Effects of Preoperative Anxiety Status on Haemodynamic Changes during and after Tooth Extraction in Middle-aged and Elderly Patients with Hypertension: a Prospective Repeated-Measures Cohort Study.

OBJECTIVE: To investigate the effects of dental anxiety on fluctuations in blood pressure (BP) and heart rate (HR) during tooth extraction in hypertensive patients under local anaesthesia, and how they are influenced by various confounding variables. METHODS: This is a prospective repeated-measures cohort study involving 600 patients successively recruited from Peking University School and Hospital of Stomatology, Beijing, China. BP and HR were repeatedly measured at rest (T0), before anaesthesia (T1), during tooth extraction (T2) and after tooth extraction (T3). Anxiety status was measured prior to local anaesthesia using a modified dental anxiety scale (MDAS). Three groups were assigned: mild anxiety (Corah DAS score of 4 to 8), moderate anxiety (score of 9 to 12) and severe anxiety (score of 13 to 20). We used a generalised linear mixed model (GLMM) to analyse the effects of dental anxiety on fluctuations in BP and HR. Interaction analysis was used to further explore the correlationship between these interactive factors. RESULTS: The mean anxiety scale score was 9.63 ± 2.88. Severe preoperative anxiety (score of 14 to 20) was associated with significantly increased HR during administration of anaesthesia. Patients with severe anxiety also displayed a significantly greater increase in HR during anaesthetic administration (P < 0.001). When analysing the joint effects of different anxiety statuses over time, blood pressure was significantly elevated in all patients with moderate and severe anxiety during tooth extraction at T2 (ß = 1.25, 95% CI 0.24 to 2.27). We also observed a significant decrease in HR in the moderate anxiety group at T3 (ß = -1.51, 95% CI -2.38 to -0.63) and a significant increase in HR in the severe anxiety group at T1, T2 and T3 (ß = 2.52, 95% CI 1.12 to 3.93; ß = 3.84, 95% CI 2.30 to 5.38; ß = 4.57, 95% CI 3.03 to 6.11, respectively). CONCLUSION: This study indicates that the effects of dental anxiety on BP and HR in middle-aged and elderly patients with hypertension during local anaesthesia and tooth extraction were influenced by various confounding variables.

[Impaired effect of BHC80 gene knock-down on the cardiac development in zebrafish].

The effect of BHC80 (a component of BRAF-HDAC complex) on development was not well studied, because BHC80 gene knock-out mice died in one day after birth. Interestingly, zebrafish embryos can live, even if their important organs like cardiac system has severe dysfunction, as 25%-40% O2 are supplied through their skin. Therefore, a model of BHC80 gene knock-down zebrafish embryos was established to explore the effect of BHC80 on the early embryonic development. BHC80-morpholino antisense oligonucleotides 2 (BHC80-MO2) was designed and injected into zebrafish embryos to interrupt the correct translation of BHC80 mRNA at one or two cells stage, which was proved by RT-PCR analysis. Two control groups, including non-injection group and control-MO (con-MO) injection group, and four different doses of BHC80-MO2 injection groups, including 4 ng, 6 ng, 8 ng and 10 ng per embryo were set up. The embryonic heart phenotype and cardiac function were monitored, analyzed and compared between con-MO and BHC80-MO2 groups by fluorescence microscope in vmhc:gfp transgenic zebrafish which express green fluorescent protein in ventricle. The results showed that BHC80-MO2 microinjection effectively knocked down the BHC80 gene expression, because the BHC80-MO2 group emerged a new 249 bp band which reduced 51 bp compared to 300 bp band of con-MO group in RT-PCR analysis, and the 51 bp was the extron 10. The abnormal embryo rate rose with the increase of BHC80-MO2 dosage. The proper BHC80-MO2 injection dosage was 8 ng per embryo, as minor embryos had abnormal phenotype in 4 ng and 6 ng per embryo groups and most embryos died in 10 ng per embryo group. BHC80-MO2 embryos exhibited abnormal cardiac phenotype, including imbalance of the proportion of heart ventricle to atrium, incomplete D-loop, even tubular heart, slow heart rates and cardiac dysfunction. The results from a model of BHC80 gene knock-down zebrafish embryos show that the abnormal cardiac phenotype and cardiac dysfunction of BHC80-MO2 embryos may be one of the probable reasons for the BHC80 gene knock-out mice death, which would provide a good research model to clarify the mechanism of cardiac development.

MicroRNA-200a promotes anoikis resistance and metastasis by targeting YAP1 in human breast cancer.

PURPOSE: The process of metastases involves the dissociation of cells from the primary tumor, penetration into the basement membrane, invasion, and exiting from the vasculature to seed and colonize distant tissues. miR-200a is involved in this multistep metastatic cascade. This study aimed to test the hypothesis that miR-200a promotes metastasis through increased anoikis resistance in breast cancer. EXPERIMENTAL DESIGN: Breast cancer cells transfected with mimic or inhibitor for miR-200a were assayed for anoikis in vitro. miR-200a expression was assessed by quantitative real-time PCR (qRT-PCR). Luciferase assays, colony formation assays, and animal studies were conducted to identify the targets of miR-200a and the mechanism by which it promotes anoikis resistance. RESULTS: We found that overexpression of miR-200a promotes whereas inhibition of miR-200a suppresses anoikis resistance in breast cancer cells. We identified Yes-associated protein 1 (YAP1) as a novel target of miR-200a. Our data showed that targeting of YAP1 by miR-200a resulted in decreased expression of proapoptotic proteins, which leads to anoikis resistance. Overexpression of miR-200a protected tumor cells from anoikis and promoted metastases in vivo. Furthermore, knockdown of YAP1 phenocopied the effects of miR-200a overexpression, whereas restoration of YAP1 in miR-200a overexpressed breast cancer cells reversed the effects of miR-200a on anoikis and metastasis. Remarkably, we found that YAP1 expression was inversely correlated with miR-200a expression in breast cancer clinical specimens, and miR-200a expression was associated with distant metastasis in patients with breast cancer. CONCLUSIONS: Our data suggest that miR-200a functions as anoikis suppressor and contributes to metastasis in breast cancer.

[Tumor-targeting expression of a new tumor suppressor gene HCCS1 and its tumor-selective inhibitory effects on hepatocellular carcinoma].

OBJECTIVE: To construct a tumor-targeting recombinant adenovirus vector containing hepatocellular carcinoma suppressor gene HCCS1 to enhance the safety of tumor treatment. METHODS: CCK-8 assay was used to observe different inhibitory effects on normal and malignant liver cells with high expressions of HCCS1 protein. The relative transcriptional activity of PEG-3p was quantified by luciferase assay. Recombinant adenovirus Ad-PEG-3p-HCCS1 was packaged with AdEasy system and confirmed by PCR. The tumor-targeted expression of HCCS1 protein in cells infected with Ad-PEG-3p-HCCS1 was determined by Western blot. Crystal violet assay and MTT assay were applied to observe the selective anti-tumor effects of the newly constructed virus in vitro. RESULTS: A higher inhibitory rate of about 60% was found in BEL-7404 and SW-620 than that in L02 and NHLF 96 h after the high expression of HCCS1. Luciferase assay showed 3.9-, 4.7-, and 1.5-fold transcriptional activity in BEL-7404, BEL-7405 and QGY-7703 respectively, in comparison with that in L02. Ad-PEG-3p-HCCS1 was constructed successfully and was verified by PCR. Western blot indicated that high expression of HCCS1 could be induced in BEL-7404 and QGY-7703 but not in L02. Crystal violet assay and MTT assay showed that it remarkably reduced the toxicity to L02 but still had enough antitumoral effect on Ad-CMV-HCCS1. CONCLUSIONS: With high expression of HCCS1 the tumor cells we used are being inhibited more. PEG-3p has the tumor-selective driving function in malignant liver cells. Our recombinant adenovirus Ad-PEG-3p-HCCS1 can tumor-targeting induce HCCS1 expression in tumor cells, which can improve the safety of gene therapy with HCCS1.

[Genotyping and polymorphism analysis of Plasmodium falciparum isolates by nest-PCR and PCR-RFLP].

OBJECTIVE: To analyze the efficiency and specificity of MSP2 alleles genotyping for Plasmodium falciparum isolates by Nest-PCR and PCR-RFLP. METHODS: MSP2 alleles from Plasmodium falciparum isolates of Yunnan and Hainan were genotyped by Nest-PCR and PCR-RFLP, respectively, and the efficiency and specificity of the two methods were analyzed. RESULTS: The conventional Nest-PCR method could detect 79.8% (166/208) alleles of MSP2, and 65.7% (65/99) for 3D7 family, but could not identify the type of any allele. While PCR-RFLP showed 25.3% higher genotyping efficiency than Nest-PCR. Moreover, this method could identify the allele types. CONCLUSION: PCR-RFLP genotyping technique is more efficient and specific than conventional Nest-PCR, and it is a convenient tool in the study on molecular epidemiology of malaria.

[The anti-tumour effect of Wuxing soup and its mechanism in inducing apoptosis of tumour cells mediated by calcium].

OBJECTIVE: To confirm the anti-cancer effect and mechanism of Wuxing soup. METHODS: Inhibition of cellular growth under Wuxing soup treatment was observed by MTT; Apoptosis was detected by gel electrophoresis, transmission electron microscopy and FACS; The concentration of calcium was measured by fluorescence probe. RESULTS: After SGC-7901 cell being treated by Wuxing soup, it showed that: 1) Wuxing soup could specifically inhibit cancer cells proliferation in a time and dose dependent manner; 2) Typical apoptotic morphological changes and DNA ladder of SGC-7901 cells were observed; 3) calcium inhibitor Bapta AM could reduce the apoptotic rate and protect SGC-7901 cells in a dose dependent manner. CONCLUSION: Wuxing soup has an effective inhibition on cancer cells, and can induce SGC-7901 cells to apoptosis by calcium.

[Preparation of anti-human integrin beta 3 monoclonal antibody and study on its antitumor activity].

AIM: To prepare the monoclonal antibody (mAb) against human integrin beta 3, and explore its role in tumor therapy. METHODS: Total RNA was isolated from human lymphocytes, and the DNA fragment encoding extra-cellular domain of human integrin beta 3 was amplified by RT-PCR. The integrin beta 3 gene was cloned into the prokaryotic expression vector pQE30, and the expression plasmid pQE30-beta 3 was transformed into E.coli M15. The expression of beta 3 protein was induced by IPTG, and the expressed beta 3 protein was purified by Ni-affinity agarose. BALB/c mice were immunized with the purified beta 3 protein, and mAb was prepared by hybridoma technique. The specificity of the mAbs was identified by Western blot. The mAbs were screened by their inhibitory effect on the tumor growth in vivo. The inhibition of the mAb to HUVEC cell growth was detected by MTT assay. The role of the mAb in inducing HUVEC apoptosis was analyzed by flow cytometry (FCM). RESULTS: The extra-cellular gene fragment of human integrin beta 3 was amplified by RT-PCR, and the expression plasmid pQE30-beta 3 was constructed. Human integrin beta 3 protein was expressed and purified, and used for immunization. Eight clones of mAb against human integrin beta 3 were successfully prepared. The mAb 4F12 was found to inhibit tumor growth in vivo and reduce blood vessels in tumor. Furthermore, the mAb 4F12 could inhibit HUVEC growth and induce apoptosis in vitro. CONCLUSION: The human integrin beta 3 protein was obtained and the mAbs against it have been prepared successfully. The mAb 4F12 can inhibit tumor growth in vivo and reduce blood vessels in tumor. One of the mechanisms of the antitumor effect is inhibition of growth and induction of apoptosis of endothelial cells of blood vessels in tumor.

[Experimental induction of immunity by anti-idiotypic vaccines against small-cell-lung cancer].

OBJECTIVE: To investigate the efficacy of anti-idiotype antibody 3F6 and its single-chain variable fragment (3F6 ScFv) to induce humoral and cellular immune responses against small-cell-lung cancer (SCLC). METHODS: 3F6 and 3F6 ScFv (Ab2) were used to immunize BALB/c mice. The reaction of antibodies (Ab3) with specific antigen on NCI-H128 cells was tested by ELISA and Western blot, and the antibody binding inhibition assays were performed by competitive Western blot. Cellular immunity against SCLC induced by Ab2 was detected by a delayed-type hypersensitivity response and mouse lymphocyte proliferation assay. RESULTS: The sera immunized with Ab2 showed significant reaction (P < 0.001, as compared to control sera) with SCLC-specific antigen on NCI-H128 cells and specifically competed the binding of 2F7 (Ab1) to the specific antigen. DTH responses challenged with NCI-H128 cells were significantly (P < 0.001) stronger in mice immunized with Ab2 as compared to mice immunized with normal mouse IgG. T cell proliferation was significantly higher in Ab2-immunized mice (P < 0.05) than in control mice. CONCLUSION: The two kinds of anti-idiotypic antibodies successfully mimic the SCLC-specific antigen on NCI-H128 cell and induce strong humoral and cellular immune responses to SCLC-specific antigen in syngeneic mice. They may become novel vaccines against human small-cell-lung cancer and worthy of further investigation.