Objective To investigate the efficacy of discontinuous density gradient combined with swim-up method for bacterial removal in frozen semen,and to provide experimental evidence for the safe application of frozen semen in assisted reproductive technology.Methods A total of 208 frozen semen samples containing non-pathogenic bacteria,cryopreserved in our human sperm bank from January 2019 to December 2023,were selected.The thawed semen samples were processed using discontinuous density gradient combined with swim-up method.To simulate in vitro fertilization procedures,the processed sperm suspensions were subjected to 5-day in vitro culture for bacterial residue monitoring.Prior to cryopreservation,all semen samples underwent routine bacterial culture to exclude pathogenic microorganisms.Post-thaw semen samples,processed sperm suspensions,and 5-day culture media were sent to an independent third-party laboratory for comprehensive bacterial identification.Results Among the 208 frozen semen samples containing non-pathogenic bacteria,41 bacterial species were identified,with 1～4 species per sample.After processing with discontinuous density gradient combined with swim-up method,the sterility rate of the sperm suspension reached 89.42%(186/208),with residual bacteria including Enterococcus faecalis,Corynebacterium glucuronolyticum,Group B Streptococcus,and Corynebacterium glycinophilum.After 5-day in vitro culture,the sterility rate increased to 93.75%(195/208),with residual bacteria limited to Entero-coccus faecalis and Group B Streptococcus.Conclusions The combined method of discontinuous density gradient and swim-up technique effectively eliminates most bacteria in frozen semen.However,the clearance rate for Entero-coccus faecalis and Group B Streptococcus remains below 90%.Optimization of pre-freezing bacterial screening and post-thaw processing techniques is necessary to enhance the safety of frozen semen in assisted reproductive technology.

Objective To explore the effect of community pharmacy outpatient service on patients with type 2 diabetes mellitus. Methods A non-randomized controlled study was conducted, and type 2 diabetes patients managed in the community were divided into an intervention group of 112 cases and a control group of 110 cases. The control group received routine medication guidance during general practice outpatient visits, while the intervention group received comprehensive pharmacy outpatient service intervention based on routine medication guidance in general practice. Follow-up visits were conducted every 3 months. Repeated measurement analysis of variance and multivariate linear regression analysis were used to evaluate the intervention effect of the pharmacy outpatient service. Results Fasting blood glucose and glycosylated hemoglobin levels in the intervention group showed a decreasing trend with the increase of intervention time compared to pre-intervention time （P<0.01）, with increased duration of weekly exercise, decreased staple food intake, increased vegetable intake, and increased medication adherence score （P<0.01）. After adjusting for confounding factors through multivariate linear regression model, pharmacy outpatient intervention was found to be an independent protective factor for fasting blood glucose level （β=−0.891, P<0.01） and glycosylated hemoglobin level （β=−0.760, P<0.01） in the study subjects. Conclusion The community pharmacy outpatient service could enhance the self-management ability of patients with type 2 diabetes mellitus, and effectively improve patients’ fasting blood glucose and glycosylated hemoglobin.

Objective To investigate the efficacy of discontinuous density gradient combined with swim-up method for bacterial removal in frozen semen,and to provide experimental evidence for the safe application of frozen semen in assisted reproductive technology.Methods A total of 208 frozen semen samples containing non-pathogenic bacteria,cryopreserved in our human sperm bank from January 2019 to December 2023,were selected.The thawed semen samples were processed using discontinuous density gradient combined with swim-up method.To simulate in vitro fertilization procedures,the processed sperm suspensions were subjected to 5-day in vitro culture for bacterial residue monitoring.Prior to cryopreservation,all semen samples underwent routine bacterial culture to exclude pathogenic microorganisms.Post-thaw semen samples,processed sperm suspensions,and 5-day culture media were sent to an independent third-party laboratory for comprehensive bacterial identification.Results Among the 208 frozen semen samples containing non-pathogenic bacteria,41 bacterial species were identified,with 1～4 species per sample.After processing with discontinuous density gradient combined with swim-up method,the sterility rate of the sperm suspension reached 89.42%(186/208),with residual bacteria including Enterococcus faecalis,Corynebacterium glucuronolyticum,Group B Streptococcus,and Corynebacterium glycinophilum.After 5-day in vitro culture,the sterility rate increased to 93.75%(195/208),with residual bacteria limited to Entero-coccus faecalis and Group B Streptococcus.Conclusions The combined method of discontinuous density gradient and swim-up technique effectively eliminates most bacteria in frozen semen.However,the clearance rate for Entero-coccus faecalis and Group B Streptococcus remains below 90%.Optimization of pre-freezing bacterial screening and post-thaw processing techniques is necessary to enhance the safety of frozen semen in assisted reproductive technology.

BACKGROUND:Digital three-dimensional reconstruction technology is gradually applied to orthopedic diseases with the advantages of visualization,accuracy and non-invasiveness,but there is less evidence-based support for its use in artificial hip hemiarthroplasty for intertrochanteric fractures of the femur in the elderly. OBJECTIVE:To investigate the application value and economic effects of digital three-dimensional reconstruction techniques in artificial hip hemiarthroplasty of intertrochanteric fractures of the femur in the elderly. METHODS:One hundred and thirty elderly patients with intertrochanteric femur fractures admitted to Zunyi First People's Hospital from January 2019 to December 2022 were selected and randomly divided into a control group(n=65)and an observation group(n=65).Artificial hip hemiarthroplasty was performed in both groups.The control group adopted the film template measurement method for manual preoperative planning while the observation group adopted a digital three-dimensional reconstruction technique.Preoperative planning and intraoperative actual application of prosthesis compliance rate,fibrinogen,D-dimer,bilateral femoral eccentric distance difference,bilateral lower limb length difference,Harris hip function score,visual analog scale score,excellent and good rate of hip function,complications,and hospitalization cost were observed in both groups. RESULTS AND CONCLUSION:(1)The proportion of acetabular side and femoral side prosthesis in grade 0(fully compliant)was higher in the observation group than that in the control group(P<0.05).(2)Fibrinogen and D-dimer levels in the observation group were lower than those in the control group 3 days after surgery(P<0.05).(3)The difference in bilateral femoral eccentric distance and the difference in bilateral lower limb length in the observation group were smaller than those in the control group immediately after surgery(P<0.05).The differences in Harris and visual analog scale scores were not significantly different between the two groups preoperatively,6 and 12 months postoperatively(P>0.05).There was no significant difference in excellent and good rate of hip function between the two groups 12 months postoperatively(P>0.05).(4)There was no significant difference in the complication rate between the two groups(P>0.05).The hospitalization cost of the observation group was higher than that of the control group(P<0.05).(5)It is indicated that digital three-dimensional reconstruction technology applied in artificial hip hemiarthroplasty of intertrochanteric femoral fracture in the elderly can not only accurately determine the prosthesis type before surgery,but also accurately reconstruct the bilateral lower limbs offline,but its hospitalization cost is high.

OBJECTIVE：To predict the active components and potential target of volatile oil of Citri reticulatae preventing and treating Alzheimer ’s disease （AD）. METHODS ：The volatile oil of C. reticulatae was determined by GC-MS ，and identified according to NIST 11.L database and manual data analysis. The active components and targets of volatile oil of C. reticulatae were predicted through TCMSP and PharmMapper database. The related targets of AD were obtained by using GeneCards and OMIM databases. Venny 2.1.0 software mapping was used to obtain the direct targets of volatile oil of C. reticulatae against AD. Core nodes were mined with STRING database and Cytoscape 7.2.1 software，and the indirect targets of volatile oil of C. reticulatae against AD were obtained by mapping and duplication coith Venny 2.1.0 software. With the help of DAVID 6.7 database，the above direct and indirect targets （i.e. action targets ）were used for gene ontology （GO）function enrichment analysis and KEGG pathway enrichment analysis. Using Cytoscape 7.2.1 software， topology analysis was conducted for the network of “active components-acting targets ”of volatile oil of C. reticulatae ，with node degree value ，betweenness centrality and closeness centrality as indexes ，then key components and key targets were mined. RESULTS & CONCLUSIONS ：A total of 40 chemical components in volatile oil were identified by GC-MS ，all of them were active components ，including D-limonene，γ-terpinene，etc. A total of 151 active components-corresponding targets and 1 291 AD-related targets were mined ，including 48 direct targets and 41 indirect targets. The above 89 targets were mainly concentrated in cell fraction ，axon，cytosol and other cell components ；intracellular signaling cascade ，response to organic substance and other biological processes ；protein kinase activity and amine receptor activity and other molecular functions ；as well as cancer pathway ，calcium signaling pathway and neurotrophin signaling pathway （P＜ 0.05）. A total of 10 key components including α-terpinene，β-elemen，thymol and （－）-4-terpineol，as well as 21 key targets such as androgen receptor ，prostaglandin G/H synthase 2，mitogen activated protein kinase 14，muscarinic acetylcholine receptor M 1 were excavated ，indicating the effect of volatile oil of C. reticulatae against AD had the characteristics of multi-component ， multi-target and multi-channel.

[Objective]To observe the dynamic changes of serum LH levels in different GnRH-along protocols ,and investigate the relationship between GnRH-adosage and LH levels ,and compare the clinical outcomes among different GnRH-a long protocols.[Methods]In this retrospective study,1.0 mg,0.8 mg,0.375 mg long-acting and 0.1 mg/d,0.05 mg/d short-acting GnRH-a long protocols were included from January to June in 2015 at the Reproductive Medicine Center of the First Affiliated Hospital of Sun Yat-Sen University. Serum LH levels were evaluated from a total of 250 women at the four time points,on the day before gonadotropin stimulation(Gn0),the fourth day of Gn stimulation(Gn4),the seventh day of Gn stimulation(Gn7),and the HCG administration day(HCG day),then the relationship between serum LH levels and the dosage of GnRH-a were analyzed. The number of oocytes retrieved,fertilization rate,good quality embryos rate,blastocyst transferred rate,the number of transferred embryos,implantation rate and clinical pregnancy rate were also compared.[Results]Among the long-acting groups,LH levels in 0.375 mg group were higher than those in 1.0 mg and 0.8 mg groups at the four time points(P<0.05). Total Gn dose,duration of Gnstimualtion and HMG dosage of 1.0 mg protocol and 0.8 mg protocol were significantly higher than those of 0.375 mg group(P < 0.05). In addition,the implantation rate showed an increasing tendency when the level of LH increases(P>0.05). In short-acting groups,the LH levels in 0.05 mg/d protocol were significantly lower than those in the 0.1 mg/d group at Gn0,Gn7 and HCG day(P<0.05). Total Gn dose,du?ration of Gn stimulation and HMG dosage did not show significant difference between 0.05mg and 0.1mg group. What′s more,the im?plantation rate in 0.1 mg/d group was higher than in 0.05 mg/d group(P>0.05).[Conclusion]Among the long-acting groups,the smaller amount of GnRH-a was administrated ,the higher LH levels during ovarian stimulation and implantation rate the patients ob?tained. As to the short-acting groups,the LH level and implantation rate in 0.1 mg/d group is higher than those in 0.05 mg/d group.

Objective: To investigate the changes in protein expression in patients with 1-bromopropane (1-BP) poisoning using high-throughput proteomic technique and to screen out protein markers. Methods: Serum samples were collected from 3 patients with 1-BP poisoning and 15 controls. The label-free proteomic tech-nique was used for the quantitation and identification of proteins expressed in these samples, and the results were compared between the patients with 1-BP poisoning and the control population. The bioinformatics tools were used to analyze the function of differentially expressed proteins. Results: Compared with the control popula-tion, the patients with 1-BP poisoning had >2-fold upregulation of 38 proteins and >2-fold downregulation of 68 proteins. The differentially expressed proteins were mainly involved in immune response, signal transduction, and stress response. Conclusion The proteins screened out may be potential protein markers for 1-BP poison-ing, which provides reliable and precise methods and thoughts for the diagnosis of 1-BP poisoning.

This study investigated the intracellular localization of asparagine synthetase (ASNS) in the relation with chemoresistance in leukemia. pIRES-GFP-ASNS-Flag/Neo expression vector was transiently tansfected into SK-N-MC cells and 297T cells respectively. Immunofluorescence and Western blot analysis were performed for cellular localization of ASNS respectively. U937 cells were treated with L-asparaginase for 48 h and examined for endogenous ASNS expression on plasma membrane by immunofluorescence staining. Immunofluorescence staining showed that the transiently expressed ASNS was partly localized on transfected-SK-N-MC cell surface. Moreover, Western blotting exhibited that ASNS expressed both in cytosol and on plasma membrane of transfected-293T cells. Immunofluorescence staining with anti-ASNS-specific monoclonal antibody revealed that endogenous ASNS was localized on the plasma membrane of U937 cells, except for its distribution in the cytosol. In addition, ASNS exhibited a higher expression on plasma membrane after treatment with L-asparaginase as compared with the untreated cells. It was concluded that the subcellular translocation of ASNS may play an important role in L-asparaginase resistance in leukemia cells.

This study investigated the intracellular localization of asparagine synthetase (ASNS) in the relation with chemoresistance in leukemia.pIRES-GFP-ASNS-Flag/Neo expression vector was transiently tansfected into SK-N-MC cells and 297T cells respectively.Immunofluorescence and Western blot analysis were performed for cellular localization of ASNS respectively.U937 cells were treated with L-asparaginase for 48 h and examined for endogenous ASNS expression on plasma membrane by immunofluorescence staining.Immunofluorescence staining showed that the transiently expressed ASNS was partly localized on transfected-SK-N-MC cell surface.Moreover,Western blotting exhibited that ASNS expressed both in cytosol and on plasma membrane of transfected-293T cells.Immunofluo-rescence staining with anti-ASNS-specific monoclonal antibody revealed that endogenous ASNS was localized on the plasma membrane of U937 cells,except for its distribution in the cytosol.In addition,ASNS exhibited a higher expression on plasma membrane after treatment with L-asparaginase as compared with the untreated cells.It was concluded that the subcellular translocation of ASNS may play an important role in L-asparaginase resistance in leukemia cells.

Objective To prospectively evaluate the sensitivity and specificity of ankle brachial index (ABI) in the diagnosis of lower extremity arterial disease(LEAD)by using conventional digital subtraction angiography (DSA) as the reference standard, and to research the threshold value of ABI screening test for diagnosis. Methods A total of 383 consecutive patients (245 men and 138 women, mean age (64.1±11.7) years] underwent conventional DSA and ABI measurement. Receiver operator characteristics (ROC) analysis was performed to assess possible threshold values for predicting the LEAD in these patients. Results Conventional DSA was used as the gold standard in defining≥50% luminal stenosis for the diagnosis of LEAD. 0. 95 was the overall cutoff of ABI which was associatcd with 93.0% sensitivity, 85.0% specificity, 8.81 positive likelihood ratio(+LR) and 0. 23 negative likelihood ratio(-LR) for detection of hemodynamically significant stenosis (lesions>≥50%) in all 383 subjects (P<0.01). The area under the ROC curve was 0. 953(95%CI 0.920～0.985). Conclusions ABI measurement is an accurate and reliable non-invasive alternative to conventional DSA in the diagnosis of lower extremity arterial disease. And the cut-off 0.95 is the threshold ABI value for detecting LEAD in Chinese population.