosphamide(CTX)-induced immunosuppression in mice.METHODS Forty-eight male C57BL/6J mice were randomly divided into the blank control group,the model group,the levamisole hydrochloride group(40 mg/kg)and the low-dose,medium-dose and high-dose CFM groups(3.75,7.5,10 g/kg),with 8 mice in each group,and given respective intervention orally once daily for 14 days.On the 5th to 7th day of administration,with the blank control group given normal saline intraperitoneally,the other groups underwent intraperitoneal CTX injections(80 mg/kg).24 hours after the last administration,organ indices of thymus and spleen were calculated;splenic histopathological alterations were assessed by HE staining;serum levels of IL-2,IL-6 and IgG were quantified using ELISA;splenic CD4+,CD8+T lymphocytes,alongside CD86+and CD206+macrophages populations were analyzed by flow cytometry;and splenic expression of CD4,CD8 and F4/80 was evaluated by immunohistochemical staining.RESULTS In CTX-treated mice,CFM administration mitigated body weight loss;enhanced thymus weight and thymic index;ameliorated splenic immune cell populations,elevated serum levels of cytokines IL-2,IL-6 and IgG in serum;and upregulated splenic levels of CD45+CD3+T lymphocytes and F4/80+CD11b+macrophages,alongside increasing the expression of CD4,CD8 and F4/80 surface markers.CONCLUSION CFM alleviates CTX-induced immunosuppression state in mice by modulating immune cells,restoring immune function and enhancing anti-inflammatory and tissue repair capabilities.

Objective:To analyze the incidence and risk factors of malignant tumors among HIV-infected patients in Taizhou, Zhejiang Province.Methods:The data were collected from the China Information System for Disease Control and Prevention and the Taizhou Chronic Disease Information Management System. A retrospective cohort study design was used. The subjects were HIV-infected patients who had their household registration in Taizhou from 2005 to 2023 and participated in the follow-up. The observation period was until December 31, 2024. The standardized incidence ratios (SIR) of malignant tumors among HIV-infected patients were analyzed. Cox proportional hazards regression model was used to analyze the influencing factors of malignant tumor incidence.Results:A total of 3 593 HIV-infected patients were included, of whom 292 had malignant tumors. The proportions of AIDS-defining malignancies and non-AIDS-defining malignancies were 12.33% (36/292) and 87.67% (256/292), respectively. The proportion of malignant tumors before and after AIDS confirmation was 43.49% (127/292) and 56.51% (165/292), respectively. 3 466 HIV-infected patients were included in the follow-up cohort, with a total follow-up of 24 968.59 person-years. The incidence rate of malignant tumors in patients with HIV infection was 658.46 per 100 000 (SIR=1.89, 95% CI: 1.61-2.20). The SIR of malignant tumors showed an upward trend with the increase of time. The results of Cox proportional hazards regression model analysis showed that HIV-infected patients in the age groups of 45-59 and ≥60 years (a HR=2.58, 95% CI: 1.26-5.28; a HR=5.00, 95% CI: 2.38-10.51) were more likely to develop malignant tumors. HIV-infected patients with an educational level of senior high school or above (a HR=0.52, 95% CI: 0.29-0.95) and those in the first CD4 +T lymphocyte/CD8 +T lymphocyte count ratio ≥0.5 (a HR=0.52, 95% CI: 0.28-0.97) were less likely to develop malignant tumors. Conclusions:From 2005 to 2023, the incidence of malignant tumors among HIV-infected people in Taizhou was higher than that of the general population, and most of them were non-AIDS-defining malignancies. It is necessary to strengthen the early screening and diagnosis of malignant tumors among HIV-infected patients.

Objective:To understand the related factors of new-type drugs use and high-risk behaviors in online-dating men who have sex with men (MSM) in Shandong Province, and provide evidence for the development of targeted HIV prevention and intervention strategies.Methods:A cross-sectional study was conducted from April to July 2023 in eight sentinel surveillance sites across cities in Shandong, recruiting MSM participants. Each city enrolled a sample of 400 individuals. Face-to-face questionnaire surveys were conducted to collect data on socio-demographic characteristics, drug use and sexual behaviors, risk perception, and online-dating practices of the MSM, and χ2 test was used to compare the differences between online-dating MSM who used new-type drugs and those who didn't use. Logistic regression model was used to analyze the related factors of group sex behavior and the lack of HIV detection before sexual behavior in the online-dating MSM. Statistical analysis was performed by using software SPSS 29.0. Results:A total of 3 235 MSM were surveyed, in whom 2 787 (86.15%) used online-dating platforms to find partners in the past six months. The majority were those who were aged 25-34 years (39.18%, 1 092/2 787), unmarried/divorced/widowed (71.51%, 1 993/2 787), had an education level of college or above (69.36%, 1 933/2 787), and primarily identified as homosexual gays (77.97%, 2 173/2 787), 37.78% (1 053/2 787) believed that they were at low risk for HIV infection and 10.88% (303/2 786) believed that using new-type drugs was less harmful and 57.59% (1 605/2 787) reported new-type drugs use. Multivariate logistic regression analysis indicated that MSM who used new-type drugs had higher risk for group sex in the past six months (a OR=1.31, 95% CI: 1.06-1.63) and sexual activity without pre-HIV testing (a OR=1.57, 95% CI:1.14-2.15) compared with those who didn't use new-type drugs.MSM who believed that the risk of using new-type drugs was general, higher and very high had higher risks for group sex in the past six months (a OR=1.54, 95% CI: 1.09-2.18) and sexual activity withou pre-HIV testing (a OR=2.02, 95% CI:1.34-3.05) compared with those who believed using new-type drugs was less harmful. Conclusions:The use of new-type drugs was relatively common in the online-dating MSM in Shandong, with widespread high-risk behaviors and poor awareness of risks to health. A gap between knowledge awareness and behavior exists. It is necessary to strengthen the health education and promotion of HIV informed dating practices in MSM.

This study aims to integrate data mining techniques of single cell transcriptomics and spatial transcriptomics, along with animal experiment validation, so as to systematically characterize the protective effects of Jianpi Tongluo Formula(JTF) on the cartilage in knee osteoarthritis(KOA) and elucidate the underlying molecular mechanisms. Single cell transcriptomics and spatial transcriptomics datasets(GSE254844 and GSE255460) of the cartilage tissue obtained from KOA patients were analyzed to map the single cell-spatial heterogeneity and identify key pathogenic factors. After that, a KOA rat model was established via knee joint injection of papain. The intervention effects of JTF on the expression features of these key factors were assessed through real-time quantitative polymerase chain reaction(PCR), Western blot, and immunohistochemical staining. As a result, the integrated single cell and spatial transcriptomics data identified distinct cell subsets with different pathological changes in different regions of the inflamed cartilage tissue in KOA, and their differentiation trajectories were closely related to the inflammatory fibrosis-like pathological changes of chondrocytes. Accordingly, the expression levels of the two key effect targets, namely nuclear receptor coactivator 4(NCOA4) and high mobility group box 1(HMGB1) were significantly reduced in the articular surface and superficial zone of the inflamed joints when JTF effectively alleviated various pathological changes in KOA rats, thus reversing the abnormal chondrocyte autophagy level, relieving the inflammatory responses and fibrosis-like pathological changes, and promoting the repair of chondrocyte function. Collectively, this study revealed the heterogeneous characteristics and dynamic changes of inflamed cartilage tissue in different regions and different cell subsets in KOA patients. It is worth noting that NCOA4 and HMGB1 were crucial in regulating chondrocyte autophagy and inflammatory reaction, while JTF could reverse the regulation of NCOA4 and HMGB1 and correct the abnormal molecular signal axis in the target cells of the inflamed joints. The research can provide a new research idea and scientific basis for developing a personalized therapeutic schedule targeting the spatiotemporal heterogeneity characteristics of KOA.
Animals ; Drugs, Chinese Herbal/administration & dosage* ; Rats ; Osteoarthritis, Knee/pathology* ; Humans ; Male ; Cartilage, Articular/metabolism* ; Chondrocytes/metabolism* ; Rats, Sprague-Dawley ; Female ; Protective Agents/administration & dosage* ; Single-Cell Analysis ; Middle Aged ; HMGB1 Protein/metabolism*

A combination of the "disease-syndrome-symptom" approach was used to study the syndrome characterization and features of dampness-heat obstruction syndrome in papain-induced knee osteoarthritis(KOA) model rats during the disease process. Forty-eight male SD rats were randomly divided into sham and model groups. The KOA model was established by injecting a mixture of papain and L-cysteine into the joint cavity on days 1, 3, and 5. During the 8 weeks following model establishment, the rats were assessed weekly for the plantar mechanical pain threshold, knee joint diameter, local skin temperature of the knee joint, weight-bearing difference between the two hind feet, and the modified Lequesne MG score of the knee joint. Samples were collected at 1, 2, 4, 6, and 8 weeks after model establishment to observe the gross lesions in cartilage and synovium. Histopathological changes in joint tissues were examined using hematoxylin-eosin, Masson's trichrome, and Senna red O-solid green staining. ELISA and immunohistochemical analysis were performed to detect the levels of interleukin(IL)-1β, IL-6, tumor necrosis factor(TNF)-α, prostaglandin E2(PGE2), and the expression of aquaporins(AQP) 1 and 3 in serum and synovium. The results showed that the ink score of articular cartilage in the model group significantly increased from 4 to 8 weeks, the cartilage Mankin's score and the percentage of Masson-positive area in cartilage increased significantly from 1 to 8 weeks. The percentage of red-stained area for cartilage proteoglycans decreased significantly from 1 to 8 weeks. The synovitis score from 1 to 6 weeks and the percentage of blue-stained collagen fibers in the synovium from 1 to 8 weeks increased significantly, with statistically significant differences compared to the sham group. The mechanical pain threshold in the model group significantly decreased from 1 to 8 weeks, the knee joint diameter significantly increased from 1 to 6 weeks, and the local skin temperature of the knee joint, the weight-bearing difference between the two hind feet, and the modified Lequesne MG score from 1 to 5 weeks significantly increased, all with statistically significant differences compared to the sham group. The levels of IL-1β, IL-6, TNF-α, and PGE2 in serum and synovium of the model group significantly increased from 1 to 6 weeks. Serum TNF-α and PGE2, and synovial IL-1β, also significantly increased at 8 weeks. The levels of cartilage AQP1 and AQP3 significantly increased from 1 to 4 weeks, while synovial AQP1 and AQP3 increased significantly from 1 to 6 weeks, with all differences statistically significant compared to the sham group. In conclusion, papain-induced KOA rats exhibited pathological changes, including articular cartilage degeneration and synovial inflammation, within 1 week of induction. The KOA rats showed characteristics of dampness-heat obstruction syndrome, such as joint pain, swelling, elevated skin temperature, and decreased function, as well as increased inflammatory factors and AQP1、AQP3 in serum and joint tissues within 5 to 6 weeks of disease onset. These results provide an experimental model for studying the syndromes of KOA with dampness-heat obstruction syndrome.
Animals ; Male ; Rats, Sprague-Dawley ; Rats ; Osteoarthritis, Knee/physiopathology* ; Disease Models, Animal ; Humans ; Interleukin-1beta/metabolism* ; Interleukin-6/metabolism* ; Tumor Necrosis Factor-alpha/metabolism* ; Knee Joint/pathology*

This study aims to systematically characterize the targeted effects of Quanduzhong Capsules on cartilage lesions in knee osteoarthritis by integrating spatial transcriptomics data mining and animal experiments validation, thereby elucidating the related molecular mechanisms. A knee osteoarthritis model was established using Sprague-Dawley(SD) rats, via a modified Hulth method. Hematoxylin and eosin(HE) staining was employed to detect knee osteoarthritis-associated pathological changes in knee cartilage. Candidate targets of Quanduzhong Capsules were collected from the HIT 2.0 database, followed by bioinformatics analysis of spatial transcriptomics datasets(GSE254844) from cartilage tissues in clinical knee osteoarthritis patients to identify spatially specific disease genes. Furthermore, a "formula candidate targets-spatially specific genes in cartilage lesions" interaction network was constructed to explore the effects and major mechanisms of Quanduzhong Capsules in distinct cartilage regions. Experimental validation was conducted through immunohistochemistry using animal-derived biospecimens. The results indicated that Quanduzhong Capsules effectively inhibited the degenerative changes in the cartilage of affected joints in rats, which was associated with the regulation of Quanduzhong Capsules on the thioredoxin-interacting protein(TXNIP)-NOD-like receptor family pyrin domain containing 3(NLRP3)-bone morphogenetic protein receptor type 2(BMPR2)-fibronectin 1(FN1)-matrix metallopeptidase 2(MMP2) signal axis in the articular cartilage surface and superficial zones, subsequently inhibiting cartilage matrix degradation leading to oxidative stress and inflammatory diffusion. In summary, this study clarifies the spatially specific targeted effects and protective mechanisms of Quanduzhong Capsules within pathological cartilage regions in knee osteoarthritis, providing theoretical and experimental support for the clinical application of this drug in the targeted therapy on the inflamed cartilage.
Animals ; Osteoarthritis, Knee/metabolism* ; Drugs, Chinese Herbal/administration & dosage* ; Rats, Sprague-Dawley ; Rats ; Male ; Humans ; Capsules ; Female ; Disease Models, Animal

Objective To investigate the effects of scutellarin(SCU)on apoptosis of nucleus pulposus cells and the Janus kinas 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway in intervertebral disc degeneration(IVDD)model rats.Methods IVDD rats were randomly divided into IVDD group,SCU low and high dose groups(SCU-L,SCU-H groups),SCU high dose+JAK2 activator bispecific protein phosphatase 19(DUSP19)group(SCU-H+DUSP19 group)and control group.Enzyme-linked immunosorbent assay(ELISA)method was applied to detect inflammatory factors and oxidative stress level in intervertebral disc tissues.Eosin stai-ning(HE)staining microscopy was used to observe pathological damage of intervertebral disc tissue.Transferase mediated dUTP notch end labeling(TUNEL)staining microscopy was applied to detect apoptosis of nucleus pulpo-sus cells in intervertebral disc tissue.Western blot was applied to detect the expression of JAK2/STAT3 signaling pathway related proteins.Results Animals in the IVDD group were suffered from partial rupture of the fibrous ring in the intervertebral disc tissue,abnormal shrinkage of the nucleus pulposus structure with reduction of nucleus pul-posus cells and blurred boundary with the fibrous ring.The level of TNF-α,IL-1β,ROS and apoptosis rate of nu-cleus pulposus cells,the expression of Bax,cleaved caspase-3,p-JAK2/JAK2,p-STAT3/STAT3 were all elevated and the expression of SOD,type Ⅱ collagen,and polysaccharides decreased(P＜0.05).The morphology,cell structure,density of fibrous ring and nucleus pulposus in the intervertebral disc tissue of the SCU-L and SCU-H groups were improved as compared to IVDD group.The degeneration was alleviated to varying degrees.The level of TNF-α,IL-1β,ROS,apoptosis rate of nucleus pulposus cells and the expression of Bax,cleaved caspase-3,p-JAK2/JAK2,p-STAT3/STAT3 were all reduced and the expression of SOD,type Ⅱ collagen,and polysaccha-rides significantly increased(P＜0.05).DUSP19 partially reverses the effect of SCU on apoptosis of IVDD rats.Conclusions SCU may alleviate apoptosis of nucleus pulposus cells in IVDD rats.

This research investigated the contamination level,distribution of drug-resistant strains,and molecular epidemiologi-cal characteristics of Campylobacter,and further explored transmission pathways and prevention strategies.Cecum,chicken carcass,chicken product,and environmental samples,as well as swabs from workers'hands,were collected from a slaughterhouse in a large broiler group in the Jiaodong area between August 2023 and July 2024.Quantitative contamination assessment of Campylobacter in chicken carcasses and chicken products was performed.After microbial mass spectrometry identification,the representative strains of different links were selected for drug resistance testing and whole genome sequencing(WGS).On the basis of the sequencing results,the resistance genes,virulence genes,multilocus sequence typing(MLST),and phylogenetic characteristics of representative strains were analyzed.Homology comparisons were performed between isolates and strains from patients with diarrhea in the NCBI database.A total of 297 Campylobacter strains were isolated from 806 samples,and the overall detection rate was 36.85%.The detection rate of Campylobacter was highest in the evisceration process(47.33%),followed by the cutting process(35.64%).Overall,the Campylo-bacter detection rate first increased,then decreased,and subsequently increased.Drug sensitivity testing revealed that 90 isolates were resistant to nalidixic acid and ciprofloxacin,and 94.97%of isolates were resistant to tetracycline.WGS showed that both Campylo-bacter jejuni(C.jejuni)and Campylobacter coli(C.coli)carried many drug resistance and virulence genes.ST-14176 of C.jejuni was isolated for the first time herein.The predominant ST-8261 strain of C.jejuni and ST-860,ST-829,and ST-1586 strains of C.coli are known to cause human diarrhea.LOS expression genes associated with Guillain-Barré syndrome(GBS)were detected in both C.jejuni isolates from the slaughter chain and patients with GBS.Some strains exhibited close genetic relatedness to human-derived Campylo-bacter strains from the NCBI database.The detection rate of Campylobacter in the slaughterhouse first increased,then decreased,and subsequently increased,and the quantitative contamination level of each link was similar to the detection rate.Quantitative analysis of chicken carcasses/products revealed that the average bacterial load was highest in eviscerated carcasses(102.80 cfu/g),and the high-est amount of Campylobacter in chicken products reached 451.80 cfu/g.Abundant drug resistance genes and virulence genes were iden-tified,and the drug resistance genes were highly correlated with the drug resistance rate.Therefore,surveillance intensity and control measures for Campylobacter in slaughter processes should be strengthened.