Background and purpose:Glioblastoma is one of the most common intracranial tumors, the morbidity and mortality are both high, and the molecular biological mechanism of the disease is still unclear. In this study, we detected the gene expression of tyrosine kinase receptor (TKR) pathway in primary glioblastoma(GBM) with low-density array, furthermore we analyzed the significance of the gene expression change. Methods:We detected 26 genes of RTK pathway in 10 primary GBM tissues and 9 normal brain tissues (gained from the decompression operation of brain trauma), and analyzed the different expressions of these two kinds of tissues by statistic method.Results:The Ct values of MAP2K1 and MAP2K4 in normal brain tissues were 1.6?1.7 and 2.2?2.1, the Ct values of MAP2K1 and MAP2K4 in primary GBM tissues were 3.9?1.5 and 5.0?2.0, and the Ct different values between normal brain tissues and primary GBM tissues of both genes were -2.3 and -2.8(P

OBJECTIVETo detect FLG gene mutations in 10 families affected with ichthyosis vulgaris and to explore mutational hot spot of the FLG gene in Chinese Han population.

METHODSPCR and direct sequencing were carried out to identify potential mutations of the FLG gene in above families. One hundred healthy individuals were analyzed as normal controls.

RESULTSThree mutations (3321delA, 5757delCCAG and S2706X) were identified in 7 families. A homozygous mutation 3321delA was also detected in two unrelated patients. No mutations were found in the remaining three families. Neither of the null mutations (5757delCCAG and S2706X) was found in the 100 controls. However, for 3321delA, a heterozygous mutation was also found in two of the controls.

CONCLUSIONThree FLG mutations have been identified in the selected families with ichthyosis vulgaris, and the 3321delA mutation was most prevalent (46.9%). Mutations 5757delCCAG and S2706X were first found in patients with ichthyosis vulgaris. Other candidate genes may underlie the disease in those without a FLG mutation.

Asian Continental Ancestry Group ; Base Sequence ; China ; Female ; Genotype ; Humans ; Ichthyosis Vulgaris ; genetics ; Intermediate Filament Proteins ; genetics ; Male ; Mutation ; Pedigree ; Phenotype

OBJECTIVETo detect the genomic deletion mutation in the NEMO gene of a family with incontinentia pigmenti (IP; MIM 308310).

METHODSA pedigree of IP was investigated. By using long PCR, the Delta4-10 deletion in NEMO gene was tested with specific primers In2/JF3R, and Delta4-10 deletion in pseudogene DeltaNEMO was investigated with primers Rev-2/JF3R. NEMO gene of 80 normal controls was also tested.

RESULTSThe deletion of exons 4-10 in both NEMO gene and the pseudogene DeltaNEMO was detected in all the patients in the family, but was not found in the normal individuals in this IP family and 80 unrelated controls.

CONCLUSIONThe study showed that the family with IP, which showed anticipation, was caused by NEMODelta4-10 deletion in the NEMO gene. Long PCR analysis is proven to be an efficient tool for identification of NEMO rearrangements. It could provide useful information for the genetic counseling of the family involved.

Adolescent ; Child ; Electrophoresis ; Exons ; genetics ; Family ; Female ; Humans ; I-kappa B Kinase ; genetics ; Incontinentia Pigmenti ; genetics ; Infant ; Male ; Pseudogenes ; genetics ; Sequence Deletion

PURPOSE: c-Met and its ligand, hepatocyte growth factor (HGF), play a critical role in oncogenesis and metastatic progression. The aim of this study was to identify inhibited enzymogram and to test the antitumor activity of SIM-89 (a c-Met receptor tyrosine kinase inhibitor) in non-small cell lung cancer. MATERIALS AND METHODS: Z′-LYTE kinase assay was employed to screen the kinase enzymogram, and mechanism of action (MOA) analysis was used to identify the inhibited kinases. Cell proliferation was then analyzed by CCK8 assay, and cell migration was determined by transwell assay. The gene expression and the phosphorylation of c-Met were examined by realtime-PCR and western blotting, respectively. Finally, the secretion of HGF was detected by ELISA assay. RESULTS: c-Met, activated protein kinase (AMPK), and tyrosine kinase A (TRKA) were inhibited by SIM-89 with the IC₅₀ values of 297 nmol/L, 1.31 µmol/L, and 150.2 nmol/L, respectively. SIM-89 exerted adenosine triphosphate (ATP) competitive inhibition on c-Met. Moreover, the expressions of STAT1, JAK1, and c-Met in H460 cells were decreased by SIM-89 treatment, and c-Met phosphorylation was suppressed in A549, H441, H1299, and B16F10 cells by the treatment. In addition, SIM-89 treatment significantly decreased the level of HGF, which accounted for the activation of c-Met receptor tyrosine kinase. Finally, we showed cell proliferation inhibition and cell migration suppression in H460 and H1299 cells after SIM-89 treatment. CONCLUSION: In conclusion, SIM-89 inhibits tumor cell proliferation, migration and HGF autocrine, suggesting it's potential antitumor activity.
Adenosine Triphosphate ; Blotting, Western ; Carcinogenesis ; Carcinoma, Non-Small-Cell Lung* ; Cell Movement ; Cell Proliferation ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; Hepatocyte Growth Factor ; Lung Neoplasms ; Phosphorylation ; Phosphotransferases ; Protein Kinases ; Protein-Tyrosine Kinases

Background and objective hTe aim of this study is to establish a HRM (high resolution melting curve) method for detection of deletion in human BIM gene and to detect this site deletion with the above method in 30 lung cancer samples and 30 normal samples. Methods The primers for detection of BIM deletion were designed and synthesized. The HRM method for geng deletion was established. And select the part of samples to detect BIM delection by normal PCR and sequencing assay. hTe Tm value of wild type PCR products was higher than that of the deletion PCR products. hTe difference of the corresponding Tm value is 2.5 oC. Results By detection with HRM methods, 1 samples were conifrmed to be mutant, 7 samples were conifrmed to be heterozygous and the other 22 samples were all wild type in the lung cancer samples. 2 samples were conifrmed to be heterozygous and the other 28 samples were all wild type in the normal samples. Conclusion hTe HRM method for detection of BIM deletion established in this study is a sensitive, accurate, simple and high throughput method.

OBJECTIVETo detect mutations of ATP2A2 gene in a pedigree and a sporadic case with Darier disease (DD) and explore the underlying molecular mechanism.

METHODSClinical data of the pedigree and the sporadic case were collected. Genomic DNA was extracted from blood samples of four members from the pedigree (including three patients and one healthy member), the sporadic case and 100 healthy controls. PCR was performed to amplify all coding exons of the ATP2A2 gene. And the products were directly sequenced to detect mutations.

RESULTSA missense mutation c.1484C>T (p.S495L) in exon 12 was detected in all patients of the pedigree. For the sporadic case, a novel splicing mutation c.325-2A>G was detected at the junction between intron 4 and exon 5. The same mutations were not found in the 100 healthy controls.

CONCLUSIONMutations of the ATP2A2 gene may lead to the occurrence of DD in both familial and sporadic cases with DD.

Aged ; Alternative Splicing ; genetics ; Base Sequence ; Child ; DNA Mutational Analysis ; Darier Disease ; genetics ; Family Health ; Female ; Genetic Predisposition to Disease ; genetics ; Humans ; Male ; Mutation, Missense ; Pedigree ; Point Mutation ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics

Objective:To investigate the role of cholinergic anti-inflammatory pathway in the regulation of peptide transporter 1 (PepT1) expression in small intestinal epithelium of septic rats by Ghrelin.Methods:One hundred adult male Sprague-Dawley (SD) rats were randomly divided into sham operation group, sepsis group, sepsis+vagotomy group, sepsis+Ghrelin group, and sepsis+vagotomy+Ghrelin group, with 20 rats in each group. In the sham operation group, the cecum was separated after laparotomy, without ligation and perforation. In the sepsis group, the rats received cecal ligation puncture (CLP). In the sepsis+vagotomy group, the rats received CLP and vagotomy after laparotomy. In the sepsis+Ghrelin group, 100 μmol/L Ghrelin was intravenously injected after CLP immediately. The rats in the sepsis+vagotomy+Ghrelin group received CLP and vagotomy at the same time, then the Ghrelin was intravenously injected immediately with the same dose as the sepsis+Ghrelin group. Ten rats in each group were taken to observe their survival within 7 days. The remaining 10 rats were sacrificed 20 hours after the operation to obtain venous blood and small intestinal tissue. The condition of the abdominal intestine was observed. The injury of intestinal epithelial cells was observed with transmission electron microscopy. The contents of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in serum and small intestinal tissue were detected by enzyme-linked immunosorbent assay (ELISA). The brush border membrane vesicle (BBMV) was prepared, the levels of mRNA and protein expression of PepT1 in the small intestinal epithelium were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and Western blotting.Results:All rats in the sham operation group survived at 7 days after operation. The 7-day cumulative survival rate of rats in the sepsis group was significantly lower than that in the sham operation group (20% vs. 100%, P < 0.05). The cumulative survival rate of rats after Ghrelin intervention was improved (compared with sepsis group: 40% vs. 20%, P < 0.05), but the protective effect of Ghrelin was weakened after vagotomy (compared with sepsis+Ghrelin group: 10% vs. 40%, P < 0.05). Compared with the sham operation group, in the sepsis group, the small intestine and cecum were dull red, the intestinal tubules were swollen and filled with gas, the intestinal epithelial cells were seriously injured under transmission electron microscopy, the levels of TNF-α and IL-1β in serum and small intestinal were significantly increased, and the expression levels of PepT1 mRNA and protein in the small intestinal epithelium were significantly decreased. It indicated that the sepsis rat model was successfully prepared. After vagotomy, the intestinal swelling and gas accumulation became worse in septic rats, leading to the death of all rats. Compared with the sepsis group, the abdominal situation in the sepsis+Ghrelin group was improved, the injury of intestinal epithelial cells was alleviated, the serum and small intestinal TNF-α and IL-1β were significantly decreased [serum TNF-α (ng/L): 253.27±23.32 vs. 287.90±19.48, small intestinal TNF-α (ng/L): 95.27±11.47 vs. 153.89±18.15, serum IL-1β (ng/L): 39.16±4.47 vs. 54.26±7.27, small intestinal IL-1β (ng/L): 28.47±4.13 vs. 42.26±2.59, all P < 0.05], and the expressions of PepT1 mRNA and protein in the small intestinal epithelium were significantly increased [PepT1 mRNA (2 -ΔΔCt): 0.66±0.05 vs. 0.53±0.06, PepT1 protein (PepT1/GAPDH): 0.80±0.04 vs. 0.60±0.05, both P < 0.05]. Compared with the sepsis+Ghrelin group, after vagotomy in the sepsis+vagotomy+Ghrelin group, the effect of Ghrelin on reducing the release of inflammatory factors in sepsis rats was significantly reduced [serum TNF-α (ng/L): 276.58±19.88 vs. 253.27±23.32, small intestinal TNF-α (ng/L): 144.28±12.99 vs. 95.27±11.47, serum IL-1β (ng/L): 48.15±3.21 vs. 39.16±4.47, small intestinal IL-1β (ng/L): 38.75±4.49 vs. 28.47±4.13, all P < 0.05], the up-regulated effect on the expression of PepT1 in small intestinal epithelium was lost [PepT1 mRNA (2 -ΔΔCt): 0.58±0.03 vs. 0.66±0.05, PepT1 protein (PepT1/GAPDH): 0.70±0.02 vs. 0.80±0.04, both P < 0.05], and the injury of small intestinal epithelial cells was worse. Conclusion:Ghrelin plays a protective role in sepsis by promoting cholinergic neurons to inhibit the release of inflammatory factors, thereby promoting the transcription and translation of PepT1.

Objective:To analyze and summarize the implementation effect of basic essential surgical training (BEST) course of laparoscopic skills over the past 10 years and the practical experience in updating course content and models.Methods:The pre-class assessment questionnaires, basic laparoscopic operation assessment results, and post-class assessment questionnaires of the students who participated in the BEST course of laparoscopic skills were collected. According to the period of the course construction, the students were divided into two groups, namely students who used the course of single training system in the early stage (traditional group) and students who used the course integrating a variety of training systems after the course model was updated in the later stage (test group). The two groups were compared for the scores of track circle moving, tunnel crossing, and high and low columns, as well as their subjective evaluation of course setting and implementation effect. The t-test, Wilcoxon test, or chi-square test was conducted according to the data type using SPSS 13.0. Results:The time for 150 traditional group students to complete track circle moving, tunnel crossing, and high and low columns was 1.08 min (0.81 min, 1.60 min), 2.20 min (1.60 min, 3.27 min), and 4.86 min (3.28 min, 6.36 min), respectively, while the time for 75 test group students to complete the three operations was 1.27 min (0.87 min, 1.83 min), 2.57 min (1.58 min, 4.07 min), and 4.35 min (2.90 min, 6.42 min), respectively, with no significant difference between the two groups ( P>0.05). In terms of students' subjective evaluation of the course, a higher percentage of the test group students were satisfied with classroom environment, teaching method arrangement, training equipment, training opportunities, helping clinical work, and meeting pre-class expectations than those in the traditional group. Conclusion:The constantly updated BEST course can ensure the training quality of trainees and obtain their higher satisfaction. The benefits of this course in clinical practice can be further verified through long-term follow-up of these trainees.