ObjectiveIdentification and analysis, animal regression test, and drug susceptibility study were conducted on a pathogenic strain causing diarrhea in cynomolgus monkeys, aiming to provide a practical basis for the clinical treatment of Citrobacter freundii. MethodsFresh fecal samples were collected from nine diarrheal cynomolgus monkeys and streaked on Salmonella-Shigella (SS) medium, LB medium, and Columbia blood agar medium, and incubated at 37 ℃ for 24 h. Subsequently, the isolated strain was identified by colony morphology observation, Gram staining, biochemical tests and 16S rRNA gene sequencing, and PCR was used to detect its major virulence genes. After propagation, the isolate was intragastrically administered to healthy cynomolgus monkeys and C57BL/6 mice for animal regression tests to evaluate its pathogenicity. Finally, the disk-diffusion method was used to detect the antimicrobial susceptibility of the isolate. ResultsA single bacterial strain, designated MF071743, was isolated from 9 fecal samples collected from diarrheal cynomolgus monkeys. The isolate formed pink, smooth, round colonies on SS medium; translucent, smooth, moist, shiny colonies with neat edges on LB medium; and smooth, moist, off-white colonies on Columbia blood agar medium. Gram staining revealed that the isolate was a Gram-negative, spore-free short bacillus. Biochemical tests showed that the isolate was positive for motility test, mannitol test, hydrogen sulfide test, methyl red test, citrate utilization test, gas production from glucose test, raffinose test, sorbitol test, and D-xylose test, but negative for phenylalanine test, gluconate test, indole test, Voges Proskauer (VP) test, urease test, lysine test, ornithine test, and adonitol test. The results of 16S rRNA gene sequencing showed that the gene sequence similarity between strain MF071743 and Citrobacter freundii was 99.0%. PCR results showed that this strain carries genes encoding the urease accessory proteins UreD, UreE, and UreF. Animal intragastric administration assays demonstrated that the strain caused loose stools in cynomolgus monkeys, and all C57BL/6 mice died within 72 h. Drug susceptibility test results indicated that the isolate was sensitive to 16 antibiotics, including ceftriaxone, amikacin, gentamicin, ceftazidime, and levofloxacin, while it exhibited resistance to 5 antibiotics, including ampicillin, cefazolin, vancomycin, erythromycin, and cephalexin. ConclusionA strain of Citrobacter freundii with certain drug resistance was isolated from the feces of diarrheal cynomolgus monkeys. The results of this study provide a reference for the diagnosis and treatment of gastrointestinal diseases in captive laboratory monkeys.

Objective To investigate changes in the pro-inflammatory mediator S100A8/9 and NLRP3/Caspase-1/IL-1β pathway in a rat kidney-aging model induced by D-galactose.Methods Twelve SD rats were divided into control and D-galactose groups,and injected subcutaneously in the back of the neck with D-galactose(150 mg/kg)to establish a rat model of kidney aging.Kidney samples were collected under anesthesia after 8 weeks.Kidneys were stained for senescence-associated beta-galactosidase(SA-β-Gal),mRNA expression levels of the aging-related genes p21,p16,and p53 were detected by quantitative reverse transcription-polymerase chain reaction(qRT-PCR),and histopathological changes were observed by hematoxylin-eosin(HE)and Masson staining.Serum urea nitrogen and creatinine,and catalase(CAT),glutathione peroxidase(GSH-PX),superoxide dismutase(SOD),and malondialdehyde(MDA)levels in the kidney tissues were detected.Reactive oxygen species(ROS)were detected by dihydroethdium staining and protein expression levels of collagen Ⅲ,α-smooth muscle actin(α-SMA),Protein expression of S100A8/9 was detected by immunofluorescence,and transforming growth factor(TGF)-β1 levels in kidney tissues and key factors in the NLRP3/Caspase-1/IL-1β inflammatory pathway were detected by Western Blot.A renal senescence model using HK-2 cells was constructed using H2O2 in vitro,and expression levels of the senescence proteins p21 and p16 and mRNA expression levels of the inflammatory factors IL-18 and tumor necrosis factor-α(TNF-α)were detected.Cell senescence was observed by SA-β-Gal staining.The effects of the S100A8/9 inhibitor paquinimod on expression levels of S100A8/9 and NLRP3/Caspase-1/IL-1β pathway-related proteins in the aging model were also detected.Results mRNA levels of the aging genes p21,p16,and p53 in kidney tissues were significantly increased in rats in the D-galactose group compared with the control group(P＜0.01),and SA-β-Gal staining showed a significant increase in senescent cells(P＜0.01).Serum blood urea nitrogen and creatinine levels increased(P＜0.05),CAT,GSH-PX,and SOD activities decreased(P＜0.01),while MDA activity increased in the D-galactose group(P＜0.01).Collagen Ⅲ,α-SMA,and TGFβ1 expression and the ROS content in tissues increased(P＜0.05).Glomeruli were atrophied or absent in the D-galactose group,the lumens of the renal sacs and renal tubules were enlarged,the nuclei were deeply stained and constricted,and numerous collagen fibers were deposited.Levels of S100A8 and S100A9 protein(P＜0.01),as well as NLRP3,Caspase-1,and IL-1β increased(P＜0.05).Paquinimod alleviated HK-2 cell senescence and decreased expression levels of the senescence proteins p21 and p16,and mRNA levels of the inflammatory factors IL-18 and TNF-α(P＜0.05,P＜0.01).The number of senile cells was also decreased,shown by SA-β-Gal staining(P＜0.01).Paquinimod also inhibited the protein expression of S100A8 and S100A9(P＜0.01)and NLRP3,Caspase-1,and IL-1β(P＜0.05 or P＜0.01).Conclusions S100A8/9 participates in the chronic inflammatory response by activating the NLRP3/Caspase-1/IL-1β pathway,thereby promoting D-galactose-induced renal aging.

Objective:To construct and validate a prognostic model of colon cancer based on differentially expressed anoikis-related genes, and to preliminarily investigate the relationship between anoikis-related genes and the tumor immune microenvironment of colon cancer.Methods:A total of 472 cancer tissues samples of patients with colon cancer, RNA sequencing data and clinical data of 41 normal tissues samples were downloaded from the Cancer Genome Atlas (TCGA) database between the establishment time and July in 2024. A total of 919 genes related to anoikis were screened out from GeneCards database, and the common genes were selected from the RNA sequencing gene datasets of colon cancer and normal colon tissues in the TCGA database, among which the differentially expressed anoikis-related genes of colon cancer and normal colon tissues were screened out based on P < 0.05. Furthermore, genes related to the prognosis of 446 colon cancer patients with prognostic data in the TCGA database were screened by using univariate Cox proportional risk model; the genes with P < 0.05 were further screened out and a colon cancer prognosis model was constructed by using LASSO-Cox proportional risk model. The risk score of the above 446 colon cancer patients in the TCGA database was calculated according to the prognostic model, and the patients were divided into high-risk (≥ median value) group and low-risk (< median value) group according to the median risk score, and the overall survival of the 2 groups was analyzed by using the Kaplan-Meier method. The risk score based on R software-based time ROC program package was used to predict 1-year, 2-year, 3-year overall survival therapeutic efficacy of colon cancer patients in the TCGA database. According to the median risk score of colon cancer patients in the TCGA database, the patients in the International Cancer Genome Consortium (ICGC) database were divided into high-risk group and low-risk group. Kaplan-Meier method and receiver operating characteristic (ROC) curve were used to verify the predictive effect of the prognostic model. The differentially expressed genes between low-risk group and high-risk group stratified by prognostic model risk score in the TCGA database were used to perform single sample gene set enrichment analysis (ssGESA) of immune cells and immune function by using R software related programs. The differences in risk scores of patients with different immunophenotypes (including inflammator response type, wound healing type, interferon gamma dominant type and lymphocyte depletion type) were compared; and correlation analysis of infiltration and risk scores between immune cells and stromal cells in tumor microenvironment was made. Based on the tumor immune function and rejection (TIDE) database, the relationship between the prognostic model risk score and programmed death receptor ligand 1 (PD-L1) gene expression level was analyzed. Results:Based on anoikis-related genes in the GeneCards database, 236 differentially expressed anoikis-related genes between colon cancer tissues and normal tissues were obtained from the TCGA database. LASSO Cox regression was applied to establish a prognostic model constructed by 7 differentially expressed anoikis-related genes in cancer tissues and normal colon tissues related to the prognosis of colon cancer. Risk score = 0.366×TIMP1-0.404×NAT1+0.207×LTB4R2+0.075×INHBB+0.140×CD36-0.109×MMP3+2.994×OFCC1. The median risk score of 446 colon cancer patients in the TCGA database was 1.754 719 545. Survival analysis showed that the overall survival of colon cancer patients in high-risk group of the TCGA database was worse than that in low-risk group ( P < 0.001); ROC curve analysis showed that the area under the curve for predicting 1-year, 2-year and 3-year overall survival of patients in the TCGA database based on the prognostic model risk score was 0.705, 0.731 and 0.723, respectively. Kaplan-Meier method analysis showed that in the ICGC database, the overall survival of colon cancer patients in high-risk group was worse than that in low-risk group ( P = 0.041); ROC curve analysis showed that the area under the curve of prognostic model risk score for predicting 1-year and 2-year overall survival of colon cancer patients in the ICGC database was 0.663 and 0.966, respectively. ssGESA analysis showed that macrophage level in high-risk group was higher than that in low-risk group, helper T (Th) 1 cell and Th2 cell levels in high-risk group were lower than those in low-risk group (all P < 0.01). In terms of immune function, the cell killing activity and histocompatibility complex Ⅰ level in high-risk group were lower than those in low-risk group, and type Ⅱ interferon response score in high-risk group was higher than that in low-risk group (all P < 0.05). The analysis of immunophenotype showed that the risk score of inflammatory response type was higher than that of wound healing type ( P < 0.05), and there was no statistically significant difference in risk score between the other 2 types (all P > 0.05). Risk score was positively correlated with stromal cell infiltration score ( R = 0.340, P < 0.001) and immune cell infiltration score ( R = 0.148, P < 0.05); the expression level of PD-L1 in high-risk group was higher than that in low-risk group in the TCGA database ( P = 0.048), and the expression level of PD-L1 was positively correlated with risk score ( R = 0.130, P = 0.009). Conclusions:A prognostic model of colon cancer constructed by anoikis-related genes can better predict the prognosis of colon cancer patients, and anoikis-related genes may play an important role in tumor immunity of colon cancer.

Objective:Exploring the predictive value of sarcopenia based on preoperative CT diagnosis for the prognosis of patients with pancreatic cancer.Methods:A retrospective analysis was conducted on the clinical,pathological,and imaging data of 488 patients who underwent radical resection for pancreatic cancer,to analyze the correlation between sarcopenia and the prognosis of patients with pancreatic cancer.Results:Among the 488 patients included,281 had sarcopenia before surgery and 207 did not.The COX proportional hazards regression model showed that sarcopenia was an independent risk factor for overall survival in pancreatic cancer patients[hazard ratio 1.43(95%confidence interval:1.02-1.99);P=0.04],while preoperative sarcopenia was not associated with postoperative disease-free survival(P>0.05).Conclusion:Sarcopenia diagnosed based on preoperative CT is significantly correlated with the overall survival of patients with pancreatic cancer after surgery and is an independent risk factor for the overall survival of patients with pancreatic cancer after surgery.

Objective: To explore effects and maintenance of school based sexual abuse prevention programs for minors, so as to provide scientific evidences for optimizing intervention design and policy making. Methods: Six Chinese and English databases were searched, including CNKI, Wanfang Database, Medline (via PubMed), Embase, Cochrane Library and Web of Science, with the time frame set from database inception to December 31, 2024. Studies on school based sexual abuse prevention programs for minors were selected, and data on knowledge, attitudes and skills related to sexual abuse prevention were extracted. Meta analysis was performed using Stata 17. Results: A total of 26 studies were included. The Meta analysis results showed that school based sexual abuse prevention programs improved participants knowledge ( SMD=1.24, 95%CI =0.96-1.52), attitudes ( SMD=0.62, 95%CI =0.19-1.04) and skills ( SMD=0.66, 95%CI =0.50-0.83) (all P <0.01). During the overall follow up, the maintenance rates for knowledge, attitudes, and skills were 0.97(95% CI =0.95-1.00), 0.99(95% CI =0.95-1.04) and 1.01(95% CI =0.99-1.04), respectively, with no statistically significant differences (all P >0.05). However, knowledge retention declined significantly when follow up exceeded three months ( R=0.91, 95%CI=0.83-0.99, P <0.01), while skills retention ( R=0.94, 95%CI=0.87-1.02, P = 0.23) remained higher than knowledge and attitudes ( R=0.98, 95%CI=0.96-1.00, P =0.13), demonstrating stronger long term effects. Conclusion School based sexual abuse prevention programs are effective in enhancing participants knowledge, attitudes and skills, but the intervention effects diminish over time, particularly in knowledge retention.

Objective:Exploring the predictive value of sarcopenia based on preoperative CT diagnosis for the prognosis of patients with pancreatic cancer.Methods:A retrospective analysis was conducted on the clinical,pathological,and imaging data of 488 patients who underwent radical resection for pancreatic cancer,to analyze the correlation between sarcopenia and the prognosis of patients with pancreatic cancer.Results:Among the 488 patients included,281 had sarcopenia before surgery and 207 did not.The COX proportional hazards regression model showed that sarcopenia was an independent risk factor for overall survival in pancreatic cancer patients[hazard ratio 1.43(95%confidence interval:1.02-1.99);P=0.04],while preoperative sarcopenia was not associated with postoperative disease-free survival(P>0.05).Conclusion:Sarcopenia diagnosed based on preoperative CT is significantly correlated with the overall survival of patients with pancreatic cancer after surgery and is an independent risk factor for the overall survival of patients with pancreatic cancer after surgery.

Objective To investigate changes in the pro-inflammatory mediator S100A8/9 and NLRP3/Caspase-1/IL-1β pathway in a rat kidney-aging model induced by D-galactose.Methods Twelve SD rats were divided into control and D-galactose groups,and injected subcutaneously in the back of the neck with D-galactose(150 mg/kg)to establish a rat model of kidney aging.Kidney samples were collected under anesthesia after 8 weeks.Kidneys were stained for senescence-associated beta-galactosidase(SA-β-Gal),mRNA expression levels of the aging-related genes p21,p16,and p53 were detected by quantitative reverse transcription-polymerase chain reaction(qRT-PCR),and histopathological changes were observed by hematoxylin-eosin(HE)and Masson staining.Serum urea nitrogen and creatinine,and catalase(CAT),glutathione peroxidase(GSH-PX),superoxide dismutase(SOD),and malondialdehyde(MDA)levels in the kidney tissues were detected.Reactive oxygen species(ROS)were detected by dihydroethdium staining and protein expression levels of collagen Ⅲ,α-smooth muscle actin(α-SMA),Protein expression of S100A8/9 was detected by immunofluorescence,and transforming growth factor(TGF)-β1 levels in kidney tissues and key factors in the NLRP3/Caspase-1/IL-1β inflammatory pathway were detected by Western Blot.A renal senescence model using HK-2 cells was constructed using H2O2 in vitro,and expression levels of the senescence proteins p21 and p16 and mRNA expression levels of the inflammatory factors IL-18 and tumor necrosis factor-α(TNF-α)were detected.Cell senescence was observed by SA-β-Gal staining.The effects of the S100A8/9 inhibitor paquinimod on expression levels of S100A8/9 and NLRP3/Caspase-1/IL-1β pathway-related proteins in the aging model were also detected.Results mRNA levels of the aging genes p21,p16,and p53 in kidney tissues were significantly increased in rats in the D-galactose group compared with the control group(P＜0.01),and SA-β-Gal staining showed a significant increase in senescent cells(P＜0.01).Serum blood urea nitrogen and creatinine levels increased(P＜0.05),CAT,GSH-PX,and SOD activities decreased(P＜0.01),while MDA activity increased in the D-galactose group(P＜0.01).Collagen Ⅲ,α-SMA,and TGFβ1 expression and the ROS content in tissues increased(P＜0.05).Glomeruli were atrophied or absent in the D-galactose group,the lumens of the renal sacs and renal tubules were enlarged,the nuclei were deeply stained and constricted,and numerous collagen fibers were deposited.Levels of S100A8 and S100A9 protein(P＜0.01),as well as NLRP3,Caspase-1,and IL-1β increased(P＜0.05).Paquinimod alleviated HK-2 cell senescence and decreased expression levels of the senescence proteins p21 and p16,and mRNA levels of the inflammatory factors IL-18 and TNF-α(P＜0.05,P＜0.01).The number of senile cells was also decreased,shown by SA-β-Gal staining(P＜0.01).Paquinimod also inhibited the protein expression of S100A8 and S100A9(P＜0.01)and NLRP3,Caspase-1,and IL-1β(P＜0.05 or P＜0.01).Conclusions S100A8/9 participates in the chronic inflammatory response by activating the NLRP3/Caspase-1/IL-1β pathway,thereby promoting D-galactose-induced renal aging.

Objective:To construct and validate a prognostic model of colon cancer based on differentially expressed anoikis-related genes, and to preliminarily investigate the relationship between anoikis-related genes and the tumor immune microenvironment of colon cancer.Methods:A total of 472 cancer tissues samples of patients with colon cancer, RNA sequencing data and clinical data of 41 normal tissues samples were downloaded from the Cancer Genome Atlas (TCGA) database between the establishment time and July in 2024. A total of 919 genes related to anoikis were screened out from GeneCards database, and the common genes were selected from the RNA sequencing gene datasets of colon cancer and normal colon tissues in the TCGA database, among which the differentially expressed anoikis-related genes of colon cancer and normal colon tissues were screened out based on P < 0.05. Furthermore, genes related to the prognosis of 446 colon cancer patients with prognostic data in the TCGA database were screened by using univariate Cox proportional risk model; the genes with P < 0.05 were further screened out and a colon cancer prognosis model was constructed by using LASSO-Cox proportional risk model. The risk score of the above 446 colon cancer patients in the TCGA database was calculated according to the prognostic model, and the patients were divided into high-risk (≥ median value) group and low-risk (< median value) group according to the median risk score, and the overall survival of the 2 groups was analyzed by using the Kaplan-Meier method. The risk score based on R software-based time ROC program package was used to predict 1-year, 2-year, 3-year overall survival therapeutic efficacy of colon cancer patients in the TCGA database. According to the median risk score of colon cancer patients in the TCGA database, the patients in the International Cancer Genome Consortium (ICGC) database were divided into high-risk group and low-risk group. Kaplan-Meier method and receiver operating characteristic (ROC) curve were used to verify the predictive effect of the prognostic model. The differentially expressed genes between low-risk group and high-risk group stratified by prognostic model risk score in the TCGA database were used to perform single sample gene set enrichment analysis (ssGESA) of immune cells and immune function by using R software related programs. The differences in risk scores of patients with different immunophenotypes (including inflammator response type, wound healing type, interferon gamma dominant type and lymphocyte depletion type) were compared; and correlation analysis of infiltration and risk scores between immune cells and stromal cells in tumor microenvironment was made. Based on the tumor immune function and rejection (TIDE) database, the relationship between the prognostic model risk score and programmed death receptor ligand 1 (PD-L1) gene expression level was analyzed. Results:Based on anoikis-related genes in the GeneCards database, 236 differentially expressed anoikis-related genes between colon cancer tissues and normal tissues were obtained from the TCGA database. LASSO Cox regression was applied to establish a prognostic model constructed by 7 differentially expressed anoikis-related genes in cancer tissues and normal colon tissues related to the prognosis of colon cancer. Risk score = 0.366×TIMP1-0.404×NAT1+0.207×LTB4R2+0.075×INHBB+0.140×CD36-0.109×MMP3+2.994×OFCC1. The median risk score of 446 colon cancer patients in the TCGA database was 1.754 719 545. Survival analysis showed that the overall survival of colon cancer patients in high-risk group of the TCGA database was worse than that in low-risk group ( P < 0.001); ROC curve analysis showed that the area under the curve for predicting 1-year, 2-year and 3-year overall survival of patients in the TCGA database based on the prognostic model risk score was 0.705, 0.731 and 0.723, respectively. Kaplan-Meier method analysis showed that in the ICGC database, the overall survival of colon cancer patients in high-risk group was worse than that in low-risk group ( P = 0.041); ROC curve analysis showed that the area under the curve of prognostic model risk score for predicting 1-year and 2-year overall survival of colon cancer patients in the ICGC database was 0.663 and 0.966, respectively. ssGESA analysis showed that macrophage level in high-risk group was higher than that in low-risk group, helper T (Th) 1 cell and Th2 cell levels in high-risk group were lower than those in low-risk group (all P < 0.01). In terms of immune function, the cell killing activity and histocompatibility complex Ⅰ level in high-risk group were lower than those in low-risk group, and type Ⅱ interferon response score in high-risk group was higher than that in low-risk group (all P < 0.05). The analysis of immunophenotype showed that the risk score of inflammatory response type was higher than that of wound healing type ( P < 0.05), and there was no statistically significant difference in risk score between the other 2 types (all P > 0.05). Risk score was positively correlated with stromal cell infiltration score ( R = 0.340, P < 0.001) and immune cell infiltration score ( R = 0.148, P < 0.05); the expression level of PD-L1 in high-risk group was higher than that in low-risk group in the TCGA database ( P = 0.048), and the expression level of PD-L1 was positively correlated with risk score ( R = 0.130, P = 0.009). Conclusions:A prognostic model of colon cancer constructed by anoikis-related genes can better predict the prognosis of colon cancer patients, and anoikis-related genes may play an important role in tumor immunity of colon cancer.

The Rh blood grouping system is a critical standardized test in transfusion medicine,espe-cially for the cases related to haemolytic transfusion reactions and neonatal haemolytic disease caused by clinical RhD blood group incompatibility.In the present case report,we presented two cases with the un-common RHD gene variation RHD*DEL37.The blood samples of the two subjects were mistakenly iden-tified as RhD-negative through conventional serological testing.Firstly,both blood samples were tested negative for the RhD antigen using traditional tube test and gel microcolumn methods.The phenotyping of RhCE were identified as ccEe and ccee for each sample,respectively.Secondly,genetic analysis was performed using polymerase chain reaction-sequence specific prime(PCR-SSP)which revealed that nei-ther sample belonging to the several common RHD gene variants which was found in Asia.Moreover,they turned out to be positive for the RHD haplotype,which indicated that exons 1-10 on one of the RHD al-leles were entirely absent.In addition,a T＞C mutation was observed at bases 1154-31 in intron 8 of the other allele,which was located at the intron 8 breakpoint.This result was obtained after further Sanger sequencing of exons 1-10 of the RHD gene.The mutant allele was designated as RHD*DEL37 by the International Society of Blood Transfusion(ISBT)and was identified as D-elute(Del)by phenotype ana-lysis.Both samples were genotyped as RHD*DEL37 and showed positive results.In summary,the true genotype of the two blood samples,of which the screening results only using serological testing method was negative,were RHD*DEL37/RHD-(RHD*01N.01).Notably,this kind of genotype was reported for the first time in Chinese population.Moreover,the two individuals did not have ties of consanguinity,indicating that some of the Chinese individuals could be carriers of the genetic mutation.Therefore,it might be necessary to further confirm the frequency of this mutation in the Chinese population and the possibility of homozygosity for this mutation.This report identifies infrequent RHD gene mutation samples by coupling molecular biology and serological methods to prevent misclassification of blood groups.Com-bining serological and molecular biology test results to determine blood group is critical in protecting pa-tients during clinical transfusion procedures.

Chronic pancreatitis is a refractory disease that severely affects the quality of life of patients. At present, the step-by-step approach of "medicine-endoscopy-surgery" is the main mode of treatment for this disease. However, with deepening understanding of the disease, especially in-depth study of its mechanism of pain, traditional treatment strategy has also undergone novel changes. The concept and practice of early surgery have shown unique advantages in the full-course treatment of chronic pancreatitis. In this article, focusing on the efficacy of early surgical intervention and the selection of surgical approaches, the significance and value of early surgery were deeply discussed, and the optimal treatment combining with suitable surgical approaches for patients with chronic pancreatitis under different circumstances was summarized.