Objective To investigate the distribution and antimicrobial resistance profiles of clinically isolated Haemophilus influenzae and Moraxella catarrhalis in hospitals across China from 2015 to 2021,and provide evidence for rational use of antimicrobial agents.Methods Data of H.influenzae and M.catarrhalis strains isolated from 2015 to 2021 in CHINET program were collected for analysis,and antimicrobial susceptibility testing was performed by disc diffusion method or automated systems according to the uniform protocol of CHINET.The results were interpreted according to the CLSI breakpoints in 2022.Beta-lactamases was detected by using nitrocefin disk.Results From 2015 to 2021,a total of 43 642 strains of Haemophilus species were isolated,accounting for 2.91％of the total clinical isolates and 4.07％of Gram-negative bacteria in CHINET program.Among the 40 437 strains of H.influenzae,66.89％were isolated from children and 33.11％were isolated from adults.More than 90％of the H.influenzae strains were isolated from respiratory tract specimens.The prevalence of β-lactamase was 53.79％in H.influenzae strains.The H.influenzae strains isolated from children showed higher resistance rate than the strains isolated from adults.Overall,779 strains of H.influenzae did not produce β-lactamase but were resistant to ampicillin(BLNAR).Beta-lactamase-producing strains showed significantly higher resistance rates to these antimicrobial agents than the β-lactamase-nonproducing strains.Of the 16 191 M.catarrhalis strains,80.06％were isolated from children and 19.94％isolated from adults.M.catarrhalis strains were mostly susceptible to both amoxicillin-clavulanic acid and cefuroxime,evidenced by resistance rate lower than 2.0％.Conclusions The emergence of antibiotic-resistant H.influenzae due to β-lactamase production poses a challenge for clinical anti-infective treatment.Therefore,it is very important to implement antibiotic resistance surveillance for H.influenzae and guide rational antibiotic use.All local clinical microbiology laboratories should actively improve antibiotic susceptibility testing and strengthen antibiotic resistance surveillance for H.influenzae.

Objective To investigate the distribution and antimicrobial resistance profiles of clinically isolated Haemophilus influenzae and Moraxella catarrhalis in hospitals across China from 2015 to 2021,and provide evidence for rational use of antimicrobial agents.Methods Data of H.influenzae and M.catarrhalis strains isolated from 2015 to 2021 in CHINET program were collected for analysis,and antimicrobial susceptibility testing was performed by disc diffusion method or automated systems according to the uniform protocol of CHINET.The results were interpreted according to the CLSI breakpoints in 2022.Beta-lactamases was detected by using nitrocefin disk.Results From 2015 to 2021,a total of 43 642 strains of Haemophilus species were isolated,accounting for 2.91％of the total clinical isolates and 4.07％of Gram-negative bacteria in CHINET program.Among the 40 437 strains of H.influenzae,66.89％were isolated from children and 33.11％were isolated from adults.More than 90％of the H.influenzae strains were isolated from respiratory tract specimens.The prevalence of β-lactamase was 53.79％in H.influenzae strains.The H.influenzae strains isolated from children showed higher resistance rate than the strains isolated from adults.Overall,779 strains of H.influenzae did not produce β-lactamase but were resistant to ampicillin(BLNAR).Beta-lactamase-producing strains showed significantly higher resistance rates to these antimicrobial agents than the β-lactamase-nonproducing strains.Of the 16 191 M.catarrhalis strains,80.06％were isolated from children and 19.94％isolated from adults.M.catarrhalis strains were mostly susceptible to both amoxicillin-clavulanic acid and cefuroxime,evidenced by resistance rate lower than 2.0％.Conclusions The emergence of antibiotic-resistant H.influenzae due to β-lactamase production poses a challenge for clinical anti-infective treatment.Therefore,it is very important to implement antibiotic resistance surveillance for H.influenzae and guide rational antibiotic use.All local clinical microbiology laboratories should actively improve antibiotic susceptibility testing and strengthen antibiotic resistance surveillance for H.influenzae.

Aim To investigate the promotive effects and mechanisms of the combined use of brucine(Bru)and lumbrokinase(LK),active ingredient derived from Longma formula,in promoting chondrocyte proliferation via the Wnt/β-catenin signaling pathway.Methods The extracted primary rat chondrocytes were divided in-to the following groups:Control group,Bru,LK alone group,and Bro+LK combination group.The optimal drug concentration and intervention time were deter-mined using CCK-8 assay,followed by cell proliferation validation through EdU and phalloidin staining.The expression levels of collagen Ⅱ,aggrecan and SRY-re-lated high-mobility group box gene 9(SOX9)in chon-drocytes following intervention with the combination of Bru and LK were detected by Western blotting.Addi-tionally,the regulatory effects of these proteins on the Wnt/β-catenin signaling pathway were also investiga-ted.Results The optimal combination concentration of Longma formula active ingredients(Bru 0.025 mg·L-1+LK 5 mg·L-1)significantly enhanced chondro-cyte viability compared to control,Bru,or LK alone at 48 h.This combination increased the S-phase ratio,promoted the aggregation of intracellular actin fila-ments,and upregulated the expression of collagen Ⅱ and aggrecan.Furthermore,it activated the Wnt/β-catenin pathway,leading to increased SOX9 expres-sion.Conclusions The optimal combination of Bru and LK(Bru 0.025 mg·L-1+LK 5 mg·L-1)de-rived from Longma formula significantly maintains chondrocyte phenotype and promotes cellular prolifera-tion through the activation of the Wnt/β-catenin signa-ling pathway,which subsequently upregulates the downstream target SOX9.

Objective To explore the possibility and genetic identification method of constructing a hepatocyte-specific NLRP3 gene knockout mouse model by using Cre-LoxP system gene knockout technology.Methods Phase one:mice specifically expressing the albumin promoter-Cre(AlbCre)recombinase in hepatocytes were mated with NLRP3flox/flox mice,and the hepatocyte-specific NLRP3 gene knockout mice with the genotype of NLRP3flox/flox/AlbCre+/-(hepatocyte NLRP3 knockout group)and the control mice in the same litter with the genotype of NLRP3flox/flox/AlbCre-/-(control group in the same litter)were obtained after two generations of selection and mating.The second stage was the mass reproduction stage.Mating NLRP3flox/flox/AlbCre+/-target mice with NLRP3flox/flox mice could quickly obtain a large number of experimental target mice and control mice in the same litter.The DNA was extracted from the tails of mice after numbering,and the offspring genotype was identified by PCR.qPCR and Western blot were used to detect the mRNA and protein expression levels of NLRP3 gene in the liver tissue.HE staining was used to observe the morphological changes in liver tissues,and serum liver transaminases and inflammatory factors were detected.The changes in body weight,liver-to-body ratio and special circumstances during reproduction and development of mice in the two groups were observed.Results The offspring genotype of the target mice in the F2 generation was consistent with theoretical result of NLRP3flox/flox/AlbCre+/-.The mRNA and protein levels of NLRP3 in liver tissues of mice in the hepatocyte NLRP3 knockout group were significantly lower than those in the control group in the same litter(P<0.05).The mice in the hepatocyte NLRP3 knockout group was not affected in terms of growth,development and reproduction after the NLRP3 gene knockout.There were no statistically significant differences in the body weight,liver-to-body ratio,liver tissue morphology,serum liver transaminase or inflammatory factors between the hepatocyte NLRP3 knockout group and the control group in the same litter(P>0.05).Conclusion The Cre-LoxP gene knockout technology can be used to successfully construct a hepatocyte-specific NLRP3 gene knockout mouse model,providing an important technical support for the next step of studying the function of the NLRP3 gene in the liver at the animal level.

Objective:To explore the pattern of lymph node metastasis and prognosis in locally advanced gastric cancer after neoadjuvant immunotherapy combined with chemotherapy (NICT).Methods:This retrospective study included pathologically confirmed gastric adenocarcinoma (cT3-4aN+) patients who underwent radical resection after ≥2 cycles of PD-1 inhibitor-based chemotherapy with complete postoperative pathology. Exclusions: distant/other metastases, non-R0 resection, Her-2+ with targeted therapy, microsatellite instability, or esophagogastric junction cancer invading >1 cm into lower esophagus. From January 2020 to December 2024, a total of 343 consecutive gastric cancer patients who received NICT treatment were admitted to Tianjin Medical University Cancer Institute and Hospital. According to the above criteria, 324 cases were included in the lymph node metastasis analysis, and 302 cases were included in the survival analysis. The median age of all patients was 58 years, with 245 males (75.6%) and a median body mass index (BMI) of 22.9 kg/m2. There were 170 cases (52.5%) at T3 stage and 154 cases (47.5%) at T4a stage; the median number of cycles of neoadjuvant immunotherapy combined with chemotherapy was 3 cycles. The primary outcome measure was the positive lymph node metastasis rate (number of metastatic cases in the group / total number of dissected cases in the group×100%). A positive lymph node metastasis rate >10% was defined as high metastasis, and <5% as low metastasis. The secondary outcome measures were high-risk factors for lymph node metastasis and influencing factors related to patient prognosis. Lymph node grouping was performed according to the 8th edition of the American Joint Committee on Cancer (AJCC) guidelines. The positive lymph node metastasis rate was statistically analyzed by stratification based on surgical methods (total gastrectomy, proximal gastrectomy, distal gastrectomy). Multivariate analysis of risk factors for lymph node metastasis were performed with logistic regression analysis, and survival analysis were performed with the Kaplan-Meier method and Cox regression model.Results:The postoperative pathological complete response rate (pCR) of all patients was 21.0% (68/324), and the overall positive lymph node metastasis rate was 48.8% (158/324). A total of 150 patients underwent total gastrectomy, 42 underwent proximal gastrectomy, and 132 underwent distal gastrectomy.In the total gastrectomy group: the high metastasis subgroups were No.1 (19.3%, 29 cases), No.2 (14.7%, 22 cases), No.3 (28.0%, 42 cases), No.7 (12.7%, 19 cases), No.8a (16.0%, 24 cases), and No.9 (17.3%, 26 cases); the low metastasis subgroups were No.5 (4.7%, 7 cases), No.10 (3.3%, 5 cases), No.11d (1.3%, 2 cases), and No.12a (4.0%, 6 cases).In the proximal gastrectomy group: the high metastasis subgroups were No.3 (14.3%, 6 cases), No.7 (23.8%, 10 cases), and No.11p (11.9%, 5 cases); the low metastasis subgroups were No.4d (2.4%, 1 case) and No.10 (2.4%, 1 case).In the distal gastrectomy group: the high metastasis subgroups were No.3 (25.8%, 34 cases), No.6 (26.5%, 35 cases), No.7 (11.4%, 15 cases), and No.11p (11.4%, 15 cases); the low metastasis subgroups were No.4sb (3.8%, 5 cases) and No.12a (4.5%, 6 cases).Results of multivariate analysis showed that TRG grade (HR: 5.938, 95%CI: 3.028-11.646, P<0.001) was an independent factor affecting lymph node metastasis in patients with locally advanced gastric cancer after neoadjuvant immunotherapy combined with chemotherapy. The median follow-up time was 26.0 (6.0-54.3) months, and the 3-year overall survival (OS) of all patients was 78.1%. Results of multivariate Cox analysis showed that ypT (HR=1.744, 95%CI: 1.300-2.338, P<0.001), ypN (HR=1.998, 95%CI: 1.503-2.655, P<0.001), and postoperative complications (HR=1.913, 95%CI: 1.111-3.294, P=0.019) were independent factors affecting the overall survival of patients with locally advanced gastric cancer after neoadjuvant immunotherapy combined with chemotherapy. Conclusion:NICT significantly changes the pattern of lymph node metastasis in LAGC. ypT and ypN stages are core indicators for survival prognosis. The necessity of dissection for lymph node groups with a metastasis rate <5% needs to be carefully evaluated.

Aim To investigate the promotive effects and mechanisms of the combined use of brucine(Bru)and lumbrokinase(LK),active ingredient derived from Longma formula,in promoting chondrocyte proliferation via the Wnt/β-catenin signaling pathway.Methods The extracted primary rat chondrocytes were divided in-to the following groups:Control group,Bru,LK alone group,and Bro+LK combination group.The optimal drug concentration and intervention time were deter-mined using CCK-8 assay,followed by cell proliferation validation through EdU and phalloidin staining.The expression levels of collagen Ⅱ,aggrecan and SRY-re-lated high-mobility group box gene 9(SOX9)in chon-drocytes following intervention with the combination of Bru and LK were detected by Western blotting.Addi-tionally,the regulatory effects of these proteins on the Wnt/β-catenin signaling pathway were also investiga-ted.Results The optimal combination concentration of Longma formula active ingredients(Bru 0.025 mg·L-1+LK 5 mg·L-1)significantly enhanced chondro-cyte viability compared to control,Bru,or LK alone at 48 h.This combination increased the S-phase ratio,promoted the aggregation of intracellular actin fila-ments,and upregulated the expression of collagen Ⅱ and aggrecan.Furthermore,it activated the Wnt/β-catenin pathway,leading to increased SOX9 expres-sion.Conclusions The optimal combination of Bru and LK(Bru 0.025 mg·L-1+LK 5 mg·L-1)de-rived from Longma formula significantly maintains chondrocyte phenotype and promotes cellular prolifera-tion through the activation of the Wnt/β-catenin signa-ling pathway,which subsequently upregulates the downstream target SOX9.

Objective To explore the possibility and genetic identification method of constructing a hepatocyte-specific NLRP3 gene knockout mouse model by using Cre-LoxP system gene knockout technology.Methods Phase one:mice specifically expressing the albumin promoter-Cre(AlbCre)recombinase in hepatocytes were mated with NLRP3flox/flox mice,and the hepatocyte-specific NLRP3 gene knockout mice with the genotype of NLRP3flox/flox/AlbCre+/-(hepatocyte NLRP3 knockout group)and the control mice in the same litter with the genotype of NLRP3flox/flox/AlbCre-/-(control group in the same litter)were obtained after two generations of selection and mating.The second stage was the mass reproduction stage.Mating NLRP3flox/flox/AlbCre+/-target mice with NLRP3flox/flox mice could quickly obtain a large number of experimental target mice and control mice in the same litter.The DNA was extracted from the tails of mice after numbering,and the offspring genotype was identified by PCR.qPCR and Western blot were used to detect the mRNA and protein expression levels of NLRP3 gene in the liver tissue.HE staining was used to observe the morphological changes in liver tissues,and serum liver transaminases and inflammatory factors were detected.The changes in body weight,liver-to-body ratio and special circumstances during reproduction and development of mice in the two groups were observed.Results The offspring genotype of the target mice in the F2 generation was consistent with theoretical result of NLRP3flox/flox/AlbCre+/-.The mRNA and protein levels of NLRP3 in liver tissues of mice in the hepatocyte NLRP3 knockout group were significantly lower than those in the control group in the same litter(P<0.05).The mice in the hepatocyte NLRP3 knockout group was not affected in terms of growth,development and reproduction after the NLRP3 gene knockout.There were no statistically significant differences in the body weight,liver-to-body ratio,liver tissue morphology,serum liver transaminase or inflammatory factors between the hepatocyte NLRP3 knockout group and the control group in the same litter(P>0.05).Conclusion The Cre-LoxP gene knockout technology can be used to successfully construct a hepatocyte-specific NLRP3 gene knockout mouse model,providing an important technical support for the next step of studying the function of the NLRP3 gene in the liver at the animal level.

Objective:To explore the pattern of lymph node metastasis and prognosis in locally advanced gastric cancer after neoadjuvant immunotherapy combined with chemotherapy (NICT).Methods:This retrospective study included pathologically confirmed gastric adenocarcinoma (cT3-4aN+) patients who underwent radical resection after ≥2 cycles of PD-1 inhibitor-based chemotherapy with complete postoperative pathology. Exclusions: distant/other metastases, non-R0 resection, Her-2+ with targeted therapy, microsatellite instability, or esophagogastric junction cancer invading >1 cm into lower esophagus. From January 2020 to December 2024, a total of 343 consecutive gastric cancer patients who received NICT treatment were admitted to Tianjin Medical University Cancer Institute and Hospital. According to the above criteria, 324 cases were included in the lymph node metastasis analysis, and 302 cases were included in the survival analysis. The median age of all patients was 58 years, with 245 males (75.6%) and a median body mass index (BMI) of 22.9 kg/m2. There were 170 cases (52.5%) at T3 stage and 154 cases (47.5%) at T4a stage; the median number of cycles of neoadjuvant immunotherapy combined with chemotherapy was 3 cycles. The primary outcome measure was the positive lymph node metastasis rate (number of metastatic cases in the group / total number of dissected cases in the group×100%). A positive lymph node metastasis rate >10% was defined as high metastasis, and <5% as low metastasis. The secondary outcome measures were high-risk factors for lymph node metastasis and influencing factors related to patient prognosis. Lymph node grouping was performed according to the 8th edition of the American Joint Committee on Cancer (AJCC) guidelines. The positive lymph node metastasis rate was statistically analyzed by stratification based on surgical methods (total gastrectomy, proximal gastrectomy, distal gastrectomy). Multivariate analysis of risk factors for lymph node metastasis were performed with logistic regression analysis, and survival analysis were performed with the Kaplan-Meier method and Cox regression model.Results:The postoperative pathological complete response rate (pCR) of all patients was 21.0% (68/324), and the overall positive lymph node metastasis rate was 48.8% (158/324). A total of 150 patients underwent total gastrectomy, 42 underwent proximal gastrectomy, and 132 underwent distal gastrectomy.In the total gastrectomy group: the high metastasis subgroups were No.1 (19.3%, 29 cases), No.2 (14.7%, 22 cases), No.3 (28.0%, 42 cases), No.7 (12.7%, 19 cases), No.8a (16.0%, 24 cases), and No.9 (17.3%, 26 cases); the low metastasis subgroups were No.5 (4.7%, 7 cases), No.10 (3.3%, 5 cases), No.11d (1.3%, 2 cases), and No.12a (4.0%, 6 cases).In the proximal gastrectomy group: the high metastasis subgroups were No.3 (14.3%, 6 cases), No.7 (23.8%, 10 cases), and No.11p (11.9%, 5 cases); the low metastasis subgroups were No.4d (2.4%, 1 case) and No.10 (2.4%, 1 case).In the distal gastrectomy group: the high metastasis subgroups were No.3 (25.8%, 34 cases), No.6 (26.5%, 35 cases), No.7 (11.4%, 15 cases), and No.11p (11.4%, 15 cases); the low metastasis subgroups were No.4sb (3.8%, 5 cases) and No.12a (4.5%, 6 cases).Results of multivariate analysis showed that TRG grade (HR: 5.938, 95%CI: 3.028-11.646, P<0.001) was an independent factor affecting lymph node metastasis in patients with locally advanced gastric cancer after neoadjuvant immunotherapy combined with chemotherapy. The median follow-up time was 26.0 (6.0-54.3) months, and the 3-year overall survival (OS) of all patients was 78.1%. Results of multivariate Cox analysis showed that ypT (HR=1.744, 95%CI: 1.300-2.338, P<0.001), ypN (HR=1.998, 95%CI: 1.503-2.655, P<0.001), and postoperative complications (HR=1.913, 95%CI: 1.111-3.294, P=0.019) were independent factors affecting the overall survival of patients with locally advanced gastric cancer after neoadjuvant immunotherapy combined with chemotherapy. Conclusion:NICT significantly changes the pattern of lymph node metastasis in LAGC. ypT and ypN stages are core indicators for survival prognosis. The necessity of dissection for lymph node groups with a metastasis rate <5% needs to be carefully evaluated.

【Objective】 To compare the killing effects of different concentrations of gentamicin (0, 10, 20, 50, 100, and 200 μg/mL) on uropathogenic Escherichia coli (UPEC) and its cytotoxicities on urinary urothelial cells and inflammatory cells such as macrophages in vitro. 【Methods】 The killing effects of different concentrations of gentamicin on different amounts (10⁸, 10⁷, and 10⁶) of UPEC strain J96 were compared. The cytotoxicities of different concentrations of gentamicin on primary cultured male C57BL/6 mouse renal tubular epithelial cells, mouse macrophages and human bladder epithelial cell line J82 at different time points (2 h and 24 h) were detected by CCK-8 assay. According to the experiments above, we chose appropriate gentamicin concentrations and incubation time in in vitro cell culture experiments to verify J96 adhesion and invasion to mouse renal tubular epithelial cells or phagocytosis and clearance of J96 by mouse macrophages. 【Results】 The killing effect of gentamicin (≥10 μg/mL) on J96 was stronger than that of 1% P/S (P<0.000 1). High concentrations of gentamicin (≥100 μg/mL) could kill up to 10⁸ J96 within 30 min. 50 μg/mL gentamincin treatment for 2 h was cytotoxic for human bladder epithelial cell line J82 (P<0.05). 【Conclusion】 The appropriate concentration and duration of gentamicin treatment for different cells in vitro were determined. Urothelial cells, especially human bladder epithelial cell line J82, were more sensitive to gentamicin.

Objective:To investigate the current situation of the use of transjugular intrahepatic portosystemic shunt (TIPS) for portal hypertension, which should aid the development of TIPS in China.Methods:The China Portal Hypertension Alliance (CHESS) initiated this study that comprehensively investigated the basic situation of TIPS for portal hypertension in China through network research. The survey included the following: the number of surgical cases, main indications, the development of Early-TIPS, TIPS for portal vein cavernous transformation, collateral circulation embolization, intraoperative portal pressure gradient measurement, commonly used stent types, conventional anticoagulation and time, postoperative follow-up, obstacles, and the application of domestic instruments.Results:According to the survey, a total of 13 527 TIPS operations were carried out in 545 hospitals participating in the survey in 2021, and 94.1% of the hospital had the habit of routine follow-up after TIPS. Most hospitals believed that the main indications of TIPS were the control of acute bleeding (42.6%) and the prevention of rebleeding (40.7%). 48.1% of the teams carried out early or priority TIPS, 53.0% of the teams carried out TIPS for the cavernous transformation of the portal vein, and 81.0% chose routine embolization of collateral circulation during operation. Most of them used coils and biological glue as embolic materials, and 78.5% of the team routinely performed intraoperative portal pressure gradient measurements. In selecting TIPS stents, 57.1% of the hospitals woulel choose Viator-specific stents, 57.2% woulel choose conventional anticoagulation after TIPS, and the duration of anticoagulation was between 3-6 months (55.4%). The limitation of TIPS surgery was mainly due to cost (72.3%) and insufficient understanding of doctors in related departments (77.4%). Most teams accepted the domestic instruments used in TIPS (92.7%).Conclusions:This survey shows that TIPS treatment is an essential part of treating portal hypertension in China. The total number of TIPS cases is far from that of patients with portal hypertension. In the future, it is still necessary to popularize TIPS technology and further standardize surgical indications, routine operations, and instrument application.