OBJECTIVE To uncover novel targets for the treatment of type 2 diabetes (T2D) by investigating rare variants with large effects in monogenic forms of the disease. RESEARCH DESIGN AND METHODS We performed whole-exome sequencing in a family with diabetes. We validated the identified gene using Sanger sequencing in additional families and diabetes- and community-based cohorts. Wild-type and variant gene transgenic mouse models were used to study the gene function. RESULTS Our analysis revealed a rare variant of the metallothionein 1E (MT1E) gene, p.C36Y, in a three-generation family with diabetes. This risk allele was associated with T2D or prediabetes in a community-based cohort. MT1E p.C36 carriers had higher HbA1c levels and greater BMI than those carrying the wild-type allele. Mice with forced expression of MT1E p.C36Y demonstrated increased weight gain, elevated postchallenge serum glucose and liver enzyme levels, and hepatic steatosis, similar to the phenotypes observed in human carriers of MT1E p.C36Y. In contrast, mice with forced expression of MT1E p.C36C displayed reduced weight and lower serum glucose and serum triglyceride levels. Forced expression of wild-type and variant MT1E demonstrated differential expression of genes related to lipid metabolism. CONCLUSIONS Our results suggest that MT1E could be a promising target for drug development, because forced expression of MT1E p.C36C stabilized glucose metabolism and reduced body weight, whereas MT1E p.C36Y expression had the opposite effect. These findings highlight the importance of considering the impact of rare variants in the development of new T2D treatments.

Cell adhesion molecules (CAMs) determine the behavior of cancer cells during metastasis. Although some CAMs are dysregulated in certain types of cancer and are associated with cancer progression, to the best of our knowledge, a comprehensive study of CAMs has not been undertaken, particularly in endometrial cancer (EC). In the present study the expression of 225 CAMs in EC patients with various clinicopathological phenotypes were evaluated by statistical analysis using publicly available data from The Cancer Genome Atlas database. The Kaplan-Meier method, and univariate and multivariate Cox proportional hazards regression models were used for survival analyses. Among the differentially expressed CAMs that were associated with aggressive clinicopathological phenotypes, 10 CAM genes were independent prognostic factors compared with other clinicopathological prognostic factors, including stage, grade, age, lymph node status, peritoneal cytology and histological subtype. A total of six genes (L1 cell adhesion molecule, mucin 15, cell surface associated, cell adhesion associated, oncogene regulated, immunoglobulin superfamily member 9B, protocadherin 9 and protocadherin β1) were selected for integrative analysis. The six-gene signature was demonstrated to be an independent prognostic factor and could effectively stratify patients with different risks. Patients with more high-expression CAMs had a higher risk of poor overall survival (OS) rate. The mortality risk for patients with elevation of >4 CAMs was 11 times of that in those without elevation of these 6 CAMs. Similar results were obtained when relapse-free survival (RFS) time was used during the analysis. Prognostic reliability of the six-gene model was validated using data of an independent cohort from the International Cancer Genome Consortium. In conclusion, a combination of CAM alterations contributed to progression and aggressiveness of EC. The six-gene signature was effective for predicting worse OS and RFS in patients with EC and could be complementary to the present clinical prognostic criteria.

1. Imatinib is widely used for the treatment of hematologic malignancies. It is common that imatinib is clinically co-prescribed with azole antifungal agents since these patients are more prone to invasive antifungal infection. The present study was to investigate the effects of azole antifungal drugs, including ketoconazole, fluconazole, voriconazole, itraconazole and posaconazole on imatinib metabolism. 2. The main metabolites, 1-OH midazolam and N-desmethyl imatinib, were determined in the absence and in the presence of various levels of ketoconazole, fluconazole, voriconazole, itraconazole and posaconazole. The relevant assay was also performed to screen mechanism-based inhibitors (MBI). 3. The inhibition ability of 1-OH midazolam formation from midazolam based on IC50 values was ketoconazole (0.09 mu M)>itraconazole (0.31 mu M)> posaconazole (0.68 mu M)>voriconazole (2.10 mu M) > fluconazole (8.90 mu M). Similarly, the rank order of inhibitory effects on formation of N-desmethyl imatinib from imatinib was ketoconazole (4.58 mu M)>itraconazole (17.45 mu M)> posaconazole (31.02 mu M)> voriconazole (367.9 mu M) >fluconazole (1.11 mM). Posaconazole and itraconazole displayed evidence of MBI. Additionally, imatinib was also shown as a MBI of CYP3A with IC50 value of 5.40 mu M against the midazolam. 4. The significant difference in IC50 values of midazolam and imatinib inhibited by azole antifungal agents was observed. The role of CYP2C8 in imatinib metabolism and imatinib autoinhibits CYP3A activity may explain this difference. Our findings suggest that the azole antifungal agents might have limited impacts on imatinib exposure by CYP3A activity.

Studies have shown hydroxyfasudil-mediated inhibition of Rho-kinase (ROCK) has efficacy in rodent models of focal ischemia and in in vitro systems that recapitulate stroke conditions. However, the mechanisms underlying the neuroprotective effects of the ROCK inhibitor on stroke are not well understood. In this study, we examined the role of γ-aminobutyric acid (GABA) interneurons in the effects of hydroxyfasudil on transient middle cerebral artery occlusion (tMCAO) induced acute ischemia-reperfusion injury to explore the mechanisms. tMCAO rats developed marked neurological deficits and impaired long-term potentiation; these effects were attenuated by hydroxyfasudil. Expression of GABAA and GABAB receptors after ischemia was decreased to various extents in three brain regions: the cortex, hippocampus, and striatum. Hydroxyfasudil up-regulated expression in the cortex and hippocampus but not in the striatum. Moreover, hydroxyfasudil could suppress the activation of nuclear factor-κB (NF-κB) in these regions during the acute ischemia. We further found that in tMCAO rats, hydroxyfasudil was able to abolish the decrease in GABAA and GABAB receptor phosphorylation triggered by high-frequency stimulation as well as the activation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). In summary, our results suggest ROCK inhibition improved neurological function and mitigated synaptic plasticity deficits caused by transient focal cerebral ischemia through the inactivation of two distinct pathways: (1) a ROCK-NF-κB pathway that leads to the suppression of GABAA and GABAB expression, and (2) a ROCK-PTEN pathway that decreases the phosphorylation of GABAA and GABAB, thereby affecting receptor function by triggering receptor endocytosis and the degradation of internalized receptors.

FAM3A plays important roles in regulating hepatic glucose/lipid metabolism and the proliferation of VSMCs. This study determined the role and mechanism of FAM3A in the adipogenesis of 3T3-L1 preadipocytes. During the adipogenesis of 3T3-L1 preadipocytes, FAM3A expression was significantly increased. FAM3A overexpression enhanced 3T3-L1 preadipocyte adipogenesis with increased phosphorylated Akt (pAkt) level, whereas FAM3A silencing inhibited 3T3-L1 preadipocyte adipogenesis with reduced pAkt level. Moreover, FAM3A silencing reduced the expression and secretion of adipokines in 3T3-L1 cells. FAM3A protein is mainly located in mitochondrial fraction of 3T3-L1 cells and mouse adipose tissue. FAM3A overexpression increased, whereas FAM3A silencing decreased ATP production in 3T3-L1 preadipocytes. FAM3A-induced adipogenesis of 3T3-L1 preadipocytes was blunted by inhibitor of P2 receptor. In white adipose tissues of db/db and HFD-fed obese mice, FAM3A expression was reduced. One-month rosiglitazone administration upregulated FAM3A expression, and increased cellular ATP content and pAkt level in white adipose tissues of normal and obese mice. In conclusion, FAM3A enhances the adipogenesis of preadipocytes by activating ATP-P2 receptor-Akt pathway. Under obese condition, a decrease in FAM3A expression in adipose tissues plays important roles in the development of adipose dysfunction and type 2 diabetes.

OBJECTIVETo investigate novel gene sets related to breast cancer (BC) using differential co-expression and differential expression (DECODE).METHODST statistics was used to quantify the degree of DE of each gene, and then Z was adopted to quantify the correlation difference between expression levels of two genes. Two optimal thresholds for defining substantial change in DE and DC were selected for each gene using chi-square maximization, and the corresponding gene was defined as the optimal gene. Based on the optimal thresholds, genes were categorized into four partitions with either high or low DC and DE characteristics. Finally, we evaluated the functional relevance of a gene partition with high DE and high DC, and the gene set with best association was considered as the optimal functional gene set.RESULTSThe optimal thresholds for DC and DE were respective 2.254 and 1.616, and the optimal gene was UBE2Q2L. Based on the optimal thresholds, genes were divided into four partitions including HDE-HDC (875 genes), HED-LDC (8038 genes), LDE-HDC (678 genes), and LDE-LDC (10516 genes). The best associated gene set was ``fatty acid catabolic process'' with 34 HDC and HDE partitions. Among these partitions, UBE2Q2L attained the highest minimum FI gain of 18.973.CONCLUSIONUBE2Q2L and fatty acid catabolic process might be potentially useful signatures in diagnostic purposes for BC.

目的 研究9种黄酮类化合物和5种芪类化合物对血脑屏障上p-糖蛋白(P-gp)外排沙奎那韦的影响.方法 以过表达P-gp的马丁达比犬肾上皮细胞(MDCKII-MDR1)作为血脑屏障体外模型.药物干预分为16组(n=4),包括阴性对照组、阳性对照组、黄酮类实验Ⅰ-Ⅸ组和芪类实验组Ⅰ-Ⅴ组.药物干预分别为50 μmol·L-1沙奎那韦(阴性对照组);50μmol·L-1沙奎那韦联用50μmol·L-1维拉帕米(阳性对照组);50μmol·L-1沙奎那韦联用25 μmol·L-1黄酮类(黄酮类实验Ⅰ-Ⅸ组)或联用25 μ.mol·L-1芪类化合物(芪类实验组Ⅰ-Ⅴ组).药物干预时间均为4h.用液相色谱质谱联用技术测定沙奎那韦在各组细胞内的积聚量.结果 细胞内沙奎那韦积聚量:与阴性对照组(101.46±22.00) ng·mg-1相比,柚皮素组(229.41 ±74.86) ng·mg-1、桑黄素组(149.89±30.38)ng·mg-1、白杨黄素组(209.75±65.35)ng ·mg-1、氧化白藜芦醇组(178.54±44.40) ng·mg-1、染料木黄酮组(294.49±66.35)ng·mg-1、表儿茶素组(212.27±49.70) ng·mg-1、三乙酰白藜芦醇组(290.94±45.54)ng·mg-1和白藜芦醇三甲醚组(205.55±35.16)ng·mg-1,均显著升高,差异有统计学意义(P＜0.05);异槲皮苷组(61.98±11.02)ng·mg-1、槲皮素组(41.54±4.92) ng·mg-1和白藜芦醇组(29.59±4.61)ng·mg-细胞内沙奎那韦积聚量,均显著降低,差异有统计学意义(P＜0.05).结论 一些黄酮类、芪类化合物可能对P-gp介导的沙奎那韦在血脑屏障上的转运过程产生调节作用.

The aim of this study was to identify an effective flavonoid that could improve the intracellular accumulation of ritonavir in human brain-microvascular endothelial cells (HBMECs). An in vivo experiment on Sprague-Dawley rats was then designed to further determine the flavonoid's impact on the pharmacokinetics and tissue distribution of ritonavir. In the accumulation assay, the intracellular level of ritonavir was increased in the presence of 25 mmol L-1 of flavonoids in HBMECs. Quercetin showed the strongest effect by improving the intracellular accumulation of ritonavir by 76.9 %. In the pharmacokinetic study, the presence of quercetin in the co-administration group and in the pretreatment group significantly decreased the area under the plasma concentration-time curve (AUC(0-t)) of ritonavir by 42.2 % (p < 0.05) and 53.5 % (p < 0.01), and decreased the peak plasma concentration (c(max)) of ritonavir by 23.1 % (p < 0.05) and 45.8 % (p < 0.01), respectively, compared to the control group (ritonavir alone). In the tissue distribution study, the ritonavir concentration in the brain was significantly increased 2-fold (p < 0.01), during the absorption phase (1 h) and was still significantly higher (p < 0.05) during the distribution phase (6 h) in the presence of quercetin.

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) using an electrospray ionization (ESI) method was developed and validated to determine the concentration of diethylstilbestrol in human plasma. Samples were extracted with methyl tert-butyl ether after alkalified by sodium tetraborate solution, using 3,5-dihydroxy-4-isopropyl toluylene as the internal standard (IS). Separation was performed on a Restek C-18 column (2.1mmx150mm, 5.0 mu m) guarded by a Hypersil Gold pre-column (2.1mmx10mm, 3 mu m). The mobile phase consisted of acetonitrile-water (85:15, containing 0.2mmol/L ammonium acetate) at a flow rate of 0.3mL/min. Detection was performed using negative multiple reaction monitoring (MRM) mode. The mass transitions monitored were [M-H](-)m/z267.2237.2 and [M-H](-)m/z253.2211.0 for diethylstilbestrol and IS, respectively. The method was fully validated using total error theory, which is based on -expectation tolerance intervals and include trueness and intermediate precision. The method was found to be accurate over a concentration range of 0.1-50ng/mL. The main advantages of this method were the high sensitivity (a lower limit of quantification, LLOQ, of 0.1ng/mL) and the short analysis time (total run time 5min per sample). The measurement uncertainty based on -expectation tolerance intervals was assessed at each concentration level of the validation standards. The method is specific, sensitive, and accuracy, and it is suitable for plasma concentration determination of diethylstilbestrol.

BACKGROUND:Brain hypoplasia and mental retardation in Down syndrome (DS) can be attributed to a severe and selective disruption of neurogenesis. Secondary disruption of the transcriptome, as well as primary gene dosage imbalance, is responsible for the phenotype. MicroRNA (miRNA) expression is relatively abundant in brain tissue. Perturbed miRNA expression might contribute to the cellular events underlying the pathology in DS.METHODS:MiRNA expression profiles in the cerebrum of Ts65Dn mice, a DS model, were examined with a real-time RT-PCR array. MiRNA target gene expression was detected by real-time quantitative PCR and Western blotting. Based on the prediction of their cerebrum-specific targets, the functions of the misregulated miRNAs were annotated by Gene Ontology (GO) enrichment analysis.RESULTS:A total of 342 miRNAs were examined. Among them, 20 miRNAs showed decreased expression in the brains of Ts65Dn mice, and some of these belonged to the same family. Two known targets of the miR-200 family, Lfng and Zeb2, were specifically selected to compare their expression in the cerebrum of Ts65Dn mice with those of euploids. However, no significant difference was found in terms of mRNA and protein expression levels of these genes. By enrichment analysis of the cerebrum-specific targets of each miRNA, we found that 15 of the differential miRNAs could significantly affect target genes that were enriched in the GO biological processes related to nervous system development.CONCLUSION:Perturbed expression of multiple functionally cooperative miRNAs contributes to the cellular events underlying the pathogenesis of DS.

Recent studies have indicated that telomerase activity promotes cancer invasion and metastasis, but the underlying mechanism remains unclear. Several studies have shown that expression of exogenous human telomerase reverse transcriptase (hTERT) can promote motility and invasiveness among telomerase-negative tumor cells, and inhibition of endogenous telomerase activity can reduce invasiveness in tumor cells. However, whether overexpression of hTERT can further enhance the motility and invasiveness of telomerase‑positive tumor cells has yet to be determined. In the present study, we showed that stable overexpression of hTERT can increase telomerase activity and telomere length, which significantly promotes the invasive and metastatic potential of telomerase‑positive HepG2 cells but does not affect cell proliferation. Further analysis suggested that enhanced invasiveness and metastasis may act through corresponding upregulation of mRNA and protein expression of matrix metalloproteinase 9 (MMP9) and Ras homolog gene family member C (RhoC). Our study indicated that exogenous expression of hTERT may promote invasiveness and metastasis through upregulation of MMP9 and RhoC.

bcl-2是Tsujimoto在Ⅱ型B细胞淋巴瘤中发现的一种基因,其过度表达能抑制撤退生长因子、射线、热休克、化疗药物、病毒及促凋亡基因等多种因素诱导的细胞凋亡 .bcl-2蛋白高表达赋予肿瘤细胞对多种化疗药物的抗性,导致化疗失败或复发.逆转bcl -2的表达可能提高化疗效果.我们将bcl-2的编码序列反向插入逆转录病毒表达载体中并转导实体瘤胃癌细胞株MGC-803,观察bcl-2反义序列对实体瘤肿瘤细胞的作用.