Ultra-Deep Sequencing Reveals the microRNA Expression Pattern of the Human Stomach

Background: While microRNAs (miRNAs) play important roles in tissue differentiation and in maintaining basal physiology, little is known about the miRNA expression levels in stomach tissue. Alterations in the miRNA profile can lead to cell deregulation, which can induce neoplasia. Methodology/Principal Findings: A small RNA library of stomach tissue was sequenced using high-throughput SOLiD sequencing technology. We obtained 261,274 quality reads with perfect matches to the human miRnome, and 42% of known miRNAs were identified. Digital Gene Expression profiling (DGE) was performed based on read abundance and showed that fifteen miRNAs were highly expressed in gastric tissue. Subsequently, the expression of these miRNAs was validated in 10 healthy individuals by RT-PCR showed a significant correlation of 83.97% (P<0.05). Six miRNAs showed a low variable pattern of expression (miR-29b, miR-29c, miR-19b, miR-31, miR-148a, miR-451) and could be considered part of the expression pattern of the healthy gastric tissue. Conclusions/Significance: This study aimed to validate normal miRNA profiles of human gastric tissue to establish a reference profile for healthy individuals. Determining the regulatory processes acting in the stomach will be important in the fight against gastric cancer, which is the second-leading cause of cancer mortality worldwide.

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

Background: Considering 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. Results: Here 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. Conclusion: Altogether, 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.

Differential expression of E-cadherin gene in human neuroepithelial tumors

Cadherins are cell-to-cell adhesion molecules that play an important role in the establishment of adherent-type junctions by mediating calcium-dependent cellular interactions. The CDH1 gene encodes the transmembrane glycoprotein E-cadherin which is important in maintaining homophilic cell-cell adhesion in epithelial tissues. E-cadherin interacts with catenin proteins to maintain tissue architecture. Structural defects or loss of expression of E-cadherin have been reported as a common feature in several human cancer types. This study aimed to evaluate the expression of E-cadherin and their correlation with clinical features in microdissected brain tumor samples from 81 patients, divided into 62 astrocytic tumors grades I to IV and 19 medulloblastomas, and from 5 white matter non-neoplasic brain tissue samples. E-cadherin (CDH1) gene expression was analyzed by quantitative real-time polymerase chain reaction. Mann-Whitney, Kruskal-Wallis, Kaplan-Meir, and log-rank tests were performed for statistical analyses. We observed a decrease in expression among pathological grades of neuroepithelial tumors. Non-neoplasic brain tissue showed a higher expression level of CDH1 gene than did neuroepithelial tumors. Expression of E-cadherin gene was higher in astrocytic than embryonal tumors (P = 0.0168). Low-grade malignancy astrocytomas (grades I-II) showed higher CDH1 expression than did high-grade malignancy astrocytomas (grades III-IV) and medulloblastomas (P < 0.0001). Non-neoplasic brain tissue showed a higher expression level of CDH1 gene than grade I malignancy astrocytomas, considered as benign tumors (P = 0.0473). These results suggest that a decrease in E-cadherin gene expression level in high-grade neuroepithelial tumors may be a hallmark of malignancy in dedifferentiated tumors and that it may be possibly correlated with their progression and dissemination.

Tumor slices as a model to evaluate doxorubicin in vitro treatment and expression of trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 in canine mammary gland cancer

Background: In women with breast cancer submitted to neoadjuvant chemotherapy based in doxorubicin, tumor expression of groups of three genes (PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2) have classified them as responsive or resistant. We have investigated whether expression of these trios of genes could predict mammary carcinoma response in dogs and whether tumor slices, which maintain epithelial-mesenchymal interactions, could be used to evaluate drug response in vitro. Methods: Tumors from 38 dogs were sliced and cultured with or without doxorubicin 1 mu M for 24 h. Tumor cells were counted by two observers to establish a percentage variation in cell number, between slices. Based on these results, a reduction in cell number between treated and control samples >= 21.7%, arbitrarily classified samples, as drug responsive. Tumor expression of PRSS11, MTSS1, CLPTM1 and SMYD2, was evaluated by real time PCR. Relative expression results were then transformed to their natural logarithm values, which were spatially disposed according to the expression of trios of genes, comprising PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. Fisher linear discrimination test was used to generate a separation plane between responsive and non-responsive tumors. Results: Culture of tumor slices for 24 h was feasible. Nine samples were considered responsive and 29 non-responsive to doxorubicin, considering the pre-established cut-off value of cell number reduction = 21.7%, between doxorubicin treated and control samples. Relative gene expression was evaluated and tumor samples were then spatially distributed according to the expression of the trios of genes: PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. A separation plane was generated. However, no clear separation between responsive and non-responsive samples could be observed. Conclusion: Three-dimensional distribution of samples according to the expression of the trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 could not predict doxorubicin in vitro responsiveness. Short term culture of mammary gland cancer slices may be an interesting model to evaluate chemotherapy activity.

Notes on Population Dynamics of Amblyomma Ticks (Acari: Ixodidae) in Brazil

Previous population dynamics data, generated for Amblyomma parvum Aragao and Aniblyomma cajennense (Fabricius) in Argentina and southeastern Brazil, have indicated that these ticks complete I generation per year, with larvae predominating in autumn, nymphs in winter, and mostly adults during spring and summer. The present study reports population dynamics data for free-living Amblyomma spp. ticks in northern Brazil (Amazon forest, latitude 10 degrees S, 63 degrees W), and for Amblyomma spp. ticks collected oil birds in Southeastern Brazil (latitude 23 degrees S, 45 degrees W). In northern Brazil, adult ticks predominated from mid-spring to mid-autumn, larvae predominated in early winter, and nymphs from mid-winter to mid-spring. Seven Amblyomma spp. were identified, although A. cajannense predominated in I of the 2 sites sampled. In southeastern Brazil, larval infestations on birds peaked in autumn, followed by a nymphal infestation peak in late winter. At least 32% and 75% of these larvae and nymphs, respectively, were identified as Amblyomma longirostre (Koch). Similar to previous work, the present study showed that Amblyomma spp. larvae and nymphs predominated during autumn-winter months, and mostly adults during spring-summer months, a pattern compatible with 1 genration/yr, even at latitude 10 degrees S in northern Brazil.

Vascular endothelial growth factor expression and microvascular density in soft tissue sarcomas in dogs

The aim of the current study was to evaluate the expression of vascular endothelial growth factor (VEGF) and the microvascular density in canine soft-tissue sarcomas. Immunohistochemistry for VEGF expression was performed on 20 canine neoplasms by the streptavidin-biotin-peroxidase method using an anti-VEGF mouse monoclonal antibody (ab-119). The Volume fraction of microvessels in the sarcomas was quantified in hematoxylin and eosin-stained tissue sections. At least 10 fields of view (40x magnification) per neoplasm were analyzed by positioning a grid with 100 points and counting the microvessels that fell into the intersection points. This percentage was considered the volume fraction of these microvessels in the tumor section. VEGF expression was detected in 65% of the neoplasms. In 92.3% of the neoplasms, the expression occurred in the peritumor region; in 46.15%, in the intratumor region; and in 38.46%, the expression was present in both regions. The cells responsible for VEGF expression were fibroblasts and macrophages in the peritumor region or in the pseudocapsule and neoplastic cells in the intratumor region. Greater intratumoral VEGF was expressed in hemangiopericytomas (P = 0.04). No difference was present in the volume fraction of tumor microvessels between VEGF-positive and VEGF-negative neoplasms (P = 0.3416) or for the different types of neoplasms (P = 0.5). The results of this study suggest that VEGF participates in the angiogenesis of soft-tissue sat-coma in dogs. Additional research will be necessary to elucidate the contribution of VEGF to the progression of malignancy.

A New Mouse Model for Marfan Syndrome Presents Phenotypic Variability Associated with the Genetic Background and Overall Levels of Fbn1 Expression

Marfan syndrome is an autosomal dominant disease of connective tissue caused by mutations in the fibrillin-1 encoding gene FBN1. Patients present cardiovascular, ocular and skeletal manifestations, and although being fully penetrant, MFS is characterized by a wide clinical variability both within and between families. Here we describe a new mouse model of MFS that recapitulates the clinical heterogeneity of the syndrome in humans. Heterozygotes for the mutant Fbn1 allele mg Delta(loxPneo), carrying the same internal deletion of exons 19-24 as the mg Delta mouse model, present defective microfibrillar deposition, emphysema, deterioration of aortic wall and kyphosis. However, the onset of a clinical phenotypes is earlier in the 129/Sv than in C57BL/6 background, indicating the existence of genetic modifiers of MFS between these two mouse strains. In addition, we characterized a wide clinical variability within the 129/Sv congenic heterozygotes, suggesting involvement of epigenetic factors in disease severity. Finally, we show a strong negative correlation between overall levels of Fbn1 expression and the severity of the phenotypes, corroborating the suggested protective role of normal fibrillin-1 in MFS pathogenesis, and supporting the development of therapies based on increasing Fbn1 expression.

Differential expression of HIF-1 alpha in CD44(+)CD24(-/) (low) breast ductal carcinomas

Background: Cancer stem cell (CSC) hypothesis postulates that tumors are maintained by a self-renewing CSC population that is also capable of differentiating into non-self-renewing cell populations that constitute the bulk of tumor. Stem cells renewal and differentiation can be directly influenced by the oxygen levels of determined tissues, probably by the reduction of oxidative DNA damage in hypoxic regions, thus leading to a friendlier microenvironment, regarding to clonal expansion and for resistance to chemotherapeutic regimens. Furthermore, there have been strong data indicating a pivotal role of hypoxic niche in cancer stem cells development. There are evidence that hypoxia could drive the maintenance of CSC, via HIF-1 alpha expression, but it still to be determined whether hypoxia markers are expressed in breast tumors presenting CD44(+)CD24(-/low) immunophenotype. Methods: Immunohistochemical analysis of CD44(+)CD24(-/low) expression and its relationship with hypoxia markers and clinical outcome were evaluated in 253 samples of breast ductal carcinomas. Double-immunolabeling was performed using EnVision Doublestain System (Dako, Carpinteria, CA, USA). Slides were then scanned into high-resolution images using Aperio ScanScope XT and then, visualized in the software Image Scope (Aperio, Vista, CA, USA). Results: In univariate analysis, CD44(+)CD24(-/low) expression showed association with death due to breast cancer (p = 0.035). Breast tumors expressing CD44(+)CD24(-/low) immunophenotype showed relationship with HIF-1 alpha (p = 0.039) and negativity for HER-2 (p = 0.013). Conclusion: Considering that there are strong evidences that the fraction of a tumour considered to be cancer stem cells is plastic depending upon microenvironmental signals, our findings provide further evidence that hypoxia might be related to the worse prognosis found in CD44(+)CD24(-/low) positive breast tumors.

In Vivo Effects on the Expression of Vascular Endothelial Growth Factor-A(165) Messenger Ribonucleic Acid of an Infrared Diode Laser Associated or Not with a Visible Red Diode Laser

Objective: This study investigated and correlated the kinetic expression of vascular endothelial growth factor (VEGF)-A(165) messenger ribonucleic acid (mRNA) with the associated use or not of an infrared laser and a visible red laser during the wound healing in rats. Background Data: There is a lack of scientific evidence demonstrating the influence of low-level laser therapy (LLLT) on the expression of VEGF mRNA in vivo. Materials and Methods: Forty-five Wistar rats were randomly allocated to one of three groups: I (n = 5, nonoperated animals), II (n = 25, operated animals), and III (n = 25, animals operated and subjected to laser irradiation). A surgical wound was performed using a scalpel in the right side of the tongue of operated animals. In group III, two sessions of laser irradiation were performed, one right after the surgical procedure (infrared laser, 780 nm, 70mW, 35 J/cm(2)) and the other 48 h later (visible red laser, 660 nm, 40mW, 5J/cm(2)). Five animals each were sacrificed 1, 3, 5, and 7 days postoperatively in groups II and III, and samples of tongue tissue were obtained. The animals of group I were sacrificed on day 7. Total RNA was extracted using guanidine-isothiocyanate-phenol-chloroform method. The results of horizontal electrophoresis after reverse transcription polymerase chain reaction permitted the ratio of VEGF-A(165) mRNA and glyceraldehyde 3-phosphate dehydrogenase mRNA expression for groups I, II, and III to be assessed (two-way analysis of variance and Tukey test, p<0.05). Results: The expression of VEGF-A(165) mRNA in group II (0.770 +/- 0.098) was statistically greater than that observed in groups I (0.523 +/- 0.164) and III (0.504 +/- 0.069) in the first day after surgery (p<0.05). Significant differences between the groups were not observed in other time periods. Conclusion: LLLT influenced the expression of VEGF-A(165) mRNA during wound healing after a surgical procedure on the tongue of Wistar rats.

RNA interference-mediated knockdown of CD49e (alpha 5 integrin chain) in human thymic epithelial cells modulates the expression of multiple genes and decreases thymocyte adhesion

Background: The thymus is a central lymphoid organ, in which bone marrow-derived T cell precursors undergo a complex process of maturation. Developing thymocytes interact with thymic microenvironment in a defined spatial order. A component of thymic microenvironment, the thymic epithelial cells, is crucial for the maturation of T-lymphocytes through cell-cell contact, cell matrix interactions and secretory of cytokines/chemokines. There is evidence that extracellular matrix molecules play a fundamental role in guiding differentiating thymocytes in both cortical and medullary regions of the thymic lobules. The interaction between the integrin alpha 5 beta 1 (CD49e/CD29; VLA-5) and fibronectin is relevant for thymocyte adhesion and migration within the thymic tissue. Our previous results have shown that adhesion of thymocytes to cultured TEC line is enhanced in the presence of fibronectin, and can be blocked with anti-VLA-5 antibody. Results: Herein, we studied the role of CD49e expressed by the human thymic epithelium. For this purpose we knocked down the CD49e by means of RNA interference. This procedure resulted in the modulation of more than 100 genes, some of them coding for other proteins also involved in adhesion of thymocytes; others related to signaling pathways triggered after integrin activation, or even involved in the control of F-actin stress fiber formation. Functionally, we demonstrated that disruption of VLA-5 in human TEC by CD49e-siRNA-induced gene knockdown decreased the ability of TEC to promote thymocyte adhesion. Such a decrease comprised all CD4/CD8-defined thymocyte subsets. Conclusion: Conceptually, our findings unravel the complexity of gene regulation, as regards key genes involved in the heterocellular cell adhesion between developing thymocytes and the major component of the thymic microenvironment, an interaction that is a mandatory event for proper intrathymic T cell differentiation.

Sequential analysis of global gene expression profiles in immature and in vitro matured bovine oocytes: potential molecular markers of oocyte maturation

Background: Without intensive selection, the majority of bovine oocytes submitted to in vitro embryo production (IVP) fail to develop to the blastocyst stage. This is attributed partly to their maturation status and competences. Using the Affymetrix GeneChip Bovine Genome Array, global mRNA expression analysis of immature (GV) and in vitro matured (IVM) bovine oocytes was carried out to characterize the transcriptome of bovine oocytes and then use a variety of approaches to determine whether the observed transcriptional changes during IVM was real or an artifact of the techniques used during analysis. Results: 8489 transcripts were detected across the two oocyte groups, of which similar to 25.0% (2117 transcripts) were differentially expressed (p < 0.001); corresponding to 589 over-expressed and 1528 under-expressed transcripts in the IVM oocytes compared to their immature counterparts. Over expression of transcripts by IVM oocytes is particularly interesting, therefore, a variety of approaches were employed to determine whether the observed transcriptional changes during IVM were real or an artifact of the techniques used during analysis, including the analysis of transcript abundance in oocytes in vitro matured in the presence of a-amanitin. Subsets of the differentially expressed genes were also validated by quantitative real-time PCR (qPCR) and the gene expression data was classified according to gene ontology and pathway enrichment. Numerous cell cycle linked (CDC2, CDK5, CDK8, HSPA2, MAPK14, TXNL4B), molecular transport (STX5, STX17, SEC22A, SEC22B), and differentiation (NACA) related genes were found to be among the several over-expressed transcripts in GV oocytes compared to the matured counterparts, while ANXA1, PLAU, STC1and LUM were among the over-expressed genes after oocyte maturation. Conclusion: Using sequential experiments, we have shown and confirmed transcriptional changes during oocyte maturation. This dataset provides a unique reference resource for studies concerned with the molecular mechanisms controlling oocyte meiotic maturation in cattle, addresses the existing conflicting issue of transcription during meiotic maturation and contributes to the global goal of improving assisted reproductive technology.

Imprinted gene expression in in vivo- and in vitro-produced bovine embryos and chorio-allantoic membranes

Cloning by nuclear transfer is often associated with poor results due to abnormal nuclear reprogramming of somatic donor cells and altered gene expression patterns. We investigated the expression patterns of imprinted genes IGF2 and IGF2R in 33- to 36-day bovine embryos and chorio-allantoic membranes derived from in vivo- and in vitro-produced embryos by somatic cell nuclear transfer (SCNT), parthenogenetic activation, and in vitro fertilization (IVF). There was a lower IGF2 expression rate in the SCNT (0.19) and parthenogenetic (0.02) groups when compared to in vivo and IVF embryos (2.01; P < 0.05). In the chorio-allantoic membranes, IGF2 showed a baseline expression pattern (P < 0.05) in parthenotes (0.001) when compared to in vivo, IVF (3.13), and SCNT (0.98) groups. IGF2R was less expressed (P < 0.05) in SCNT chorio-allantoic membranes (0.25) when compared to the in vivo group. The low expression of IGF2 in parthenogenetic embryos and chorio-allantoic membranes confirms its imprinted status in cattle. Alterations in the relative frequency of IGF2 and IGF2R transcripts were observed in SCNT-derived bovine embryos and chorioallantoic membranes, respectively, supporting the hypothesis that abnormalities in the expression of imprinted genes are causes of the low efficiency of SCNT procedures in this species.

The structure and dynamics of Abell 1942

Aims. We present a dynamical analysis of the galaxy cluster Abell 1942 based on a set of 128 velocities obtained at the European Southern Observatory. Methods. Data on individual galaxies are presented and the accuracy of the determined velocities as some properties of the cluster are discussed. We have also made use of publicly available Chandra X-ray data. Results. We obtained an improved mean redshift value z = 0.22513 +/- 0.0008 and velocity dispersion sigma = 908(139)(+147) km s(-1). Our analysis indicates that inside a radius of similar to 1.5 h(70)(-1) Mpc (similar to 7 arcmin) the cluster is well relaxed, without any remarkable features and the X-ray emission traces the galaxy distribution fairly well. Two possible optical substructures are seen at similar to 5 arcmin from the centre in the northwest and the southwest directions, but are not confirmed by the velocity field. These clumps are, however, kinematically bound to the main structure of Abell 1942. X-ray spectroscopic analysis of Chandra data resulted in a temperature kT = 5.5+/-0.5 keV and metal abundance Z = 0.33 +/- 0.15 Z(circle dot). The velocity dispersion corresponding to this temperature using the T(X-sigma) scaling relation is in good agreement with the measured galaxy velocities. Our photometric redshift analysis suggests that the weak lensing signal observed to the south of the cluster and previously attributed to a ""dark clump"" is produced by background sources, possibly distributed as a filamentary structure.

Dynamics and constraints of the massive graviton dark matter flat cosmologies

We discuss the dynamics of the Universe within the framework of the massive graviton cold dark matter scenario (MGCDM) in which gravitons are geometrically treated as massive particles. In this modified gravity theory, the main effect of the gravitons is to alter the density evolution of the cold dark matter component in such a way that the Universe evolves to an accelerating expanding regime, as presently observed. Tight constraints on the main cosmological parameters of the MGCDM model are derived by performing a joint likelihood analysis involving the recent supernovae type Ia data, the cosmic microwave background shift parameter, and the baryonic acoustic oscillations as traced by the Sloan Digital Sky Survey red luminous galaxies. The linear evolution of small density fluctuations is also analyzed in detail. It is found that the growth factor of the MGCDM model is slightly different (similar to 1-4%) from the one provided by the conventional flat Lambda CDM cosmology. The growth rate of clustering predicted by MGCDM and Lambda CDM models are confronted to the observations and the corresponding best fit values of the growth index (gamma) are also determined. By using the expectations of realistic future x-ray and Sunyaev-Zeldovich cluster surveys we derive the dark matter halo mass function and the corresponding redshift distribution of cluster-size halos for the MGCDM model. Finally, we also show that the Hubble flow differences between the MGCDM and the Lambda CDM models provide a halo redshift distribution departing significantly from the those predicted by other dark energy models. These results suggest that the MGCDM model can observationally be distinguished from Lambda CDM and also from a large number of dark energy models recently proposed in the literature.

Characterization of new IS elements and studies of their dispersion in two subspecies of Leifsonia xyli

Background: Leifsonia xyli is a xylem-inhabiting bacterial species comprised of two subspecies: L. xyli subsp. xyli (Lxx) and L. xyli subsp. cynodontis (Lxc). Lxx is the causal agent of ratoon stunting disease in sugarcane commercial fields and Lxc colonizes the xylem of several grasses causing either mild or no symptoms of disease. The completely sequenced genome of Lxx provided insights into its biology and pathogenicity. Since IS elements are largely reported as an important source of bacterial genome diversification and nothing is known about their role in chromosome architecture of L. xyli, a comparative analysis of Lxc and Lxx elements was performed. Results: Sample sequencing of Lxc genome and comparative analysis with Lxx complete DNA sequence revealed a variable number of IS transposable elements acting upon genomic diversity. A detailed characterization of Lxc IS elements and a comparative review with IS elements of Lxx are presented. Each genome showed a unique set of elements although related to same IS families when considering features such as similarity among transposases, inverted and direct repeats, and element size. Most of the Lxc and Lxx IS families assigned were reported to maintain transposition at low levels using translation regulatory mechanisms, consistent with our in silico analysis. Some of the IS elements were found associated with rearrangements and specific regions of each genome. Differences were also found in the effect of IS elements upon insertion, although none of the elements were preferentially associated with gene disruption. A survey of transposases among genomes of Actinobacteria showed no correlation between phylogenetic relatedness and distribution of IS families. By using Southern hybridization, we suggested that diversification of Lxc isolates is also mediated by insertion sequences in probably recent events. Conclusion: Collectively our data indicate that transposable elements are involved in genome diversification of Lxc and Lxx. The IS elements were probably acquired after the divergence of the two subspecies and are associated with genome organization and gene contents. In addition to enhancing understanding of IS element dynamics in general, these data will contribute to our ongoing comparative analyses aimed at understanding the biological differences of the Lxc and Lxx.