Staphylococcus aureus has the ability to invade bovine mammary epithelial cells and cause persistent intracellular infections.For diagnosing mastitis in cows caused by the bacterial in-fections,it is necessary to determine whether there is a persistent intracellular infection by S.au-reus within mammary epithelial cells,and to establish scientific treatment strategies and imple-ment proactive management decisions.The content of this study is the establishment of a brand new diagnostic method aimed at confirming this type of bovine disease efficiently and cost-effec-tively.The diagnostic procedure is as follows:Add 100 μL S.aureus liquid culture with a concentra-tion of 8.5 ×1010 CFU/mL to the well-grown bovine mammary epithelial cell line and co-culture at 37 degrees Celsius for 2 h to establish an intracellular infection model;After the co-cultivation is completed,add 100 μL of lysostaphin to the dish to thoroughly eliminate extracellular S.aureus.It can be confirmed that the model has been successfully established that black spherical bacteria with a diameter of approximately 1μm are observed within the sample cells using a transmission e-lectron microscope.Intracellular infection is evidenced by the observation of DNA-staining positive material within the cytoplasm of DAPI-stained sample cells,as observed through a laser confocal microscope.The sample is subjected to gram staining after being treated with 200 μL of 0.5％Tri-tonX-100 for 15 min,and intracellular infection is observed within the cytoplasm of the sample cells with Gram-positive results under oil immersion microscopy.The results of the novel"Tri-tonX-100 & gram staining method"validate that the presence of Staphylococcus aureus individu-als within bovine mammary epithelial cells is consistent with images observed under transmission electron microscopy and laser confocal microscopy.The verification results indicate that the"Tri-tonX-100 & gram staining method"is suitable for the diagnosis of S.aureus persistent intracellu-lar infection in bovine mastitis.Later,somatic cells from milk were extracted by gradient centrifu-gation and purified to obtain bovine mammary epithelial cells for clinical testing of 70 S.aureus positive mastitis cattle,which showed a positive rate of 82.9％and a negative rate of 17.1％.

Objective To investigae microbial community structure of lower respiratory tract in severe hospital-acquired pneumonia(SHAP)patients in intensive care unit(ICU)using metagenomic next-generation sequencing(mNGS).Methods mNGS was performed on bronchoalveolar lavage fluid(BALF)of 84 patients with SHAP.Patients were grouped based on age,smoking status,underlying diseases and duration of artificial airway.The differences in α diversity,β diversity,microbial composition and community structure of airway microbiota were compared between different groups.The differential airway microbiota associated with artificial airway were screened,and microbial co-occurrence networks was constructed to observe the interaction in microorganisms.Results Results of α diversity analysis revealed that diversity and evenness of the microbial community were higher in young adults compared to those of middle-aged patients,while microbial diversity and evenness were significantly reduced in patients with comorbid stroke.In the group of artificial airway treatment,the diversity and uniformity of microorganisms decreased as the duration of artificial airway treatment increased.The diversity was the lowest when the artificial airway treatment lasted for more than 3 days.β diversity analysis confirmed that there were significant differences in the distinct microbial community structures between the>3 days support group and the non-intubated and≤3 days support cohorts.Acinetobacter baumannii was dominant in all groups.The bacterial diversity was significantly higher in the middle-aged group,the non-smoking group,the group without artificial airway therapy,the group without diabetes mellitus,the group with artificial airway therapy≥3 days and the group with chronic lung disease than those of other groups.In particular,pseudomonas aeruginosa,corynebacterium striatum and veillonella parvula were enriched in these groups.Difference analysis showed that there were significant differences in pseudomonas aeruginosa and corynebacterium striatum between the group with artificial airway treatment>3 days,the group without artificial airway therapy and the group with artificial airway treatment≤3 days.Network co-occurrence showed that there may be synergistic or antagonistic relationships between some microorganisms.Conclusion The microbial diversity of the lower respiratory tract in patients with SHAP significantly decreases in the elderly,those with concurrent stroke and those receiving artificial airway treatment.For these groups,rational use of antibiotics should be adopted to guide precise anti-infection treatment.

Objective To investigae microbial community structure of lower respiratory tract in severe hospital-acquired pneumonia(SHAP)patients in intensive care unit(ICU)using metagenomic next-generation sequencing(mNGS).Methods mNGS was performed on bronchoalveolar lavage fluid(BALF)of 84 patients with SHAP.Patients were grouped based on age,smoking status,underlying diseases and duration of artificial airway.The differences in α diversity,β diversity,microbial composition and community structure of airway microbiota were compared between different groups.The differential airway microbiota associated with artificial airway were screened,and microbial co-occurrence networks was constructed to observe the interaction in microorganisms.Results Results of α diversity analysis revealed that diversity and evenness of the microbial community were higher in young adults compared to those of middle-aged patients,while microbial diversity and evenness were significantly reduced in patients with comorbid stroke.In the group of artificial airway treatment,the diversity and uniformity of microorganisms decreased as the duration of artificial airway treatment increased.The diversity was the lowest when the artificial airway treatment lasted for more than 3 days.β diversity analysis confirmed that there were significant differences in the distinct microbial community structures between the>3 days support group and the non-intubated and≤3 days support cohorts.Acinetobacter baumannii was dominant in all groups.The bacterial diversity was significantly higher in the middle-aged group,the non-smoking group,the group without artificial airway therapy,the group without diabetes mellitus,the group with artificial airway therapy≥3 days and the group with chronic lung disease than those of other groups.In particular,pseudomonas aeruginosa,corynebacterium striatum and veillonella parvula were enriched in these groups.Difference analysis showed that there were significant differences in pseudomonas aeruginosa and corynebacterium striatum between the group with artificial airway treatment>3 days,the group without artificial airway therapy and the group with artificial airway treatment≤3 days.Network co-occurrence showed that there may be synergistic or antagonistic relationships between some microorganisms.Conclusion The microbial diversity of the lower respiratory tract in patients with SHAP significantly decreases in the elderly,those with concurrent stroke and those receiving artificial airway treatment.For these groups,rational use of antibiotics should be adopted to guide precise anti-infection treatment.

OBJECTIVE To investigate the age-group differences and seasonal distribution patterns of epidemiologi-cal characteristics of hospital-associated infection in elderly inpatients in a three-A hospital.METHODS Based on the Xinglin Real-time Monitoring System,a retrospective analysis was conducted on clinical data of 67 157 patients hospitalized in the Air Force Hospital of Eastern Theater Command from Jan.2020 to Dec.2024.Elderly patients were divided into three age groups:young-age group(60-69 years),middle-age group(70-79 years)and ol-dest-age(≥80 years).The epidemiological characteristics and seasonal distribution patterns of the three groups were analyzed.RESULTS From 2020 to 2024,there were 1 524 cases of hospital-associated infection,with an inci-dence rate of 2.27％.The infection rate was higher in the oldest-age group(6.08％)than those in the young-age group(1.77％)and the middle-age group(2.52％)(P＜0.001).The top three departments with the highest in-cidence rates of hospital-associated infection were the geriatric department(10.97％),intensive care unit(9.65％)and respiratory department(4.96％).The lower respiratory tract was the main site of infection,and the infection rate was higher in winter than in other seasons.The pathogens were predominantly gram-negative bacteria(60.35％),with Pseudomonas aeruginosa being the most frequently detected.CONCLUSIONS Advanced age,male gender and hospitalization in winter are key risk factors for hospital-associated infection.Seasonal pre-vention and control strategies should be implemented in departments with high incidence of respiratory infections,and monitoring of multidrug-resistant bacteria should be enhanced.

Staphylococcus aureus has the ability to invade bovine mammary epithelial cells and cause persistent intracellular infections.For diagnosing mastitis in cows caused by the bacterial in-fections,it is necessary to determine whether there is a persistent intracellular infection by S.au-reus within mammary epithelial cells,and to establish scientific treatment strategies and imple-ment proactive management decisions.The content of this study is the establishment of a brand new diagnostic method aimed at confirming this type of bovine disease efficiently and cost-effec-tively.The diagnostic procedure is as follows:Add 100 μL S.aureus liquid culture with a concentra-tion of 8.5 ×1010 CFU/mL to the well-grown bovine mammary epithelial cell line and co-culture at 37 degrees Celsius for 2 h to establish an intracellular infection model;After the co-cultivation is completed,add 100 μL of lysostaphin to the dish to thoroughly eliminate extracellular S.aureus.It can be confirmed that the model has been successfully established that black spherical bacteria with a diameter of approximately 1μm are observed within the sample cells using a transmission e-lectron microscope.Intracellular infection is evidenced by the observation of DNA-staining positive material within the cytoplasm of DAPI-stained sample cells,as observed through a laser confocal microscope.The sample is subjected to gram staining after being treated with 200 μL of 0.5％Tri-tonX-100 for 15 min,and intracellular infection is observed within the cytoplasm of the sample cells with Gram-positive results under oil immersion microscopy.The results of the novel"Tri-tonX-100 & gram staining method"validate that the presence of Staphylococcus aureus individu-als within bovine mammary epithelial cells is consistent with images observed under transmission electron microscopy and laser confocal microscopy.The verification results indicate that the"Tri-tonX-100 & gram staining method"is suitable for the diagnosis of S.aureus persistent intracellu-lar infection in bovine mastitis.Later,somatic cells from milk were extracted by gradient centrifu-gation and purified to obtain bovine mammary epithelial cells for clinical testing of 70 S.aureus positive mastitis cattle,which showed a positive rate of 82.9％and a negative rate of 17.1％.

Objective:To screen the key genetic,diagnostic and therapeutic targets of chronic obstructive pulmonary disease(COPD)patients by using microarray datasets and Mendelian randomization(MR)method,and to provide the evidence for clinical diagnosis and treatment of COPD.Methods:Four COPD gene expression profile datasets were obtained from the Gene Expression Omnibus(GEO)database.The data were processed and normalized using R software,and differentially expressed genes(DEGs)were screened.MR analysis was performed to explore the causal relationship between COPD and expression quantitative trait loci(eQTL),intersection with DEGs was taken to identify potential key targets.Gene Set Enrichment Analysis(GSEA),Gene Ontology(GO)functional enrichment analysis,and Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analysis were conducted to investigate the functional roles and pathways of the key targets,external datasets were used to validate their expression.Results:A total of 1 571 DEGs were screened,including 820 upregulated genes and 751 downregulated genes.MR analysis identified 286 COPD-related genes,and intersection with DEGs revealed 3 upregulated genes:diacylglycerol kinase gamma(DGKG),neurofilament heavy polypeptide(NEFH),and Fc receptor like B(FCRLB);and 6 downregulated genes:STEAP4 metalloreductase(STEAP4),pleckstrin homology domain containing family F member 2(PLEKHF2),CD3d molecule(CD3D),transgelin 2(TAGLN2),tripartite motif containing 22(TRIM22),and ribosomal protein L9(RPL9).The biological function analysis results indicated that these genes were mainly involved in pathways such as iron ion transport into the cells,oxidoreductase activity,primary immunodeficiency,and Th1 and Th2 cell differentiation.The MR analysis results confirmed the causal relationship between these targets and COPD.The external validation results showed that compared with healthy controls,the expression level of FCRLB in COPD samples was significantly increased(P<0.01),while the expression levels of CD3D and RPL9 were significantly decreased(P<0.05 or P<0.01),which was consistent with the MR analysis results,highlighting the reliability of this study.Conclusion:DGKG,NEFH,FCRLB,STEAP4,PLEKHF2,CD3D,TAGLN2,TRIM22,and RPL9 may serve as important regulatory factors and clinical diagnostic/therapeutic targets in the pathogenesis of COPD,providing clues for early screening,diagnosis,and targeted treatment of COPD.

OBJECTIVE To investigate the age-group differences and seasonal distribution patterns of epidemiologi-cal characteristics of hospital-associated infection in elderly inpatients in a three-A hospital.METHODS Based on the Xinglin Real-time Monitoring System,a retrospective analysis was conducted on clinical data of 67 157 patients hospitalized in the Air Force Hospital of Eastern Theater Command from Jan.2020 to Dec.2024.Elderly patients were divided into three age groups:young-age group(60-69 years),middle-age group(70-79 years)and ol-dest-age(≥80 years).The epidemiological characteristics and seasonal distribution patterns of the three groups were analyzed.RESULTS From 2020 to 2024,there were 1 524 cases of hospital-associated infection,with an inci-dence rate of 2.27％.The infection rate was higher in the oldest-age group(6.08％)than those in the young-age group(1.77％)and the middle-age group(2.52％)(P＜0.001).The top three departments with the highest in-cidence rates of hospital-associated infection were the geriatric department(10.97％),intensive care unit(9.65％)and respiratory department(4.96％).The lower respiratory tract was the main site of infection,and the infection rate was higher in winter than in other seasons.The pathogens were predominantly gram-negative bacteria(60.35％),with Pseudomonas aeruginosa being the most frequently detected.CONCLUSIONS Advanced age,male gender and hospitalization in winter are key risk factors for hospital-associated infection.Seasonal pre-vention and control strategies should be implemented in departments with high incidence of respiratory infections,and monitoring of multidrug-resistant bacteria should be enhanced.

Community-acquired pneumonia （CAP） is a common respiratory infectious disease in the elderly， and traditional Chinese medicine （TCM） and integrated TCM and western medicine showed effectiveness. According to the clinical characteristics of CAP， the staging concepts of CAP infection and recovery are proposed， and the prevention and treatment goals and strategies of staging and grading are put forward in combination with the disease seve-rity of mild， moderate and severe infections. The main objectives are to increase the cure rate of mild and moderate pneumonia， to reduce the case fatality rate of severe pneumonia， and to reduce the incidence of new upper and lower respiratory tract infections during the recovery period， respectively. TCM prevention and treatment strategies： for mild pneumonia， treatments should scatter and dissipate external pathogens， diffuse and descend lung qi， dispel dampness and dissolve phlegm， or clear lung and dissolve phlegm； for moderate pneumonia， treatments should clear lungs and resolve toxins， dry dampness and dissolve phlegm， and supplementing with tonifying lungs and strengthen spleen， or benefit qi and nourish yin； for severe pneumonia， the treatment of dispelling pathogen and supporting healthy qi should be emphasised according to the primary and secondary levels of excess pathogen and deficiency healthy qi； and for the recovery period， treatments should mainly support healthy qi， and supplement with dispelling pathogen. Relevant clinical studies have been conducted for practical verification， and the results showed that integrated TCM and Western medicine treatment can improve the cure rate of mild to moderate pneumonia， decrease the case fatality rate of severe pneumonia， and reduce the re-hospitalisation of pneumonia during the recovery period， indicating that the staged and graded prevention and treatment strategy has important guiding value for improving the diagnosis and treatment of CAP.

ObjectiveTo analyze the development status and quality of clinical practice guidelines for the treatment of dominant diseases with Chinese patent medicines （CPMs）. MethodsDatabases were searched from Jan. 2019 to Dec.2023 to collect the published clinical practice guidelines of CPMs for the treatment of dominant diseases. The information about the title， the participants， clinical problems， outcomes， evidence grade， recommendations， and recommendation strength in the included clinical practice guidelines were collected， for which the development status was analyzed， and the quality was evaluated with the Scientific， Transparent and Applicable Rankings （STAR） tool for clinical practice guidelines. ResultsTotally， 34 guidelines were included， involving 273 kinds of CPMs. One to ten （with the medium five） clinical problems were proposed from 29 clinical practice guidelines respectively. All the guidelines divided the evidence into four grades according to Grade of Recommendation Assessment， Deve-lopement an Evaluation. And 28 guidelines had five levels of recommendation strength. A total of 344 recommendations were extracted， including 86 strong-recommendations， 191 weak-recommendations （including 36 weak recommendations only based on expert consensus） and 67 recommendations with unclear recommendation strength. All guidelines had high scores in the three areas of “clinical questions （94.20%）”， “evidence （91.45%）” and “recommendations （89.06%）”， while the scores in the three areas of “registry （22.06%）”， “protocol （19.00%）” and “accessibility （31.51%）” were low. The STAR recommended stars of 8 guidelines were 5.0~4.0 stars， while that of 18 guidelines were 3.5~2.5 stars， and 8 guidelines were 2.0~1.0 stars. The three guidelines with the highest recommended stars were depressive disorder， community-acquired pneumonia， and influenza in adult. ConclusionThere is a certain gap in the quality of the published clinical practice guidelines of CPMs， and the quality of the guidelines could be further improved in registry， protocols， funds， and accessibility.

TCM believes that spleen deficiency is the root cause of emotional abnormalities in chronic fatigue syndrome (CFS), and clinical treatment often involves the heart, liver and kidney. TCM therapy has a significant efficacy in CFS emotional abnormalities. It is mostly treated with oral administration of TCM, acupuncture, moxibustion and massage therapy. It may play a therapeutic role by improving oxidative stress and immune inflammation, regulating nerve-endocrine, controlling energy metabolism and other ways. It is suggested to establish the syndrome differentiation standard of CFS emotional abnormality in the future, so as to improve the accuracy of syndrome differentiation and treatment; form a perfect treatment guide or expert consensus to guide the standardized application of various internal and external treatment methods; explore objective indicators based on the pathogenesis, and focus on the morphological and functional changes of disease target brain regions with the help of neuroimaging techniques, so as to improve the diagnosis and prognosis evaluation of CFS; based on the guidance of TCM theory, improve the CFS emotional abnormal animal modeling method.