MicroRNA-145 Is Downregulated in Glial Tumors and Regulates Glioma Cell Migration by Targeting Connective Tissue Growth Factor

Glioblastomas (GBM), the most common and aggressive type of malignant glioma, are characterized by increased invasion into the surrounding brain tissues. Despite intensive therapeutic strategies, the median survival of GBM patients has remained dismal over the last decades. In this study we examined the expression of miR-145 in glial tumors and its function in glioma cells. Using TCGA analysis and real-time PCR we found that the expression of miR-145/143 cluster was downregulated in astrocytic tumors compared to normal brain specimens and in glioma cells and glioma stem cells (GSCs) compared to normal astrocytes and neural stem cells. Moreover, the low expression of both miR-145 and miR-143 in GBM was correlated with poor patient prognosis. Transfection of glioma cells with miR-145 mimic or transduction with a lentivirus vector expressing pre-miR 145 significantly decreased the migration and invasion of glioma cells. We identified connective tissue growth factor (CTGF) as a novel target of miR-145 in glioma cells; transfection of the cells with this miRNA decreased the expression of CTGF as determined by Western blot analysis and the expression of its 3′-UTR fused to luciferase. Overexpression of a CTGF plasmid lacking the 3′-UTR and administration of recombinant CTGF protein abrogated the inhibitory effect of miR-145 on glioma cell migration. Similarly, we found that silencing of CTGF decreased the migration of glioma cells. CTGF silencing also decreased the expression of SPARC, phospho-FAK and FAK and overexpression of SPARC abrogated the inhibitory effect of CTGF silencing on cell migration. These results demonstrate that miR-145 is downregulated in glial tumors and its low expression in GBM predicts poor patient prognosis. In addition miR-145 regulates glioma cell migration by targeting CTGF which downregulates SPARC expression. Therefore, miR-145 is an attractive therapeutic target for anti-invasive treatment of astrocytic tumors.     

Catechin treatment improves cerebrovascular flow-mediated dilation and learning abilities in atherosclerotic mice

Severe dyslipidemia and the associated oxidative stress could accelerate the age-related decline in cerebrovascular endothelial function and cerebral blood flow (CBF), leading to neuronal loss and impaired learning abilities. We hypothesized that a chronic treatment with the polyphenol catechin would prevent endothelial dysfunction, maintain CBF responses, and protect learning abilities in atherosclerotic (ATX) mice. We treated ATX (C57Bl/6-LDLR(-/-)hApoB(+/+); 3 mo old) mice with catechin (30 mg · kg(-1) · day(-1)) for 3 mo, and C57Bl/6 [wild type (WT), 3 and 6 mo old] mice were used as controls. ACh- and flow-mediated dilations (FMD) were recorded in pressurized cerebral arteries. Basal CBF and increases in CBF induced by whisker stimulation were measured by optical coherence tomography and Doppler, respectively. Learning capacities were evaluated with the Morris water maze test. Compared with 6-mo-old WT mice, cerebral arteries from 6-mo-old ATX mice displayed a higher myogenic tone, lower responses to ACh and FMD, and were insensitive to NOS inhibition (P < 0.05), suggesting endothelial dysfunction. Basal and increases in CBF were lower in 6-mo-old ATX than WT mice (P < 0.05). A decline in the learning capabilities was also observed in ATX mice (P < 0.05). Catechin 1) reduced cerebral superoxide staining (P < 0.05) in ATX mice, 2) restored endothelial function by reducing myogenic tone, improving ACh- and FMD and restoring the sensitivity to nitric oxide synthase inhibition (P < 0.05), 3) increased the changes in CBF during stimulation but not basal CBF, and 4) prevented the decline in learning abilities (P < 0.05). In conclusion, catechin treatment of ATX mice prevents cerebrovascular dysfunctions and the associated decline in learning capacities.     

Pathway-directed weighted testing procedures for the integrative analysis of gene expression and metabolomic data

We explore the utility of p-value weighting for enhancing the power to detect differential metabolites in a two-sample setting. Related gene expression information is used to assign an a priori importance level to each metabolite being tested. We map the gene expression to a metabolite through pathways and then gene expression information is summarized per-pathway using gene set enrichment tests. Through simulation we explore four styles of enrichment tests and four weight functions to convert the gene information into a meaningful p-value weight. We implement the p-value weighting on a prostate cancer metabolomic dataset. Gene expression on matched samples is used to construct the weights. Under certain regulatory conditions, the use of weighted p-values does not inflate the type I error above what we see for the un-weighted tests except in high correlation situations. The power to detect differential metabolites is notably increased in situations with disjoint pathways and shows moderate improvement, relative to the proportion of enriched pathways, when pathway membership overlaps.     

"Omics" data and levels of evidence for biomarker discovery

With the development of new technologies for assaying biological activity on a global basis in experimental samples, various new "-omics" signatures have been developed to predict disease progression. Such signatures hold the potential to alter the nature of clinical management of human disease. In this article, we describe some necessary statistical considerations needed to take these signatures from the discovery phase to a clinically useful assay. Much of the work discussed is in the area of cancer.     

Docosahexaenoic acid ethyl esters from Isochrysis galbana

In order to produce docosahexaenoic acid (DHA), a culture of the microalgal strain Isochrysis galbana was implemented. In Erlenmeyer flasks, a natural seawater medium, the Provasoli 1/3 medium, was compared to the classical Jones medium for DHA production. The Provasoli 1/3 medium stimulated growth (0.44 d(-1)), but influenced DHA accumulation negatively (0.240 pg cell(-1)). However, DHA production per liter of culture medium were of the same order of magnitude with both media (0.961 mg l(-1)). In a 2-l bioreactor, DHA production per liter of culture medium did not increase significantly between 4 and 8 days of culture. With a view to optimize DHA productivity, cells should be harvested at the end of exponential phase i.e. after 4 days of culture. Two strategies were then attempted to produce DHA ethyl esters. First, lipids from I. galbana were submitted to lipase-catalyzed transesterification with ethanol. Secondly, fatty acids from I. galbana were submitted to lipase catalyzed esterification with ethanol. In both cases, lipase from Candida antarctica was shown to be the best candidate, among the five tested, with conversion yields of 20 and 60% after 24 h of transesterification and esterification respectively.     

Protein aggregation in Huntington's disease

The presence of an expanded polyglutamine produces a toxic gain of function in huntingtin. Protein aggregation resulting from this gain of function is likely to be the cause of neuronal death. Two main mechanisms of aggregation have been proposed: hydrogen bonding by polar-zipper formation and covalent bonding by transglutaminase-catalyzed cross-linking. In cell culture models of Huntington's disease, aggregates are mostly stabilized by hydrogen bonds, but covalent bonds are also likely to occur. Nothing is known about the nature of the bonds that stabilize the aggregates in the brain of patients with Huntington's disease. It seems that the nature of the bond stabilizing the aggregates is one of the most important questions, as the answer would condition the therapeutic approach to Huntington's disease.     

Effects of chronic paroxetine treatment on dialysate serotonin in 5-HT1B receptor knockout mice

The role of serotonin (5-HT)1B receptors in the mechanism of action of selective serotonin re-uptake inhibitors (SSRI) was studied by using intracerebral in vivo microdialysis in conscious, freely moving wild-type and 5-HT1B receptor knockout (KO 5-HT1B) mice in order to compare the effects of chronic administration of paroxetine via osmotic minipumps (1 mg per kg per day for 14 days) on extracellular 5-HT levels ([5-HT]ext) in the medial prefrontal cortex and ventral hippocampus. Basal [5-HT]ext values in the medial prefrontal cortex and ventral hippocampus, approximately 20 h after removing the minipump, were not altered by chronic paroxetine treatment in both genotypes. On day 15, in the ventral hippocampus, an acute paroxetine challenge (1 mg/kg i.p.) induced a larger increase in [5-HT]ext in saline-pretreated mutant than in wild-type mice. This difference between the two genotypes in the effect of the paroxetine challenge persisted following chronic paroxetine treatment. Conversely, in the medial prefrontal cortex, the paroxetine challenge increased [5-HT]ext similarly in saline-pretreated mice of both genotypes. Such a challenge produced a further increase in cortical [5-HT]ext compared with that in saline-pretreated groups of both genotypes, but no differences were found between genotypes following chronic treatment. To avoid the interaction with raphe 5-HT1A autoreceptors, 1 micro m paroxetine was perfused locally through the dialysis probe implanted in the ventral hippocampus; similar increases in hippocampal [5-HT]ext were found in acutely or chronically treated wild-type mice. Systemic administration of the mixed 5-HT1B/1D receptor antagonist GR 127935 (4 mg/kg) in chronically treated wild-type mice potentiated the effect of a paroxetine challenge dose on [5-HT]ext in the ventral hippocampus, whereas systemic administration of the selective 5-HT1A receptor antagonist WAY 100635 did not. By using the zero net flux method of quantitative microdialysis in the medial prefrontal cortex and ventral hippocampus of wild-type and KO 5-HT1B mice, we found that basal [5-HT]ext and the extraction fraction of 5-HT were similar in the medial prefrontal cortex and ventral hippocampus of both genotypes, suggesting that no compensatory response to the constitutive deletion of the 5-HT1B receptor involving changes in 5-HT uptake capacity occurred in vivo. As steady-state brain concentrations of paroxetine at day 14 were similar in both genotypes, it is unlikely that differences in the effects of a paroxetine challenge on hippocampal [5-HT]ext are due to alterations of the drug's pharmacokinetic properties in mutants. These data suggest that there are differences between the ventral hippocampus and medial prefrontal cortex in activation of terminal 5-HT1B autoreceptors and their role in regulating dialysate 5-HT levels. These presynaptic receptors retain their capacity to limit 5-HT release mainly in the ventral hippocampus following chronic paroxetine treatment in mice.     

Depletion of delta 9 desaturase (EC 1.14.99.5) activity in lactating rat during protein restriction

The effects of protein restriction on the activity of delta9 desaturase (EC 1.14.99.5) were investigated in lactating rats. A control group was fed a balanced diet (20% casein) for 14 days, whereas the experimental groups were fed a low-protein diet (8% casein), supplemented with or without L-methionine (0.4%), for 14 days. The enzyme activity was measured by incubations of hepatic microsomal pellets with (1-14C) stearic acid. Results showed a decreased delta9 desaturase activity, after 2,7 and 14 days of depleted diet, of -50, -40 and -33% respectively, compared with control. The supplementation of the low-protein diet with 0.4% methionine, which favours food consumption as well as growth, did not improve the altered delta9 desaturase activity. Our data evidenced that delta9 desaturase activity is depleted by protein restriction during lactation, when the protein needs are high for the biosynthesis of animal tissues. This change has to be considered as a sign of depressed delta9 desaturase biosynthesis or modifications of enzymatic properties, or both.