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 protective effect of liraglutide against myocardial ischemia-reperfusion injury(MIRI)in rats and its relationship with pyroptosis.Methods Forty SPF-grade male SD rats were randomly divided into five groups:the sham operation group(Sham group),the MIRI model group(I/R group),the pyroptosis inhibitor group(Dis+I/R group),the liraglutide group(Lir+I/R group),and li-raglutide+pyroptosis inhibitor group(Lir+Dis+I/R group),with 8 rats in each group.Except for the Sham group,MIRI models were established in all other groups,followed by corresponding treatments.Myocardial infarct size was measured by 2,3,5-triphenyltetrazolium chloride(TTC)staining.Superoxide dismutase(SOD)activity and malondialdehyde(MDA)level were detected using assay kits.Serum levels of nucleotide-binding oligomerization domain-like receptor protein 3(NLRP3)and IL-1β were measured by ELISA.Protein expression of apoptosis-associated speck-like protein containing a CARD(ASC),caspase-1,and cleaved N-ter-minal gasdermin D(GSDMD-N)was detected by Western blot.Results Compared with the I/R group,myo-cardial infarct size was reduced in the Lir+I/R and Dis+I/R groups(P＜0.05),with a further reduction in the Lir+Dis+I/R group compared to the Lir+I/R group(P＜0.05).SOD activity increased and MDA levels decreased in the Lir+I/R and Dis+I/R groups compared to the I/R group(P＜0.05),while SOD activity was further elevated in the Lir+Dis+I/R group compared to the Dis+I/R group and Lir+I/R group(P＜0.05).NLRP3 and IL-1β protein expression levels were reduced in the Lir+I/R and Dis+I/R groups compare to the I/R group(P＜0.05),and IL-1β expression level was further reduced in the Lir+Dis+I/R group com-pared to the Dis+I/R group and Lir+I/R group(P＜0.05).The protein expression levels of ASC and GSD-MD-N in the Lir+I/R group were lower than those in the I/R group(P＜0.05);the protein expression levels of ASC,caspase-1 and GSDMD-N in the Dis+I/R group were lower than those in the I/R group(P＜0.05).ASC expression level further reduced in the Lir+Dis+I/R group compared to both the Dis+I/R and Lir+I/R groups(P＜0.05).Conclusion Liraglutide exerts a protective effect in MIRI,potentially by inhibi-ting pyroptosis.

Objective:To investigate the expression of squamous cell heat shock protein 53(CRNN)in esophageal squamous cell carcinoma(ESCC),and toevaluate its impact on the biological behavior of ESCC cells Eca9706.Methods:Immunohistochemical method was used to detect the expression of CRNN protein in 93 ESCC tissues and 101 normal esophageal epithelial tissues adjacent to cancer,and the associations of CRNN expression levels with the clinical pathological characteristics and survival prognosis of ESCC patients were analyzed.Receiver operating characteristic(ROC)curve was used to analyze the predictive performance of CRNN expression level on ESCC.The Eca9706 cells were divided into control group and CRNN group(overexpression of CRNN).Cell counting kit-8(CCK-8)assay was used to detect the proliferation activities of Eca9706 cells in two groups;Transwell chamber assay was used to detect the numbers of migration cells of Eca9706 cells in two groups;plate clone formation assay was used to assess the numbers of clone formation of Eca9706 cells in two groups;flow cytometry was used to detect the apoptotic rates of Eca9706 cells in two groups.Results:Compared with adjacent normal esophageal epithelial tissue,the expression intensity of CRNN protein in ESCC tissue was significantly decreased(x2=23.476,P＜0.001).The downregulation of CRNN protein expression in ESCC patients was associated with tumor location(x2=5.353,P=0.021)and histological grade(x2=4.434,P=0.035),but not with age(x2=0.102,P=0.750),gender(x2=0.050,P=0.822),tumor stage(x2=0.047,P=0.828)or lymph node metastasis(x2=0.553,P=0.457).Survival analysis showed that ESCC patients in high expression of CRNN protein group had better prognosis than those in low expression of CRNN protein group(P=0.013).Univariable Cox proportional hazards regression analysis showed the associations between overall survival rate in ESCC patients and the expression level of CRNN protein[hazard ratio(HR)=0.198,95％confidence interval(CI):0.047-0.842,P=0.028]and tumor stage(HR=2.479,95％CI:1.247-4.929,P=0.010).Multivariable Cox regression analysis showed that the expression level of CRNN protein(HR=0.213,95％CI:0.050-0.895,P=0.035)and tumor stage(HR=2.391,95％CI:1.198-4.772,P=0.013)were independent factors for the prognosis of ESCC.Compared with control group,the proliferation activity of cells in CRNN group was significantly decreased(P=0.004),the number of clone formation was decreased(P=0.002),the number of migration cells was decreased(P=0.002),and the apoptotic rate was significantly increased(P=0.006).Conclusion:Low expression level of CRNN protein suggests poor prognosis for the ESCC patients.Overexpression of CRNN may inhibit the proliferation,migration and invasion abilities of ESCC cells,and promote their apoptosis.

Objective:To discuss the expression of Rh family C glycoprotein(RHCG)in the esophageal squamous cell carcinoma(ESCC)tissue and its effect on the malignant biological behavior of ESCC cells,and to clarify the value of RHCG as a diagnostic and prognostic marker for the ESCC patients.Methods:A total of 143 ESCC tissue samples and 105 adjacent normal tissue samples were collected.Using immunohistochemical staining method,141 ESCC samples were divided into two groups:RHCG low expression group(immunohistochemistry score≤6)and RHCG high expression group(immunohistochemistry score>6).Immunohistochemical method was used to detect the RHCG protein expression in 143 ESCC tissues and 105 normal tissues,and the relationship between the clinicopathological characteristics of the ESCC patients was analyzed.Receiver operating characteristic(ROC)curve and Kaplan-Meier survival analysis were used to evaluate the value of RHCG in diagnosis and prognosis of the ESCC patients;univariate and multivariate COX regression analysis were used to determine the independent risk factors affecting the prognosis of the ESCC patients.Gene Expression Profiling Interactive Analysis(GEPIA2)database was used to analyze the expression of RHCG mRNA in various tumor tissues.The ESCC TE-1 cells were cultured and transfected in to 6-well cell culture plates with different Lipofectamine2000∶RHCG ratios;the cells in RHCG transfection group were transfected with weights of 2.0,2.5,and 3.0 μg for 24 and 48 h,respectively,and the cells in NC group transfected with empty vector as control.Western blotting method was used to detect the RHCG protein expression level in the TE-1 cells in various groups after transfection at different concentrations and verify the optimal transfection conditions;cell counting kit-8(CCK-8)assay was used to detect the proliferation activities of the TE-1 cells;plate clone formation assay was used to detect the colony formation numbers of the TE-1 cells;Transwell chamber assay was used to detect the numbers of migrating TE-1 cells.Results:Compared with adjacent normal tissue,the RHCG gene expression level in various cancer tissues including ESCC,glioblastoma multiforme,and head and neck squamous cell carcinoma was significantly decreased(P<0.05).RHCG protein was mainly located on the cell membrane of normal esophageal squamous epithelial cells;the RHCG protein expression intensity in ESCC tissues was lower than that in adjacent normal esophageal tissue(χ2=109.373,P<0.001),and the patients in RHCG low expression group had poorer differentiation than those in RHCG high expression group(P=0.041).The area under the curve(AUC)value of RHCG for diagnosing ESCC was 0.86,with sensitivity and specificity of 95.1%and 75.0%,respectively;the Kaplan-Meier survival analysis results showed that compared with high RHCG expression group,the patients in low RHCG expression group had shorter survival time and poorer prognosis[harard ratio(HR)=0.269,95%confidence interval(CI):0.113-0.639,P=0.020];the COX regression analysis results showed that low RHCG expression could serve as an independent risk factor affecting the prognosis of ESCC[HR=4.569,95%CI=1.315-15.877,P=0.017)].The Western blotting results verified that the optimal transfection condition was 3.0 μg RHCG plasmid for 48 h,at which time RHCG overexpression was optimal and RHCG protein expression level was highest.The CCK-8 assay results showed that compared with control group,the proliferation activity in RHCG overexpression group was decreased on the 4th day after cell seeding(P<0.001).In the TE-1 cells,the colony formation number of the TE-1 cells in RHCG over-expression group was lower than that in control group(t=17.70,P<0.001).The Transwell chamber assay results showed that compared with control group,the number of migrating cells in RHCG over-expression group was decreased(t=23.74,P<0.001).Conclusion:RHCG expression is decreased in ESCC tissues and associated with poor prognosis in ESCC patients;overexpression of RHCG can inhibit the proliferation and migration of the TE-1 cells,providing a theoretical basis for RHCG as a novel diagnostic and prognostic marker and therapeutic target for ESCC.

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 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.

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 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.

Liver fibrosis is a common pathological process associated with multiple chronic liver diseases. Recent advancements have significantly improved the non-invasive assessment of liver fibrosis. This article focuses on the exploration of hot topics, namely how noninvasive indicators can evaluate liver fibrosis reversal and predict clinical outcomes. Concurrently, it indicates that attention should be paid to the dynamic changes of noninvasive indicators, and population screening efforts should be strengthened to achieve early diagnosis and treatment of liver fibrosis so as to improve long-term clinical outcomes.

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.