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.

Objective:To investigate the role and possible mechanism of triggering receptor expressed on myeloid cells-1(TREM-1)in the inflammatory response of rat vascular smooth muscle cells(VSMCs)mediated by trimethylamine N-oxide(TMAO).Methods:VSMCs were treated with TMAO at different concentrations(0,100,300,600,900,1 200,1 500 μmol/L)for 24 hours.VSMCs were transfected with the siRNA interference plasmid targeting the TREM-1 gene(si-TREM-1)and its negative control interference plas-mid(si-NC),and 600 μmol/L TMAO was used to induce inflammatory response in VSMCs,combined with the intervention with the nuclear factor-kappa B(NF-κB)activator phorbol myristate acetate(PMA)for 24 hours.CCK-8 assay was used to measure cell prolif-erative activity;ELISA was used to measure the levels of interleu-kin-1β(IL-1β),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)in cell supernatant;qRT-PCR was used to measure the mRNA expression levels of TREM-1,cyclooxygenase-2(COX-2),intercellular adhesion molecule-1(ICAM-1),IL-1β,IL-6,and TNF-α in cells,and Western blot was used to measure the protein expression levels of TREM-1,COX-2,ICAM-1,NF-κB p65,and p-NF-κB p65(Ser536)in cells.Results:Treatment with TMAO at different concentrations had no significant impact on the prolifera-tive activity of VSMCs(P=0.375),but it significantly upregulated the levels of IL-1β,IL-6,and TNF-α in supernatant and the mRNA and protein expression levels of TREM-1,COX-2,and ICAM-1 in cells(all P<0.01)in a concentration-dependent manner.Silencing of the TREM-1 gene significantly inhibited the increases in the levels of IL-1β,IL-6,and TNF-α in the supernatant of VSMCs in-duced by TMAO,and it also suppressed the upregulation of the mRNA expression levels of COX-2,ICAM-1,IL-1β,IL-6,and TNF-α and the ratio of p-NF-kB p65/NF-kB p65 in VSMCs(all P<0.01).However,PMA intervention significantly reversed the role of si-lencing the TREM-1 gene on TMAO-induced inflammatory response in VSMCs.Conclusion:Silencing of the TREM-1 gene can in-hibit TMAO-induced inflammatory response in VSMCs,possibly by inhibiting the activation of the NF-κB pathway.

Objective To investigate the cardiac protective effect of Taohong Tongluo granules on coronary microvascular dysfunction(CMD)rats.Methods SD rats were randomly assigned to sham-operated group,CMD group,nicorandil group(5 mg/kg),or Taohong Tongluo granule group(50 mg/kg).Animals were administered corresponding drugs for 7 d according to the grouping,and the CMD model was prepared 2 h after the last administration.The rat CMD model was induced by injecting embolization microspheres(diameter 40-120 μm,approximately 1 000 microspheres)into the left ventricular cavity.Twenty-four hours after modeling,echocardiography was performed to measure the left ventricular ejection fraction(EF),fractional shortening(FS),and end-diastolic volume(EDV).The damaged myocardial area was assessed by 2,3,5-triphenyltetrazolium chloride(TTC)staining.Myocardial morphological changes were observed by hematoxylin-eosin(H-E)staining.The protein expression levels of NOD-like receptor family pyrin domain containing protein 3(NLRP3),apoptosis-associated speck-like protein(ASC),and cysteine aspartic acid specific protease(caspase)-1 in rat myocardial tissue were detected by immunohistochemical staining and Western blotting.Results Echocardiography showed that the EF and FS values in the Taohong Tongluo granule group,CMD group,and nicorandil group were significantly lower than those in the sham-operated group(all P＜0.001).The EF and FS values in the Taohong Tongluo granule group and nicorandil group were significantly higher than those in the CMD group(all P＜0.01).However,there were no significant differences in EDV among the groups(all P＞0.05).H-E staining showed no abnormalities in the myocardium in the sham-operated group.The CMD group exhibited microsphere embolism in the myocardium,myocardial cell dissolution and rupture,and inflammatory infiltration.The lesions in the nicorandil group and the Taohong Tongluo granule group were relatively milder,and the number of thrombi in both groups was lower than that in the CMD group(both P＜0.01).The results of TTC staining indicated that the areas of damaged myocardial regions in both the nicorandil group and the Taohong Tongluo granule group were smaller than that in the CMD group(P＜0.05 or P＜0.01).Moreover,the area in the Taohong Tongluo granule group was smaller than that in the nicorandil group(P＜0.05).The results of immunohistochemical staining showed that in the CMD model,the expression of ASC and caspase-1 proteins,as well as the number of positive cells for these proteins,was increased and was distributed in myocardial and interstitial cells.The numbers of ASC and caspase-1 positive cells in the Taohong Tongluo granule group were lower than that in the CMD group(both P＜0.01).The Western blotting showed that the expression levels of NLRP3,ASC,and caspase-1 proteins in the Taohong Tongluo granule group were all lower than those in the CMD group(all P＜0.05).Conclusion Taohong Tongluo granules can improve cardiac function,ameliorate hemodynamic parameters,and reduce myocardial infarction area in rats with CMD induced by microsphere embolism.The mechanism is related to the inhibition of myocardial inflammasome activation,thereby attenuating the myocardial injuries.

Objective To clarify the potential risk factors of disinfection and supply room,and explore the application effect of strengthening quality control management,so as to improve the service quality of disinfection and supply room and reduce the risk of medical accidents.Methods From January 2022 to December 2023,800 medical devices processed in a disinfection supply room were selected as the study subjects.They were randomly divided into two groups of 400 each:a control group receiv-ing conventional management and an observation group receiving enhanced quality control management.Statistical analysis was conducted on potential risk factors,and the work quality of the two groups was compared.Results Univariate and multivariate logistic regression analyses revealed that potential risk factors included insufficient professional competence of staff,deficiencies in the management system,infrequent departmental training,and a noisy work environment(P＜0.05).The observation group showed significantly higher qualified rates in various indicators,such as biological monitoring,compared to the control group(P＜0.05).Furthermore,the observation group experienced significantly fewer risk events than the control group(P＜0.05),indicating a statistically significant difference.Conclusion The primary potential risk factors in disinfection supply rooms in-clude systemic issues,staff quality,work environment,and training frequency.Addressing these factors by strengthening quality control management can improve staff professional competence,basic quality,and work attitude,thereby enhancing management quality,reducing the likelihood of risk events,and ultimately improving management safety.

Objective:To study the growth inhibition of Thunberg Fritillary extract on non-small cell lung cancer.Methods:The Thunberg Fritillary extract was prepared and characterized by UPLC-QE/MS.Replicated Lewis lung carcinoma ectopic tumor-bear-ing mouse model,yeast injection induced acute inflammation,compared the effect of Thunberg Fritillary extract combination with acute inflammation on the growth,tumor volume and tumor suppression rate of Lewis lung carcinoma mice,and determine the content of inflammatory factors by the flow CBA method(IL-6,IL-1β,IL-1α,IL-10,IL-27,IL-17A,IL-12p70,IL-23,TNF-α,IFN-γ,IFN-β,GM-CSF,MCP-1).Results:The inhibition of Lewis lung carcinoma mice was similar to that of cisplatin alone,and the tumor suppression rate was 35%;the tumor suppression rate of Thunberg Fritillary extract combined with acute inflammatory stimulation of yeast was 62%,1.8 times that of cisplatin alone.The decrease in the expressions of cytokines IL-23,MCP-1 after acute inflammatory stimulation in yeast was associated with tumor suppression;while the increased expressions of IL-6,IL-1β,IL-1α,IL-10,IL-27,IL-17A,IL-12p70,TNF-α,IFN-γ,IFN-β and GM-CSF cytokines were associated with tumor suppression.Conclusion:The Thun-berg Fritillary extract combination with acute inflammation can play a positive role against non-small cell lung cancer,which will pro-vide new research ideas and methods for the prevention and treatment of non-small cell lung cancer.

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.

Objective:To investigate the effect of TCF1+CD8+T cells on the prognosis of nasopharyngeal carcinoma(NPC)pa-tients undergoing immunotherapy.Methods:A retrospective study was conducted on 108 NPC patients admitted to Anyang Cancer Hospital from January 2018 to April 2020.Pathological findings and clinical data were collected,and multiple immunofluorescence staining was utilized to detect TCF1+CD8+T cell levels in tumor tissue.Cell counts and the proportion of positive cells were measured,and the optimal cut-off value was determined using a time-dependent receiver operating characteristic(ROC)curve.Patients were clas-sified into high expression group and low expression group according to the cut-off value.The general information and survival status were compared between two groups,and univariate and multivariate Cox regression analysis were performed to screen the influencing factors of patient prognosis.Results:The optimal cut-off value was determined using ROC curve.A cut-off value of 0.34%for TCF1+CD8+T cell area under the curve(AUC)=0.653 resulted in a sensitivity of 73.5%and a specificity of 43.7%.Based on the opti-mal cut-off value,33 patients with TCF1+CD8+T cell beyond 0.34%were included in high expression group,and the remaining 75 pa-tients were included in low expression group.The proportion of TCF1+CD8+T cells was(0.43±0.09)%in high expression group and(0.21±0.08)%in low expression group.Comparison of general data yielded that the gender,age,body mass index(BMI),clinical stage,and pathological classification showed no statistical difference between two groups(P>0.05),while the percentage of patients with T stage of T3 and T4,N stage of N2 and N3,and M stage of M1 in high expression group were smaller than that in low expression group(P<0.05).The 24-month survival rate was 81.82%(27/33)in high expression group and 74.67%(56/75)in low expression group,with no statistical difference between two groups(χ2=0.658 8,P=0.417 0).Univariate and multivariate Cox regression analysis found that T stage,N stage and M stage were independent risk factors affecting the prognosis of patients,and TCF1+CD8+T expression was a protective factor.ROC curve indicated that the AUC,sensitivity and specificity of TCF1+CD8+T expression in predicting death of NPC patients undergoing immunotherapy was 0.674,69.7%and 65.3%,while the AUC,sensitivity and specificity of TCF1+CD8+T ex-pression combined with TNM stage in predicting death was 0.809,75.8%and 84.0%,respectively.Conclusion:NPC patients with higher proportion of TCF1+CD8+T cells and lower TNM stage have higher 24-month survival rate after immunotherapy,and the above parameters are of great predictive value for patient prognosis.

Background::The prevalence of type 2 diabetes mellitus (T2DM) has been rapidly growing in Chinese populations in recent decades, and the shift in eating habits is a key contributing factor to this increase. Eating out of home (EOH) is one of the major shifts in eating habits during this period. However, the influence of EOH on the incidence of T2DM among Chinese urban workers is unknown.Methods::The cross-sectional study involved an analysis of 13,904 urban workers recruited from 11 health examination centers in the major cities of China to explore the relationship between EOH and T2DM between 2013 September and 2016 March.Results::Average weekly EOH frequency ≥10 times was positively associated with increased incidence of T2DM in the sampled population (OR: 1.31 [1.11-1.54], p < 0.01), most notably in participants ≤45 years old (OR: 1.41[1.11-1.80], p < 0.01]) and in males (OR:1.26 [1.06-1.51], p < 0.01). An EOH frequency of 5 times/week appears as a threshold for a significant increase in the odds of T2DM. Weekly EOH frequency ≥5 times was associated with increased odds of T2DM in a dose-response manner in the total population and almost all subgroups ( poverall association < 0.05 and pnonlinearity ≤ 0.05). Conclusion::This study showed that a frequency of EOH (≥5 times/week) was associated with a frequency-dependent increase in the odds of T2DM urban workers in China. More nutrition promotion is needed to improve the eating behavior of Chinese urban workers to reduce T2DM risk.

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.

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.