Genome-wide analysis of genetic alterations in testicular primary seminoma using high resolution single nucleotide polymorphism arrays

Testicular germ cell tumors (TGCT) represent the most common malignancy among young males. To our knowledge no comprehensive Copy Number Variation (CNVs) studies of TGCT using high-resolution Single Nucleotide Polymorphism (SNP) array have been performed. By a genome-wide analysis of CNV and loss of heterozygosity (LOH) in 25 primary seminomas, we confirmed several previously reported genomic alterations and discovered eight novel genomic alterations including amplifications and homozygous deletions. Moreover, a comparison of genomic alterations of early and late stage seminoma identified CNVs that correlate with progression, which included deletions in chromosomes 4q, 5p, 9q, 13q and 20p and amplifications in chromosomes 9q and 13q. We compared previously perform Affymetrix expression analysis in a subset of samples and found robust correlation between expression and genomic alterations. Furthermore, high correlations (40-75%) were observed between CNV by SNP analysis and quantitative PCR. Our findings may lead to better understanding of TGTC's pathogenesis.     

Heavy Metals in Hair of Wild Canids from the Brazilian Cerrado

In this study, we aimed to assess whether free-ranging wild canids are exposed to heavy metals in one of the most developed and populated regions of Brazil. Hair of 26 wild canids (maned wolves Chrysocyon brachyurus, crab-eating foxes Cerdocyon thous, and hoary foxes Lycalopex vetulus) from the Cerrado biome in Southeast Brazil were analyzed by spectrophotometry to detect cadmium, chromium, and lead, and also the essential copper, iron, manganese, and zinc traces. All samples showed traces of copper, iron, manganese, and zinc. Non-essential lead was detected in 57% (2.35 ± 0.99 mg/kg), and chromium in 88% (2.98 ± 1.56 mg/kg) of samples. Cadmium traces (detection limit 0.8 mg/kg) were not found. Crab-eating foxes had more copper, iron, and manganese in hair than maned wolves. Correlations among element levels differed between maned wolves and crab-eating foxes. Concentrations of chromium and lead were outstandingly higher than in wild canids from other areas. Addressing the causes of such levels and the impacts of the heavy metal pollution in Neotropical ecosystems is urgent for animal health and conservation purposes. We argue that heavy metal pollution should be considered as dangerous threats to wildlife health in Brazil and recommend hair sampling as a biomonitoring tool for heavy metals in Neotropical terrestrial mammals.     

Correlation between BRAF mutation and promoter methylation of TIMP3, RARβ2 and RASSF1A in thyroid cancer

Our aim was to comprehensively analyze promoter hypermethylation of a panel of novel and known methylation markers for thyroid neoplasms and to establish their relationship with BRAF mutation and clinicopathologic parameters of thyroid cancer. A cohort of thyroid tumors, consisting of 44 cancers and 44 benign thyroid lesions, as well as 15 samples of adjacent normal thyroid tissue, was evaluated for BRAF mutation and promoter hypermethylation. Genes for quantitative methylation specific PCR (QMSP) were selected by a candidate gene approach. Twenty-two genes were tested: TSHR, RASSF1A, RARβ2, DAPK, hMLH1, ATM, S100, p16, CTNNB1, GSTP1, CALCA, TIMP3, TGFßR2, THBS1, MINT1, CTNNB1, MT1G, PAK3, NISCH, DCC, AIM1 and KIF1A. The PCR-based “mutector assay” was used to detect BRAF mutation. All p values reported are two sided. Considerable overlap was seen in the methylation markers among the different tissue groups. Significantly higher methylation frequency and level were observed for KIF1A and RARß2 in cancer samples compared with benign tumors. A negative correlation between BRAF mutation and RASSF1A methylation, and a positive correlation with RARß2 methylation were observed in accordance with previous results. In addition, positive correlation with TIMP3 and a marginal correlation with DCC methylation were observed. The present study constitutes a comprehensive promoter methylation profile of thyroid neoplasia and shows that results must be analyzed in a tissue-specific manner to identify clinically useful methylation markers. Integration of genetic and epigenetic changes in thyroid cancer will help identify relevant biologic pathways that drive its development.     

Courtship Behavior of Zaprionus indianus (Gupta) (Diptera: Drosophilidae) from Populations Colonizing South America

We describe for the first time the sexual behavior and the courtship song of males of the African fly Zaprionus indianus (Gupta), a recent invader of South America. The male courtship song is formed by monocyclic pulses and the courtship behavior is simple when compared to that of species of Drosophila. Two interpulse interval (IPI) distributions were observed: pre-mounting and mounting. No significant difference was observed between the pre-mounting IPIs of males that descended from three geographical populations from South America. We also observed the songs produced by females and the homosexual behavior exhibited by males. A sequence of bursts is produced by females as a refusal signal against males, while males emit a characteristic song that identifies sex genus, which differs from the courtship song. The short courtship and mating latencies recorded reveal vigorous males and receptive females, respectively.     

Over-Expression of DSCR1 Protects against Post-Ischemic Neuronal Injury

Background and Purpose

The Down syndrome candidate region 1 (DSCR1) gene is located on human chromosome 21 and its protein is over-expressed in brains of Down syndrome individuals. DSCR1 can modulate the activity of calcineurin, a phosphatase abundant in the brain, but its influence on stroke outcome is not clear. We compared stroke outcome in wildtype (WT) and transgenic (DSCR1-TG) mice which over-express isoform 1 of human DSCR1.

Methods

Transient cerebral ischemia was produced by occlusion of the middle cerebral artery for 0.5 h. After 23.5 h reperfusion, we assessed neurological impairment, brain infarct and edema volume, leukocyte infiltration and markers of inflammation. Intrinsic resistance to apoptosis following glucose deprivation was also assessed in primary cultures of WT and DSCR1-TG neurons.

Results

In contrast to WT, DSCR1-TG mice had an improved neurological deficit score, greater grip strength, attenuated infarct volume and brain swelling, and lacked hippocampal lesions after stroke. Expression of mouse DSCR1-1, but not DSCR1-4, mRNA and protein was increased by ischemia in both WT and DSCR1-TG. Brain calcineurin activity was increased to a similar degree after ischemia in each genotype. DSCR1-TG mice had fewer infiltrating neutrophils and activated microglia compared with WT, in association with an attenuated upregulation of several pro-inflammatory genes. Neurons from DSCR1-TG mice were more resistant than WT neurons to apoptotic cell death following 24 h of glucose deprivation.

Conclusions

Over-expression of DSCR1 in mice improves outcome following stroke. Mechanisms underlying this protection may involve calcineurin-independent, anti-inflammatory and anti-apoptotic effects mediated by DSCR1 in neurons.     

Organization and evolution of the alcohol dehydrogenase gene in Drosophila

The alcohol dehydrogenase (Adh) gene was isolated from Drosophila simulans and D. mauritiana, and the DNA sequence of a 4.6-kb region, containing the structural gene and flanking sequence, was determined for each. These sequences were compared with the Adh region of D. melanogaster to characterize changes that occur in the Drosophila genome during evolution and to identify conserved sequences of functional importance. Drosophila simulans and D. mauritiana Adh are organized in a manner similar to that of D. melanogaster Adh, including the presence of two promoters for the single Adh gene. This study identified conserved flanking elements that, in conjunction with other studies, suggest regions that may be involved in the control of Adh expression. Inter- and intraspecies comparisons revealed differences in the kinds of sequence changes that have accumulated. Sequence divergence in and around the Adh gene was used to assess inter- and intraspecies evolutionary relationships. Finally, there appears to be an unrelated structural gene located directly 3' of the Adh transcribed region.      

Mutant p53R175H upregulates Twist1 expression and promotes epithelial–mesenchymal transition in immortalized prostate cells

A mutation within one allele of the p53 tumor suppressor gene can inactivate the remaining wild-type allele in a dominant-negative manner and in some cases can exert an additional oncogenic activity, known as mutant p53 ‘gain of function'' (GOF). To study the role of p53 mutations in prostate cancer and to discriminate between the dominant-negative effect and the GOF activity of mutant p53, we measured, using microarrays, the expression profiles of three immortalized prostate epithelial cultures expressing wild-type, inactivated p53 or mutated p53. Analysis of these gene expression profiles showed that both inactivated p53 and p53^R175H mutant expression resulted in the upregulation of cell cycle progression genes. A second group, which was upregulated exclusively by mutant p53^R175H, was predominantly enriched in developmental genes. This group of genes included the Twist1, a regulator of metastasis and epithelial–mesenchymal transition (EMT). Twist1 levels were also elevated in metastatic prostate cancer-derived cell line DU145, in immortalized lung fibroblasts and in a subset of lung cancer samples, all in a mutant p53-dependent manner. p53^R175H mutant bearing immortalized epithelial cells showed typical features of EMT, such as higher expression of mesenchymal markers, lower expression of epithelial markers and enhanced invasive properties in vitro. The mechanism by which p53^R175H mutant induces Twist1 expression involves alleviation of the epigenetic repression. Our data suggest that Twist1 expression might be upregulated following p53 mutation in cancer cells.     

A new technique for staining mast cells using ferroin

We describe here a new method for specific staining of mast cells using ferroin. Different hamster tissues were fixed in 4% formalin and processed for paraffin embedding. Sections were stained with hematoxylin followed by ferroin acidified with 2.5 N sulfuric acid to pH 4.0. Mast cells stained an intense orange color that contrasted markedly with bluish violet nuclei. High contrast was also observed when ferroin colored sections were counterstained with light green instead of hematoxylin. To evaluate the specificity of the stain, hamster cheek pouch sections were stained with toluidine blue, alcian blue-safranin O, and ferroin. Quantitative evaluation of mast cells stained with the three techniques showed no statistical difference. The simplicity and selectivity of this method is sufficient for image analysis of mast cells.