Hippocampal Gene Network Analysis in an Experimental Model of Posttraumatic Epilepsy

In the present study, we performed comprehensive gene expression and gene network analyses using a DNA microarray to elucidate the molecular events responsible for the pathology of posttraumatic epilepsy at the partial seizure stage. We used an experimental posttraumatic epilepsy model of amygdalar focal FeCl₃-injected rats and compared gene expression profiles in the hippocampus at the partial seizure stage (less than stage 3 on Racine’s convulsion scale) and that of sham-operated animals. At the partial seizure stage, upregulation of phospholipase A2 (PLA2) and lipid metabolism were observed, which have been reported to be caused by brain injury and seizures in previous studies. Furthermore, significant upregulation of genes related to inflammation and the immune system was observed. These molecular changes in PLA2 and lipid metabolism may be related to seizure propagation.     

Expression of Lipid Metabolism-Related Proteins in Metastatic Breast Cancer

Purpose

The tumor biology of metastatic breast cancers differ according to the metastatic sites, and the features of cancer metabolism may also be different. The aim of this study is to investigate the expression of lipid metabolism-related proteins in metastatic breast cancer according to metastatic site and discuss the clinical significance thereof.

Methods

Immunohistochemical staining for lipid metabolism-related proteins [fatty acid synthase (FASN), hormone-sensitive lipase (HSL), carnitine palmitoyltransferase IA (CPT-1A), acyl-CoA oxidase 1 (ACOX1), fatty acid binding protein 4 (FABP4,) and perilipin 1 (PLIN1)] was performed using a tissue microarray of 149 cases of metastatic breast cancer (bone metastasis = 39, brain metastasis = 37, liver metastasis = 21, and lung metastasis = 52).

Results

The expression levels of ACOX1 (p = 0.009) and FASN (p = 0.007) varied significantly according to metastatic site, with the highest expression in brain metastasis and the lowest expression in liver metastasis. ACOX1 positivity (p = 0.005) and FASN positivity (p = 0.003) correlated with HER-2 positivity. The expression of FASN was significantly higher in HER-2 type breast cancer, and lower in luminal A and TNBC type breast cancer (p<0.001). Among lipid metabolism-related proteins, PLIN1 positivity was found to be an independent poor prognostic factor on multivariate analysis (Hazard ratio: 4.979, 95% CI: 1.054–22.59, p = 0.043).

Conclusion

Different expression levels of lipid metabolism-related proteins were observed according to metastatic site. The expression of ACOX1 and FASN was highest in brain metastasis. These results suggest that the metastatic site should be considered when using lipid metabolism inhibitors for targeted therapy.     

Expression of SHANK3 in the Temporal Neocortex of Patients with Intractable Temporal Epilepsy and Epilepsy Rat Models

SH3 and multiple ankyrin (ANK) repeat domain 3 (SHANK3) is a synaptic scaffolding protein enriched in the postsynaptic density of excitatory synapses. SHANK3 plays an important role in the formation and maturation of excitatory synapses. In the brain, SHANK3 directly or indirectly interacts with various synaptic molecules including N-methyl-D-aspartate receptor, the metabotropic glutamate receptor (mGluR), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor. Previous studies have shown that Autism spectrum disorder is a result of mutations of the main SHANK3 isoforms, which may be due to deficit in excitatory synaptic transmission and plasticity. Recently, accumulating evidence has demonstrated that overexpression of SHANK3 could induce seizures in vivo. However, little is known about the role of SHANK3 in refractory temporal lobe epilepsy (TLE). Therefore, we investigated the expression pattern of SHANK3 in patients with intractable temporal lobe epilepsy and in pilocarpine-induced models of epilepsy. Immunofluorescence, immunohistochemistry, and western blot analysis were used to locate and determine the expression of SHANK3 in the temporal neocortex of patients with epilepsy, and in the hippocampus and temporal lobe cortex of rats in a pilocarpine-induced epilepsy model. Double-labeled immunofluorescence showed that SHANK3 was mainly expressed in neurons. Western blot analysis confirmed that SHANK3 expression was increased in the neocortex of TLE patients and rats. These results indicate that SHANK3 participates in the pathology of epilepsy.     

Global Gene Expression Profiling Reveals SPINK1 as a Potential Hepatocellular Carcinoma Marker

Background

Liver cirrhosis is the most important risk factor for hepatocellular carcinoma (HCC) but the role of liver disease aetiology in cancer development remains under-explored. We investigated global gene expression profiles from HCC arising in different liver diseases to test whether HCC development is driven by expression of common or different genes, which could provide new diagnostic markers or therapeutic targets.

Methodology and Principal Findings

Global gene expression profiling was performed for 4 normal (control) livers as well as 8 background liver and 7 HCC from 3 patients with hereditary haemochromatosis (HH) undergoing surgery. In order to investigate different disease phenotypes causing HCC, the data were compared with public microarray repositories for gene expression in normal liver, hepatitis C virus (HCV) cirrhosis, HCV-related HCC (HCV-HCC), hepatitis B virus (HBV) cirrhosis and HBV-related HCC (HBV-HCC). Principal component analysis and differential gene expression analysis were carried out using R Bioconductor. Liver disease-specific and shared gene lists were created and genes identified as highly expressed in hereditary haemochromatosis HCC (HH-HCC) were validated using quantitative RT-PCR. Selected genes were investigated further using immunohistochemistry in 86 HCC arising in liver disorders with varied aetiology. Using a 2-fold cut-off, 9 genes were highly expressed in all HCC, 11 in HH-HCC, 270 in HBV-HCC and 9 in HCV-HCC. Six genes identified by microarray as highly expressed in HH-HCC were confirmed by RT qPCR. Serine peptidase inhibitor, Kazal type 1 (SPINK1) mRNA was very highly expressed in HH-HCC (median fold change 2291, p = 0.0072) and was detected by immunohistochemistry in 91% of HH-HCC, 0% of HH-related cirrhotic or dysplastic nodules and 79% of mixed-aetiology HCC.

Conclusion

HCC, arising from diverse backgrounds, uniformly over-express a small set of genes. SPINK1, a secretory trypsin inhibitor, demonstrated potential as a diagnostic HCC marker and should be evaluated in future studies.     

Gene Expression Profiling in the Type 1 Diabetes Rat Diaphragm

Background

Respiratory muscle contractile performance is impaired by diabetes, mechanisms of which included altered carbohydrate and lipid metabolism, oxidative stress and changes in membrane electrophysiology. The present study examined to what extent these cellular perturbations involve changes in gene expression.

Methodology/Principal Findings

Diaphragm muscle from streptozotocin-diabetic rats was analyzed with Affymetrix gene expression arrays. Diaphragm from diabetic rats had 105 genes with at least ±2-fold significantly changed expression (55 increased, 50 decreased), and these were assigned to gene ontology groups based on over-representation analysis using DAVID software. There was increased expression of genes involved in palmitoyl-CoA hydrolase activity (a component of lipid metabolism) (P = 0.037, n = 2 genes, fold change 4.2 to 27.5) and reduced expression of genes related to carbohydrate metabolism (P = 0.000061, n = 8 genes, fold change −2.0 to −8.5). Other gene ontology groups among upregulated genes were protein ubiquitination (P = 0.0053, n = 4, fold change 2.2 to 3.4), oxidoreductase activity (P = 0.024, n = 8, fold change 2.1 to 6.0), and morphogenesis (P = 0.012, n = 10, fold change 2.1 to 4.3). Other downregulated gene groups were extracellular region (including extracellular matrix and collagen) (P = 0.00032, n = 13, fold change −2.2 to −3.7) and organogenesis (P = 0.032, n = 7, fold change −2.1 to −3.7). Real-time PCR confirmed the directionality of changes in gene expression for 30 of 31 genes tested.

Conclusions/Significance

These data indicate that in diaphragm muscle type 1 diabetes increases expression of genes involved in lipid energetics, oxidative stress and protein ubiquitination, decreases expression of genes involved in carbohydrate metabolism, and has little effect on expression of ion channel genes. Reciprocal changes in expression of genes involved in carbohydrate and lipid metabolism may change the availability of energetic substrates and thereby directly modulate fatigue resistance, an important issue for a muscle like the diaphragm which needs to contract without rest for the entire lifetime of the organism.     

Temporal Colonic Gene Expression Profiling in the Recurrent Colitis Model Identifies Early and Chronic Inflammatory Processes

The recurrent TNBS-colitis model in BALB/c mice has been proposed as a model of Inflammatory Bowel Disease with a shifting pattern of local cytokines with the expression of Th1 cytokines during the early phase, Th17 cytokines during the intermediate phase and Th2 cytokines during late fibrotic stages. In this study, we evaluated the development of pathology in time–in conjunction with genome-wide gene expression in the colons–in response to three weekly intrarectal instillations of TNBS. During this time-frame mice develop colitis with extensive cellular infiltration of (sub)mucosa and mildly to moderately affected crypt architecture. These pathological processes were sensitive to local treatment with budesonide. Gene expression profiling confirmed an acute phase response after each intrarectal TNBS-challenge. In addition, a chronic inflammatory process developed over time particularly evident from a gradual increase in expression of mast cell related genes. The changes in pathological hallmarks were consistent with a temporal expression of mRNA encoding a selection of chemokines. In conclusion, the early stages of the recurrent TNBS-colitis model reflect several aspects of inflammatory bowel disease which are sensitive to immunomodulation.     

Gene Expression Profiling Reveals Epithelial Mesenchymal Transition (EMT) Genes Can Selectively Differentiate Eribulin Sensitive Breast Cancer Cells

Objectives

Eribulin mesylate is a synthetic macrocyclic ketone analog of the marine sponge natural product halichondrin B. Eribulin is a mechanistically unique inhibitor of microtubule dynamics. In this study, we investigated whether selective signal pathways were associated with eribulin activity compared to paclitaxel, which stabilizes microtubules, based on gene expression profiling of cell line panels of breast, endometrial, and ovarian cancer in vitro.

Results

We determined the sets of genes that were differentially altered between eribulin and paclitaxel treatment in breast, endometrial, and ovarian cancer cell line panels. Our unsupervised clustering analyses revealed that expression profiles of gene sets altered with treatments were correlated with the in vitro antiproliferative activities of the drugs. Several tubulin isotypes had significantly lower expression in cell lines treated with eribulin compared to paclitaxel. Pathway enrichment analyses of gene sets revealed that the common pathways altered between treatments in the 3 cancer panels were related to cytoskeleton remodeling and cell cycle regulation. The epithelial-mesenchymal transition (EMT) pathway was enriched in genes with significantly altered expression between the two drugs for breast and endometrial cancers, but not for ovarian cancer. Expression of genes from the EMT pathway correlated with eribulin sensitivity in breast cancer and with paclitaxel sensitivity in endometrial cancer. Alteration of expression profiles of EMT genes between sensitive and resistant cell lines allowed us to predict drug sensitivity for breast and endometrial cancers.

Conclusion

Gene expression analysis showed that gene sets that were altered between eribulin and paclitaxel correlated with drug in vitro antiproliferative activities in breast and endometrial cancer cell line panels. Among the panels, breast cancer provided the strongest differentiation between eribulin and paclitaxel sensitivities based on gene expression. In addition, EMT genes were predictive of eribulin sensitivity in the breast and endometrial cancer panels.     

Differential Expression of Lipid Metabolism-Related Proteins in Different Breast Cancer Subtypes

Purpose

This study aimed to determine the expression and clinical significance of proteins that are involved in lipid metabolism in human breast tumors.

Methods

Tumors from 476 breast cancer patients were used to construct tissue microarrays. Then, immunohistochemistry (IHC) for hormone-sensitive lipase (HSL), Perilipin 1 (PLIN1), fatty acid binding protein 4 (FABP4), carnitine palmitoyltransferase IA (CPT-1A), acyl-CoA oxidase 1 (ACOX-1), and fatty acid synthase (FASN) was performed on these microarrays.

Results

Breast tumors were classified into 4 subtypes: luminal A (n = 242; 50.8%), luminal B (n = 134; 28.2%), human epidermal growth factor receptor 2 (HER2) (n = 50; 10.5%), and triple negative breast cancer (TNBC) (n = 50; 10.5%). The expression of PLIN1 (p < 0.001), FABP4 (p = 0.029), CPT-1A (p = 0.001), ACOX-1 (p < 0.001), and FASN (p < 0.001) differed significantly among these tumor subtypes. Notably, PLIN1, CPT-1A, and FASN expression was highest in HER2 tumors and lowest in TNBC tumors. Similarly, the expression of FABP4 and ACOX-1 was highest in HER2 tumors and lowest in luminal A tumors. In addition, ACOX-1 positivity was associated with significantly shorter overall survival (p = 0.018). When tumor subtype was considered, FABP4 positivity was associated with significantly shorter disease-free survival (p = 0.005) and overall survival (p = 0.041) in TNBC.

Conclusion

Lipid metabolism-related proteins are differentially expressed in different IHC subtypes of breast cancer and some are associated with decreased survival rates.     

Cell-Type-Specific Gene Expression Profiling in Adult Mouse Brain Reveals Normal and Disease-State Signatures

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基因芯片在骨骼肌老龄化相关基因表达谱中的应用

对老龄组大鼠 (30月龄 )和年轻对照组大鼠 (3月龄 )的腓肠肌超微结构进行观察 ,可以看到前者肌肉肌纤维萎缩伴有线粒体空泡变性。并进行总RNA抽提、mRNA纯化、探针制备 ,应用基因芯片筛选老龄化相关基因 ,两组大鼠骨骼肌重复出现的差异表达基因 12 7个 ,下调基因涉及能量代谢、信号转导 ,上调基因涉及蛋白质分解、细胞凋亡     

Gene Expression Profiling of Rat Cerebral Cortex Development Using cDNA Microarrays

A large amount of genetic information is devoted to brain development. In this study, the cortical development in rats at eight developmental time points (four embryonic [E15, E16, E18, E20] and four postnatal [P0, P7, P14, P21]) was studied using a rat brain 10K cDNA microarray. Significant differential expression was observed in 467 of the 9,805 genes represented on the microarray. Two major Gene Ontology classes—cell differentiation and cell–cell signaling—were found to be important for cortical development. Genes for ribosomal proteins, heterogeneous nuclear ribonucleoproteins, and tubulin proteins were up-regulated in the embryonic stage, coincidently with extensive proliferation of neural precursor cells as the major component of the cerebral cortex. Genes related to neurogenesis, including neurite regeneration, neuron development, and synaptic transmission, were more active in adulthood, when the cerebral cortex reached maturity. The many developmentally modulated genes identified by this approach will facilitate further studies of cortical functions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

伴海马硬化的颞叶癫痫患者海马组织内BDNF表达变化

目的：研究伴海马硬化的难治性颞叶癫痫（TLE）患者海马组织内脑源性神经营养因子（brain derivedneurotrophic factor,BDNF）的表达变化,探讨其在难治性颞叶癫痫发病机制中的作用。方法：采集5例伴海马硬化的难治性TLE患者手术中切除的海马组织,用逆转录-聚合酶链反应（RT—PCR）法检测BDNFmRNA表达,并与3例非海马硬化TLE患者对照。结果：与非海马硬化组比较,伴海马硬化的难治性TLE患者海马组织中的BDNFmRNA表达明显增加（P〈0．01）。结论：伴海马硬化的难治性TLE患者海马组织中BDNFmRNA表达表达增高,可能在海马硬化和难治性颞叶癫痫发生、发展中具有重要作用。     

Notch Signaling Regulates Microglial Activation and Inflammatory Reactions in a Rat Model of Temporal Lobe Epilepsy

The inflammatory response mediated by microglia in the central nervous system is closely related to epilepsy. Notch signaling plays an important role in the microglial activation during hypoxia. This study aimed to investigate whether Notch signaling is involved in microglial activation and subsequent inflammation-related neuronal injury during the process of epileptogenesis in a rat model of temporal lobe epilepsy. By using western blotting, real-time quantitative PCR, immunohistochemistry and immunofluorescence labeling, we found that the expression of Notch signaling increased after status epilepticus and that a γ-secretase inhibitor could significantly inhibit the upregulation of Notch signaling, the activation of microglia, and the release of proinflammatory cytokines. Likewise, the neuronal apoptosis and loss in the hippocampus after SE were attenuated by the γ-secretase inhibitor. These results suggest that Notch signaling plays a key role in neuroinflammation and inflammation-related neuronal damage in epilepsy, and γ-secretase inhibitors may become a novel prospective therapeutic agent for epilepsy.     

Abnormal Expression of FBXL20 in Refractory Epilepsy Patients and a Pilocarpine-Induced Rat Model

E3 ubiquitin ligases are important protein-modifying enzymes involved in the pathogenesis of a variety of neurodegenerative diseases. F-box and leucine-rich repeat protein 20 (FBXL20), an E3 ubiquitin ligase widely expressed in the central nervous system, plays an important role in the ubiquitin-dependent degradation of regulating synaptic membrane exocytosis 1 (RIM1), which is an important factor in the release of synaptic vesicles. FBXL20 has been associated with a variety of neurodegenerative diseases; thus, we hypothesized that FBXL20 is involved in the development of epilepsy. Herein, we used immunofluorescence staining, immunohistochemistry and western blotting to determine the expression pattern of FBXL20 in temporal lobe epilepsy patients and pilocarpine-induced epilepsy animal models. We also injected SD rats with lentivirus-vector mediated overexpression of FBXL20. The results showed that FBXL20 is expressed in the membrane and the cytoplasm of cortical neurons, and overexpression of FBXL20 decreased the onset level of spontaneous seizure, the frequency and duration of seizures. Additionally, FBXL20 protein level was decreased but RIM1 protein level was increased in the epileptic group compared with the LV-FBXL20 and LV-GFP group. These findings in humans were consistent with the results from a pilocarpine-induced animal model of chronic epilepsy. Thus, abnormal expression of FBXL20 might play an important role in the development of epilepsy.