Metabolic Syndrome is an Independent Risk Factor for Fuhrman Grade and TNM Stage of Renal Clear Cell Carcinoma.

BACKGROUND: More and more evidences show that metabolic syndrome (MS) is closely related to clear cell renal cell carcinoma (ccRCC), but the impact of MS on Fuhrman grade and TNM stage of ccRCC is rarely reported. PURPOSE: To explore the relationship between MS and its components of Fuhrman grade and TNM stage in ccRCC. OBJECTIVE: The clinical data of 247 patients with ccRCC diagnosed in our hospital from January 2016 to November 2020 were retrospectively collected and analyzed. Based on diagnostic criteria of MS, the patients were divided into MS and non-MS group. Logistic regression analysis was used to analyze the independent risk factors of ccRCC. RESULTS: The incidence of MS was 32.79% (81/247). There was no significant difference in age, gender, smoking and drinking between MS group and non-MS group (P > 0.05). In MS group, BMI ≥25kg/m2, hypertension, diabetes, hyperlipidemia, tumor diameter, poorly differentiated renal cancer, high-stage renal cancer, triglyceride, fasting blood glucose, glycated hemoglobin, fasting insulin and homeostasis model assessment index were significantly higher than those in non-MS group (P < 0.001), while in high density lipoprotein cholesterol (p < 0.005), islet beta cell secretory index (P < 0.001), well-differentiated renal cell carcinoma (P= 0.009), and low-stage renal cell carcinoma (P = 0.019) were significantly lower than that of non-MS group. Logistic regression analysis showed that hypertension (P = 0.005), diabetes (P = 0.012), hyperlipidemia (P = 0.021) are independent risk factors for Fuhrman grade of ccRCC, while diabetes (P = 0.002), hyperlipidemia (P = 0.007) are independent risk factors for TNM staging of ccRCC. CONCLUSION: The patients with ccRCC and MS had higher Fuhrman grade and TNM stage. MS is an independent risk factor for Fuhrman grade and TNM stage of ccRCC.

Item Anomaly Detection Based on Dynamic Partition for Time Series in Recommender Systems.

In recent years, recommender systems have become an effective method to process information overload. However, recommendation technology still suffers from many problems. One of the problems is shilling attacks-attackers inject spam user profiles to disturb the list of recommendation items. There are two characteristics of all types of shilling attacks: 1) Item abnormality: The rating of target items is always maximum or minimum; and 2) Attack promptness: It takes only a very short period time to inject attack profiles. Some papers have proposed item anomaly detection methods based on these two characteristics, but their detection rate, false alarm rate, and universality need to be further improved. To solve these problems, this paper proposes an item anomaly detection method based on dynamic partitioning for time series. This method first dynamically partitions item-rating time series based on important points. Then, we use chi square distribution (χ2) to detect abnormal intervals. The experimental results on MovieLens 100K and 1M indicate that this approach has a high detection rate and a low false alarm rate and is stable toward different attack models and filler sizes.

[Effect of p53-regulated apoptosis-inducing protein 1 transfection on the biological characteristics of PC-3M human prostate cancer cells].

OBJECTIVE: To investigate the effect of p53-regulated apoptosis-inducing protein 1 (p53AIP1) gene on the proliferation, cell cycle, apoptosis, invasion and migration of PC-3M human prostate cancer cells in vitro. METHODS: The eukaryotic expression vector pDC316-p53AIP1 was constructed and confirmed by double enzyme digestion and PCR analysis. Then it was transfected into PC-3M human prostate cancer cells by Lipofectamine (TM) 2000. The expression of p53AIP1 protein was detected by Western blotting. The proliferation of PC-3M cells was tested by CCK-8 assay; the cell cycle and apoptosis rate were analyzed by flow cytometry combined with annexin V-FITC/PI staining; the effect of p53AIP1 on the cell invasion and migration was detected by Transwell(TM) assay. RESULTS The eukaryotic expression vector pDC316-p53AIP1 was constructed successfully. After transfected into PC-3M cells, Western blotting demonstrated that the protein p53AIP1 was effectively expressed. CCK-8 assay revealed that the proliferation of PC-3M cells was significantly inhibited by p53AIP1 (P<0.05). Flow cytometry indicated that the cells were arrested at S/G2-M phase (P<0.05) and cell apoptosis was evidently promoted (P<0.05). Transwell(TM) chamber experiments showed that p53AIP1 decreased the abilities of both invasion and migration of the cells (P<0.05). CONCLUSION: The p53AIP1 inhibits the proliferation of PC-3M cells, arrests cell cycle at S/G2-M phase, decreases the abilities of invasion and migration and promotes cell apoptosis.

[Expression and activity identification of a fusion protein for promoting adenovirus infection efficiency of dendritic cells].

OBJECTIVE: To construct a prokaryotic expression plasmid for CT40L, express the target protein in E. coli, purity the CT40L fusion protein and verify its antigenicity. METHODS: Gene sequences of Coxsackie and adenovirus receptor (CAR), bacteriophage T4 fibritin and mouse CD40L were found out in GenBank. Then functional domains of three molecules were linked to form a fusion sequence which was then optimized for prokaryotic expression. The optimized sequence was cloned into prokaryotic expression vector pET42a(+) to construct the recombinant expression vector pET42a-CT40L. The recombinant vector was transformed into BL21 (DE3) and the fusion protein CT40L/GST was induced by IPTG. The fusion protein was then subjected to purification using GST affinity chromatography and to identification of the immune activity using Western blotting and ELISA. RESULTS: The recombinant expression vector was verified correct by double digestion with Nco I and EcoR I. After IPTG induction, SDS-PAGE showed that the relative molecular mass of the fusion protein was about 78 kDa and that the purity of the purified protein reached 90%. Western blotting and ELISA demonstrated that the purified fusion protein had a valid antigenicity. CONCLUSION: The prokaryotic expression plasmid pET42a-Ct40L was successfully constructed and expressed in E. coli, and the purified fusion protein was proved to have a good antigenicity.

[Over-expression of human SPRY2 promotes the proliferation and survival of HEK293T cells].

OBJECTIVE: To construct a recombinant expression vector pDC315/hSPRY2 carrying human SPRY2 (hSPRY2) gene, and transfect the recombinant plasmid into human embryonic kidney HEK293T cells to express the target protein, and thus explore preliminarily the effect of hSPRY2 on the proliferation and survival of HEK293T cells. METHODS: The recombinant adenoviral vector expressing hSPRY2 was constructed and transfected into HEK293T cells in vitro. The expression of hSPRY2 target protein in the transfected cells was identified by Western blot analysis. The effect of hSPRY2 on the proliferation and survival of HEK293T cells in a serum-free condition was tested using CCK-8 assay. RESULTS: The recombinant expression vector pDC315/hSPRY2 was constructed, and the target protein hSPRY2 was expressed in the transfected HEK293T cells. The proliferation and survival rates of HEK293T cells transfected with pDC315/hSPRY2 were significantly higher than those transfected with pDC315/EGFP (P<0.05). CONCLUSION: The recombinant expression vector for hSPRY2 was successfully constructed and its expression was demonstrated in the transfected HEK293T cells. Over-expression of SPRY2 promoted the proliferation and survival of HEK293T cells.

[Cloning, prokaryotic expression, and antigenicity identification of extracellular domain of mouse CD40].

OBJECTIVE: To construct a prokaryotic expression plasmid for extracellular domain of mouse CD40 (mCD40), express the mCD40/GST recombinant protein in E.coli, purify the mCD40/GST recombinant protein and characterize its antigenicity. METHODS: Extracellular domain of mouse CD40 was amplified by PCR from cell line DC2.4 and then was cloned into prokaryotic expression vector pGEX-6P-1 to construct the recombinant expression vector pGEX-6P-1-mCD40. The expression vector was transformed into E.coli BL21 (DE3) and the fusion protein mCD40/GST was induced by IPTG. The fusion protein was purified through sepharose 4B. Then antigenicity of the purified mCD40/GST protein was verified by Western blotting and ELISA. RESULTS: The PCR product was verified by DNA sequencing to be consistent with the sequence of mouse CD40 on GenBank. The recombinant plasmid was identified by double digestion successfully. SDS-PAGE analysis showed the relative molecular mass of the fusion protein induced by IPTG was 45 000. Western blotting and ELISA demonstrated that the purified mCD40/GST protein had a good antigenicity. CONCLUSION: The prokaryotic expression plasmid pGEX-6P-1-mCD40 was constructed successfully. In E.coli BL21 (DE3) transformed with the plasmid, the mCD40/GST fusion protein was expressed by IPTG induction. The purified mCD40/GST fusion protein had a high antigenicity, which provides a strong support for the future study of CD40.

[Influence of electroporation on immunogenicity of the DNA vaccine pVAX-tG250FcGB].

OBJECTIVE: To investigate the influence of electroporation on the immunogenicity of the DNA vaccine pVAX- tG250FcGB. METHODS: The DNA vaccine pVAX-tG250FcGB was constructed by inserting the coding gene of tG250 fusion genes into the expression vector pVAX. The DNA vaccine was delivered in BALB/c mouse by electroporation or intramuscular injection, and the induced antigen specific immune responses were compared. RESULTS: The vaccine delivered by electroporation and intramuscular injection both induced immune responses in BALB/c mouse, but electroporation produced an obviously stronger effect than intramuscular injection. CONCLUSION: Electroporation-mediated DNA vaccine delivery can produce strong immune response in mice and is an effective means for studying the immunogenic effect of DNA vaccine pVAX-tG250FcGB.

[Construction and identification of a recombinant replication-defective adenovirus vector encoding p53AIP1 and its expression in human HeLa cells].

OBJECTIVE: To construct a recombinant replication-defective adenovirus vector carrying p53AIP1 (p53-regulated apoptosis-inducing protein 1) gene and observe its expression in human HeLa cells. METHODS: Specific primers were designed according to p53AIP1 gene sequence and inserted specific enzyme cutting sites. P53AIP1 gene was amplified by PCR and cloned into the adenovirus shuttle plasmid pDC316 to construct the recombinant vector pDC316-p53AIP1, which was co-transfected with helper plasmid pBHGloxδE1, 3Cre into HEK293 cells by Lipofectamine(TM); 2000. The recombinant replication-defective adenovirus (Ad-p53AIP1) was generated by means of homologous recombination of the two plasmids in HEK293 cells with the Cre-loxP recombinase system and harvested after 12 days. Ad-p53AIP1 and Ad-null were respectively transfected into HeLa cells at MOI=100. Then the expression of p53AIP1 gene was detected by Western blotting. RESULTS: pDC316-p53AIP1 was constructed successfully. The new constructed vector was confirmed by PCR analysis, double enzyme digestion and DNA sequencing. The results were in conformity with the expected. Western blotting demonstrated that the target p53AIP1 proteins were effectively expressed in the transfected HeLa cells. CONCLUSION: The recombinant adenovirus vector of Ad-p53AIP1 was successfully established, and it was proved to have a strong ability of infectivity.

[Prokaryotic expression, purification and antigenicity identification of recombinant human survivin protein].

OBJECTIVE: To construct a prokaryotic expression plasmid pET28a-survivin, optimize the recombinant protein expression conditions in E.coli, and purify the survivin recombinant protein and identify its antigenicity. METHODS: Survivin cDNA segment was amplified by PCR and cloned into prokaryotic expression vector pET28a(+) to construct the recombinant expression vector pET28a-survivin. The expression vector was transformed into BL21 (DE3) and the fusion protein survivin/His was induced by IPTG. The fusion protein was purified through Ni affinity chromatography. The antigenicity of the purified survivin protein was identified by Western blotting and ELISA. RESULTS: The recombinant expression vector was verified successfully by BamHI and HindIII. The fusion protein induced by IPTG was obtained with Mr; about 24 000. The purity of the purified protein reached 90% by SDS-PAGE analysis. And the antigenicity of the survivin protein was validated by Western blotting and ELISA. CONCLUSION: The prokaryotic expression plasmid pET28a-survivin was successfully constructed and the survivin protein was expressed and purified in E.coli. The antigenicity of the purified survivin protein was demonstrated desirable.

[The expression, purification and characterization of ß-hCG protein in E.coli].

OBJECTIVE: To obtain ß-chain human chorionic gonadotropin (ß-hCG) fusion protein (ß-hCG/GST) and identify its antigenicity. METHODS: The full-length gene of ß-hCG was amplified by PCR. The PCR product was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-ß-hCG, and then it was transformed into BL21 (DE3) for ß-hCG/GST fusion protein expression under IPTG induction. After SDS-PAGE assay, the fusion protein was purified by affinity chromatography and identified by Western blotting. The antigenicity of the purified fusion protein was characterized by ELISA. RESULTS: The ß-hCG gene we obtained had an identical sequence to that retrieved in GenBank. The prokaryotic expression vector pET-42a-ß-hCG was successfully constructed as confirmed by enzyme digestion and DNA sequencing. Both Western blotting and ELISA demonstrated that the purified ß-hCG fusion protein had satisfactory antigenicity. CONCLUSION: The purified ß-hCG/GST fusion protein with satisfactory antigenicity has been obtained, which will facilitate further study on active anti-tumor immunotherapy targeting ß-hCG.

[Prokaryotic expression, purification and antigenicity identification of mouse VEGFR2 extracellular 1-4 IgG-like domains].

OBJECTIVE: To obtain 1-4 IgG-like domains of mouse vascular endothelial growth factor receptor 2 (VEGFR2) fusion protein (mVEGFR2D1-4/GST) and identify its antiginicity and biological activity. METHODS: The gene of mVEGFR2D1-4 was amplified by RT-PCR from 14-days embryos of Balb/c mice. The PCR product was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-mVEGFR2D1-4, which was transformed into E. coli BL21 (DE3) strain for mVEGFR2D1-4/GST expression. The fusion protein was identified by SDS-PAGE and Western blotting, and the antigenicity of the protein purified by affinity chromatography was characterized by ELISA. The VEGF blocking effect of the purified protein in human umbilical vein endothelial cells (HUVECs) were evaluated in in vitro cell cultures. RESULTS: The mVEGFR2D1-4 gene was obtained, which had an identical sequence to that retrieved in GenBank. The prokaryotic expression vector for mVEGFR2D1-4 was successfully constructed as confirmed by enzyme digestion and DNA sequencing. Both Western blotting and ELISA demonstrated the antigenicity of the purified mVEGFR2D1-4 fusion protein, which obviously blocked the effect of VEGF in promoting HUVEC proliferation in vitro. CONCLUSION: The mVEGFR2D1-4/GST fusion protein obtained shows a strong antigenicity and biological activity to facilitate further study of active anti-tumor immunotherapy targeting VEGFR2.