The aim of this investigation was to determine the optimal storage medium for testicular hypothermic transportation and identify the ideal concentration for the application of the protective agent 5-aminolevulinic acid (5-ALA). Furthermore, this study aimed to explore the underlying mechanism of the protective effects of 5-ALA. First, we collected and stored mouse testicular fragments in different media, including Hank's balanced salt solution (HBSS; n = 5), Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12; n = 5), and alpha-minimum essential medium (αMEM; n = 5). Storage of testicular tissue in HBSS preserved the integrity of testicular morphology better than that in the DMEM/F12 group ( P < 0.05) and the αMEM group ( P < 0.01). Testicular fragments were subsequently placed in HBSS with various concentrations of 5-ALA (0 [control], 1 mmol l -1 , 2 mmol l -1 , and 5 mmol l -1 ) to determine the most effective concentration of 5-ALA. The 2 mmol l -1 5-ALA group ( n = 3) presented the highest positive rate of spermatogonial stem cells compared with those in the control, 1 mmol l -1 , and 5 mmol l -1 5-ALA groups. Finally, the tissue fragments were preserved in HBSS with control ( n = 3) and 2 mmol l -1 5-ALA ( n = 3) under low-temperature conditions. A comparative analysis was performed against fresh testes ( n = 3) to elucidate the underlying mechanism of 5-ALA. Gene set enrichment analysis (GSEA) for WikiPathways revealed that the p38 mitogen-activated protein kinase (MAPK) signaling pathway was downregulated in the 2 mmol l -1 5-ALA group compared with that in the control group (normalized enrichment score [NES] = -1.57, false discovery rate [FDR] = 0.229, and P = 0.019). In conclusion, these data suggest that using 2 mmol l -1 5-ALA in HBSS effectively protected the viability of spermatogonial stem cells upon hypothermic transportation.
Male ; Animals ; Testis/cytology* ; Aminolevulinic Acid/pharmacology* ; Mice ; Organ Preservation/methods* ; Organ Preservation Solutions/pharmacology* ; Cryopreservation/methods*

Eightly-four novel thioheterocyclic nucleoside derivatives were designed, synthesized, and evaluated for antitumor activity in vitro and in vivo. Most of the compounds inhibited the growth of HCT116 and HeLa cancer cells in vitro, among them 33a and 36b exhibited potent activity against HCT116 cells (IC₅₀ = 0.27 and 0.49 μmol/L, respectively). Both compounds 33a and 36b inhibited cell metastasis, arrested the cell cycle in the G₂/M phase, and induced apoptosis in vitro. Mechanistic studies revealed that 33a and 36b increased ROS levels, led to DNA damage, ER stress, and mitochondrial dysfunction, and inhibited autophagy in HCT116 cells. Biological information analysis, RNA-sequencing, Gene Set Enrichment Analysis (GSEA), drug affinity responsive target stability (DARTS) assay, cellular thermal shift assay (CETSA), and SPR experiments identified that compounds 33a and 36b showed antitumor activity by suppressing the c-MYC pathway. c-MYC silencing assays indicated that c-MYC proteins participated in 33a-mediated anticancer activities in HCT116 cells. More importantly, compound 33a presented favorable pharmacokinetic properties in mice (T _1/2 = 6.8 h) and showed significant antitumor efficacy in vivo without obvious toxicity, showing promising potential for further clinical development.

This work was aimed at analyzing the protein characteristics of Coiled-Coil Domain-Containing Protein 115(CCDC115)and using Ccdc115-deficient mouse monocyte-macrophage leukemia cells(RAW264.7)to explore the influence of CCDC115 on the intracellular survival of Salmonella Typhimurium.Bioinformatics analysis was conducted to examine the fundamental attributes of CCDC115,which was determined to be an unstable protein consisting of two α-helices and an intervening disordered re-gion,devoid of any transmembrane structural domains.A RAW264.7-Ccdc115-KO cell line was successfully established with CRISPR/Cas9 gene-editing technology.To elucidate the effects of CCDC115 on the intracellular survival of Salmonella Typhimurium,we infected RAW264.7 cells with Salmonella Typhimurium.The expression of CCDC115 was found to be upregulated at both the mRNA and protein levels post-infection,according to RT-qPCR and western blot analysis.Via counting of colony-forming units(CFU),the proliferation rate of Salmonella Typhimurium within RAW264.7-Ccdc115-KO cells was found to be 1.5-fold higher than that in RAW264.7 cells.Acidification imaging studies indicated that,whereas Salmonella Typhimurium phagosomes underwent acidifi-cation in RAW264.7 cells,this process was absent in RAW264.7-Ccdc115-KO cells.In conclusion,the study successfully estab-lished a RAW264.7-Ccdc115-KO cell line and demonstrated that the expression of CCDC115 is elevated during Salmonella Ty-phimurium infection,thus potentially inhibiting the intracellular survival of Salmonella Typhimurium by facilitating phagosome acidifi-cation.This study lay a theoretical foundation for functional studies of CCDC115 and the investigation of mechanisms regulating the survival of intracellular Salmonella Typhimurium.

Objective To analyze the effect of Hsa-circ-0101216 on gemcitabine(GEM)chemotherapy resistance in pancreatic cancer and its mechanism.Methods Differentially expressed circRNAs between GEM-resistant pancreatic cancer cells and parent cells were screened using the GEO database.Pancreatic cancer GEM resistant cell lines(BxPC-3-GEM and Capan-1-GEM)were constructed by intermittent concentration gradient method.qRT-PCR was used to detect the expression of Hsa-circ-0101216 in cells.GEM resistant pancreatic cancer cell lines were taken and divided into sh-circ-0101216 group(knockdown of circ-0101216),sh-NC group(transfected with sh-NC),and blank control group(untreated).CCK-8 assay and EdU proliferation assay were used to detect the half inhibitory concentration(IC50)of GEM and proliferation ability of cells in each group.Western blotting was performed to detect the expression of multidrug resistance-related protein 1(MRP1),breast cancer resistance protein(BCRP),and human equilibrative nucleoside transporter-1(hENT-1).A subcutaneous xenograft tumor model of human pancreatic cancer in nude mice was constructed,and sh-NC+GEM group and sh-circ-0101216+GEM group(n=6)were set up.The volume and weight of xenograft tumor in nude mice were compared between the two groups.Western blotting and immunohistochemistry were used to detect the expression of MRP1,BCRP,and hENT-1 proteins in xenograft tumor tissues,and EDU proliferation assay was used to detect the proliferation ability of tumor cells.Results The GEO database screening showed that Hsa-circ-0101216 was up-regulated in GEM-resistant pancreatic cancer cell lines.Pancreatic cancer GEM-resistant cell lines were successfully constructed,and the expression levels of Hsa-circ-0101216 and the IC50 value in GEM-resistant pancreatic cancer cells BxPC-3-GEM and Capan-1-GEM were significantly higher than those in parental cells(P<0.05).In sh-circ-0101216 group,the IC50 values of GEM,cell viability,EdU positivity rate,and the expression levels of MRP1 and BCRP proteins in GEM-resistant pancreatic cancer cells BxPC-3-GEM and Capan-1-GEM were significantly lower than those in blank control group and sh-NC group,while the expression level of hENT-1 protein was significantly higher(P<0.05 or P<0.001).In sh-circ-0101216+GEM group,the weight and volume of subcutaneous xenograft tumors in nude mice,the expression levels and positive expression rates of MRP1 and BCRP proteins in tumor tissues,and the EdU positive rate were significantly lower than those in sh-NC+GEM group,while the expression level and positive expression rate of hENT-1 protein were significantly higher(P<0.05).Conclusions Hsa-circ-0101216 is highly expressed in GEM-resistant pancreatic cancer cell lines.Its knockdown can inhibit the proliferation of pancreatic cancer cells and enhance the chemosensitivity of pancreatic cancer cells to GEM.The mechanism may be related to the regulation of transmembrane transporter protein expression.

Objective To establish and evaluate a nomogram prediction model for spontaneous peritonitis in HBV-related primary liver cancer.Methods A retrospective study was conducted on 1298 patients with HBV-related primary liver cancer hospitalized in the Kunming Third People's Hospital from January 2012 to December 2022.General data and serological indicators were collected,and patients were divided into infection group(n=262)and control group(n=1036)based on the occurrence of spontaneous peritonitis.Univariate and LASSO regression analyses were used to screen variables,followed by binary logistic regression to analyze the influencing factors of spontaneous peritonitis in HBV-related primary liver cancer patients,leading to the establishment of a nomogram prediction model.Finally,the Hosmer-lemeshow(H-L)goodness of fit test,receiver operating characteristic(ROC)curve,calibration curve,decision curve analysis(DCA)and clinical impact curve(CIC)were utilized to evaluate the fit degree,accuracy,calibration,and clinical practicability of the nomogram prediction model.Results Single factor analysis revealed significant differences between infection group and control group in portal vein cancer thrombus(PVTT),Child-Pugh grade,China Liver Cancer Staging(CNLC)stage,alcohol consumption history,smoking history,white blood cell count(WBC),neutrophil count(NE),hemoglobin(Hb),fibrinogen(FIB),abnormal prothrombin(PIVKA-Ⅱ),aspartate aminotransferase(AST),alanine aminotransferase(ALT),total protein(TP),prealbumin(PA),γ-glutamyltransferase(GGT),alkaline phosphatase(ALP),cholinesterase(CHE),total bile acid(TBA),total cholesterol(TC),low density lipoprotein(LDL),creatinine(Cr),HBV DNA,CD3+T cells count,CD4+T cells count,CD8+T cells count,CD4+T cells/CD8+T cells ratio,procalcitonin(PCT),serum amyloid A(SAA),interleukin-6(IL-6),high-sensitivity C-reactive protein(hs-CRP),alpha-fetoprotein(AFP),and IL-4(P＜0.05).LASSO regression analysis identified 5 variables:Child-Pugh grade,PVTT,WBC,CHE and hs-CRP.Binary logistic regression analysis indicated that Child-Pugh grade(Grade B:OR=5.780,95％CI 3.271-10.213,P＜0.001;Grade C:OR=14.818,95％CI 7.697-28.526,P＜0.001),PVTT(OR=2.893,95％CI 2.037-4.108,P＜0.001),WBC(OR=1.088,95％CI 1.031-1.148,P=0.002),and hs-CRP(OR=1.005,95％CI 1.001-1.010,P=0.026)were the independent risk factors of spontaneous peritonitis in HBV-related primary liver cancer patients.Using these 4 variables,a nomogram prediction model was constructed and evaluated.The P-value of the H-L goodness of fit test was 0.760.Moreover,the area under ROC curve(AUC)was 0.866,with a sensitivity of 0.870 and a specificity of 0.716.The average absolute error of the calibration curve is 0.022.DCA and CIC analyses demonstrated that the nomogram prediction model possessed some clinical utility.Conclusion The nomogram prediction model for spontaneous peritonitis in HBV-related primary liver cancer patients,constructed using Child-Pugh grade,PVTT,WBC and hs-CRP,exhibits a high fitting degree and accuracy,with the prediction probability highly consistent with the actual occurrence probability,and possesses certain clinical practicability.

Objective To explore the clinical and genetic characteristics of children with holocarboxylase synthetase(HLCS)deficiency.Methods A retrospective analysis was conducted on the clinical data of 3 children with HLCS deficiency who were admitted to Children's Hospital Affiliated to Zhengzhou University from December 2014 to January 2024.Relevant literature indexed in CNKI,Wanfang Data,PubMed and other databases was reviewed to summarize the clinical characteristics and HLCS gene mutations of children with HLCS deficiency.Results All 3 children were male,with onset age of 4-6 months.The main clinical manifestations included shortness of breath,vomiting,diarrhea,and poor mental state,and partial cases were complicated by growth retardation and neurological symptoms.Laboratory tests showed metabolic acidosis in all cases,blood amino acid and acylcarnitine profiles as well as urinary organic acid analysis suggested multiple carboxylase deficiency.Genetic testing revealed compound heterozygous mutation in the HLCS gene of all 3 children,among which the c.1892delT(p.L631X)mutation was previously unreported.According to the guidelines of the American College of Medical Genetics and Genomics(ACMG),the c.1892delT(p.L631X)mutation was rated as pathogenic mutation(PVS1+PM2_supporting+PM3).Biotin supplementation was effective in all cases.Literature review included 27 English literatures and 29 Chinese literatures,reporting a total of 133 children with HLCS deficiency caused by HLCS gene mutation.Common clinical manifestations included metabolic acidosis,skin lesions,vomiting,feeding difficulties,dyspnea,diarrhea,and neurological symptoms,etc.Conclusions Blood amino acid and acylcarnitine profiles,urine organic acid analysis,and gene testing are helpful for the diagnosis of HLCS deficiency.Timely biotin supplementation leads to a good prognosis.The mutation of HLCS gene is considered as the genetic etiology of HLCS deficiency in 3 children,among which the c.1892delT(p.L631X)mutation is a newly discovered mutation.

High-performance liquid chromatography(HPLC) was used to determine the content of three major components in Citri Reticulatae Semen(CRS), including limonin, nomilin, and obacunone. The chromaticity of the CRS sample during salt processing and stir-frying was measured using a color difference meter. Next, the relationship between the color and content of the salt-processed CRS sample was investigated through correlation analysis. By integrating the oil bath technique for processing simulation with HPLC, the changes in the relative content of nomilin and its transformation products were analyzed, with its structural transformation pattern during processing identified. Additionally, RAW264.7 cells were induced with lipopolysaccharides(LPSs) to establish an inflammatory model, and the anti-inflammatory activity of nomilin and its transformation product, namely obacunone was evaluated. The results indicated that as processing progressed, E~*ab and L~* values showed a downward trend; a~* values exhibited a slow increase over a certain period, followed by no significant changes, and b~* values remained stable with no significant changes over a certain period and then started to decrease. The limonin content remained barely unchanged; the nomilin content decreased, and the obacunone increased significantly. The changing trends in content and color parameters during salt-processing and stir-frying were basically consistent. The content of nomilin and obacunone was significantly correlated with the colorimetric values(L~*, a~*, b~*, and E~*ab), while limonin content showed no significant correlation with these values. By analyzing HPLC patterns of nomylin at different heating temperatures and time, it was found that under conditions of 200-250 ℃ for heating of 5-60 min, the content of nomilin significantly decreased, while the obacunone content increased pronouncedly. The in vitro anti-inflammatory activity results indicated that compared to the model group, the group with a high concentration of nomilin and the groups with varying concentrations of obacunone showed significantly reduced release of nitric oxide(NO)(P<0.01). When both were at the same concentration, obacunone showed better performance in inhibiting NO release. In this study, the obvious correlation between the color and content of major components during the processing of CRS samples was identified, and the dynamic patterns of quality change in CRS samples during processing were revealed. Additionally, the study revealed and confirmed the transformation of nomilin into obacunone during processing, with the in vitro anti-inflammatory activity of obacunone significantly greater than that of nomilin. These findings provided a scientific basis for CRS processing optimization, tablet quality control, and its clinical application.
Mice ; Animals ; Drugs, Chinese Herbal/pharmacology* ; RAW 264.7 Cells ; Limonins/chemistry* ; Chromatography, High Pressure Liquid ; Citrus/chemistry* ; Color ; Benzoxepins/chemistry* ; Anti-Inflammatory Agents/chemistry*

This work was aimed at analyzing the protein characteristics of Coiled-Coil Domain-Containing Protein 115(CCDC115)and using Ccdc115-deficient mouse monocyte-macrophage leukemia cells(RAW264.7)to explore the influence of CCDC115 on the intracellular survival of Salmonella Typhimurium.Bioinformatics analysis was conducted to examine the fundamental attributes of CCDC115,which was determined to be an unstable protein consisting of two α-helices and an intervening disordered re-gion,devoid of any transmembrane structural domains.A RAW264.7-Ccdc115-KO cell line was successfully established with CRISPR/Cas9 gene-editing technology.To elucidate the effects of CCDC115 on the intracellular survival of Salmonella Typhimurium,we infected RAW264.7 cells with Salmonella Typhimurium.The expression of CCDC115 was found to be upregulated at both the mRNA and protein levels post-infection,according to RT-qPCR and western blot analysis.Via counting of colony-forming units(CFU),the proliferation rate of Salmonella Typhimurium within RAW264.7-Ccdc115-KO cells was found to be 1.5-fold higher than that in RAW264.7 cells.Acidification imaging studies indicated that,whereas Salmonella Typhimurium phagosomes underwent acidifi-cation in RAW264.7 cells,this process was absent in RAW264.7-Ccdc115-KO cells.In conclusion,the study successfully estab-lished a RAW264.7-Ccdc115-KO cell line and demonstrated that the expression of CCDC115 is elevated during Salmonella Ty-phimurium infection,thus potentially inhibiting the intracellular survival of Salmonella Typhimurium by facilitating phagosome acidifi-cation.This study lay a theoretical foundation for functional studies of CCDC115 and the investigation of mechanisms regulating the survival of intracellular Salmonella Typhimurium.

Objective To summarize the clinical and genetic characteristics of children with sodium taurocholate co-transporting polypeptide(NTCP)deficiency.Methods Clinical data of children with NTCP deficiency diagnosed and treated at the First People's Hospital of Yunnan Province from July 2022 to March 2025 were retrospectively analyzed.Results A total of 14 children were included(6 males,8 females),all with normal growth and development.Reasons for initial consultation included elevated serum bile acids in 7 cases,jaundice in 4 cases,cholestatic hepatitis in 1 case,and one case each of pneumonia and cow's milk protein allergy.At the first visit,all patients had elevated serum total bile acids beyond the normal range,with a mean of 152.5 μmol/L.Elevated alanine aminotransferase was observed in 1 case,elevated aspartate aminotransferase in 2 cases,and elevated total bilirubin in 10 cases.Genetic sequencing revealed that all children carried the homozygous SLC10A1 variant c.800C>T(p.Ser267Phe),classified as likely pathogenic.Conclusions NTCP deficiency often lacks obvious clinical symptoms and signs.Some children present with transient hyperbilirubinemia,cholestasis,or other liver function abnormalities.Persistent isolated elevation of serum bile acids warrants suspicion for this disease.Biallelic pathogenic variants in SLC10A1 constitute the basis for definitive diagnosis.There is no specific treatment for this disease,and management is mainly symptomatic.

Objective:To explore and analyze the correlation between the expression of mismatch repair(MMR)protein and programmed death ligand 1(PD-L1)in colorectal cancer(CRC)tissue and clinical pathological characteristics.Methods:The study period were from July 2021 to December 2024,200 cases of colorectal cancer patients treated in our hospital were selected as the research subjects.Collected all patient lesion tissue samples(lesion group)and adjacent tissue samples(located ≥5 cm from the tumor edge,adjacent group),and used the immunohistochemistry(IHC)method to detect the expression of Mismatch repair(MMR)(MLH1,MSH2,MSH6,and PMS2)protein and PD-L1 protein.Investigated the clinical and pathological characteristics of patients and conducted correlation analysis.Results:The positive rates of dMMR and pMMR protein expression in the lesion group were 39.00％and 70.00％,respectively,while those in the adjacent cancer group were 11.00％and 89.00％,respectively.There were significant difference compared between the lesion group and the adjacent cancer group(P＜0.05).The positive rate of PD-L1 protein expression in the lesion group were 25.00％,while that in the adjacent cancer group were 80.00％.The expression of PD-L1 protein in the lesion group were significantly lower than that in the adjacent cancer group(P＜0.05).There were significant differences in the positive rates of MMR and PD-L1 protein expression among patients with different histological differentiation,clinical stages,and lymph node metastasis(P＜0.05),while there were no significant differences in the positive rates of MMR and PD-L1 protein expression among patients of different genders,ages,and disease sites(P＞0.05).Spearman analysis showed there were correlation between the expression of MMR and PD-L1 proteins in colorectal cancer tissues and clinical pathological features such as histological differentiation,clinical staging,and lymph node metastasis(P＜0.05).Conclusion:Low expression of MMR and PD-L1 proteins are commonly observed in colorectal cancer tissues.The expression of MMR and PD-L1 proteins in colorectal cancer tissues are correlated with clinical pathological features such as histological differentiation,clinical staging,and lymph node metastasis.