Objective To investigate the factors influencing the efficacy of intravesical Bacille Calmette-Guérin(BCG)instillation after transurethral resection of bladder tumor(TURBT)in patients with intermediate-and high-risk non-muscle invasive bladder cancer(NMIBC),and to construct a prediction model for recurrence after BCG treatment.Methods A retrospective cohort study was conducted on the subjected patients diagnosed with intermediate-and high-risk NMIBC undergoing TURBT followed by standard BCG instillation.The 110 patients treated in Department of Urology of Army Medical Center of PLA from January 2018 to December 2023 were assigned into a training set,while the 52 patients treated at Department of Urology of General Hospital of Central Theater Command from January 2015 to December 2020 were into an external validation set.A total of 17 variables were included and analyzed.Univariate and multivariate Cox regression analyses were performed to identify factors associated with recurrence after BCG instillation,and nomograms were plotted to predict 1-year,3-year,and 5-year recurrence-free survival(RFS).Calibration curve,decision curve analysis(DCA),and receiver operating characteristic(ROC)curve analysis were conducted for internal and external validation to evaluate the predictive performance and clinical utility of the model.Results In the training set,26 patients(23.64%)experienced recurrence during the follow-up period,with a median RFS of 32.00(18.00～50.50)months.Univariate Cox regression analysis suggested that platelet count,eosinophil to lymphocyte ratio(ELR),neutrophil to lymphocyte ratio(NLR),platelet to lymphocyte ratio(PLR),systemic immune inflammation(SII)index,and neutrophil-monocyte to lymphocyte ratio(NMLR),pathological T1 stage(pT1)tumor and hemoglobin,albumin,lymphocyte,and platelet(HALP)score were potential factors influencing recurrence after BCG instillation.Multivariate Cox regression analysis identified high HALP score(HR=0.185,95%CI:0.046～0.736,P=0.017)as an independent protective factor,while high ELR(HR=3.599,95%CI:1.505～8.608,P=0.004)and pT1 stage(HR=3.240,95%CI:1.191～8.818,P=0.021)were independent risk factors for recurrence.Based on this,a nomogram prediction model was constructed.The calibration curves demonstrated good agreement between predicted and actual 1-,3-,and 5-year recurrence risks.Decision curve analysis indicated clinical utility across a wide threshold probability range.In the training set,the model showed strong predictive performance for 1-(AUC=0.842),3-(AUC=0.847),and 5-year(AUC=0.887)recurrence risks,which was further validated in the external cohort.Conclusion Higher HALP score prior to BCG instillation therapy is a protective factor against tumor recurrence,while higher ELR and pT1 stage are risk factors.Our nomogram prediction model based on HALP score,ELR and pathological T stage,can identify individuals at high risk of recurrence after BCG instillation therapy.

Objective To investigate risk factors for residual lesions after initial transurethral resection of bladder tumors(TURBT)and risk factors for tumor recurrence after second TURBT in patients with non-muscle-invasive bladder cancer(NMIBC)in order to provide reference for clinical management.Methods A case-control study design was adopted to include 120 NMIBC patients who underwent initial TURBT and then second surgery within 2～8 weeks in our department from January 2017 to January 2025.Based on the presence of residual lesions after the initial TURBT or not,the patients were divided into a residual lesion group(n=34)and a non-residual lesion group(n=86).Chi-square test and multivariate logistic regression analysis were performed to identify potential risk factors for residual lesions following the initial TURBT.Univariate and multivariate Cox regression models were used to analyze potential risk factors for tumor recurrence after the second TURBT.Results The residual lesion rate after initial TURBT was 28.33％.Chi-square test analysis revealed that tumor stage T1(Chi-square=5.756,P=0.016)and broad tumor base(Chi-square=4.331,P=0.037)were factors influencing residual lesions after initial TURBT.Multivariate logistic regression analysis identified tumor stage T1(OR=3.047,95％CI:1.128～8.226,P=0.028)as an independent risk factor for residual lesions after initial TURBT.The tumor recurrence rate after second TURBT was 17.5％.Multivariate Cox regression analysis identified tumor stage T1(OR=4.258,95％CI:1.248～14.532,P=0.021),intravesical chemotherapy instillation after second TURBT(OR=3.539,95％CI:1.284～9.752,P=0.015),history of urinary system tumors(OR=3.002,95％CI:1.145～7.873,P=0.025)and high platelet-to-lymphocyte(PLR)ratio(OR=2.798,95％CI:1.115～7.023,P=0.028)as independent risk factors for tumor recurrence after second TURBT.Conclusion Tumor stage T1 and broad tumor base are risk factors for residual lesions after initial TURBT,while tumor stage T1,intravesical chemotherapy instillation after second TURBT,history of urinary system tumors and high PLR ratio are risk factors for tumor recurrence after second TURBT.Comprehensive analysis on above 4 indicators can effectively assess the risk of tumor recurrence in NMIBC patients following second TURBT,and timely early medical intervention is beneficial for improving patient outcomes.

Objective To study anti-aging mechanism of skin with water extract of Codonopsis Radix by applying computational biology and animal experiments.Methods A total of 50 SPF 8-week-old C57BL/6 mice were selected and then randomly divided into five groups.D-galactose-induced aging mice model was constructed,and different doses of water extract of Codonopsis Radix were used for intervention.Hematoxylin-eosin staining(HE)and Masson staining were used to observe the pathological changes of mouse skin tissue.The content of hydroxyproline(HYP),superoxide dismutase(SOD),and malondialdehyde(MDA)in mouse skin were measured by biochemical detection.Transmission electron microscopy was used to observe the ultrastructural changes of mouse skin tissue.The chip data of skin aging from GEO database was obtained to screen skin-aging differential genes.TCMSP and UniProt databases were used to search for active ingredients and targets of Codonopsis Radix.The intersection of targets for Codonopsis Radix-skin aging was obtained by integrating the above data.A protein interaction network of all core gene proteins for Codonopsis Radix intervention in skin aging was constructed through the STRING database.Then,quantitative real-time PCR(RT-qPCR)and Western Blotting were used to verify target genes expression and pathway-related protein expression after the intervention of Codonopsis Radix in the aging model.Results Compared with the normal control group,the skin tissue structure of mice in senile model group were damaged significantly,the damage of skin tissue structure was improved significantly after the intervention of Codonopsis Radix.Compared with the normal control group,the content of HYP and SOD in the skin tissue of mice in senile model group were significantly reduced(P＜0.05),while MDA was significantly increased(P＜0.05).After the intervention of Codonopsis Radix,the content of HYP and SOD were increased,while MDA was decreased(P＜0.05)compared with senile model group.It was found that matrix metalloproteinase 9(MMP9)was the core target for the intervention of Codonopsis Radix on skin aging in computational biology.Experiments have shown that the expression of MMP9 was significantly increased in the skin of aging model mice compared to normal control group(P＜0.05).After the intervention of Codonopsis Radix,the expression of MMP9 is significantly reduced(P＜0.05),the expression of the key protein including inhibitory subunit of NF-kappa B alpha(IκBα)、IκB kinase-alpha(IKKα)、nuclear factor kappa-B(NF-κB)P65 of NF-κB signaling pathway were significantly changed(P＜0.05).Conclusion Codonopsis Radix water extract can effectively alleviate skin aging in aging model mice by inhibiting the protein expression of IκBα、IKKα、NF-κB P65 of NF-κB signaling pathway,reducing the expression of downstream gene MMP9,and ultimately alleviate skin collagen damage and resist skin aging.

Objective:To investigate the effect of tumor necrosis factor-α (TNF-α) on the differentiation of orbital fibroblasts (OF) in thyroid-associated ophthalmopathy (TAO) and its regulation mechanism.Methods:Six patients (six eyes) diagnosed with TAO were collected in Tianjin Medical University Eye Hospital from December 2019 to August 2020.Adipose connective tissue was collected during the orbital decompression surgery.OF was isolated and cultured using the tissue block method and vimentin was identified by immunofluorescence.Lipogenic differentiation of OF was induced and identified by oil red O staining.Complete culture medium containing 0, 0.1, 1.0 and 10.0 μg/L TNF-α was used to induce the dedifferentiation of orbital mature adipocytes.Primary culturing cells, 14-day differentiation cells and 20-day dedifferentiation cells were collected.The relative mRNA expression levels of peroxisomal proliferation-activated receptor (PPARγ), extracellular regulatory protein kinase1 (ERK1), ERK2 and fat-coated protein1 (perilipin1) were detected by real-time fluorescent quantitative PCR.The relative protein expression levels of PPARγ, P-ERK1/2 and perilipin1 were detected by Western blot.Results:Human TAO-derived OF were successfully cultured in vitro, spindle-shaped or polygonal, tightly arranged in a vortex pattern, and immunofluorescence staining for vimentin was positive.After OF adipogenic differentiation, lipid droplet structures could be seen in the cytoplasm of some cells, and the stained lipid droplet structures in the cytoplasm could be seen by oil red O staining, which confirmed that the cells obtained after differentiation were adipocytes.Dedifferentiation of adipocytes was induced by 0.1, 1.0, and 10.0 μg/L TNF-α.With the extension of induction time, the volume of lipid droplets in the cytoplasm and the number of cells containing lipid droplets decreased.Lipid droplets disappeared in the cytoplasm on the 20th day of dedifferentiation, and the cells became long spindle-shaped and tightly arranged, dedifferentiated into fibroblast-like cells.Real-time fluorescence quantitative PCR detection results showed that the relative expression levels of PPARγ, ERK1, ERK2 and perilipin1 mRNA in 14-day differentiation group were 4.26±0.09, 2.01±0.09, 3.23±0.10 and 8.69±0.33, respectively, which were significantly higher than 1.00±0.09, 1.05±0.19, 1.00±0.10 and 1.05±0.07 in primary group, and 1.06±0.03, 1.15±0.11 and 6.27±0.09 in 20-day dedifferentiation group (all at P<0.05). Western blot analysis showed that the expression levels of PPARγ, ERK1/2 and perilipin1 proteins in 14-day differentiation group were 1.07±0.03, 1.00±0.03 and 1.13±0.02, respectively, which were significantly higher than 0.37±0.02, 0.29±0.02 and 0.00±0.00 in primary group, and 0.20±0.02, 0.38±0.06 and 0.00±0.00 in 20-day dedifferentiation group (all at P<0.001). Conclusions:TNF-α has a dedifferentiation effect on TAO orbital adipocytes.The mechanism may be related to the downregulation of ERK1/2-PPARγ-perilipin1 signaling pathway.

[Abstract] Objective: To explore the mechanism of glucose transport protein-1(Glut-1) promoting the migration of osteosarcoma MG63 cells through Wnt/β-catenin pathway. Methods: RNA interference recombinant adenovirus targeting Glut-1 gene (Ad-Glut-siRNA) and control recombinant adenovirus (Ad-GFP) were constructed and transfected into MG63 cells to silence Glut-1 gene expression. The cell migration ability of Blank group, Ad-AFP group, Ad-Glut-siRNA group and AZD2858 (inhibitor of GSK-3) group were detected by Transwell chamber migration assay. Immunofluorescence assay was used to detect the expression of E-cadherin and vimentin in each group and the nuclear translocation of β-catenin. The expression of MMP-2 and MMP-9 in each group and FZD7, β-catenin, Dsh protein in Blank group, Ad-AFP group, Ad-Glut-siRNA group were detected by Western blotting assay. Results: The migration ability of MG63 cells was significantly decreased (P<0.05) after Glut-1 gene silencing, which was restored afterAZD2858 treatment (P <0.05). Compared with Blank group and Ad-GFP group, the E-cadherin level in MG63 cells in Ad-Glut-siRNA group was significantly increased (P<0.05), while the expressions of vimentin, MMP-2, MMP-9, FZD7, β-catenin and Dsh protein were significantly reduced (all P<0.05). Compared with Ad-Glut-siRNA group, E-cadherin expression of AZD2858 group was significantly reduced, while the expressions of vimentin, MMP-2, MMP-9 were significantly up-regulated (P<0.05). Conclusion: The high expression of Glut-1 gene is closely related to the invasion and metastasis of MG63 cells. The possible mechanism is that the high expression of Glut-1 leads to the activation of Wnt/β-catenin pathway, which leads to the decrease of EMT-related protein E-cadherin, and the increase of vimentin and MMP-2, MMP-9, and further promotes the migration of MG63 cells.

Objective To look for more serum biomarkers supporting the diagnosis of multiple system atrophy ( MSA) and providing more evidence for early treatment.Methods All patients and healthy controls were enrolled from January 2011 to March 2015 in the First Affiliated Hospital of Zhengzhou University.Demographic features and biochemical examination results were collected.The t test was used to compare the lipid levels between MSA patients and controls.LSD-t test was used to compare the lipid levels among subtypes of MSA patients.Multivariate Logistic regression analysis was conducted to analyze the influencing factors.The relevance between lipid levels and onset age, disease duration and Hoehn & Yahr stage was calculated by Spearman correlation coefficients.Results Participants included 195 MSA patients and 195 age-and gender-matched controls with no neurological diseases.The levels of total cholesterol ((4.33 ±0.90) mmol/L), triglyceride ((1.27 ±0.71) mmol/L), low-density lipoprotein (LDL;(2.70 ±0.76) mmol/L) were significantly lower in patients than in controls ((4.52 ±0.85), (1.47 ± 0.86), (2.85 ±0.71) mmol/L ,t=2.056,2.528 and 2.149 respectively, all P<0.05).The levels of total cholesterol ((4.28 ±0.96) mmol/L) and triglyceride ((1.20 ±0.64) mmol/L) were significantly lower in MSA-P patients than in control group ((4.52 ±0.85), (1.47 ±0.86) mmol/L;LSD-t=1.983, 2.566, both P<0.05).After adjusting for age, gender and histories, the odds ratio ( OR) was 0.31 (95%CI 0.15-0.65, P =0.002 ) for MSA patients in the highest quartile of triglyceride and 0.38 (95%CI 0.17 -0.83,P=0.016) for those in the highest quartile of high-density lipoprotein (HDL), compared with the lowest quartiles.And HDL level was in a significantly positive correlation with onset age (r=0.15, P=0.039).Conclusion Our data suggest that triglyceride and HDL may be associated with the prevalence of MSA, and the lower levels of HDL, the earlier onset of MSA.

The main commercial production of fructooligosaccharides (FOS) comes from enzymatic transformation using sucrose as substrate by microbial enzyme fructosyltransferase. A fructosyltransferase genomic DNA was isolated from Aspergillus niger QU10 by PCR. The nucleotide sequence showed a 1 941 bp size, and has been submitted to GenBank (KF699529). The cDNA of the fructosyltransferase, containing an open reading frame of 1 887 bp, was further cloned by RT-PCR. The fructosyltransferase gene from Aspergillus niger was functionally expressed both in Escherichia coli and Pichia pastoris GS 115. The highest activity value for the construction with the α-factor signal peptide reached 431 U/mL after 3 days of incubation. The recombinant enzyme is extensively glycosylated, and the active form is probably represented by a homodimer with an apparent molecular mass of 200 kDa as judged from mobility in seminative PAGE gels. The extracellular recombinant enzyme converted sucrose mostly to FOS, mainly 1-kestose and nystose, liberating glucose. FOS reached a maximal value and represented about 58% of total sugars present in the reaction mixture after 4 h reaction. The results suggest that the availability of recombinant Pichia pastoris as a new source of a FOS-producing enzyme might result of biotechnology interest for industrial application.
Aspergillus niger ; enzymology ; genetics ; Base Sequence ; Cloning, Molecular ; DNA, Complementary ; Escherichia coli ; Fungal Proteins ; genetics ; metabolism ; Glycosylation ; Hexosyltransferases ; genetics ; metabolism ; Molecular Sequence Data ; Molecular Weight ; Pichia ; Sucrose ; metabolism ; Trisaccharides ; metabolism

In this study, we investigated the mechanism of transformation by Bacillus subtilis strain 168 by proteomic analysis. B. subtilis strain 168 was able to stereoselectively transform cis-propenylphosphonic acid (cPPA) to fosfomycin. The maximal fosfomycin production was 816.6 microg/mL after two days cultivation, with a conversion rate of 36.05%. We separated the whole cellular proteins by two-dimensional gel electrophoresis (2-DE) method, and 562 protein spots were detected in the presence of cPPA in the medium, while 527 protein spots were detected in the absence of cPPA. Of them, 98 differentially expressed protein spots were found. Among them, 52 proteins were up-regulated whereas 20 were down-regulated in the presence of cPPA in the medium, and 26 induced at the presence of cPPA. The differentially expressed proteins were analyzed by combined MS and MS/MS methods. Eighty protein spots, including 45 up-regulated proteins, 17 down-regulated proteins, and 18 induced by cPPA were identified. Based on the results of proteomic analysis, we postulated two steps of transformation: in the first step, cPPA was hydrated to 2-hydroxypropylphosphonic acid; in the second step, 2-hydroxypropylphosphonic acid was transformed to fosfomycin via a dehydrogenation reaction.
Bacillus subtilis ; genetics ; growth & development ; metabolism ; Bacterial Proteins ; metabolism ; Biotransformation ; Fosfomycin ; metabolism ; Organophosphorus Compounds ; metabolism ; Proteome ; metabolism ; Proteomics