[The role and mechanism of TRAF2 in the biological behavior of gastric cancer].

Objective: To clarify the effect of TRAF2 in the biological behavior of gastric cancer and explore the mechanism. Methods: TRAF2 stably depleted AGS cell was established. Cell growth was monitored by x-CELLigence system. Cell proliferation was detected using cell viability assay. The apoptosis and cell cycle were detected by flow cytometry. The difference of migration and invasion abilities were measured by real-time xCELLigence system and Transwell. The expression and activity of NF-κB signaling pathway were measured by western blot and TransAM assay. The expression of TRAF2 in gastric cancer tissue and its clinical significance were detected by immunohistochemistry. Results: The cell index of AGS-siTRAF2 cells was significantly lower than that of AGS-sictrl cells at 8 h. In the cell viability assay, the A values of AGS-siTRAF2 cells were 51 296.00±2 631.06, 68 389.25±6 703.21 and 65 559.50±6 339.22 at 24 h, 48 h and 72 h. The values of the viability of AGS-siTRAF2 cells were significantly lower than those of AGS-sictrl cells (P<0.001). The results of flow cytometry showed that the apoptosis rates of AGS-siTRAF2 cells were (1.42±0.07)%, (2.98±0.11)% and (1.56±0.03)% at 24 h, 48 h and 72 h, respectively, which were significantly higher than those of AGS-sictrl cells (all P<0.05). The distribution of S phase in AGS-siTRAF2 cells was (23.57±1.12)%, while that in the AGS-sictrl cells was (19.49±1.19)%. The difference was statistically significant (P=0.012). AGS-siTRAF2 cells migrated much slower than AGS-sictrl cells from 3 h and the number of migrated AGS-sictrl cells was 121.7±6.7 while that of AGS-siTRAF2 cells was 84.0±6.6 (P=0.002). The cell index of AGS-siTRAF2 cells was less than that of AGS-sictrl cells from 3 h. In Transwell assay, the number of invaded AGS-sictrl cells was 109.3±3.1 after 24 h of culture, significantly higher than 79.0±6.2 of AGS-siTRAF2 cells (P=0.002). Western blot analysis showed that the expression levels of RelA, RelB, p50 and p52 in AGS-siTRAF2 cells were significantly lower than those in AGS-sictrl cells. The activities of RelA, RelB, p50 and p52 in AGS-siTRAF2 cells were 0.01±0.00, 0.01±0.01, 0.92±0.01 and 0.53±0.03, respectively, significantly lower than those of AGS-sictrl cells (all P<0.001). High TRAF2 expression (TRAF2-high) was found in 53.0% of GC samples, while TRAF2-high was only observed in 38.0% of the paired adjacent tissues (P=0.033). The expression of TRAF2 was significantly higher in the tubular adenocarcinoma, poor differentiation advanced T, advanced N, and clinical staging (P<0.05). The median survival time were 17 months and 78 months in the TRAF2 high-expression and low-expression groups, respectively, and the difference was statistically significant (P=0.010). Conclusions: Depletion of TRAF2 inhibits the AGS cell growth, migration and invasion. The expression of TRAF2 is increased in gastric tumor tissue. The expression of TRAF2 is associated with the prognosis of gastric cancer.

A mutational signature associated with alcohol consumption and prognostically significantly mutated driver genes in esophageal squamous cell carcinoma.

Background: Esophageal squamous cell carcinoma (ESCC) is often diagnosed at an advanced and incurable stage. Information on driver genes and prognosticators in ESCC remains incomplete. The objective was to elucidate significantly mutated genes (SMGs), mutational signatures, and prognosticators in ESCC. Patients and methods: Three MutSig algorithms (i.e. MutSigCV, MutSigCL and MutSigFN) and '20/20+' ratio-metric were employed to identify SMGs. Nonnegative matrix factorization was used to decipher mutational signatures. Kaplan-Meier survival analysis, multivariate Cox and logistic regression models were applied to analyze association between mutational features and clinical parameters. Results: We identified 26 SMGs, including 8 novel (NAV3, TENM3, PTCH1, TGFBR2, RIPK4, PBRM1, USP8 and BAP1) and 18 that have been previously reported. Three mutational signatures were identified to be prevalent in ESCC including clocklike C>T at CpG, APOBEC overactive C>T at TpCp[A/T], and a signature featured by T>C substitution. The T>C mutational signature was significantly correlated with alcohol consumption (OR: 3.59; 95% CI: 2.30-5.67; P < 0.001). This alcohol consumption signature was also observed in liver cancer and head and neck squamous cell carcinoma, and its mutational activity was substantially higher in samples with mutations in TP53. Survival analysis revealed that TENM3 mutations (HR: 5.54; CI: 2.68-11.45; P < 0.001) and TP53 hotspot mutation p.R213* (HR: 3.37; CI: 1.73-8.06; P < 0.001) were significantly associated with shortened survival outcome. The association remained statistically significant after controlling for age, gender, TNM stage and tumor grade. Conclusions: We have uncovered several new SMGs in ESCC and defined an alcohol consumption related mutational signature. TENM3 mutations and the TP53 hotspot mutation p.R213* are independent prognosticators for poor survival in ESCC.

Two-step identification of taro (Colocasia esculenta cv. Xinmaoyu) using specific psbE-petL and simple sequence repeat-sequence characterized amplified regions (SSR-SCAR) markers.

Colocasia esculenta cv. Xinmaoyu is an eddoe-type taro cultivar local to Taicang, Jiangsu Province, China; it is characterized by its pure flavor, glutinous texture, and high nutritional value. Due to its excellent qualities, the Trademark Office of the State Administration for Industry and Commerce of the People's Republic of China awarded Xinmaoyu, a geographical indication certification in 2014. Therefore, there is an urgent need to develop an efficient molecular marker for the specific identification of this cultivar, which would greatly facilitate the conservation and utilization of this unique germplasm resource. In the present study, amplifying the psbE-petL fragment from two dasheen-type and seven eddoe-type taro cultivars revealed three conserved insertions/deletions among sequences from the two taro types. Based on these sequence differences, a pair of site-specific primers was designed targeting the psbE-petL sequence from the dasheen-type taro, which specifically amplified a DNA band in all individuals from cultivars of this type, but not in those from the seven eddoe-type cultivars. To discriminate Xinmaoyu from the other eddoe-type taro cultivars, a pair of simple sequence repeat-sequence characterized amplified region (SSR-SCAR) primers was further developed to specifically amplify a DNA band from all Xinmaoyu individuals, but not from individuals of other eddoe-type taro cultivars. In conclusion, through a two-step-screening procedure using psbE-petL and SSR-SCAR markers, we developed a pair of primers that could specifically discriminate Xinmaoyu from nine taro cultivars commonly cultivated in Jiangsu Province and Fujian Province.

Thermal stability studies of immunoglobulins using capillary isoelectric focusing and capillary zone electrophoretic methods.

The affinity of an antibody towards its antigen is highly specific to its conformation, in order to have optimal antibody-antigen interaction. The increase of temperature might cause changes in antibody conformations. The change of structure conformations may be reflected in the isoelectric points (pI values), peak shape and absorbance of the antibody. In this study, a monoclonal antibody was heated over a period of time. Capillary isoelectric focusing (cIEF) and capillary zone electrophoresis (CZE) were used to monitor the change in the antibody. The longer the heating period, the lower the pI values were under cIEF conditions. CZE also showed changes in peak shapes and decreases in absorbance of the antibody with heating.

Separation and quantitation of monoclonal antibodies in cell growth medium using capillary zone electrophoresis.

IgG1 is separated from its impurities in cell growth medium under simple CZE conditions without specific sample pretreatment. Linearity, limit of quantitation, limit of detection, precision and accuracy for the method are demonstrated. The quantitation for IgG1 in the cell growth medium is obtained by generating a calibration curve and by using standard additions. This CE method can offer a good alternative to conventional HPLC methods. Attempts are also made to separate the heterogeneous species in monoclonal antibodies using both CZE and MECC.

Decidual natural killer cytotoxicity decreased in normal pregnancy but not in anembryonic pregnancy and recurrent spontaneous abortion.

PROBLEM: The natural killer (NK) cell activity is depressed in the decidua of early normal pregnancy. Recently Morii et al. (Am J Reprod Immunol 1993;29:1-4) found that all early intradecidual CD3+ T cells expressed either T cell receptor (TCR) alpha/beta or gamma/delta but that the expression of the CD3+/TCR complex was down-regulated. METHOD: To test whether these changes in decidual cellular immunity are different among normal pregnancy, anembryonic pregnancy and recurrent spontaneous abortion, we examined the immune cell subpopulations in the decidua from these three types of pregnancy using flow cytometry and an NK cytotoxicity assay. RESULTS: Intradecidual CD3+ T cells expressed either TCR alpha/beta or gamma/delta, and the level of expression of the CD3/TCR complex was down-regulated in normal pregnancy, anembryonic pregnancy, and recurrent spontaneous abortion. Although the relative proportion of decidual NK cells was increased to approximately the same extent in all three types of pregnancy, decidual NK activity was higher in anembryonic pregnancies and in recurrent spontaneous abortions than it was in normal pregnancies. CONCLUSION: Decidual NK cell responses are different in anembryonic pregnancies and in recurrent spontaneous abortions than in normal pregnancies. Whether this difference is pathogenic or is the response to a dead embryo remains to be elucidated.

[Synthesis of "danshensu" derivatives].

This paper describes the synthesis of alpha-nicotinyloxy-beta-(substituted phenyl) propionic acid and alpha-(o-acetoxy)benzoyloxy-beta-phenyl propionic acid. They were obtained in six steps starting from benzaldehyde or substituted benzaldehyde. The preliminary pharmacological tests show that all compounds were effective for inhibition of rabbit platelet aggregation.

Antitumor agents, 107. New cytotoxic 4-alkylamino analogues of 4'-demethyl-epipodophyllotoxin as inhibitors of human DNA topoisomerase II.

A series of analogues of etoposide, the C-4 amino- and alkylamino-substituted 4'-demethyl-epipodophyllotoxins, have been synthesized and studied for their activity to inhibit type II human DNA topoisomerase as well as their activity in causing cellular protein-linked DNA breakage. Substitution of the glycosidic moiety of 1 by a 2"-hydroxyethylamino or 2"-methoxyethylamino chain at the C-4 beta position resulted in potent inhibitors of the human DNA topoisomerase II. This inhibitory activity correlates reasonably well with their activity in causing protein-linked DNA breakage in KB cells. The in vitro cytotoxicity (KB) appears to have no correlation with the inhibitory activity of the human DNA topoisomerase II.

Antitumor agents, 99. Synthetic ring C aromatized podophyllotoxin analogues as potential inhibitors of human DNA topoisomerase II.

Several ring C aromatized analogues of podophyllotoxin were synthesized for testing against human DNA topoisomerase II. The results indicate that aromatization of ring C gave rise to no inhibition of this enzyme at 200 microM. A comparison of the cytotoxicity among these compounds also demonstrates that a free hydroxyl group at C-4 contributes to significant cytotoxicity.