Activation of Mir-29a in Activated Hepatic Stellate Cells Modulates Its Profibrogenic Phenotype through Inhibition of Histone Deacetylases 4

Background

Recent studies have shown that microRNA-29 (miR-29) is significantly decreased in liver fibrosis and that its downregulation influences the activation of hepatic stellate cells (HSCs). In addition, inhibition of the activity of histone deacetylases 4 (HDAC4) has been shown to strongly reduce HSC activation in the context of liver fibrosis.

Objectives

In this study, we examined whether miR-29a was involved in the regulation of HDAC4 and modulation of the profibrogenic phenotype in HSCs.

Methods

We employed miR-29a transgenic mice (miR-29aTg mice) and wild-type littermates to clarify the role of miR-29a in cholestatic liver fibrosis, using the bile duct-ligation (BDL) mouse model. Primary HSCs from both mice were treated with a miR-29a mimic and antisense inhibitor in order to analyze changes in profibrogenic gene expression and HSC activation using real-time quantitative RT-PCR, immunofluorescence staining, western blotting, and cell proliferation and migration assays.

Results

After BDL, overexpression of miR-29a decreased collagen-1α1, HDAC4 and activated HSC markers of glial fibrillary acidic protein expression in miR-29aTg mice compared to wild-type littermates. Overexpression of miR-29a and HDAC4 RNA-interference decreased the expression of fibrotic genes, HDAC4 signaling, and HSC migration and proliferation. In contrast, knockdown of miR-29a with an antisense inhibitor increased HDAC4 function, restored HSC migration, and accelerated HSC proliferation.

Conclusions

Our results indicate that miR-29a ameliorates cholestatic liver fibrosis after BDL, at least partially, by modulating the profibrogenic phenotype of HSCs through inhibition of HDAC4 function.     

Effect of Common Genetic Variants of Growth Arrest-Specific 6 Gene on Insulin Resistance,Obesity and Type 2 Diabetes in an Asian Population

Objectives

Growth arrest-specific 6 (Gas6), a vitamin K-dependent protein, has been implicated in systemic inflammation, obesity, and insulin resistance (IR). Data from recent studies suggest that polymorphisms in the Gas6 gene are associated with cardiovascular disorders and type 2 diabetes (T2D). However, the association of Gas6 gene variants with obesity, IR, and T2D development has not been explored.

Materials and Methods

Four common single nucleotide polymorphisms (SNPs) in the Gas6 gene were genotyped in 984 participants from the Stanford Asia-Pacific Program for Hypertension and Insulin Resistance (SAPPHIRe) family cohort. An insulin suppression test was performed to determine IR based on steady-state plasma glucose (SSPG). Associations between IR indices and obesity, and SNP genotypes, based on previously-reported data for this cohort (Phase I), were analyzed. In the present follow-up study (Phase II), the effects of gene variants of Gas6 on the progression to T2D were explored in individuals who were free of T2D in Phase I. The mean follow-up period for Phase II was 5.7 years.

Results

The mean age of the study population in Phase I was 49.5 years and 16.7% of individuals developed T2D during follow-up. After adjusting for covariates, three SNPs (rs8191973, rs8197974, and rs7323932) were found to be associated with SSPG levels (p = 0.007, p = 0.03, and p = 0.011, respectively). This association remained significant after multiple testing and showed a significant interaction with physical activity for SNP rs8191973. However, no other significant correlations were observed between Gas6 polymorphisms and other indices of IR or obesity. A specific haplotype, AACG (from rs8191974, rs7323932, rs7331124, and rs8191973), was positively associated with SSPG levels (p = 0.0098). None of the polymorphisms were associated with an increased risk of T2D development.

Conclusions

Our results suggest that Gas6 gene variants are associated with IR, although their effects on subsequent progression to T2D were minimal in this prospective Asian cohort.     

Quantum Dots-Based Quantitative and In Situ Multiple Imaging on Ki67 and Cytokeratin to Improve Ki67 Assessment in Breast Cancer

Background

As a marker for tumor cell proliferation, Ki67 has important impacts on breast cancer (BC) prognosis. Although immunohistochemical staining is the current standard method, variations in analytical practice make it difficult for pathologists to manually measure Ki67 index. This study was to develop a fluorescent spectrum-based quantitative analysis of Ki67 expression by quantum-dots (QDs) multiple imaging technique.

Methods

A QDs-based in situ multiple fluorescent imaging method was developed, which stained nuclear Ki67 as red signal and cytoplasmic cytokeratin (CK) as green signal. Both Ki67 and CK signals were automatically separated and quantified by professional spectrum analysis software. This technique was applied to tissue microarrays from 240 BC patients. Both Ki67 and CK values, and Ki67/CK ratio were obtained for each patient, and their prognostic value on 5-year disease free survival was assessed.

Results

This method simultaneously stains nuclear Ki67 and cytoplasmic CK with clear signal contrast, making it easy for signal separation and quantification. The total fluorescent signal intensities of both Ki67 sum and CK sum were obtained, and Ki67/CK ratio calculated. Ki67 sum and Ki67/CK ratio were each attributed into two grades by X-tile software based on the best P value principle. Multivariate analysis showed Ki67 grade (P = 0.047) and Ki67/CK grade (P = 0.004) were independent prognostic factors. Furthermore, area under curve (AUC) of ROC analysis for Ki67/CK grade (AUC: 0.683, 95%CI: 0.613–0.752) was higher than Ki67 grade (AUC: 0.665, 95%CI: 0.596–0.734) and HER-2 gene (AUC: 0.586, 95%CI: 0.510–0.661), but lower than N stage (AUC: 0.760, 95%CI: 0.696–0.823) and histological grade (AUC: 0.756, 95%CI: 0.692–0.820) on predicting the risk for recurrence.

Conclusions

A QDs-based quantitative and in situ multiple imaging on Ki67 and CK was developed to improve Ki67 assessment in BC, and Ki67/CK grade had better performance than Ki67 grade in predicting prognosis.     

Comparative Validity and Reproducibility Study of Various Landmark-Oriented Reference Planes in 3-Dimensional Computed Tomographic Analysis for Patients Receiving Orthognathic Surgery

BackgroundThree-dimensional computed tomographic imaging has become popular in clinical evaluation, treatment planning, surgical simulation, and outcome assessment for maxillofacial intervention. The purposes of this study were to investigate whether there is any correlation among landmark-based horizontal reference planes and to validate the reproducibility and reliability of landmark identification.ResultsA total of 30 patients with facial deformity and malocclusion—10 patients with facial symmetry, 10 patients with facial asymmetry, and 10 patients with cleft lip and palate—were recruited. Comparing the differences among the 5 reference planes showed no statistically significant difference among all patient groups. Regarding intraobserver reproducibility, the mean differences in the 3 coordinates varied from 0 to 0.35 mm, with correlation coefficients between 0.96 and 1.0, showing high correlation between repeated tests. Regarding interobserver reliability, the mean differences among the 3 coordinates varied from 0 to 0.47 mm, with correlation coefficients between 0.88 and 1.0, exhibiting high correlation between the different examiners.ConclusionsThe 5 horizontal reference planes were reliable and comparable for 3D craniomaxillofacial analysis. These reference planes were useful in standardizing the orientation of 3D skull models.     

Spatial and Functional Heterogeneities Shape Collective Behavior of Tumor-Immune Networks

Tumor growth involves a dynamic interplay between cancer cells and host cells, which collectively form a tumor microenvironmental network that either suppresses or promotes tumor growth under different conditions. The transition from tumor suppression to tumor promotion is mediated by a tumor-induced shift in the local immune state, and despite the clinical challenge this shift poses, little is known about how such dysfunctional immune states are initiated. Clinical and experimental observations have indicated that differences in both the composition and spatial distribution of different cell types and/or signaling molecules within the tumor microenvironment can strongly impact tumor pathogenesis and ultimately patient prognosis. How such “functional” and “spatial” heterogeneities confer such effects, however, is not known. To investigate these phenomena at a level currently inaccessible by direct observation, we developed a computational model of a nascent metastatic tumor capturing salient features of known tumor-immune interactions that faithfully recapitulates key features of existing experimental observations. Surprisingly, over a wide range of model formulations, we observed that heterogeneity in both spatial organization and cell phenotype drove the emergence of immunosuppressive network states. We determined that this observation is general and robust to parameter choice by developing a systems-level sensitivity analysis technique, and we extended this analysis to generate other parameter-independent, experimentally testable hypotheses. Lastly, we leveraged this model as an in silico test bed to evaluate potential strategies for engineering cell-based therapies to overcome tumor associated immune dysfunction and thereby identified modes of immune modulation predicted to be most effective. Collectively, this work establishes a new integrated framework for investigating and modulating tumor-immune networks and provides insights into how such interactions may shape early stages of tumor formation.     

噪声和二硫化碳对大鼠外膝体神经元光反应的影响及其联合效应

目的：观察噪声或二硫化碳（CS2）暴露对大鼠外侧膝状体（LGB）神经元光反应的影响及二者的联合效应。方法：脉冲噪声刺激和光栅刺激由计算机控制输出,CS2通过皮下注射,运用电生理学方法记录LGB神经元电活动。结果：噪声暴露后有21％的神经元光反应被抑制,当噪声持续时,抑制程度有增强的趋势,呈现出剂量效应关系。不同剂量的CS2暴露,神经元受到不同程度的抑制,亦呈剂量一效应关系。噪声和CS2同时作用时,呈一定的协同作用。结论：噪声或者CS2的单独作用可明显抑制外膝体神经元的光反应,在共同作用时存在联合效应,主要表现为协同效应。     

腺病毒介导的HIF-1α基因在缺血心肌中的表达

 研究腺病毒介导的低氧诱导因子=1α(HIF=1α)在急性心肌梗死(AMI)后缺血心肌中的表达变化.建立兔AMI模型,随机分为4组,分别心肌内注入腺病毒介导的HIF-1α基因(Ad-HIF-1α)、不含HIF-1α基因腺病毒颗粒(Ad--Blank)、HIF-1α核酶基因(Rz-HIF-1α),假手术组(Sham)为对照.RT-PCR和免疫组化方法分别检测HIF-1α及下游基因VEGF的mRNA和蛋白在不同时间点的表达.结果显示,AMI后1 d, HIF-1α、VEGF的mRNA和蛋白表达开始升高,7 d 达高峰,14 d、28 d逐渐下降,56 d时HIF-1αmRNA和蛋白无表达,而VEGF mRNA和蛋白仍有表达. 因此, 腺病毒介导的HIF-1α基因在缺血心肌能被有效转染并激活,其表达的时间窗为心肌缺血急性期至亚急性期.HIF-1α核酶基因能抑制HIF-1α及其下游基因的表达.     

Prenatal diagnosis and genetic counseling of mucopolysaccharidosis type II (Hunter syndrome)

We present prenatal diagnosis of mucopolysaccharidosis type II (MPS II) (Hunter syndrome) and demonstrate marked mucopolysaccharide deposition in multiple vital organs in a 22-gestational-week affected fetus. Level II ultrasound showed cardiomegaly and hepatomegaly. Histological examinations of the fetal vital organs manifested marked mucopolysaccharide deposition. We suggest that any therapeutic approach and counseling for prenatally diagnosed MPS II should consider the early signs of in utero marked mucopolysaccharide storage.     

SAR of a novel 'Anthranilamide Like' series of VEGFR-2, multi protein kinase inhibitors for the treatment of cancer

Novel anthranilamides were surprisingly found to exert additional activity on B-RAF. Corresponding thiophene, pyrazole, and thiazole core analogs were prepared as VEGFR-2 inhibitors with c-KIT, and B-RAF activity. Compounds in the phenyl, thiophene, and thiazole series are in vivo active.