Objective: To analyze the bioinformatics of domestic Clostridium difficile toxin B(TcdB) and prepare it to provide data support for the development of effective vaccines. Methods: Using bioinformatics software such as Snippy, Blast, Muscle, and the dist.alignment() and hclust() functions in R, 1 355 strains of Clostridium difficile from NCBI GenBank in China were compared and analyzed, and TcdB were grouped. The maximum likelihood tree and phylogenetic tree were beautified and displayed using iTOL. An online bioinformatics analysis website was used to predict and analyze the spatial structure and antigenic epitopes of the two largest subgroups, TcdB1 and TcdB2. The antigen protein TcdB was expressed and purified by prokaryotic system. Results: According to the genotype of toxin B, the 1 355 prevalent strains of Clostridium difficile in China could be roughly divided into 12 subtypes, among which TcdB1 and TcdB2 were the main subtypes, accounting for more than 93.94% of all isolated strains, and about 17.20% of the strains were nontoxigenic or lack TcdB. The antigen epitope prediction of TcdB1 and TcdB2 showed that their antigen epitope distributions were basically the same, and many of them were distributed outside the C-terminal combined repetitive oligopeptides domains. Conclusion A specialized typing system for C. difficile TcdB in China has been established, and its main subtypes have been predicted for antigenic epitopes. The screened TcdB has been expressed for recombinant preparation.

Objective To observe the value of machine learning(ML)models constructed based on pre-treatment clinical and CT features for predicting acute liver function deterioration(ALFD)in hepatocellular carcinoma(HCC)patients after the first time TACE.Methods Totally 320 HCC patients who underwent the first TACE were retrospectively enrolled and divided into training set(n=256)and test set(n=64)at the ratio of 4∶1.ALFD was evaluated according to clinical,laboratory and image findings within 2 weeks after TACE.Univariate analysis was performed to compare clinical baseline data and diameter of HCC on pre-TACE CT in training set,and parameters being statistical different between patients with and without ALFD were used to construct ML models using 9 different ML algorithms.The efficacy of each model for predicting ALFD in test set was evaluated,and the optimal model was selected.The calibration degree and clinical value of the optimal model were assessed in test set,and the contribution of each parameter was analyzed using SHAP method.Results In training set,76 cases were ALFD and 180 cases were non-ALFD,while in test set,18 cases were ALFD and 46 cases were non-ALFD.Among 9 ML models,the sensitivity,specificity,accuracy,area under the curve,F1 value and Kappa value of extreme gradient boosting(XGBoost)model in test set was 85.12%,89.34%,88.08%,0.927,0.811 and 0.725,respectively.XGBoost model was considered as the optimal one,with predicted probability in test set in good agreement with actual probability and high clinical net benefit.The contribution of patients'age,lesion diameter on pre-TACE CT,glutamic-pyruvic transaminase,glutamic-oxaloacetic transaminase and TACE time were all great for XGBoost model.Conclusion XGBoost model based on pre-treatment clinical and CT features could be used to effectively predict ALFD in HCC patients after the first time TACE.

Objective To compare the distribution characteristics of retinal capillary non-perfusion areas(NPAs)across different layers and regions in patients with neovascular glaucoma(NVG)secondary to proliferative diabetic retinop-athy(PDR)versus those with PDR alone through wide-field swept-source optical coherence tomography angiography(SS-OCTA)and to analyze the impacts of NPAs on the development of NVG.Methods This prospective cross-sectional study enrolled 33 patients with PDR(33 eyes,the PDR group)and 30 patients with NVG(30 eyes,the PDR+NVG group)diag-nosed at Affiliated Hospital of Shandong Second Medical University(formerly Weifang Medical University)from January 2022 to June 2023.The fundus examination was performed using SS-OCTA,and the NPA boundaries of the superficial capil-lary plexus(SCP)and deep capillary plexus(DCP)of the retina were manually delimited with the aid of ImageJ.The reti-na was divided based on two methods.Specifically,according to different concentric circles,the retina could be divided in-to the foveal area,parafoveal area,perifoveal area,annulus6-9,annulus9-12,annulus12-retinal boundary;besides,the ret-ina could also be divided into four quadrants(supratemporal,infratemporal,supranasal,and infranasal quadrants)based on the horizontal and vertical lines centered on the macular fovea.Based on that,the NPA area and ischemia index(ISI)in each layer and subdivision of the two groups of patients were counted.Additionally,the NPA and ISI in different concentric circles and different quadrants of the SCP and DCP were compared between the two groups.Moreover,the distribution characteristics of NPAs as well as the effect of NPAs on NVG were analyzed.Results(1)The NPA area and ISI in the DCP were larger than those in the SCP in both groups(all P＜0.001);the NPA area and ISI in the SCP and DCP of patients in the PDR+NVG group were larger than those in the PDR group(all P＜0.001).(2)In the supratemporal,infratemporal,supranasal,and infranasal quadrants,the NPA area and ISI in the SCP and DCP of patients in the PDR+NVG group were larger than those in the PDR group(all P＜0.01).The NPA area in the inferotemporal quadrant was the largest in the SCP and DCP,respectively,within each group(all P＜0.01).(3)The differences in the NPA area and ISI between the two groups were statistically significant in the annulus6-9,annulus9-12,and annulus12-retinal boundary in the SCP and DCP(all P＜0.01).The differences in the NPA area and ISI were statistically significant between different annular subdivisions in the SCP and DCP within each group(all P＜0.001).The multiple comparison results showed that the NPA area and ISI of the annulus12-retinal boundary in the SCP and DCP were larger than those in other annuli in both groups(all P＜0.05).The NPA area and ISI of the annulus9-12 were larger than those of the parafoveal and perifoveal areas;the NPA area and ISI of the annulus6-9 were larger than those of the parafoveal area(all P＜0.05).There was no statistically significant differ-ence in the NPA area and ISI in the remaining annuli(all P＞0.05).(4)The multivariate logistic regression analysis showed that the NPA area and ISI in the subnasal quadrant of the SCP were negatively correlated with the risk of NVG sec-ondary to PDR(P=0.036 and 0.038).The increased NPA area and ISI in the subnasal quadrant of the DCP were risk fac-tors for NVG secondary to PDR,and they may increase the risk of NVG(P=0.029 and 0.028).Conclusion The in-creased NPA area and ISI in the subnasal quadrant of the DCP were risk factors for secondary NVG in patients in the PDR group.

BACKGROUND:Studies have found that massage can reduce blood sugar,promote myogenic factor expression,and increase skeletal muscle content.The extracellular matrix is an important component of skeletal muscle,and association between massage and extracellular matrix and their mechanism of action are still unclear.OBJECTIVE:To explore the effect of massage on extracellular matrix collagen deposition in type 2 diabetic sarcopenia rats.METHODS:Totally 24 Wistar male rats were randomly divided into blank group,model group,and massage group.High-fat diet combined with the streptozotocin method was used to establish a type 2 diabetes mellitus and sarcopenia model.After successful model establishment,the massage group used abdominal massage combined with hind limbs.After 8 weeks of treatment,the fasting blood glucose and serum insulin levels of the rats were measured.The skeletal muscle mass was detected by dual-energy X-ray.The exhaustion time was measured by small animal treadmill.The sliding angle was measured by inclined board test.The pathological changes of skeletal muscle tissue were observed by hematoxylin-eosin staining.The skeletal muscle collagen deposition was observed by Masson staining.The mRNA and protein expressions of type Ⅰ and type Ⅲ collagen in skeletal muscle were detected by qPCR and western blot assay.RESULTS AND CONCLUSION:(1)Compared with the model group,the blood glucose(P＜0.05)and serum insulin(P＜0.01)decreased in the massage group.(2)Compared with the model group,the skeletal muscle mass,running exhaustion time,and the angle of inclined plate experiment were increased in massage group(P＜0.05).(3)Compared with the model group,the skeletal muscles of the massage group were arranged neatly,muscle atrophy was improved,and collagen fiber deposition was reduced.(4)Compared with the model group,the expression levels of type Ⅰ and type Ⅲ collagen mRNA and protein in skeletal muscle were decreased in the massage group(P＜0.05).(5)The results suggest that massage can enhance insulin sensitivity,lower blood sugar,improve skeletal muscle mass,strength and function,and diminish collagen deposition in rats with type 2 diabetes,and may be a potential target for massage to exert its therapeutic effects.

Objective:To investigate changes in the density and spatial distribution of CD69 + T cells within hepatocellular carcinoma tissues following immune checkpoint blockade (ICB) therapy, and to explore their correlation with tumor infiltrating immune cell. Methods:Tumor specimens were collected from 12 patients with hepatocellular carcinoma who were admitted to the Department of Hepatobiliary and Pancreatic Surgery of People's Hospital of Zhengzhou University and the First Affiliated Hospital of Zhengzhou University from July 2023 to July 2024. There were 10 males and 2 females, aged (58.5±5.6) years. Of the 12 patients, 6 cases underwent radical surgery directly and 6 underwent radical surgery after immunotherapy. The maximum tumor diameter and tumor volume of the immunotherapy group were measured by imaging. The density and distribution of immune cells such as CD8 + CD69 + T, CD4 + CD69 + T and programmed death-1 (PD-1) were detected by immunohistochemistry and immunofluorescence. The number of immune cells around the target cells was calculated to evaluate the effective score, and the intercellular distance was measured to evaluate the intercellular interaction. Results:The maximum tumor diameter and tumor volume of 6 patients after immunotherapy were lower than before treatment, and the differences were statistically significant (all P<0.05). The density of PD1 + cells in the immunotherapy group was 36.6 (25.9, 55.9) cells/mm 2, which was less than that in the control group 53.9 (38.3, 84.5) cells/mm 2, and the difference was statistically significant ( Z=-2.66, P=0.008). In the control group, the number of CD8 + CD69 + T cells was positively correlated with CD8 + PD1 + T cells and CD8 + PD1 + CD103 + T cells, and the correlation coefficients were 0.42 and 0.40, respectively ( P=0.001, 0.002). The effective scores of CD8 + CD69 + T cells and CD8 + PD1 + T, CD4 + CD103 + T, CD4 + PD1 + CD103 + T and CD8 + PD1 + CD103 + T cells in the above three areas in the immunotherapy group were lower than those in the control group, with statistical significance (all P<0.05). The distance between CD8 + CD69 + T and CD4 + CD69 + CD103 + T cells in the interface area of the control group was closer than that of the immunotherapy group, and the difference was statistically significant ( t=2.67, P=0.009). Conclusion:After immunotherapy in hepatocellular carcinoma patients, PD-1+ cells and immune cells around CD8 + CD69 + T cells decreased, and this change was related to the distance between CD8 + CD103 + T cells.

Objective:To analyze the risk factors for clinically relevant delayed gastric emptying (CR-DGE) following laparoscopic pancreaticoduodenectomy (LPD) and to develop a model to predict the postoperative CR-DGE after LPD using the machine-learning approach with multi-model comparison.Methods:Clinical data of 278 patients with tumors located in the pancreatic head and periampullary region undergoing LPD at People’s Hospital of Zhengzhou University from January 2019 to December 2023 were retrospectively analyzed, including 167 males and 111 females, aged 59 (53, 66) years. According to the occurrence of DGE, patients were divided into the CR-DGE group ( n=94) and the non-CR-DGE group ( n=184). Main clinical characteristics were compared between the groups, including pancreatic duct diameter, intraoperative blood loss and operative time. The perioperative indicators were selected using the least absolute shrinkage and selection operator (LASSO) algorithm. Following variable selection, 278 patients were allocated into a training set ( n=222) and a validation set ( n=56) in an 8∶2 ratio. Eight machine learning models were selected to model the training set: random forest, adaptive boosting, light gradient boosting, multilayer perceptron, support vector machine, K-nearest neighbor algorithm, decision tree and complementary set plain bayes. The area under the curve (AUC) of receiver operating characteristic curve of the validation set was utilized to identify the optimal model. The predictive performance of the optimal model was evaluated using calibration plots and decision curve analysis (DCA). The contribution of each feature to the prediction is assessed using Shapley additive explanation (SHAP). Results:Univariate analysis showed statistically significant differences between the CR-DGE and non-CR-DGE groups in terms of age [66(62, 69) years vs. 56(51, 60), years], diabetes [42.6%(40/94) vs. 11.4%(21/184)], level of fibrinogen [3.43(2.74, 4.18) g/L vs. 3.84(3.19, 4.68) g/L], pancreatic duct diameter [2.00(1.50, 2.70) mm vs. 3.40(1.60, 5.00) mm], intraoperative blood loss [300(200, 600) ml vs. 200(150, 300) ml], operative time [472(430, 502) min vs. 430(365, 475) min], clinically relevant postoperative pancreatic fistula [34.0%(32/94) vs. 3.8%(7/184)], abdominal fluid accumulation [46.8%(44/94) vs. 12.5%(23/184)], postoperative hemorrhage [20.2%(19/94) vs. 3.3%(6/184)], abdominal infection [28.7%(27/94) vs. 11.4% (21/184)] and duration of postoperative gastrointestinal decompression [4.00 (2.00, 6.00) d vs. 3.00 (2.00, 5.00) d] (all P<0.05). The eleven variables selected via LASSO were incorporated into each of the eight machine learning models. Results demonstrated that the random forest model achieved the highest performance in the validation set, with an AUC of 0.894 (95% CI: 0.800-0.985), accuracy of 0.820 and sensitivity of 0.606. Calibration plots and DCA confirmed the robustness of the random forest model. SHAP analysis indicated that age, pancreatic duct diameter and preoperative aspartate aminotransferase were important predictors in the random forest model. Conclusion:The random forest model developed in this study demonstrated a good predictive performance for CR-DGE after LPD and may assist in the early identification of high-risk patients in clinical practice.

Objective To investigate the differences in neurite density index(NDI),neurite orientation dispersion index(ODI),and volume fraction of isotropic water molecule(Viso)of subcutaneous white matter fiber tracts in patients with cerebral small ves-sel disease(CSVD),and the effect of the glymphatic system(GS)on NDI,ODI,and Viso values of white matter fiber tracts in patients with CSVD.Methods A total of 69 CSVD patients(CSVD group)were retrospectively selected.All patients underwent conventional plain MRI scans[3D-T1,T2WI,3D-T2 fluid attenuated inversion recovery(FLAIR)],spin echo-echo planar imaging(SE-EPI),and susceptibility weighted imaging(SWI)scans.The NDI,ODI,and Viso values of 29 white matter fiber tracts in the brain were meas-ured using post-processing software.Thirty-five healthy volunteers were recruited as the control group,and the independent sample t test was used to compare the differences of NDI,ODI,and Viso values between the two groups.Multiple linear regression was used to evaluate the relationship between along the perivascular space(ALPS)index and NDI,ODI,and Viso values of 29 white matter fiber tracts in CSVD patients.Results Compared with control group,CSVD group showed a statistically significant increase in Viso values in 26 white matter fiber tracts and decrease in NDI values in 14 white matter fiber tracts(PFDR<0.05).In multiple linear regression,the decrease of ALPS index in CSVD patients was found to be statistically different from that of NDI values in certain white matter fiber tracts(PFDR<0.05)and primarily located in the frontal and temporal lobes.Conclusion Neurite orientation dispersion and density imaging(NODDI)technology is more effective in identifying the biological differences,which impact the integrity of white matter fiber tracts in patients with CSVD.Furthermore,it reflects the pathophysiological changes of the brain GS on different white matter fiber tracts in CSVD patients.This provides valuable insights for recognizing the clinical manifestations of CSVD and developing appropri-ate treatment plans.

Objective:To evaluate the safety and feasibility of continuous lamellar pancreaticojejunostomy in laparoscopic pancreaticoduodenectomy (LPD).Methods:Clinical data of 313 patients with periampullary tumors undergoing LPD at the People's Hospital of Zhengzhou University from January 2017 to December 2021 were retrospectively analyzed, including 163 males and 150 females, aged (62.5±6.7) years. Patients were divided into two groups according to the type of pancreaticojejunostomy. Patients undergoing continuous lamellar pancreaticojejunostomy were included in the study group ( n=162) and those undergoing traditional pancreaticojejunostomy were included in the control group ( n=151). Clinical data such as gender, age, level of carbohydrate antigen 199 (CA199), operation time, postoperative hospital stay, and complications were collected. In order to reduce the baseline differences between groups, propensity score matching (PSM) was used. Results:There were significant differences in age and preoperative CA199 between the study group and the control group before PSM (all P<0.05). After PSM for age and CA199, 156 cases were successfully matched, with 78 cases in both the study and control group. Preoperative data were conparable between the two groups after PSM (all P>0.05). The drainage time [16.0 (14.0, 21.0)d vs. 18.0(15.8, 22.0)d] and postoperative hospital stay [20.0(15.0, 24.0)d vs. 21.5(20.0, 25.0)d] were shorter in the study group (all P<0.05). The incidence of grade B/C postoperative pancreatic fistula (POPF) in the study group was lower than that in the control group [7.7%(6/78) vs. 20.5%(16/78), P<0.05]. Conclusion:Compared with traditional pancreaticojejunostomy, continuous lamellar pancreaticojejunostomy could be safe and feasible, which might reduce the incidence of grade B/C POPF and enhance postoperative recovery.

Objective:To compare the efficacy of surgical resection and radiofrequency ablation (RFA) treatment for China liver cancer staging (CNLC) Ia hepatocellular carcinoma (HCC) at difficult-to-reach locations.Methods:A retrospective analysis was conducted on the clinical data of 114 patients with CNLC Ia HCC at Ⅶ、Ⅷ、Ⅳb or Ⅰ segments that were difficult-to-reach locations who were admitted to People's Hospital of Zhengzhou University from December 2018 to December 2023. Among the patients, 85 were males and 29 were females, aged (58.1±1.0) years. The patients were divided into two groups: a RFA group with 31 cases and a surgical resection group with 83 cases. Compare the levels of alanine transaminase (ALT) and aspartate transaminase (AST) before and after surgery, the surgical time, intraoperative blood loss, postoperative hospital stay, postoperative complications, recurrence free survival rate, and cumulative survival rate between the two groups.Results:The comparison of age, gender, ALT, and AST between the two groups showed no statistically significant differences (all P>0.05). The differences in ALT and AST levels before and after surgery in the RFA group were (134.8±38.7) U/L and (195.1±53.9) U/L, respectively, which were significantly lower than those in the surgical resection group [(226.8±17.9) U/L and (229.5±16.2) U/L] ( t=-2.45 and -1.12, P=0.016 and 0.041). The RFA group had shorter operation time [(69.2±11.7) min vs. (210.6±8.9) min], less intraoperative blood loss [(8.7±3.8) ml vs. (238.6±20.8) ml], and shorter postoperative hospital stays [(6.4±1.0) d vs. (13.1±0.4) d] compared to the surgical resection group, with all differences statistically significant (all P<0.05). The overall complication rates were 19.4% (6/31) in the RFA group and 22.9% (19/83) in the surgical resection group, showing no significant difference ( χ2=0.16, P=0.685). No statistically significant diffe-rence was found in recurrence-free survival rates between the two groups ( χ2=0.13, P=0.717). Similarly, there was no statistically significant difference in cumulative survival rates between the groups ( χ2<0.01, P=0.978). Conclusion:For HCC at CNLC Ⅰa in challenging locations, RFA demonstrated shorter operation time and postoperative hospital stay, less intraoperative bleeding, and superior liver function recovery compared with surgical resection, while no significant difference was observed in survival outcomes between the two treatment groups.

Objective:To analyze the application value of three-dimensional visualization combined with indocyanine green fluorescence staining in laparoscopic resection of patients with complex liver cancer.Methods:The data of patients with complex liver cancer (liver cancer located in liver segments Ⅶ, Ⅷ, Ⅳa, Ⅴ and caudate lobe) who underwent laparoscopic anatomical liver resection in Zhengzhou University People's Hospital from August 2018 to August 2023 were retrospectively analyzed. A total of 334 patients were enrolled, including 249 males and 85 females, with the age of (57.0±10.6) years. Among the 334 patients, patients who underwent laparoscopic liver resection using three-dimensional visualization combined with indocyanine green fluorescence staining were included in the combined group ( n=128), and the other patients who underwent traditional laparoscopic liver resection were included in the traditional group ( n=206). Propensity score was used to match the preoperative indicators and postoperative pathology between the two groups. The two groups were compared in terms of gender, age, maximum diameter of tumor, alpha-fetoprotein, number of tumors, tumor location, American Society of Anesthesiologists (ASA) grade, operation time, portal occlusion time, intraoperative blood loss, surgical margin and postoperative aspartate transaminase (ALT), alanine transaminase (AST), albumin, total bilirubin, and severe postoperative complications (Clavien-Dindo grade≥grade Ⅲ). The Kaplan-Meier method was used to analyze the prognosis, and the log-rank test was used to compare the survival rate. Results:After propensity score matching, 120 cases were included in the combined group and the traditional group, respectively. There were no significant differences in gender, age, maximum diameter of tumor, alpha-fetoprotein, number of tumors, tumor location, and ASA grade between the two groups (all P>0.05). The intraoperative blood loss in the combined group was 200.0 (150.0, 300.0) ml, and the positive surgical margin rate was 6.7% (8/120), which were lower than 300.0 (150.0, 500.0) ml and 15.8% (19/120) in the traditional group, with statistically significant differences ( Z=-2.43, P=0.015, χ2=5.05, P=0.025). There were no statistically significant differences in the operation time, portal occlusion time, and postoperative ALT, AST, albumin, and total bilirubin between the two groups (all P>0.05). Eight cases (6.7%) in the combined group and 11 cases (9.2%) in the traditional group had severe complications after surgery, and all were discharged after treatment. The 1-, 3-, and 5-year recurrence-free survival rates of the combined group were 83.3%, 61.7%, and 58.3% respectively, while that of the traditional group were 71.7%, 52.5%, and 49.2%, respectively, with significant difference ( χ2=4.57, P=0.031). There was no significant difference in the cumulative survival rate between the combined group and the traditional group ( χ2=0.66, P=0.417). Conclusion:Compared with laparoscopic liver resection, laparoscopic liver resection for patients with complex liver cancer using three-dimensional visualization technology combined with indocyanine green fluorescence staining technology can reduce intraoperative blood loss, positive margin rate, and postoperative recurrence.