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 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.

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.

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 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.

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.

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 study the distribution characteristics of current patients with Kashin-Beck disease (KBD) in Molidawa Daur Autonomous Banner (referred to as Morin Banner), and provide suggestions for service management.Methods:Information of KBD current patients in Morin Banner was collected from January 1, 2018 to June 30, 2024 using the "KBD Current Patient Survey System" provided by the Center for Endemic Disease Control, Chinese Center for Disease Control and Prevention. A descriptive study method was used to analyze the basic information and clinical data of current patients.Results:As of June 30, 2024, a total of 6 223 KBD current patients were reported in Morin Banner, and the patients were distributed in 15 townships (towns). There was a statistically significant difference in the prevalence rate of KBD among different townships (towns, χ 2 = 3 069.01, P < 0.001). The minimum age of the KBD current patients was 27 years old, and the maximum was 98 years old, mainly concentrated in the age range of 45 - 74 years old, accounting for 95.7% (5 954/6 223). There was a significant difference in the prevalence rate of KBD among different age groups (χ 2 = 5 912.76, P < 0.001). The male to female ratio was 1.00∶1.14 (2 910 ∶ 3 313), and there was a statistically significant difference in prevalence rate of KBD between genders(χ 2 = 44.38, P < 0.001). The KBD current patients mainly had a primary school education, married, and farmers, accounting for 59.2% (3 685/6 223), 89.8% (5 590/6 223), 93.2% (5 802/6 223), respectively; and the clinical grading of patients is mainly degree Ⅰ. There was a statistically significant difference in the rate of limb disability among patients with different clinical grades (χ 2 = 64.26, P < 0.001). The rate of limb disability in males was higher than that in females (χ 2 = 10.36, P = 0.001). Conclusions:The KBD current patients in Morin Banner are distributed in various township (town), with middle-aged and elderly famers being the main ones. It is necessary to strengthen monitoring of KBD, and pay attention to personalized treatment and management of KBD current patients.

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 explore the distribution characteristics of HBsAg levels in treatment-na?ve and treatment-experienced patients with chronic hepatitis B (CHB) in China.Methods:Data were obtained from the China Registry of Hepatitis B (CR-HepB) platform from the establishment of the platform to April 11, 2024. Patients with CHB who were treatment-na?ve and treatment-experienced with nucleos(t)ide analogs (NAs) were included. Relevant clinical data were collected. The distribution of hepatitis B surface antigen (HBsAg) status, as well as the levels in populations of different age groups after different antiviral treatment durations, were retrospectively analyzed. Normally and non-normally distributed measured data were represented by Mean± SD, and M( Q1, Q3). Results:A total of 13 505 treatment-na?ve patients and 6 390 treatment-experienced patients were included in the analysis. The proportions of treatment-na?ve patients with HBsAg<100, <500, and <1 500 IU/mL were 10.51%, 28.47%, and 46.85%, and the corresponding proportions of treatment-experienced patients were 12.88%, 29.84%, and 52.07%. The proportions of treatment-na?ve patients with HBsAg levels≥1 500, ≥3 000, and≥8 000 IU/mL were 53.15%, 38.17%, and 15.62%, and the corresponding proportions of treatment-experienced patients were 47.93%, 31.77%, and 10.39%. HBsAg level showed a trend of gradual decrease with the increase of antiviral treatment time. The proportion of treatment-experienced patients with HBsAg<100 IU/mL increased from 12.73% when the treatment duration was less than three years to 26.92% when the treatment duration was≥10 years, while the proportion of patients with HBsAg levels≥3 000 IU/mL or≥8 000 IU/mL decreased from 34.66% to 23.08% and from 12.19% to 5.77%, respectively. The proportion of patients with HBsAg<100, <500, and<1 500 IU/mL increased with age, while the proportion of patients with HBsAg≥1 500, ≥3 000, and ≥8 000 IU/mL decreased sequentially.Conclusions:The CR-HepB platform provides a basis for clarifying the serum HBsAg levels in treatment-na?ve and treatment-experienced CHB patients in China. The HBsAg status indicates that with a prolonged antiviral treatment duration, there is a gradual decline trend in HBsAg level.