Objective:To assess the prognostic value of methylation of interferon regulatory factor 6 ( IRF6) gene promoter in patients diagnosed with Kidney renal clear cell carcinoma (KIRC). Methods:The primary lesions of fifty KIRC patients who were diagnosed at the First Affiliated Hospital of Nanjing Medical University from January 2016 to January 2020 were collected. The expression of IRF6 protein was determined with an immunohistochemical method. The correlation between the level of IRF6 expression and survival and/or metastasis status was analyzed. The mRNA and protein levels of the IRF6 in KIRC and normal renal tissues were compared by using bioinformatic tools. The difference in the methylation rate of the IRF6 gene promoter between tumor and adjacent tissues was analyzed by searching the online databases. Statistical analysis was carried out for the methylation status of the IRF6 gene promoter region to select those negatively correlated with the overall survival (OS) among the patients. In vitro experiments were conducted with cell lines to verify the correlation between the status of promoter methylation and transcription level of the IRF6 gene. Results:The mRNA and protein levels of the IRF6 gene in KIRC tissues were significantly lower than those of the normal controls, and this was more prominent in patients who had died or developed metastasis. The extent of IRF6 gene promoter methylation in the KIRC tissues was much higher compared with that of the adjacent normal renal tissues. There was a significant negative correlation between the methylation of the IRF6 gene promoter and mRNA level of the IRF6 ( R= -0.52). The higher methylation degree in the IRF6 gene promoter regions cg12034118 and cg16030177, the shorter the OS and worse prognosis in the patients. Only twenty CpG sites in cg12034118 were confirmed to be highly methylated in KIRC cell lines. The transcription level of the IRF6 gene was upregulated in a time- and dose-dependent manner after the treatment with demethylation reagent 5-azadeoxycytidine. Conclusion:The methylation of IRF6 gene promoter in the renal tissues of KIRC patients is closely correlated with the OS. Cg12034118 may provide a promising biomarker for laboratory detection, and its high methylation rate has certain reference value for the prognosis.

[This corrects the article DOI: 10.1016/j.apsb.2021.07.006.].

Objective:To Clone the silencer sequences of human stimulator of interferon genes (STING) and evaluate its activity in HEK293T and HeLa, and to preliminarily investigate the transcriptional regulatory mechanisms, screening and verifying the possible binding elements for the silencer sequences of STING.Methods:The human STING-5-1a(-124~+ 267, transcription start site, TSS: 0) and STING-5-2a(-124~+ 168) regions were amplified by PCR, subcloned into pGL3-Basic plasmid, and the luciferase activity was detected in HEK293T and HeLa; the human STING silencer region STING-5-1b(+ 169~+ 267) was subcloned into the pGL3-Control plasmid (pGL3-C-5-1b-positive/negative) and STING-5-1b is divided into two complementary elements, subcloned into pGL3-Control vector, named pGL3-C-STING 5-1b-α(+ 169~+ 209) and pGL3-C-STING-5-1b-β(+ 210~+ 267), detecting the relative luciferase activity of the above plasmid in HEK293T and HeLa. Using bioinformatics methods to predict the transcription factor binding site of human STING silencer, make site-directed mutagenesis at the predicted binding site, and detect luciferase activity. Knockdown of THAP1 and TEAD1 by a siRNA strategy and detect the expression level of STING by western blot analysis. Chromosome immunoprecipitation assay further verified the binding of transcription factor to hSTING silencer.Results:It was verified that plasmids mentioned above were constructed correctly by nucleotide sequencing. The relative luciferase increased after truncating the STING-5-1b(+ 169~+ 267) fragment. The relative luciferase activity of pGL3-C-5-1b-positive recombinant plasmid decreased ( P<0.05), compared with pGL3-Control plasmid. Among them, STING-5-1b-β(+ 210~+ 267) fragment play a major inhibitory role. Using bioinformatics software to predict that the transcription factors PR domain zinc finger protein 4 (PRDM4), Thanatos-associated protein 1 (THAP1), TEA Domain Transcription Factor 1 (TEAD1), Nuclear receptor subfamily 4 group A member 1 (NR4A1), Krueppel-like factor 4 (KLF4) and Forkhead box protein O3(FOXO3) may bind to the human STING silencer region (+ 210~+ 267). After transfecting the mutant recombinant plasmid of the transcription factors into HEK293T and HeLa, the relative luciferase activity of THAP1-Mut and TEAD1-Mut were significantly increased, suggesting that STING silencers may contain binding sites of THAP1 and TEAD1. Knockdown of THAP1 and TEAD1 by a siRNA strategy significantly enhanced the transcription activity. Chromosome immunoprecipitation assay showed that the transcription factors TEAD1 and THAP1 combined with hSTING silencer region in the cells. Conclusions:The hSTING silencer luciferase reporter plasmid was successfully constructed. By the activity comparison, it is speculated that the core silencer region of human STING is located in the + 210~+ 267 element, which may contain several potential transcription factor binding sequences.The potential binding sites for transcription factors that may be contained in the DNA, and use Western blot and chromosome immunoprecipitation assays to further confirm the combination of transcription factors TEAD1 and THAP1 with hSTING silencer, laying the foundation for subsequent research.

Given the opposing effects of Akt and AMP-activated protein kinase (AMPK) on metabolic homeostasis, this study examined the effects of deletion of Akt2 and AMPKα2 on fat diet-induced hepatic steatosis. Akt2–Ampkα2 double knockout (DKO) mice were placed on high fat diet for 5 months. Glucose metabolism, energy homeostasis, cardiac function, lipid accumulation, and hepatic steatosis were examined. DKO mice were lean without anthropometric defects. High fat intake led to adiposity and decreased respiratory exchange ratio (RER) in wild-type (WT) mice, which were ablated in DKO but not Akt2−/− and Ampkα2−/− mice. High fat intake increased blood and hepatic triglycerides and cholesterol, promoted hepatic steatosis and injury in WT mice. These effects were eliminated in DKO but not Akt2−/− and Ampkα2−/− mice. Fat diet promoted fat accumulation, and enlarged adipocyte size, the effect was negated in DKO mice. Fat intake elevated fatty acid synthase (FAS), carbohydrate-responsive element-binding protein (CHREBP), sterol regulatory element-binding protein 1 (SREBP1), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), peroxisome proliferator-activated receptor-α (PPARα), PPARγ, stearoyl-CoA desaturase 1 (SCD-1), phosphoenolpyruvate carboxykinase (PEPCK), glucose 6-phosphatase (G6Pase), and diglyceride O-acyltransferase 1 (DGAT1), the effect was absent in DKO but not Akt2−/− and Ampkα2−/− mice. Fat diet dampened mitophagy, promoted inflammation and phosphorylation of forkhead box protein O1 (FoxO1) and AMPKα1 (Ser485), the effects were eradicated by DKO. Deletion of Parkin effectively nullified DKO-induced metabolic benefits against high fat intake. Liver samples from obese humans displayed lowered microtubule-associated proteins 1A/1B light chain 3B (LC3B), Pink1, Parkin, as well as enhanced phosphorylation of Akt, AMPK (Ser485), and FoxO1, which were consolidated by RNA sequencing (RNAseq) and mass spectrometry analyses from rodent and human livers. These data suggest that concurrent deletion of Akt2 and AMPKα2 offers resilience to fat diet-induced obesity and hepatic steatosis, possibly through preservation of Parkin-mediated mitophagy and lipid metabolism.

Cancer stem cells are currently under intensive investigation due to their capabilities for tumor initiation, self-renewal, and resistance to chemotherapy. CD133 is implicated in stemness and the malignancy of tumor cells. Here, we explored heat shock protein gp96 adjuvanted CD133 epitope vaccine against leukemia. We screened and identified three H2-Kd-restricted cytotoxic T lymphocyte (CTL) epitopes derived from CD133, CD133₄₁₉₋₄₂₈, CD133₇₀₂₋₇₁₀ and CD133₇₆₀₋₇₆₉. The immunogenicity and antitumor activity of the epitope vaccine using heat shock protein gp96 as adjuvant were further determined in CD133⁺ leukemia xenograft mice. Finally, we demonstrate that adoptive transfer of epitope-specific CTLs led to suppression of leukemia growth. Our data therefore provide the basis for designing a CD133 epitope vaccine to activate specific CTLs against CD133⁺ leukemia and other cancers.

BACKGROUND: Circulating levels of cell-free DNA increase in many pathologic conditions. However, notable discrepancies in the quantitative analysis of cell-free DNA from a large number of laboratories have become a considerable pitfall, hampering its clinical application. METHODS: We designed a novel recombinant DNA fragment that could be applied as an internal standard in a newly developed and validated duplex real-time PCR assay for the quantitative analysis of total cell-free plasma DNA, which was tested in 5,442 healthy adults and 200 trauma patients. RESULTS: Compared with two traditional methods, this novel assay showed a lower detection limit of 0.1 ng/mL, lower intra- and inter-assay CVs, and higher accuracy in the recovery test. The median plasma DNA concentration of healthy males (20.3 ng/mL, n=3,092) was significantly higher than that of healthy females (16.1 ng/mL, n=2,350) (Mann-Whitney two-sample rank sum test, P<0.0001). The reference intervals of plasma DNA concentration were 0-45.8 ng/mL and 0-52.5 ng/mL for healthy females and males, respectively. The plasma DNA concentrations of the majority of trauma patients (96%) were higher than the upper normal cutoff values and were closely related to the corresponding injury severity scores (R²=0.916, P<0.0001). CONCLUSIONS: This duplex real-time PCR assay with a new internal standard could eliminate variation and allow for more sensitive, repeatable, accurate, and stable quantitative measurements of plasma DNA, showing promising application in clinical diagnosis.
Adult ; DNA/*blood/standards ; Female ; Healthy Volunteers ; Humans ; Male ; Middle Aged ; Real-Time Polymerase Chain Reaction/*methods/standards ; Reference Values ; Wounds and Injuries/blood

Type 1 diabetes (T1D), the most prevalent human autoimmune disease, occurs in genetically susceptible individuals. Regulatory T cells (Tregs) are defective in T1D setting. Therefore, efforts to repair or restore Tregs in T1D may prevent or reverse this autoimmune disease. Here, we studied the potential role of rgp96 in preventing T1D, using non-obese diabetic (NOD) mice as an animal model. High-dose rgp96 immunization elicited efficient protection of mice against T1D, as evidenced by stable blood glucose, decreased disease incidence. Significantly increased CD4⁺ CD25⁺ Foxp3⁺ Tregs were observed in immunized mice. In vitro co-culture experiments demonstrated that rgp96 stimulation enhanced Treg proliferation and suppressive function by up-regulation of Foxp3 and IL-10. Our work shows that activation of Tregs by high-dose rgp96 immunization protects against T1D via inducing regulatory T cells and provides preventive and therapeutic potential for the development of an rgp96-based vaccine against T1D.
Animals ; Antigens, Neoplasm ; administration & dosage ; immunology ; Coculture Techniques ; Diabetes Mellitus, Type 1 ; prevention & control ; therapy ; Forkhead Transcription Factors ; Heat-Shock Proteins ; administration & dosage ; immunology ; Interleukin-10 ; immunology ; Mice ; Mice, Inbred NOD ; T-Lymphocytes, Regulatory ; immunology ; Up-Regulation ; Vaccination

Objective To improve early diagnosis of testicular torsion,reduce misdiagnosis and reduce the rate of orchiectomy.Methods The diagnosis and treatment of testicular torsion in 31 cases were retrospectively ana-lyzed.Results 54.8% misdiagnosis rate was in this group,all the 31 cases were diagnosed by color Doppler ultra-sound,including 19 cases of retained testicle and orchiectomy in 12 cases (38.7%).In 19 cases of this group retained testis,testicular torsion time within 5 hours was 2 cases,and postoperative had testicles survival.In the 6 cases of testicular torsion time within 5 hours to 10 hours,5 cases had the testis survival.In the 5 cases of torsion of testis time was 10 hours to 24 hours,3 caseshad the testis survival.In the 6 cases of testicular torsion time more than 24 hours,2 cases of testis survival.After postoperative following-up,19 cases of retained testis had no recurrence,all the 31 cases were found no contralateral testicular torsion and all cases sex hormone levels were in the normal levels. Conclusion Testicular torsion is easily misdiagnosed,color doppler ultrasound should be preferred.Early diagnosis and aggressive surgical exploration,unity and strictly control the orchiectomy surgery indications,means a lot to reduce the rate of orchiectomy.

Objective To investigate the related index of early diagnosis of acute-on-chronic liver failure caused by chronic hepatitis B.Methods 13 cases of the acute-on-chronic liver failure caused by chronic hepatitis B were collected in our department,39 cases of chronic hepatitis B were randomly paired to analyze the difference index.ROC curve analysis was conducted for the different data,then to choose the data whose AUC (area under the curve)is >0.8 to accumulate points,while the cutoff value in the light of Youden index was indentified.Each accu-mulating points data would score 1 point if it is≥cutoff value,otherwise,it would score 0 points.ROC curve analysis was conducted for the total accumulating points,the cutoff value of the total accumulating points according to Youden index was indentified,the sensitivity and specificity were calculated.Results The data of which AUC are >0.8 include glutamic-pyruvic transaminase (ALT)/Upper Limit Of Normal (ULN),neutrophil count (N),alkaline phosphatase (AKP)/ULN,glutamic-oxalacetic transaminease(AST)/ULN,direct bilirubin/total bilirubin(Dbil/Tbil),total bile acid(TBA),whose AUC were respectively 0.869,0.874,0.897,0.917,0.919 and 0.978,while their cutoff value were respectively 18.57,3.1 ×109/L,0.99,22.21,44.41%and 140.20μmol/L.The cutoff value of total accumula-ting points of the six data was 4 points,AUC was 0.987,Youden index was 0.949,and their sensitivity for ealier diag-nosis for the acute -on -chronic liver failure caused by chronic hepatitis B was 100%,specificity was 94.87%. Conclusion The accumulating points of the six data aboved (ALT/ULN,N,AKP/ULN,AST/ULN,Dbil/Tbil,TBA) will help the early diagnosis of acute-on-chronic liver failure caused by chronic hepatitis B.

Objective To investigate the optimal extraction technique and antioxidant activity of curcuminoids from Curcuma longa L. Methods The optimized extraction process of curcuminoids from Curcuma longa L. with ethanol as extracting solvent were studied based on orthogonal test.The effect of each factor such as the ethanol concentration, the amount of solvent, extraction temperature and extraction time were investigated based on a single-factor experiment.The effects of extraction yield of curcuminoids under different processing conditions were compared by HPLC method. Content of curcuminoids and antioxidant activity of the extract were used as comprehensive survey index. Free radical scavenging DPPH method was used to assess the antioxidant effect. And the extraction technique parameters were optimized. Results The condition of optimal extraction technique was: ethanol concentration 60%, amount of solvent 20 folds, extraction time 2 h, extraction temperature 90 ℃. Under these conditions, the extraction yield of curcuminoids was 3. 65%. DPPH free radical scavenging yield of the curcumin extract was 79.79%. Conclusion This optimized technology is simple and have a good reappearing rate, and the extracts of Curcuma longa L.show good antioxidant activity.