This study aimed to explore the interaction between the small molecule Lyb24 and PyrD, a key enzyme in the pyrimidine biosynthesis pathway of Klebsiella pneumoniae (KP), and the effect of Lyb24 on the catalytic activity of PyrD, thus to provide a theoretical basis for the development of novel antimicrobial agents. The pET-30a(+)-PyrD recombinant plasmid was constructed using Nde I/Xba I double digestion technology and was transformed into Escherichia coli BL21 (DE3) competent cells using the heat-shock method. The recombinant protein was induced at 16 ℃ with 0.3 mmol/L isopropyl β-D-thiogalactopyranoside (IPTG). The recombinant PyrD protein was purified using nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography to obtain a high-purity product. Surface plasmon resonance (SPR) experiments were conducted to detect the direct interaction between Lyb24 and PyrD protein, and a DCIP-based colorimetric assay was used to evaluate the effect of Lyb24 on the catalytic activity of PyrD. The pET-30a(+)-PyrD plasmid was successfully constructed, and the recombinant PyrD protein with a molecular weight of approximately 36 kD was expressed and purified to a concentration of 5.58 mg/mL. Lyb24 exhibited high-affinity direct binding to PyrD (KD = 8.83 × 10−5 mol/L) and exerted an uncompetitive inhibition effect on the catalytic activity of PyrD. This study demonstrates that Lyb24, a small-molecule compound, directly binds to PyrD and inhibits its enzymatic activity, providing crucial experimental evidence for developing PyrD-targeted antibacterial agents with value of clinical translation.

BACKGROUND:Gradient artificial bone repair scaffolds can mimic unique anatomical features in musculoskeletal tissues,showing great potential for repairing injured musculoskeletal tissues. OBJECTIVE:To review the latest research advances in gradient artificial bone repair scaffolds for tissue engineering in the musculoskeletal system and describe their advantages and fabrication strategies. METHODS:The first author of the article searched the Web of Science and PubMed databases for articles published from 2000 to 2023 with search terms"gradient,bone regeneration,scaffold".Finally,76 papers were analyzed and summarized after the screening. RESULTS AND CONCLUSION:(1)As an important means of efficient and high-quality repair of skeletal system tissues,gradient artificial bone repair scaffolds are currently designed bionically for the natural gradient characteristics of bone tissue,bone-cartilage,and tendon-bone tissue.These scaffolds can mimic the extracellular matrix of native tissues to a certain extent in terms of structure and composition,thus promoting cell adhesion,migration,proliferation,differentiation,and regenerative recovery of damaged tissues to their native state.(2)Advanced manufacturing technology provides more possibilities for gradient artificial bone repair scaffold preparation:Gradient electrospun fiber scaffolds constructed by spatially differentiated fiber arrangement and loading of biologically active substances have been developed;gradient 3D printed scaffolds fabricated by layered stacking,graded porosity,and bio-3D printing technology;gradient hydrogel scaffolds fabricated by in-situ layered injections,simple layer-by-layer stacking,and freeze-drying method;and in addition,there are also scaffolds made by other modalities or multi-method coupling.These scaffolds have demonstrated good biocompatibility in vitro experiments,were able to accelerate tissue regeneration in small animal tests,and were observed to have significantly improved histological structure.(3)The currently developed gradient artificial bone repair scaffolds have problems such as mismatch of gradient scales,unclear material-tissue interactions,and side effects caused by degradation products,which need to be further optimized by combining the strengths of related disciplines and clinical needs in the future.

OBJECTIVE To summarize the current situation of pharmaceutical clinic service in our hospital over the past five years， and explore sustainable development strategies for service models of pharmaceutical clinics. METHODS A retrospective analysis was conducted on the consultation records of patients who registered and established files at the pharmaceutical clinic in our hospital from January 2019 to December 2023. Statistical analysis was performed on patients’ general information， medication- related problems， and types of pharmaceutical services provided by pharmacists. RESULTS A total of 963 consultation records were included， among which females aged 20-39 years accounted for the highest proportion （66.04%）； obstetrics and gynecology- related consultations accounted for the largest number of cases. Additionally， 80 patients attended follow-up visits at our hospital’s pharmaceutical clinic. A total of 1 029 medication-related issues were resolved， including 538 cases of drug consultations （52.28%）， 453 medication recommendations （44.02%）， 22 medication restructuring（2.14%）， and 16 medication education （1.55%）； the most common types of medication-related problems identified were adverse drug events（70.07%）. CONCLUSIONS Although the pharmaceutical clinic has achieved recognition from clinicians and patients， challenges such as low awareness among healthcare providers and the public persist. Future efforts should focus on strengthening information technology construction， enhancing pharmacist training， and establishing various forms of outpatient pharmaceutical service models.

Objective:To investigate the effects and underlying mechanisms of silent information regulator 1(SIRT1)on the dysfunction of umbilical vein endothelial cells(HUVECs)induced by oxidized low-density lipoprotein(ox-LDL).Methods:The impact of ox-LDL on the viability of HUVEC was assessed using the Cell Counting Kit-8(CCK-8)assay, which also facilitated the determination of the optimal ox-LDL concentration.Subsequent to ox-LDL treatment, several parameters were evaluated, including reactive oxygen species(ROS)production, apoptosis, migration, and angiogenesis, utilizing a ROS detection kit, flow cytometry, a Transwell migration assay, and an angiogenesis assay, respectively.The expression levels of apoptosis-related proteins, namely cleaved caspase-3(c-caspase-3), Bcl-2-associated X protein(Bax), B-cell lymphoma-2(Bcl-2), SIRT1, and peroxisome proliferator-activated receptor γ coactivator-1α(PGC-1α), were quantified using Western blot analysis.Adenoviral vectors were employed to either overexpress or silence SIRT1, while the ROS inhibitor N-acetylcysteine(NAC)was applied to assess its effects on cell function.Additionally, PGC-1α acetylation(Ac-Lys)was investigated through co-immunoprecipitation.Results:In the oxidative model of ox-LDL-stimulated HUVECs, compared to controls, we observed a significant increase in ROS-positive cells(35.9±3.1 vs.5.4±0.9), heightened apoptosis(16.3±0.9 vs.7.6±0.7), diminished endothelial cell migration capacity, and reduced angiogenic capacity.Additionally, there was an elevation in the pro-apoptotic protein c-caspase3 and Bax, alongside a decrease in the anti-apoptotic protein bcl-2.Furthermore, SIRT1 expression was increased, as was the expression of PGC-1α.In comparison to the GFP group(28.5±1.9), the reduction in SIRT1 expression resulted in an increase in apoptosis(37.0±1.9).Conversely, overexpression of SIRT1 mitigated ox-LDL-induced apoptosis(25.2±1.6)(all P<0.05).Notably, the expression levels of PGC-1α and SIRT1 exhibited consistent changes: PGC-1α expression increased with SIRT1 overexpression and decreased when SIRT1 expression was reduced(both P<0.05).The administration of NAC to the ox-LDL-treated group led to a reduction in ROS production( t=11.18, P<0.01)and a significant enhancement in cell function.Immunoprecipitation results indicated that SIRT1 overexpression decreased ox-LDL-induced PGC-1α acetylation( t=18.18, P<0.01), whereas silencing of SIRT1 further increased PGC-1α acetylation levels( t=-19.09, P<0.01). Conclusions:SIRT1 is shown to protect against ox-LDL-induced apoptosis and dysfunction in HUVECs by deacetylating and activating PGC-1α, thereby highlighting its therapeutic potential in the context of endothelial cell injury.

Objective To investigate the predictive value of neuregulin 4(Nrg4)combined withγ-aminobutyric acid(GABA)in cognitive dysfunction among patients with severe obstructive sleep apnea-hypopnea syndrome(OSAHS).Methods A total of 169 patients with severe OSAHS were se-lected as study subjects and divided into normal cognitive function group(n=89)and cognitive dys-function group(n=80)based on cognitive function assessment results.General information of the pa-tients was collected,and the levels of Nrg4 and GABA were detected by enzyme-linked immunosor-bent assay(ELISA).Receiver operating characteristic(ROC)curve analysis was used to evaluate the predictive value of Nrg4 and GABA for cognitive dysfunction in OSAHS patients.Results The proportions of patients with a history of hypertension and diabetes,as well as the levels of diastolic blood pressure,total cholesterol(TC),triglyceride(TG),high-density lipoprotein cholesterol(HDL-C),and low-density lipoprotein cholesterol(LDL-C)were significantly higher in the cognitive dysfunction group than those in the normal cognitive function group(P＜0.05).The levels of Nrg4 and GABA were significantly lower in the cognitive dysfunction group than in the normal cognitive function group(P＜0.05).The Montreal Cognitive Assessment(MoCA)score in the cognitive dys-function group was significantly lower than that in the normal cognitive function group[(12.36±2.35)versus(28.25±1.02),P＜0.05].Multivariate Logistic regression analysis revealed that a history of hypertension and diabetes,diastolic blood pressure,TC,TG,HDL-C,and LDL-C were risk factors for cognitive dysfunction in patients with severe OSAHS(P＜0.05),while Nrg4,GABA,and MoCA scores were protective factors(P＜0.05).ROC curve analysis showed that combined de-tection of Nrg4 and GABA had a higher predictive value for cognitive dysfunction in patients with se-vere OSAHS compared with either marker alone(P＜0.05).Conclusion A history of hyperten-sion and diabetes,diastolic blood pressure,TC,TG,HDL-C,LDL-C,Nrg4,GABA,and MoCA scores are all factors influencing cognitive dysfunction in patients with severe OSAHS.Combined de-tection of Nrg4 and GABA can effectively predict cognitive dysfunction in these patients.

Objective:To investigate the survival outcomes and prognostic factors of patients with double primary breast cancer (BC) and endometrial cancer (EC).Methods:A retrospective cohort study was conducted using data for the period 1992-2018 from the Surveillance, Epidemiology, and End Results (SEER) database. There were 3 465 patients with BC as the first primary cancer (BC-EC group) and 2 804 patients with EC as the first primary cancer (EC-BC group). Kaplan-Meier analysis and cumulative incidence function were used to estimate overall mortality, breast cancer-specific mortality, and endometrial cancer-specific mortality, respectively. Cox regression and Fine-Gray regression were used to analyze the prognostic factors of overall mortality, breast cancer-specific mortality, and endometrial cancer-specific mortality, respectively.Results:During a median follow-up of 160 months, 1 616 deaths occurred in the BC-EC group, with EC being the leading cause of death (37.69%); 994 deaths occurred in the EC-BC group, with BC being the leading cause of death (28.77%). Cox regression identified patients with older ages at first primary cancer diagnosis (54-61 years: HR=1.46, 95% CI: 1.26-1.69; 62-68 years: HR=2.64, 95% CI: 2.29-3.03; ≥69 years: HR=4.89, 95% CI: 4.27-5.60), shorter time interval between the diagnoses (0-5 months: HR=6.13, 95% CI: 5.21-7.21; 6-23 months: HR=5.69, 95% CI: 4.95-6.55; 24-59 months: HR=3.44, 95% CI: 3.04-3.89; 60-119 months: HR=2.32, 95% CI: 2.07-2.59), mixed ductal-lobular BC ( HR=1.29, 95% CI: 1.11-1.48), endometrial mixed cell adenocarcinoma ( HR=1.23, 95% CI: 1.01-1.50), advanced tumor grade (grade Ⅱ BC: HR=1.13, 95% CI: 1.01-1.27; grade Ⅲ BC: HR=1.24, 95% CI: 1.10-1.41; grade Ⅱ EC: HR=1.19, 95% CI: 1.06-1.33; grade Ⅲ EC: HR=1.68, 95% CI: 1.48-1.90), advanced tumor stage of the two cancers (distant BC: HR=3.14, 95% CI: 2.50-3.94; regional EC: HR=1.53, 95% CI: 1.36-1.71; distant EC: HR=3.00, 95% CI: 2.59-3.47) had increased risk of overall mortality. Fine-Gray regression showed that compared with BC-EC patients, EC-BC patients had a higher risk of breast cancer-specific mortality [sub-distribution hazard ratio (s HR=1.24, 95% CI: 1.04-1.47], but a lower risk of endometrial cancer-specific mortality (s HR=0.37, 95% CI: 0.30-0.46). Older ages at first cancer diagnosis, shorter intervals between the diagnoses, negative ER and PR status, and advanced BC grades/stages were associated with increased breast cancer-specific mortality ( P＜0.05). Similarly, older ages, shorter intervals, endometrial serous carcinoma/mixed cell adenocarcinoma, and advanced EC grades/stages correlated with elevated endometrial cancer-specific mortality ( P＜0.05). Conclusion:The management of double primary BC and EC patients requires multidisciplinary strategies, with particular attention to patients presenting older ages at first cancer diagnosis, shorter intervals between the diagnoses, and unfavorable tumor characteristics.

Objective To investigate the biological function of the proprotein convertase subtilisin/kexin type 9(PCSK9)in prostate cancer(PCa)and its effect on ferroptosis sensitivity Methods Bioinformatics was used to analyze the relationship between the expression of PCSK9 and the prognosis of prostate cancer.The expression of PCSK9 in PCa cell lines were detected using RT-qPCR.PCa cells with PCSK9 knockdown were constructed using siRNA,and the The effect of PCSK9 on cell proliferation,migration,and invasion were detected using CCK-8 assays and Transwell assays.The Cancer Therapeutics Response Portal(CTRP)was employed to investigate the correlation between PCSK9 and ferroptosis drug sensitivity,and PCa cells with PCSK9 knockdown were treated with the ferroptosis inducer(RSL3)to detect the sensitivity to ferroptosis.Results Bioinformatics showed low expression of PCSK9 had longer disease specific survival(P<0.05).The results of the in vitro experiments showed that PCSK9 knockdown significantly inhibited the proliferation,migration,and invasion of PCa cells(P<0.001).Furthermore,CTRP analysis showed that cellular sensitivity to ferroptosis inducers correlated with the expression level of PCSK9.PCSK9 knockdown cells exhibited higher sensitivity to the ferroptosis inducer RSL3.Conclusion Knockdown of PCSK9 inhibits the proliferation,migration,and invasion of PCa cells,and increases the sensitivity of cells to ferroptosis inducers.PCSK9 may provide new insights for the treatment of PCa.

Objective To develop the Expert Consensus on Sensitive Indicators for Assessment of the Quality of Nursing in Operating Theatre and provide a scientific and practical guidance for improving the quality of nursing in operating theatre.Methods The writing team established by the Operating Room Nursing Professional Committee of Guangdong Nursing Association conducted systematic literature retrieval and screening,and used the updated clinical Guidelines for Research and Evaluation Ⅱ in UK 2017.AGREE Ⅱ and the evidence evaluation system of the Australian JBI(Joanna Briggs Institute,JBI)Evidence-Based Health Care Center evidence level system(2016 Edition)comprehensively analyzed the evidence related to the sensitive indicators for evaluating the quality of operating room nursing and the suggestions of the writing group members.The first draft was formed based on the three-dimensional quality evaluation theoretical framework of"structure-process-result".Through the Delphi method,after two rounds of expert consultations and members'votes,the first draft was deeply revised and improved.Results Based on the three-dimensional quality evaluation theoretical framework of"structure-process-outcome"proposed by American scholar Donabedian,the expert consensus finally included five primary indicators:basic nursing quality,quality indicators of patient safety,quality indicators of hospital infection control,quality indicators of medication and safety management,and quality indicators of specialised nursing in operating theatre.The secondary indicators consisted of one structural indicator(management of commonly used instrument and equipment in operating theatre)and 17 process indicators(e.g.,infusion and blood transfusion management,body temperature management,etc.).The tertiary indicators included 26 process indicators and 11 outcome indicators(e.g.,incidence of adverse reactions of infusion during surgery,incidence of intra-operative hypothermia,etc.).Conclusion The evidence-and guideline-based Expert Consensus on Sensitive Indicators for Assessment of the Quality of Nursing in Operating Theatre based on eviclence and guidelines was established through rigorous evidence-based methods.It is operational and practical,and offers theoretical support and practical guidance for the managers of operating theatre to improve the quality of nursing.

Based on the previous transcriptomic experimental data of Trichinella spiralis(T.spira-lis)in this study,the larval stage specific gene TP12446 was screened and its identity in the ubiq-uitin ligase RNF family was predicted.In the study,bioinformatics methods were used to analyze its physicochemical properties and its activity to lay the foundation for further exploring the func-tion of TP12446 gene.The physicochemical properties and protein structure of TP12446 protein were predicted by bioinformatics.Its ubiquitin ligase activity was also verified by ubiquitination re-actions in vitro.The expression characteristics of TP12446 protein in different stage of T.spiralis infection were analyzed by qPCR and Western blot.Bioinformatics analysis showed that TP12446 protein was composed of 453 amino acids and its molecular weight was 51.48 kDa.The protein had a transmembrane structure and contained signal peptides.The results indicated that it was a secre-tory protein and mainly located in the cytoplasmic membrane.The protein structure analysis re-vealed that the protein contained RING and PA domain,its secondary structure was mainly com-posed of α-helix and irregular crimp and there were 10 B cell epitopes on TP12446 protein.The prediction of glycosylation and phosphorylation sites indicated that TP12446 protein contained 38 potential phosphorylation sites.Results of PPI interaction protein prediction showed that TP12446 protein had strong interaction with Usp8,Tmem37,Otub1,Otub2,Ubox5 and CD151.The results of qPCR and Western blot showed that TP12446 gene expression was the highest in the larva stage of T.spiralis,the activity of ubiquitin ligase was verified by ubiquitination reaction in vitro.TP12446 protein was a secretory hydrophobic protein with E3 ubiquitin ligase activity,which was involved in regulating cell cycle and apoptosis.

Objective To establish a standardized supply model for ophthalmic surgical instruments using lean manage-ment methods,develop a formula for determining optimal instrument inventory levels,and improve the management efficiency of ophthalmic surgical instruments.Methods A total of 2,281 cataract instrument sets from January 2023 to December 2024 were analyzed.From January to December 2023(1 141 sets),conventional process management was implemented,while from January to December 2024(1 140 sets),a lean theory-based standardized model was applied.Key parameters including surgical time,sterilization workflow,and instrument management were recorded to establish the model.A quantitative analysis incorporating sur-gical time,turnover interval,transportation time,relaxation rate,cycle interval,and target instruments was conducted to derive the inventory formula.A two-month pilot study on cataract surgeries was performed to validate the model's feasibility.The inci-dence of special events before and after implementing the standardized model was compared.Results The formula-derived data aligned with clinical demands,demonstrating precise quantification of ophthalmic instrument sets and high process stability.The incidence of special events post-implementation was significantly lower than pre-implementation(P＜0.05).Conclusion The lean theory-based standardized supply model effectively enhances the management of ophthalmic surgical instruments.