Valeria Valente

The contribution of 700,000 ORF sequence tags to the definition of the human transcriptome

Open reading frame expressed sequences tags (ORESTES) differ from conventional ESTs by providing sequence data from the central protein coding portion of transcripts. We generated a total of 696,745 ORESTES sequences from 24 human tissues and used a subset of the data that correspond to a set of 15,095 full-length mRNAs as a means of assessing the efficiency of the strategy and its potential contribution to the definition of the human transcriptome. We estimate that ORESTES sampled over 80% of all highly and moderately expressed, and between 40% and 50% of rarely expressed, human genes. In our most thoroughly sequenced tissue, the breast, the 130,000 ORESTES generated are derived from transcripts from an estimated 70% of all genes expressed in that tissue, with an equally efficient representation of both highly and poorly expressed genes. In this respect, we find that the capacity of the ORESTES strategy both for gene discovery and shotgun transcript sequence generation significantly exceeds that of conventional ESTs. The distribution of ORESTES is such that many human transcripts are now represented by a scaffold of partial sequences distributed along the length of each gene product. The experimental joining of the scaffold components, by reverse transcription–PCR, represents a direct route to transcript finishing that may represent a useful alternative to full-length cDNA cloning.

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

BackgroundConsidering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue.ResultsHere we show that amongst seven frequently used housekeeping genes TBP and HPRT1 are adequate references for glioblastoma gene expression analysis. Evaluation of the expression levels of 12 target genes utilizing different endogenous controls revealed that the normalization method applied might introduce errors in the estimation of relative quantities. Genes presenting expression levels which do not significantly differ between tumor and normal tissues can be considered either increased or decreased if unsuitable reference genes are applied. Most importantly, genes showing significant differences in expression levels between tumor and normal tissues can be missed. We also demonstrated that the Holliday Junction Recognizing Protein, a novel DNA repair protein over expressed in lung cancer, is extremely over-expressed in glioblastoma, with a median change of about 134 fold.ConclusionAltogether, our data show the relevance of previous validation of candidate control genes for each experimental model and indicate TBP plus HPRT1 as suitable references for studies on glioblastoma gene expression.

Brief Report: The lincRNA Hotair Is Required for Epithelial-to-Mesenchymal Transition and Stemness Maintenance of Cancer Cell Lines

Hotair is a member of the recently described class of noncoding RNAs called lincRNA (large intergenic noncoding RNA). Various studies suggest that Hotair acts regulating epigenetic states by recruiting chromatin‐modifying complexes to specific target sequences that ultimately leads to suppression of several genes. Although Hotair has been associated with metastasis and poor prognosis in different tumor types, a deep characterization of its functions in cancer is still needed. Here, we investigated the role of Hotair in the scenario of epithelial‐to‐mesenchymal transition (EMT) and in the arising and maintenance of cancer stem cells (CSCs). We found that treatment with TGF‐β1 resulted in increased Hotair expression and triggered the EMT program. Interestingly, ablation of Hotair expression by siRNA prevented the EMT program stimulated by TGF‐β1, and also the colony‐forming capacity of colon and breast cancer cells. Furthermore, we observed that the colon CSC subpopulation (CD133+/CD44+) presents much higher levels of Hotair when compared with the non‐stem cell subpopulation. These results indicate that Hotair acts as a key regulator that controls the multiple signaling mechanisms involved in EMT. Altogether, our data suggest that the role of Hotair in tumorigenesis occurs through EMT triggering and stemness acquisition. Stem Cells 2013;31:2827–2832

Mitochondrial genome diversity of Native Americans supports a single early entry of founder populations into America.

There is general agreement that the Native American founder populations migrated from Asia into America through Beringia sometime during the Pleistocene, but the hypotheses concerning the ages and the number of these migrations and the size of the ancestral populations are surrounded by controversy. DNA sequence variations of several regions of the genome of Native Americans, especially in the mitochondrial DNA (mtDNA) control region, have been studied as a tool to help answer these questions. However, the small number of nucleotides studied and the nonclocklike rate of mtDNA control-region evolution impose several limitations to these results. Here we provide the sequence analysis of a continuous region of 8.8 kb of the mtDNA outside the D-loop for 40 individuals, 30 of whom are Native Americans whose mtDNA belongs to the four founder haplogroups. Haplogroups A, B, and C form monophyletic clades, but the five haplogroup D sequences have unstable positions and usually do not group together. The high degree of similarity in the nucleotide diversity and time of differentiation (i.e., approximately 21,000 years before present) of these four haplogroups support a common origin for these sequences and suggest that the populations who harbor them may also have a common history. Additional evidence supports the idea that this age of differentiation coincides with the process of colonization of the New World and supports the hypothesis of a single and early entry of the ancestral Asian population into the Americas.

Modulation of HJURP (Holliday Junction-Recognizing Protein) Levels Is Correlated with Glioblastoma Cells Survival

Background Diffuse astrocytomas are the most common type of primary brain cancer in adults. They present a wide variation in differentiation and aggressiveness, being classified into three grades: low-grade diffuse astrocytoma (grade II), anaplastic astrocytoma (grade III) and glioblastoma multiforme (grade IV), the most frequent and the major lethal type. Recent studies have highlighted the molecular heterogeneity of astrocytomas and demonstrated that large-scale analysis of gene expression could help in their classification and treatment. In this context, we previously demonstrated that HJURP, a novel protein involved in the repair of DNA double-strand breaks, is highly overexpressed in glioblastoma. Methodology/Principal Findings Here we show that HJURP is remarkably overexpressed in a cohort composed of 40 patients with different grade astrocytomas. We also observed that tumors presenting the higher expression levels of HJURP are associated with poor survival prognosis, indicating HJURP overexpression as an independent prognostic factor of death risk for astrocytoma patients. More importantly, we found that HJURP knockdown strongly affects the maintenance of glioblastoma cells in a selective manner. Glioblastoma cells showed remarkable cell cycle arrest and premature senescence that culminated in elevated levels of cell death, differently from non-tumoral cells that were minimally affected. Conclusions These data suggest that HJURP has an important role in the maintenance of extremely proliferative cells of high-grade gliomas and point to HJURP as a potential therapeutic target for the development of novel treatments for glioma patients.

The use of Open Reading frame ESTs (ORESTES) for analysis of the honey bee transcriptome

BackgroundThe ongoing efforts to sequence the honey bee genome require additional initiatives to define its transcriptome. Towards this end, we employed the Open Reading frame ESTs (ORESTES) strategy to generate profiles for the life cycle of Apis mellifera workers.ResultsOf the 5,021 ORESTES, 35.2% matched with previously deposited Apis ESTs. The analysis of the remaining sequences defined a set of putative orthologs whose majority had their best-match hits with Anopheles and Drosophila genes. CAP3 assembly of the Apis ORESTES with the already existing 15,500 Apis ESTs generated 3,408 contigs. BLASTX comparison of these contigs with protein sets of organisms representing distinct phylogenetic clades revealed a total of 1,629 contigs that Apis mellifera shares with different taxa. Most (41%) represent genes that are in common to all taxa, another 21% are shared between metazoans (Bilateria), and 16% are shared only within the Insecta clade. A set of 23 putative genes presented a best match with human genes, many of which encode factors related to cell signaling/signal transduction. 1,779 contigs (52%) did not match any known sequence. Applying a correction factor deduced from a parallel analysis performed with Drosophila melanogaster ORESTES, we estimate that approximately half of these no-match ESTs contigs (22%) should represent Apis-specific genes.ConclusionsThe versatile and cost-efficient ORESTES approach produced minilibraries for honey bee life cycle stages. Such information on central gene regions contributes to genome annotation and also lends itself to cross-transcriptome comparisons to reveal evolutionary trends in insect genomes.

Drosophila melanogaster lipins are tissue-regulated and developmentally regulated and present specific subcellular distributions

Lipins constitute a novel family of Mg2+‐dependent phosphatidate phosphatases that catalyze the dephosphorylation of phosphatidic acid to yield diacylglycerol, an important intermediate in lipid metabolism and cell signaling. Whereas a single lipin is detected in less complex organisms, in mammals there are distinct lipin isoforms and paralogs that are differentially expressed among tissues. Compatible with organism tissue complexity, we show that the single Drosophila Lpin1 ortholog (CG8709, here named DmLpin) expresses at least three isoforms (DmLpinA, DmLpinK and DmLpinJ) in a temporal and spatially regulated manner. The highest levels of lipin in the fat body, where DmLpinA and DmLpinK are expressed, correlate with the highest levels of triacylglycerol (TAG) measured in this tissue. DmLpinK is the most abundant isoform in the central nervous system, where TAG levels are significantly lower than in the fat body. In the testis, where TAG levels are even lower, DmLpinJ is the predominant isoform. Together, these data suggest that DmLpinA might be the isoform that is mainly involved in TAG production, and that DmLpinK and DmLpinJ could perform other cellular functions. In addition, we demonstrate by immunofluorescence that lipins are most strongly labeled in the perinuclear region of the fat body and ventral ganglion cells. In visceral muscles of the larval midgut and adult testis, lipins present a sarcomeric distribution. In the ovary chamber, the lipin signal is concentrated in the internal rim of the ring canal. These specific subcellular localizations of the Drosophila lipins provide the basis for future investigations on putative novel cellular functions of this protein family.

Glutamate NMDA Receptor Subunit R1 and GAD mRNA Expression in Human Temporal Lobe Epilepsy

Abstract1. Molecular mechanisms underlying increased hippocampal excitability in human temporal lobe epilepsy (TLE) are largely unknown. A disturbance of the imbalance between excitatory and inhibitory neurotransmission pathways in the epileptic hippocampus may contribute substantially to a decreased seizure threshold.2. We have extended the investigation whether TLE is associated with changes in the expression of GAD67 and NMDAR1 by assessing the relative amounts of the mRNAs in human hippocampal samples by means of semiquantitative RT-PCR. The samples included 16 hippocampal slices obtained at surgery from intractable TLE (HS, n = 14; non-HS, n = 2) and 3 postmortem control hippocampi.3. The ratio for the GAD/NMDAR1 transcripts was significantly higher in TLE cases when compared to the nonepileptic samples. Such findings are mainly a consequence of the increased amounts of GAD mRNA detected in the epileptic hippocampus. Compared with nonepileptic samples, and without correction for neuron losses, the amounts of NMDAR1 mRNA in HS are slightly reduced, and in the non-HS samples they are significantly increased, which is consistent with an increase of NMDAR1 in the hippocampal remaining neurons, as previously reported.4. Our results also contribute to the indication of GAD67 mRNA upregulation in human TLE. A possible functional implication for the increased GAD mRNA levels could be a mechanism to reduce neuronal hiperexcitability, synchronization, and/or the spread of seizure.

Increased expression of GluR2-flip in the hippocampus of the Wistar audiogenic rat strain after acute and kindled seizures.

The Wistar Audiogenic Rat (WAR) is an epileptic‐prone strain developed by genetic selection from a Wistar progenitor based on the pattern of behavioral response to sound stimulation. Chronic acoustic stimulation protocols of WARs (audiogenic kindling) generate limbic epileptogenesis, confirmed by ictal semiology, amygdale, and hippocampal EEG, accompanied by hippocampal and amygdala cell loss, as well as neurogenesis in the dentate gyrus (DG). In an effort to identify genes involved in molecular mechanisms underlying epileptic process, we used suppression‐subtractive hybridization to construct normalized cDNA library enriched for transcripts expressed in the hippocampus of WARs. The most represented gene among the 133 clones sequenced was the ionotropic glutamate receptor subunit II (GluR2), a member of the α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazoleopropionic acid (AMPA) receptor. Although semiquantitative RT‐PCR analysis shows that the hippocampal levels of the GluR2 subunits do not differ between naïve WARs and their Wistar counterparts, we observed that the expression of the transcript encoding the splice‐variant GluR2‐flip is increased in the hippocampus of WARs submitted to both acute and kindled audiogenic seizures. Moreover, using in situ hybridization, we verified upregulation of GluR2‐flip mainly in the CA1 region, among the hippocampal subfields of audiogenic kindled WARs. Our findings on differential upregulation of GluR2‐flip isoform in the hippocampus of WARs displaying audiogenic seizures is original and agree with and extend previous immunohistochemical for GluR2 data obtained in the Chinese P77PMC audiogenic rat strain, reinforcing the association of limbic AMPA alterations with epileptic seizures.

Mitochondrial genome instability in colorectal adenoma and adenocarcinoma.

Mitochondrial dysfunction is regarded as a hallmark of cancer progression. In the current study, we evaluated mitochondrial genome instability and copy number in colorectal cancer using Next Generation Sequencing approach and qPCR, respectively. The results revealed higher levels of heteroplasmy and depletion of the relative mtDNA copy number in colorectal adenocarcinoma. Adenocarcinoma samples also presented an increased number of mutations in nuclear genes encoding proteins which functions are related with mitochondria fusion, fission and localization. Moreover, we found a set of mitochondrial and nuclear genes, which cooperate in the same mitochondrial function simultaneously mutated in adenocarcinoma. In summary, these results support an important role for mitochondrial function and genomic instability in colorectal tumorigenesis.