Aim To investigate the effect of N6-methy-ladenosine(m6A)demethylase ALKBH5 on the prolif-eration and migration of cardiac fibroblasts(CFs)in-duced by high glucose.Methods Primary CFs were isolated from neonatal mouse hearts and identified u-sing optical and confocal microscopy.Cell activation was induced using a high-glucose medium(33 mmol·L-1 glucose).An ALKBH5 overexpression model was established by transfecting CFs with an ALKBH5 ex-pression vector in a high-glucose medium.The expres-sion of ALKBH5 in CFs was assessed through immuno-fluorescence staining,Western blot and RT-qPCR.Changes in m6A levels were evaluated using Dot blot a-nalysis.Additionally,Alterations in the expression of proliferating cell nuclear antigen(PCNA)and collagenⅠ,a pivotal fibrosis indicator,were measured using Western blot.The proliferation and migration ability of CFs were assessed through EdU staining and Transwell migration assay,respectively.Results Following treatment with high glucose,the expression of ALKBH5 in CFs notably decreased,while m6A level increased.This was accompanied by a significant increase in the expression of the proliferation marker PCNA and the fi-brosis marker collagen Ⅰ.Additionally,there was a sig-nificant improvement in the ability of proliferation and migration.Overexpression of ALKBH5 resulted in a significant decrease in the expressions of PCNA and collagen Ⅰ,leading to the inhibition of both proliferation and migration in CFs.Conclusion Overexpression of ALKBH5 suppresses the expression of PCNA and colla-gen Ⅰ,consequently reducing the proliferation and mi-gration of CFs,potentially through m6A methylation modification.

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.

Aim To investigate the effect of N6-methy-ladenosine(m6A)demethylase ALKBH5 on the prolif-eration and migration of cardiac fibroblasts(CFs)in-duced by high glucose.Methods Primary CFs were isolated from neonatal mouse hearts and identified u-sing optical and confocal microscopy.Cell activation was induced using a high-glucose medium(33 mmol·L-1 glucose).An ALKBH5 overexpression model was established by transfecting CFs with an ALKBH5 ex-pression vector in a high-glucose medium.The expres-sion of ALKBH5 in CFs was assessed through immuno-fluorescence staining,Western blot and RT-qPCR.Changes in m6A levels were evaluated using Dot blot a-nalysis.Additionally,Alterations in the expression of proliferating cell nuclear antigen(PCNA)and collagenⅠ,a pivotal fibrosis indicator,were measured using Western blot.The proliferation and migration ability of CFs were assessed through EdU staining and Transwell migration assay,respectively.Results Following treatment with high glucose,the expression of ALKBH5 in CFs notably decreased,while m6A level increased.This was accompanied by a significant increase in the expression of the proliferation marker PCNA and the fi-brosis marker collagen Ⅰ.Additionally,there was a sig-nificant improvement in the ability of proliferation and migration.Overexpression of ALKBH5 resulted in a significant decrease in the expressions of PCNA and collagen Ⅰ,leading to the inhibition of both proliferation and migration in CFs.Conclusion Overexpression of ALKBH5 suppresses the expression of PCNA and colla-gen Ⅰ,consequently reducing the proliferation and mi-gration of CFs,potentially through m6A methylation modification.

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 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.

OBJECTIVE: To explore and verify the effect and potential mechanism of Brucea javanica Seed Oil Emulsion Injection (YDZI) and Shengmai Injection (SMI) on peripheral microcirculation dysfunction in treatment of gastric cancer (GC). METHODS: The potential mechanisms of YDZI and SMI were explored through network pharmacology and verified by cellular and clinical experiments. Human microvascular endothelial cells (HMECs) were cultured for quantitative real-time polymerase chain reaction, Western blot analysis, and human umbilical vein endothelial cells (HUVECs) were cultured for tube formation assay. Twenty healthy volunteers and 97 patients with GC were enrolled. Patients were divided into surgical resection, surgical resection with chemotherapy, and surgical resection with chemotherapy combining YDZI and SMI groups. Forearm skin blood perfusion was measured and recorded by laser speckle contrast imaging coupled with post-occlusive reactive hyperemia. Cutaneous vascular conductance and microvascular reactivity parameters were calculated and compared across the groups. RESULTS: After network pharmacology analysis, 4 ingredients, 82 active compounds, and 92 related genes in YDZI and SMI were screened out. β-Sitosterol, an active ingredient and intersection compound of YDZI and SMI, upregulated the expression of vascular endothelial growth factor A (VEGFA) and prostaglandin-endoperoxide synthase 2 (PTGS2, P<0.01), downregulated the expression of caspase 9 (CASP9) and estrogen receptor 1 (ESR1, P<0.01) in HMECs under oxaliplatin stimulation, and promoted tube formation through VEGFA. Chemotherapy significantly impaired the microvascular reactivity in GC patients, whereas YDZI and SMI ameliorated this injury (P<0.05 or P<0.01). CONCLUSIONS YDZI and SMI ameliorated peripheral microvascular reactivity in GC patients. β-Sitosterol may improve peripheral microcirculation by regulating VEGFA, PTGS2, ESR1, and CASP9.
Humans ; Microcirculation/drug effects* ; Drugs, Chinese Herbal/administration & dosage* ; Stomach Neoplasms/physiopathology* ; Emulsions ; Male ; Plant Oils/administration & dosage* ; Brucea/chemistry* ; Middle Aged ; Female ; Drug Combinations ; Human Umbilical Vein Endothelial Cells/metabolism* ; Seeds/chemistry* ; Injections ; Vascular Endothelial Growth Factor A/metabolism* ; Aged ; Network Pharmacology

This study systematically analyzed the global research landscape, technological composition, and core patents in the field of networks target and network pharmacology, and proposes further suggestions based on the IncoPat patent citation database and VOSviewer bibliometric network visualization tool. Using patent literature metrics and scientific knowledge mapping method, technological innovation pathways, research hotspots, and future directions in this field were further revealed. In particular, this field is moving towards data-driven, intelligent, and systematic approaches. Patent analysis indicated that most patent applications in this domain focused on traditional Chinese medicine(TCM), which have provided key engineering technical approaches to explore and solve complex problems of TCM. By integrating big data and artificial intelligence technologies, network targets and network pharmacology have conferred high-precision screening and quality control of key components and targets in herbal formulations and prescriptions, accelerating the clinical translation and industrialization of TCM-based new drugs and health products with medicine-food homology. Therefore, it is essential to optimize the patent protection system and establish integrated technology platforms in this field for ensuring the competitiveness of technological achievements in research and clinical application. These efforts will advance the widespread application and high-quality development of TCM modernization, precision medicine, and innovative drug discovery.
Bibliometrics ; Patents as Topic ; Humans ; Medicine, Chinese Traditional ; Network Pharmacology/trends* ; Drugs, Chinese Herbal/pharmacology*

Objective To evaluate the management and indicators of pathogen detection before antimicrobial treat-ment for inpatients in second level and above medical institutions(MIs)in Guangdong Province,and provide direc-tion and decision-making basis for the improvement of pathogen detection quality in the region.Methods The ma-nagement status,information system functions,and pathogen detection rate indicators of secondary and above MIs in 21 cities in Guangdong Province was surveyed through online questionnaire surveys and system submission.A baseline survey on sentinel monitoring MIs was conducted from July 15th to August 8th,2023.From November 7th to 30th,a baseline survey on non-sentinel monitoring MIs was launched.Surveys on indicator information of all MIs were completed from January 15th to 30th,2024.Results A total of 265 MIs were surveyed,and the proportions of establishing special working groups(83.98％),developing special action improvement plans(79.01％),estab-lishing pathogen detection rate management systems(91.71％),and developing management assessment plans(76.80％)of tertiary MIs were all higher than that of secondary MIs,differences were all statistically significant(all P＜0.05).The proportion of tertiary MIs with various information system functions was higher than that of secondary MIs(all P＜0.05).The pathogen detection rate(61.07％)before antimicrobial treatment and health-care-associated infection(HAI)diagnosis-related pathogen detection rate(88.00％)of inpatients in tertiary MIs were both higher than those in secondary MIs(both P＜0.05).Among different types of MIs,pathogen detection rate before antimicrobial treatment of inpatients in maternal and child health MIs was higher than that in other types of MIs.HAI diagnosis-related pathogen detection rate in other specialized hospitals was the highest,and pathogen detection rate before combined use of key antimicrobial treatment in traditional Chinese medicine hospitals was the lowest,differences were all statistically significant(all P＜0.05).Conclusion Tertiary MIs have more advantages in management strategies and information technology construction than secondary MIs,secondary MIs need more guidance and support.Monitoring and analysis of pathogen detection rate indicators in MIs of different levels and types should be strengthened through special actions.

Objective:To investigate the clinical efficacy and safety of Pseudomonas aeruginosa injection in preventing reurrent urinary tract infection in women. Methods:This was a multicenter, randomized, open, positive-controlled, non-inferiority trial involving female patients with recurrent urinary tract infections (rUTIs) who were admitted to 11 medical centers in China. Inclusion criteria: ①Aged 18-70 years, with verifiable clinical data showing at least 3 episodes of acute UTIs within 1 year and at least 2 episodes within 6 months, and cured by antimicrobial therapy; ② At the time of enrollment, the patients had no obvious symptoms of urinary tract irritation, normal white blood cell count in midstream urine routine (within the normal range of laboratory standards of each unit) or ≤3HP by centrifuge microscopy, negative leucocyte esterase and nitrite, and negative urine culture; ③No abnormal urinary anatomic function (such as urinary obstruction, calculus or congenital urinary malformation) and residual urine volume ≤50 ml were detected by B-ultrasound of urinary system; ④Informed consent signed by the person or agent; ⑤Clear consciousness, able to answer questions independently, according to the requirements of the test plan to complete the research questionnaire. Exclusion criteria: ①Patients allergic to the above drugs; ②Any complex signs of urinary tract infection or pyelonephritis (manifested as low back pain, fever ≥37.3℃, systemic symptoms); ③Drugs affecting immune function were used within 7 days before randomization; ④Patients with basic diseases of urinary system such as obstruction, calculus, urinary stenosis, vesicoureteral reflux or other functional abnormalities, urine diversion, indwelling catheter or stent tube or intermittent catheterization; ⑤Combined with or existing systemic lupus erythematosus, AIDS and other diseases that can lead to systemic immune function abnormalities; ⑥Patients who are known or suspected to be pregnant, breastfeeding, or planning a pregnancy within 3 months of stopping the drug; ⑦Patients with malignant tumors and mental patients; ⑧Persons who have received any other investigational drug treatment or participated in another interventional clinical trial within 4 weeks prior to screening; ⑨Failure to comply with the trial protocol or other conditions deemed unsuitable for enrollment by the investigator. Patients were randomly divided into 2 groups. The experimental group was given Pseudomonas aeruginosa injection for 5 times, 0.5 ml for the first time, and 1 ml/ time per week for the following 4 weeks. The control group was given fosfomycin aminotriol 3g orally, once every 10 days, for 9 consecutive times. The patients were followed up for 6 to 8 months, during which urinary tract symptoms developed and routine urine tests showed abnormally elevated white blood cells, which was defined as recurrent UTIs. Urine routine, liver and kidney function, and urinary secretory immunoglobulin A(SIgA) were reviewed 0-2 days (V2) after the 5th administration of the experimental group and the 4th administration of the control group. Urine routine and urine SIgA were reviewed at (90±10) d (V3) and (180±10) d (V4) after treatment. At (270±10) d (V5) after treatment, the recurrence (re-infection caused by the same species of bacteria) or re-infection (re-infection caused by non-same species of bacteria) of the two groups were compared, and non-inferiority analysis was performed, and the non-inferiority threshold was set at 0.2. Results:From March 2021 to May 2022, a total of 152 rUTIs patients were enrolled in this study, including 80 patients in the experimental group, 71 patients in the intention-to-analysis set (ITT) and 66 patients in the protocol analysis set (PPS). In the control group, 72 cases met ITT in 69 cases and PPS in 67 cases. There were no significant differences in age, body mass index, marital status, duration of urinary tract infection, history of diabetes, history of previous major surgery, history of infection, and urinary SIgA between the two groups (all P>0.05). The recurrence rates of the experimental group and the control group at V5 time point were 44.78% (30/67) and 42.65% (29/68), respectively ( P=0.803) (ITT data set analysis results showed that the difference in recurrence rates between the two groups was 0.0213(95% CI-0.1460-0.1886, P=0.0048). PPS data set analysis showed that the difference of recurrence rate between the two groups was -0.0021(95%CI -0.1711-0.1670, P=0.0109), and the recurrence rate of the experimental group was not worse than that of the control group. At V2 time points, there were no significant differences in liver and kidney function indexes between test group and control group ( P>0.05). At V2 to V4 time points, urinary SIgA of test group and control group were 0.90 (0.37, 2.89) mg/L and 1.32 (0.34, 3.08) mg/L, 1.54 (0.44, 3.23) mg/L and 1.71 (0.27, 2.92) mg/L, 1.11 (0.65, 3.42) mg/L and 2.18 (0.43, 3.26) mg/L, there was no statistical significance ( P>0.05). The incidence of adverse events in the experimental group was 30.0% (24/80), including 14 cases of redness, pain and discomfort at the injection site, 5 cases of fever, 2 cases of allergic rash, and 1 case of urticaria, headache and constipation each. The incidence of adverse events in the control group was 5.6% (4/72), all of which were diarrhea, and the difference between the two groups was statistically significant ( P<0.01). No life-threatening serious adverse events occurred in both groups, and all adverse events were self-healing without additional intervention. Conclusions:Compared with fosfomycin aminotriol, Pseudomonas aeruginosa injection has the same clinical effect in preventing rUTI and has good safety.

Currently, clinically used drugs for the treatment of gout inflammation, such as colchicine, nonsteroidal anti-inflammatory drugs, and glucocorticoids, can only relieve the pain of joint inflammation and have severe hepatorenal toxicity and multiple organ adverse reactions. The NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome is a key complex that induces the onset of gout inflammation and has become a crucial target in the development of anti-gout drugs. This article reviews the research progress of anti-gout small molecules targeting the NLRP3 inflammasome and their bioactivity evaluation methods in the past five years, in order to provide information for the development of specific drugs for the treatment of gout inflammation.