Top PDF Response-predictive gene expression profiling of glioma progenitor cells in vitro.

Response-predictive gene expression profiling of glioma progenitor cells in vitro.

Response-predictive gene expression profiling of glioma progenitor cells in vitro.

In vitro drug testing tools for predicting treatment effects in tumor patients were undertaken for more than 30 years. None of them has reached clinical application. Limitations lay in the long- term culture of differentiated tumor cell lines with prolonged treatment periods and conventional assays as readout. It is well known that tumor lines propagated under serum-containing Figure 2. Cellular growth and proliferation under Sunitinib treatment. BTICs were incubated with 1 mM Sunitinib or 0.00025% DMSO (control), and the XTT proliferation assay was performed after 96 h. Each individual assay was performed with five replicates per treatment group. The assay was repeated at least three times for each cell line. (A) Growth pattern in a responding (BTIC-5) and a non-responding (BTIC-16) BTIC line. Representative pictures are shown for two differently responding BTIC lines. (B) The mean absorbance of Sunitinib treated cells relative to control cells obtained in an individual assay was assessed after 24 h, 96 h and 144 hours incubation period and is plotted in a dot blot graph (y-axis) against incubation time (x-axis). (C) The relative difference of the mean proliferation relative to control is blotted in a dot blot graph (y-axis) against the corresponding BTIC line (x-axis). Each data point indicates the result of an individual experiment. The assay shows the variety of effects in the investigated lines. (D) Prediction of proliferation based on gene expression 6 h after treatment in vivo. The x-axis shows cross validated predictions of proliferation response after 96 hours based on gene expression levels monitored 6 hours after treatment, while the y-axis shows the actual proliferation measurements after 96 hours. The correlation between predicted and measured proliferation is significant (p,0.01, chi-square test). (E) Failed prediction of proliferation using expression values from untreated samples. There is no significant correlation between predictions and measurements (p = 0.98).
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In vitro drug response and efflux transporters associated with drug resistance in pediatric high grade glioma and diffuse intrinsic pontine glioma.

In vitro drug response and efflux transporters associated with drug resistance in pediatric high grade glioma and diffuse intrinsic pontine glioma.

In order to elucidate the differences in effect of drug treatment observed in glioma cultures in vitro, and the clinical responses reported in literature, we investigated the role of drug efflux transporters that belong to the ATP-binding cassette (ABC) superfamily. More precisely, we determined the expres- sion of three major drug efflux transporters present in brain: P- glycoprotein/MDR1 (P-gp), Multidrug Resistant Protein 1 (MRP1), and Breast Cancer Resistance Protein 1 (BCRP1), on both the glioma cells and surrounding tissue. P-gp, encoded by the ABCB1 gene, reduces intracellular drug accumulation by acting as an active ATP-driven transmembrane drug transporter [27], and is thought to prevent toxic substances from entering the blood brain barrier (BBB) by its expression in endothelial cells [28]. Using immunohistochemical staining, we show that P- gp is expressed on a large number of endothelial cells of the tumor vasculature in half of the pHGG cultures tested, comparable to the expression in normal brain [29]. No expression of P-gp was detected in glioma cells. These results were confirmed by Western blotting, which showed a complete absence of P-gp protein in primary pHGG cultures. Similar results have been described for adult HGG, where no P-gp was detected in primary adult HGG cultures [30]. Interestingly, however, cell lines derived from human adult glioma generally
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Identification of molecular pathways facilitating glioma cell invasion in situ.

Identification of molecular pathways facilitating glioma cell invasion in situ.

Gliomas are mostly incurable secondary to their diffuse infiltrative nature. Thus, specific therapeutic targeting of invasive glioma cells is an attractive concept. As cells exit the tumor mass and infiltrate brain parenchyma, they closely interact with a changing micro-environmental landscape that sustains tumor cell invasion. In this study, we used a unique microarray profiling approach on a human glioma stem cell (GSC) xenograft model to explore gene expression changes in situ in Invading Glioma Cells (IGCs) compared to tumor core, as well as changes in host cells residing within the infiltrated microenvironment relative to the unaffected cortex. IGCs were found to have reduced expression of genes within the extracellular matrix compartment, and genes involved in cell adhesion, cell polarity and epithelial to mesenchymal transition (EMT) processes. The infiltrated microenvironment showed activation of wound repair and tissue remodeling networks. We confirmed by protein analysis the downregulation of EMT and polarity related genes such as CD44 and PARD3 in IGCs, and EFNB3 , a tissue-remodeling agent enriched at the infiltrated microenvironment. OLIG2, a proliferation regulator and glioma progenitor cell marker upregulated in IGCs was found to function in enhancing migration and stemness of GSCs. Overall, our results unveiled a more comprehensive picture of the complex and dynamic cell autonomous and tumor-host interactive pathways of glioma invasion than has been previously demonstrated. This suggests targeting of multiple pathways at the junction of invading tumor and microenvironment as a viable option for glioma therapy.
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Gene expression profiling and secretome analysis differentiate adult-derived human liver stem/progenitor cells and human hepatic stellate cells.

Gene expression profiling and secretome analysis differentiate adult-derived human liver stem/progenitor cells and human hepatic stellate cells.

In the second part of this functional analysis, we focused on cytokines implicated in the inflammatory response. Indeed, HSC are known to secrete several pro-inflammatory cytokines while ADHLSC seem to exhibit immuno-modulatory properties. Twenty-seven cytokines were analyzed, using a multiplex technology. The cytokines were classified as growth factors, chemokines, pro-inflammatory cytokines, anti-inflammatory cyto- kines and those with dual roles. Among the growth factors analyzed, the main differences were noticed for VEGF and PDGFbb for which significant higher levels were detected in ADHLSCs (Figure 5A). The increase in VEGF and PDGFbb levels was respectively 17 and 1.5 times as compared to HSCs from the same donor. Regarding the chemokines analyzed, Eotaxin (CCL11) was 14 times more secreted by ADHLSC than HSC (Figure 5A). With respect to pro-inflammatory cytokines, the major differences between the two cell populations were IP-10 (3.6 times), IL-5 (2 times), IL-7 (2 times), IL-8 (30 times), and IL-17 (4.5 times), which are highly secreted by ADHLSC. To a lesser extent, the same trend was observed for IL-9 (1.6 times), IL-12 (1.7 times), interferonc (1.5 times) and TNFa (1.6 times) (Figures 5A & 5B). Concerning the anti-inflammatory cytokines, a significant higher level of IL-13 (2.1 times) and IL-10 (1.8 times), was secreted by ADHLSCs as compared to HSCs. IL-1ra, a natural inhibitor of the pro-inflammatory effect of IL1b, and IL-4 (presenting anti- Table 4. Phenotypic characterization of ADHLSC and HSC by flow cytometry for mesenchymal stem cells, hematopoietic cells and extracellular matrix markers.
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Genetic analysis of human traits in vitro: drug response and gene expression in lymphoblastoid cell lines.

Genetic analysis of human traits in vitro: drug response and gene expression in lymphoblastoid cell lines.

To examine how much of the variability in gene expression might be demonstrably attributed to inherited DNA variation, we searched for cis-eQTLs associated with RNA expression levels in our experiment. Using HapMap Phase 2 SNPs with MAF .10% that lie within a 0.15 Mb window around each gene, we performed standard linear regression between expression values of that gene and SNP genotypes coded 0,1,2 (representing the number of minor alleles carried by the individual). In our dataset, ,9% of genes harbored a cis-eQTL that explained 5% or more of the gene’s variance in expression levels (Figure 4C, reporting the Figure 3. Biological variation in RNA expression. 49 unrelated individuals were whole-genome RNA profiled on the Affymetrix platform in two independent experiments at the Broad Institute. (same-platform biological replicates) A subset of 14 (of the 49) were also profiled independently at the WTSI on the Illumina platform (cross- platform biological replicates) and an aliquot of that RNA (‘‘WTSI RNA’’) was again profiled at the Broad Institute on the Affymetrix platform. (cross-platform technical replicates) (A) Expression values of all 3538 expressed genes were ranked in each of the 14 unrelated individuals in the two Broad Institute biological replicate experiments and ranks were compared between: the same individuals in two separate experiments (black); all pairs of unrelated individuals across two experiments (red); 5 chimpanzees assayed in the first experiment and all individuals assayed in the second experiment (blue). Plot shows that overall expression profiles in LCLs are highly similar across biological replicates, between unrelated individuals, and even across species. (B) The 49 individuals were ranked according to their relative levels of each gene in the first Broad experiment. The ranking was then independently repeated for the second Broad experiment. Ranks were compared across the two experiments for each gene and the results plotted in (green
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β-Catenin inactivation is a pre-requisite for chick retina regeneration.

β-Catenin inactivation is a pre-requisite for chick retina regeneration.

The retina houses neurons arranged in three cell layers. The innermost cell layer contains retinal ganglion cells, the inner nuclear layer has bipolar, amacrine, and horizontal cells, and the outer nuclear layer contains the bodies of photoreceptors. Mu¨ller glia cells expand throughout the different cell layers of the retina [52]. Antibodies recognizing proteins specifically expressed in each retinal cell type were used to determine that DN-Lef1 did indeed induce neuroepithelium from both the CM and RPE that was able to differentiate into all major retinal cell types including Mu¨ller glia cells as seen in previously studied FGF2 induction (Fig. 10) [8]. Figure 7. Nuclear b-catenin is absent in the CM and RPE during FGF2-induced regeneration. Co-expression of nuclear b-catenin (Nu b-cat) and Sox2 in the CM (A–F) and RPE (G–L) at 1 d PR (A–C and G–I) and 3 d PR (D–F and J–L); (B,C) show the boxed area in (A); (E, F) show the boxed area in (D); (H, I) show the boxed area in (G); and (K, L) show the boxed area in (J). Panels A, D, G, and J have DIC overlay. Panels B, E, H, and K include DIC overlay and are equivalent to C, F, I, and L respectively. b = FGF2 bead; Cr = ciliary regeneration; Td = transdifferentiation; PE: pigmented epithelium; NPE: non-pigmented epithelium; RPE: retina pigmented epithelium; L: lens. DAPI stains the nuclei in C, F, I and L. Scale bar in (A) represents 100 mm and applies to D, G and J. Scale bar in (B) represents 100 mm and applies to C, E, F, H, I, K and L.
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Profiling gene expression induced by protease-activated receptor 2 (PAR2) activation in human kidney cells.

Profiling gene expression induced by protease-activated receptor 2 (PAR2) activation in human kidney cells.

Currently ,900 human G protein-coupled receptors (GPCRs) are annotated, forming a diverse family of membrane-spanning cell-surface proteins that may account for .2% of the human genome [1,2]. Typically GPCRs are single polypeptide chains containing seven membrane-localized helices connected by three extracellular and three intracellular loops, with extracellular amino and intracellular carboxyl termini. Both extracellular and intracellular domains vary substantially in size, the former having evolved to selectively recognize many types of GPCR-activating extracellular ligands, while the latter mediate signal transduction through coupling to combinations of G proteins resulting in extensive functional diversity [3]. Protease activated receptors (PARs) are unusual GPCRs [4] with as yet no known endogenous extracellular ligands. PARs are however indirectly activated by proteases which cleave the N-terminus of at least four PAR isoforms, exposing a new N-terminus that folds back and intramolecularly self-activates PAR [5]. Short synthetic peptides corresponding to the new N-terminus can trigger PAR activation, but only at much higher concentrations than proteases [4]. The most active reported PAR2 agonist is the hexapeptide 2-furoyl- LIGRLO-NH 2 (EC 50 ,200 nM). PAR2 is activated by mainly serine proteases (e.g. trypsin, tryptase, cathepsin G) but not
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Profiling of olfactory receptor gene expression in whole human olfactory mucosa.

Profiling of olfactory receptor gene expression in whole human olfactory mucosa.

Taken together, a study of OR gene expression in the whole human olfactory mucosa (WHOM) provides an opportunity to define ORs specifically involved in olfaction, allowing choosing frequently expressed and potentially functional ORs for deorpha- nization campaigns. OR gene expression in WHOM has been seldomly studied, probably due to the difficult access to human material. Two publications have reported the characterization of the expression of the human OR gene family in 3 individuals only using DNA microarray and only in one individual using deep sequencing [45,50]. Therefore, we designed an innovative approach based on a TaqMan Low Density Array (TLDA) containing probes for 356 predicted OR loci to investigate more thoroughly the OR gene expression profile in human olfactory mucosa. Real-time reverse transcription PCR (qRT-PCR) is frequently used for gene expression quantification, at the transcriptional level, due to its reproducibility and sensitivity. The method has also become the preferred method for validating results obtained by other techniques, such as microarrays or deep sequencing.
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Neuropeptide Y is produced by adipose tissue macrophages and regulates obesity-induced inflammation.

Neuropeptide Y is produced by adipose tissue macrophages and regulates obesity-induced inflammation.

Neuropeptide Y (NPY) is induced in peripheral tissues such as adipose tissue with obesity. The mechanism and function of NPY induction in fat are unclear. Given the evidence that NPY can modulate inflammation, we examined the hypothesis that NPY regulates the function of adipose tissue macrophages (ATMs) in response to dietary obesity in mice. NPY was induced by dietary obesity in the stromal vascular cells of visceral fat depots from mice. Surprisingly, the induction of Npy was limited to purified ATMs from obese mice. Significant basal production of NPY was observed in cultured bone marrow derived macrophage and dendritic cells (DCs) and was increased with LPS stimulation. In vitro, addition of NPY to myeloid cells had minimal effects on their activation profiles. NPY receptor inhibition promoted DC maturation and the production of IL-6 and TNFa suggesting an anti-inflammatory function for NPY signaling in DCs. Consistent with this, NPY injection into lean mice decreased the quantity of M1-like CD11c + ATMs and suppressed Ly6c hi monocytes. BM chimeras generated from
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Gene expression dosage regulation in an allopolyploid fish.

Gene expression dosage regulation in an allopolyploid fish.

Interestingly, the results of the available studies on plants and invertebrates are not all coin- cident. Several showed that the majority of genes are expressed additively [48, 49], while other studies found higher levels of nonadditive expression [13, 44]. The causes for these apparent discrepancies are not yet clear [50]. Anyway, the considerable body of data gathered so far shows a possible positive correlation between size of the fraction of the nonadditively expressed genes and the magnitude of heterotic response. Also, increasing the number of diverse genome copies in an allopolyploid, usually leads to increasingly greater magnitudes of heterosis [51]. Nevertheless, there is no consensus about the amount or identity of the nonadditively express- ed genes [50, 52]. Concerning the S. alburnoides complex, the phenomenon of heterosis has been barely addressed, except for a few comparisons of growth and reproductive traits between diploid and triploid hybrids [53] and a comparative morphometric study [36], where the AA and PP parental genomotypes were not included. From these studies, mostly non-significant differences between diploid and triploid hybrids have been found, except for a marginal lon- gevity increase in triploids. Yet, the PAA genomotype is far more frequent in the natural popu- lations than PA. So, if we consider the number of non-additively expressed transcripts as an indicator of heterosis, PAA S. alburnoides are favored since the amount of additively expressed transcripts is higher than in PA genomotype. Also, according the Bateson-Dobzhansky-Muller Model a lower fitness in hybrids might result from a bad interaction between divergent ge- nomes due to the differential capacity of interaction between their proteins [54]. In this light, allopolyploid individuals have better chances to evade this weakness. Allopolyploids have more options to non-additively combine allele-specific regulated expressions and so, have higher chances to achieve an optimized and more functional expression pattern than one achieved merely additively.
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Evaluation of gene expression profiling in a mouse model of L-gulonolactone oxidase gene deficiency

Evaluation of gene expression profiling in a mouse model of L-gulonolactone oxidase gene deficiency

5.0 software, comparison analyses between WT and SFX subsets from different tissues were performed based on the Affymetrix change algorithm, in which Wilcoxon’s signed rank test is used to compute p-value of the change call, in- cluding Increase, Marginal Increase, No Change, Marginal Decrease, or Decrease. In addition, the intensity log ratio of SFX vs. WT was calculated for each probe set based on the Signal Log Ratio Algorithm. To identify differentially ex- pressed genes between different groups, a cutoff value of twofold changes was chosen, based on most published studies (Schena et al., 1995; Gu et al., 2002b, 2003). To dis- play distinct gene expression patterns and to find a possible gene interaction network based on gene expression pattern, hierarchical clustering was performed on three different data subsets using Cluster and TreeView software (Eisen et al., 1998). To estimate the biological meaning of gene ex- pression changes, Gene Ontology (GO) functional analysis was conducted, using the GenMAPP 2.0 software accord- ing to the instructions (http://www.genmapp.org).
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Regulation and gene expression profiling of NKG2D positive human cytomegalovirus-primed CD4+ T-cells.

Regulation and gene expression profiling of NKG2D positive human cytomegalovirus-primed CD4+ T-cells.

Recombinant IL-2 (proleukin) was obtained from Novartis. Recombinant human TNF-a and IL-15 were purchased from Peprotech. CFSE and dilinoleyloxycarbocyanine (DIO) were purchased from Invitrogen. PI, CHX, sucrose, methyl-b-cyclo- dextrin, ionomycin, PMA and BrefA were purchased from Sigma. The following antibodies were used for cell sorting and flow cytometry analysis: Rat anti-CCR7 (3D12, IgG2a), anti-rat IgG2a- biotinylated, and streptavidin-pacific blue were kindly provided by Dr. Federica Sallusto (Institute for Research in Biomedicine, Bellinzona, Switzerland). Anti-CD45RA-FITC (ALB11), anti- CD8-Pc5 (B9.11), anti-CD19-Pc5 (J3-119), anti-CD25-Pc5 (B1.49.9), anti-CD56-Pc5 (N901), and anti-CD4-PE (13B8.2) were purchased from Immunotech. Anti-NKG2D-APC (FAB139A), anti-NKG2D-PE (FAB139P), anti-CD71-APC (FAB2474A), anti- NKG2A-APC (FAB1059A), and anti-NKG2C-APC (FAB138A) were purchased from RnD Systems. Anti-CD4-FITC (555346), anti-CD94-PE (555889), and control mouse IgG Abs were purchased from Becton Dickinson. The following antibodies were used for stimulation of cells for cytokine measurements and/or for the redirected cytotoxicity assay: control Ab (mouse IgG) was purchased from Santa Cruz; anti-CD3 Ab (OKT3) was purchased from Imgenex; anti-CD94 Ab (DX22) and anti-NKG2D Ab (5C6) were purchased from eBioscience.
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Gene expression of corals in response to macroalgal competitors.

Gene expression of corals in response to macroalgal competitors.

As corals decline and macroalgae proliferate on coral reefs, coral-macroalgal competition becomes more frequent and ecologically important. Whether corals are damaged by these interactions depends on susceptibility of the coral and traits of macroalgal competitors. Investigating changes in gene expression of corals and their intracellular symbiotic algae, Symbiodinium, in response to contact with different macroalgae provides insight into the biological processes and cellular pathways affected by competition with macroalgae. We evaluated the gene expression profiles of coral and Symbiodinium genes from two confamilial corals, Acropora millepora and Montipora digitata, after 6 h and 48 h of contact with four common macroalgae that differ in their allelopathic potency to corals. Contacts with macroalgae affected different biological pathways in the more susceptible (A. millepora) versus the more resistant (M. digitata) coral. Genes of coral hosts and of their associated Symbiodinium also responded in species-specific and time-specific ways to each macroalga. Changes in number and expression intensity of affected genes were greater after 6 h compared to 48 h of contact and were greater following contact with Chlorodesmis fastigiata and Amphiroa crassa than following contact with Galaxaura filamentosa or Turbinaria conoides. We documented a divergence in transcriptional responses between two confamilial corals and their associated Symbiodinium, as well as a diversity of dynamic responses within each coral species with respect to the species of macroalgal competitor and the duration of exposure to that competitor. These responses included early initiation of immune processes by Montipora, which is more resistant to damage after long-term macroalgal contact. Activation of the immune response by corals that better resist algal competition is consistent with the hypothesis that some macroalgal effects on corals may be mediated by microbial pathogens.
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Orientador: Dora Maria Tuna de Oliveira Brites Investigadora Coordenadora e Professora Catedrática Convidada Faculdade de Farmácia, Universidade de Lisboa Co-orientador: Ana Sofia Iria Azeredo Falcão de Jesus Investigadora Auxiliar (Ciência 2007) Faculdad

Orientador: Dora Maria Tuna de Oliveira Brites Investigadora Coordenadora e Professora Catedrática Convidada Faculdade de Farmácia, Universidade de Lisboa Co-orientador: Ana Sofia Iria Azeredo Falcão de Jesus Investigadora Auxiliar (Ciência 2007) Faculdad

, favours brain regeneration (Chang et al., 2012; Pluchino et al., 2005). Nevertheless, the intrinsic signals are not sufficient to promote proliferation and differentiation of NSC. Therefore, regeneration could be triggered by the stimulation of endogenous repair mechanisms at sites of degeneration leading NSC to secrete a plethora of trophic factors able to protect and prevent neural cell damage, and to re-establish the functional interactions between neural and glial cells (De Feo et al., 2012; Taupin, 2006). Studies revealed that in the SVZ, newly generated neuronal cells migrate partially through the rostro-migratory stream to the sites of nerve cell degeneration (Arvidsson et al., 2002). For instance, recent studies report that selective-serotonin reuptake inhibitors, such as fluoxetine, stimulate proliferation of NSC and increase the number of cells with neuronal features. It was shown that fluoxetine promotes both proliferation and neuronal differentiation of NSC and exerts protective effects in NSC, suggesting its therapeutic usage in several neurodegenerative diseases, such as AD and PD, considering its actions on NSC (Chang et al., 2012). Despite the generation of new neuronal cells at the sites of degeneration, this is insufficient to promote functional recovery after neurological injuries. This failure results from the low number of new neurons generated, or even because they are non-functional (Joo et al., 2012; Taupin, 2006).
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IMMUNE RESPONSE IN MALIGNANT GLIOMA

IMMUNE RESPONSE IN MALIGNANT GLIOMA

The GBM-associated immune suppression can be explained on one hand with circulating T cells entering the tumor environment which can potentially respond specifically to antigens. On the other hand, most peripheral T cells are never closely exposed to tumor cells and glioblastoma patients are not globally immunocompromised (8). These observations prompt that the tumor itself might act as a driving force behind the generation of the immunosuppressive effect. The fact that in the patient with postoperative survival of 0.5 months the CD4/CD8 ratio dropped down and the number of NKT cells (CD3+/CD56+) was augmented suggests that the altered systemic immune response is essential for tumor progression. The four patients with lethal outcome showed high percentage of CD8+CD11b+ cells suggesting that the balance between T cells with suppressor and cytotoxic phenotypes is crucial for the survival of GBM patients.
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Transcriptional dysregulation in NIPBL and cohesin mutant human cells.

Transcriptional dysregulation in NIPBL and cohesin mutant human cells.

We have identified specific gene expression profiles for CdLS that are capable of classifying probands and tightly correlate with disease severity. Cohesin preferentially binds to promoter regions of the actively expressed genes suggesting a role as a general transcription factor. These binding sites are significantly reduced in NIPBL mutant CdLS samples. This result is likely due to NIPBL’s direct role in cohesin loading on chromatin, which in turn affects transcriptional regulation at specific loci and would contribute to the CdLS pathogenesis. Out of the 339 dysregulated genes with FDR ,0.01, 202 were upregulated (59.6%) and 137 were downregulated (40.4%), more genes were reactivated than inhibited with mutations in NIPBL (59.6% versus 40.4%, p = 3.44e217) suggesting that NIPBL and cohesin can result in both negative (as transcriptional repression) and positive (as transcriptional activation) effects on expression. A similar percent- Figure 5. Cohesin and CTCF colocalize and separate the active chromatin region from the repressive chromatin region. The cohesin site at this position is lost in CdLS, thus the silencing epigenetic signal from region 3 is able to migrate into region 2, which harbors ATP11A and downregulates its transcription. (A) Screen shot of ENCODE ENr132 region from the UCSC genome browser is displaying histone methylation and acetylation status, CTCF binding sites, and DNaseI sensitivity sites on this region in GM06990 cells (from Sanger Institute and University of Washington databases, respectively). hSCC1-Control and hSCC1-CdLS are custom tracks. hSCC1-Control track indicates the results of whole genome cohesin binding analysis in LCLs from controls, whereas hSCC1-CdLS track indicates the results of whole genome cohesin binding analysis in LCLs from the CdLS proband; data on CTCF_Bcell2_8 track are adapted from Wendt et al. [10]. (B) Schematic of ENr132 locus as in (A). Five genes located in three regions are displayed. Two cohesin and six CTCF binding sites are shown. Cohesin and CTCF colocalize at Chromosome 13: 112,645,000– 112,645,600, which separates region 2 from region 3. Cohesin binding at this position was lost in CdLS proband. (C) ChIP-qPCR validation in three different healthy controls ‘‘Normal,’’ ‘‘N6,’’ and ‘‘N12’’ and three additional CdLS probands ‘‘PT2,’’ ‘‘PT12,’’ and ‘‘CDL-017.’’ Cohesin bound to this locus was dramatically reduced among CdLS probands including a proband with an SMC1A mutation (CDL-017). Sites 1 and 2 are positive controls, site 8 spans Chromosome 13: 112,645,000–112,645,600.
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In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma.

In vitro analysis of integrated global high-resolution DNA methylation profiling with genomic imbalance and gene expression in osteosarcoma.

Genetic and epigenetic changes contribute to deregulation of gene expression and development of human cancer. Changes in DNA methylation are key epigenetic factors regulating gene expression and genomic stability. Recent progress in microarray technologies resulted in developments of high resolution platforms for profiling of genetic, epigenetic and gene expression changes. OS is a pediatric bone tumor with characteristically high level of numerical and structural chromosomal changes. Furthermore, little is known about DNA methylation changes in OS. Our objective was to develop an integrative approach for analysis of high-resolution epigenomic, genomic, and gene expression profiles in order to identify functional epi/genomic differences between OS cell lines and normal human osteoblasts. A combination of Affymetrix Promoter Tilling Arrays for DNA methylation, Agilent array-CGH platform for genomic imbalance and Affymetrix Gene 1.0 platform for gene expression analysis was used. As a result, an integrative high-resolution approach for interrogation of genome-wide tumour-specific changes in DNA methylation was developed. This approach was used to provide the first genomic DNA methylation maps, and to identify and validate genes with aberrant DNA methylation in OS cell lines. This first integrative analysis of global cancer-related changes in DNA methylation, genomic imbalance, and gene expression has provided comprehensive evidence of the cumulative roles of epigenetic and genetic mechanisms in deregulation of gene expression networks.
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Rev. Soc. Bras. Med. Trop.  vol.50 número2

Rev. Soc. Bras. Med. Trop. vol.50 número2

3.0 Arrays (each containing 2999 probe sets). miRNA labeling was performed using the FlashTaq Biotin HSR RNA Labeling kit (Affymetrix), 10rxn (P/N 901910). For the labeling step, a minimum of 130ng total RNA was used for poly (A) tailing. Briely, nuclease free water was used to adjust the volume of RNA to 8µL, to which, 2µL RNA Spike Control Oligos was added. The ATP mixture was diluted in 1mM Tris and the Poly (A) tailing master mix was prepared according to the Affymetrix protocol. After the addition of 5µL master mix to the 10µL RNA Spike Control Oligos, the mixture was incubated at 37°C for 15 min. Approximately 15µL of tailed RNA was utilized for the ligation step by adding 4µL 5X FlashTaq Biotin HSR ligation mixture followed by 2µL T4 deoxyribonucleic acid (DNA) ligase to each sample, then incubating at room temperature for 30 min. After the reaction was stopped through the addition of 2.5µL HSR stop solution, 23.5µL ligated sample was added. The Enzyme Linked Oligo Sorbent Assay quality control was performed prior to array hybridization according to the manufacturer’s procedure. A volume of 21.5µL biotin labeled sample was then used for hybridization on Affymetrix Gene Chip ® miRNA 3.0 Arrays. After preparing the oven, 110.5µL
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OASIS/CREB3L1 is induced by endoplasmic reticulum stress in human glioma cell lines and contributes to the unfolded protein response, extracellular matrix production and cell migration.

OASIS/CREB3L1 is induced by endoplasmic reticulum stress in human glioma cell lines and contributes to the unfolded protein response, extracellular matrix production and cell migration.

Cells were treated as described in the figure legends and washed with PBS prior to lysis in: (1% Triton X-100, 20 mM HEPES, pH 7.4, 100 mM KCl, 2 mM EDTA, 1 mM PMSF, 10 m g/ml leupeptin, and 10 m g/ml aprotinin, 10 mM NaF, 2 mM Na3VO4, and 10 nM okadaic acid) for 15–20 min on ice. The lysate was centrifuged (10 min) and protein concentration measured using the BCA protein assay kit (Pierce, Inc., Rockford, IL). Equivalent protein amounts were resolved using 10% SDS- PAGE and electro-transferred to Hybond nitrocellulose mem- branes (GE Healthcare, Piscataway, NJ). Immunodetection was performed with the following primary antibodies: rabbit anti- OASIS (Protein Tech Group, Inc., Chicago, IL), mouse anti- KDEL, mouse anti-PDI (Stressgen Bioreagents, Victoria, BC), rabbit anti-cleaved caspase 3 (Cell Signaling), anti-c-tubulin (Sigma-Aldrich, St. Louis, MO). The secondary antibodies, anti- mouse HRP (GE Healthcare) and anti-rabbit HRP (Cell Signaling Technology) were used as required and detected by ECL kit (GE Healthcare, RPN2106). Immunoblots were scanned and protein intensities were quantified using Scion Image software (Frederick, MD).
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Proteínas recombinantes ligadas a TAT e sua aplicação terapêutica na reversão de dano isquêmico de ilhotas pancreáticas: impacto em transplante

Proteínas recombinantes ligadas a TAT e sua aplicação terapêutica na reversão de dano isquêmico de ilhotas pancreáticas: impacto em transplante

motif of this kind is known as pANTP or penetratin. It corre- sponds to 16-amino acid (RQIKIWFQNRRMKWKK) residues of the third helix of the antennapedia homeotic transcription fac- tor (ANTP) from Drosophila (4). It was postulated that the positively charged pANTP associates with the charged phos- pholipids in the outer side of the cellular membrane. This is followed by destabilization of the membrane and formation of an inverted micelle that somehow penetrates the cytoplasmic compartment (8). However, recent studies performed on cells, rather than artificial phospholipid bilayers, indicate that ad- sorptive-mediated endocytosis has a role in translocation mech- anism of pANTP into cells (9). pANTP has been mostly re- stricted to the delivery of small molecules such as peptides (10) and peptide nucleic acids (11–13). pANTP-mediated transduc- tion of peptides has been successfully used to study the mech- anism of RNA transport (14). In vivo applications of pANTP- fused molecules include the topical administration of the NH2- terminal peptide of ␣-smooth muscle actin. As this inhibits the contraction of rat wound granulation tissue, this approach could help develop new therapeutic strategies for fibrocontrac- tive pathological situations (15). Moreover, the administration of pANTP fused to a 20-amino acid peptide (amino acids 84 – 103) from the p16 tumor suppressor protein suppressed pan- creatic cancer growth and extended survival in mice (16). In- hibitors of the protein kinase C-⑀ fused to the antennapedia cell penetrating peptide were used to confirm the crucial role of this isozyme in the signaling pathway associated with protective heart ischemia preconditioning (17). Two other Drosophila ho- meodomain proteins, Fushi-tarazu and Engrailed, have similar transduction properties (18). Furthermore, a new PTD, pIsl1, with translocation ability similar to that of pANTP, was re- cently discovered (19). PIsl1 originates from rat protein home- odomain of islet-1, an insulin gene enhancer. It encompasses amino acid residues 45 to 60 (RVIVWFQNKKRCKDKK) from the third helix motif. Similarly, it was recently reported that pancreatic and duodenal homeobox-1, a key transcription factor for pancreatic development and insulin transcription, contains an antennapedia-like PTD (RHIKIWFQNRRMKWKK) in the third ␣-helix of its homeodomain. Pancreatic and duodenal ho- meobox-1 is capable of in vitro transduction of pancreatic ducts and islets (20).
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