Novel primate-specific genes, RMEL 1, 2 and 3, with highly restricted expression in melanoma, assessed by new data mining tool

Melanoma is a highly aggressive and therapy resistant tumor for which the identification of specific markers and therapeutic targets is highly desirable. We describe here the development and use of a bioinformatic pipeline tool, made publicly available under the name of EST2TSE, for the in silico detection of candidate genes with tissue-specific expression. Using this tool we mined the human EST (Expressed Sequence Tag) database for sequences derived exclusively from melanoma. We found 29 UniGene clusters of multiple ESTs with the potential to predict novel genes with melanoma-specific expression. Using a diverse panel of human tissues and cell lines, we validated the expression of a subset of three previously uncharacterized genes (clusters Hs.295012, Hs.518391, and Hs.559350) to be highly restricted to melanoma/melanocytes and named them RMEL1, 2 and 3, respectively. Expression analysis in nevi, primary melanomas, and metastatic melanomas revealed RMEL1 as a novel melanocytic lineage-specific gene up-regulated during melanoma development. RMEL2 expression was restricted to melanoma tissues and glioblastoma. RMEL3 showed strong up-regulation in nevi and was lost in metastatic tumors. Interestingly, we found correlations of RMEL2 and RMEL3 expression with improved patient outcome, suggesting tumor and/or metastasis suppressor functions for these genes. The three genes are composed of multiple exons and map to 2q12.2, 1q25.3, and 5q11.2, respectively. They are well conserved throughout primates, but not other genomes, and were predicted as having no coding potential, although primate-conserved and human-specific short ORFs could be found. Hairpin RNA secondary structures were also predicted. Concluding, this work offers new melanoma-specific genes for future validation as prognostic markers or as targets for the development of therapeutic strategies to treat melanoma.     

Quantification of angiogenesis in estrogen receptor-positive and negative breast carcinoma

Background

The objective of this study was to evaluate angiogenesis according to CD34 antigen expression in estrogen receptor (ER)-positive and negative breast carcinomas.

Methods

This study comprised 64 cases of infiltrating ductal carcinoma in postmenopausal women divided into two groups: Group A: ER-positive, n = 35; and Group B: ER-negative, n = 29. The anti-CD34 monoclonal antibody was used as a marker for endothelial cells. Microvessel count was carried out in 10 fields per slide using a 40× objective lens (magnification 400×). Statistical analysis of the data was performed using Student's t-test (p < 0.05).

Results

The mean number of vessels stained with the anti-CD34 antibody in the estrogen receptor-positive and negative tumors was 23.51 ± 1.15 and 40.24 ± 0.42, respectively. The number of microvessels was significantly greater in the estrogen receptor-negative tumors (p < 0.001).

Conclusion

ER-negative tumors have significantly greater CD34 antigen expression compared to ER-positive tumors.

     

Cellular prion protein: implications in seizures and epilepsy

1. Cellular prion (PrP^c) is a plasma membrane protein involved with copper uptake, protection against oxidative stress, cell adhesion, differentiation, signaling, and survival in the central nervous system2. Deletion of PrP^c gene (Prnp) in mice enhances sensitivity to seizures in vivo and neuronal excitability in vitro which can be related to: (i) disrupted Ca⁺²-activated K⁺ currents, with loss of I _HAP conductance in hippocampus; (ii) abnormal GABA-A inhibition in the hippocampus; (iii) mossy fiber reorganization in the hippocampus; (iv) changes in ectonucleotidases in both hippocampus and neocortex; and (v) higher levels of neocortical and subcortical oxidative stress. Moreover, postnatal Prnp knockout mice showed a significant reduction of after hyperpolarization potentials in hippocampal CA1 cells.3. Taken together, these findings suggest that loss of PrP^c function contributes to the hyperexcitable and synchronized activities underlying epileptic seizures generated in neocortex and hippocampus. Hence, the role of PrP^c on human symptomatic, cryptogenic or idiopathic epileptic syndromes deserves further investigation.     

Nasu–Hakola Disease (Polycystic Lipomembranous Osteodysplasia with Sclerosing Leukoencephalopathy—PLOSL): A Dementia Associated with Bone Cystic Lesions. From Clinical to Genetic and Molecular Aspects

The authors review the clinical, radiological, electrophysiological, pathological, and molecular aspects of Nasu-Hakola disease (polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy or PLOSL). Nasu-Hakola disease is a unique disease characterized by multiple bone cysts associated with a peculiar form of neurodegeneration that leads to dementia and precocious death usually during the fifth decade of life. The diagnosis can be established on the basis of clinical and radiological findings. Recently, molecular analysis of affected families revealed mutations in the DAP12 (TYROBP) or TREM2 genes, providing an interesting example how mutations in two different subunits of a multi-subunit receptor complex result in an identical human disease phenotype. The association of PLOSL with mutations in the DAP12 or TREM2 genes has led to improved diagnosis of affected individuals. Also, the possible roles of the DAP12/TREM2 signaling pathway in microglia and osteoclasts in humans are just beginning to be elucidated. Some aspects of this peculiar signaling pathway are discussed here.     

Phosphoproteomic analysis of synaptosomes from human cerebral cortex

Protein phosphorylation is a crucial post-translational modification mechanism in the regulation of synaptic organization and function. Here, we analyzed synaptosome fractions from human cerebral cortex obtained during therapeutic surgery. To minimize changes in the phosphorylation state of proteins, the tissue was homogenized within two minutes of excision. Synaptosomal proteins were digested with trypsin and phosphopeptides were isolated by immobilized metal affinity chromatography and analyzed by liquid chromatography and tandem mass spectrometry. The method allowed the detection of residues on synaptic proteins that were presumably phosphorylated in the intact cell, including synapsin 1, syntaxin 1, and SNIP, PSD-93, NCAM, GABA-B receptor, chaperone molecules, and protein kinases. Some of the residues identified are the same or homologous to sites that had been previously described to be phosphorylated in mammals whereas others appear to be novel sites which, to our knowledge, have not been reported previously. The study shows that new phosphoproteomic strategies can be used to analyze subcellular fractions from small amounts of tissue for the identification of phosphorylated residues for research and potentially for diagnostic purposes.     

A simple method to separate base population and segregation effects in genomic relationship matrices

Background

Genomic selection and estimation of genomic breeding values (GBV) are widely used in cattle and plant breeding. Several studies have attempted to detect population subdivision by investigating the structure of the genomic relationship matrix G. However, the question of how these effects influence GBV estimation using genomic best linear unbiased prediction (GBLUP) has received little attention.

Methods

We propose a simple method to decompose G into two independent covariance matrices, one describing the covariance that results from systematic differences in allele frequencies between groups at the pedigree base (G_A^*) and the other describing genomic relationships (G_S) corrected for these differences. Using this decomposition and F_st statistics, we examined whether observed genetic distances between genotyped subgroups within populations resulted from the heterogeneous genetic structure present at the base of the pedigree and/or from breed divergence. Using this decomposition, we tested three models in a forward prediction validation scenario on six traits using Brown Swiss and dual-purpose Fleckvieh cattle data. Model 0 (M0) used both components and is equivalent to the model using the standard G-matrix. Model 1 (M1) used G_S only and model 2 (M2), an extension of M1, included a fixed genetic group effect. Moreover, we analyzed the matrix of contributions of each base group (Q) and estimated the effects and prediction errors of each base group using M0 and M1.

Results

The proposed decomposition of G helped to examine the relative importance of the effects of base groups and segregation in a given population. We found significant differences between the effects of base groups for each breed. In forward prediction, differences between models in terms of validation reliability of estimated direct genomic values were small but predictive power was consistently lowest for M1. The relative advantage of M0 or M2 in prediction depended on breed, trait and genetic composition of the validation group. Our approach presents a general analogy with the use of genetic groups in conventional animal models and provides proof that standard GBLUP using G yields solutions equivalent to M0, where base groups are considered as correlated random effects within the additive genetic variance assigned to the genetic base.     

Reference genes for quantitative reverse transcription-polymerase chain reaction expression studies in wild and cultivated peanut

Background

Molecular genetic studies on rare tumour entities, such as bone tumours, often require the use of decalcified, formalin-fixed, paraffin-embedded tissue (dFFPE) samples. Regardless of which decalcification procedure is used, this introduces a vast breakdown of DNA that precludes the possibility of further molecular genetic testing. We set out to establish a robust protocol that would overcome these intrinsic hurdles for bone tumour research.

Findings

The goal of our study was to establish a protocol, using a modified DNA isolation procedure and quality controls, to select decalcified samples suitable for array-CGH testing. Archival paraffin blocks were obtained from 9 different pathology departments throughout Europe, using different fixation, embedding and decalcification procedures, in order to preclude a bias for certain lab protocols. Isolated DNA samples were subjected to direct chemical labelling and enzymatic labelling systems and were hybridised on a high resolution oligonucleotide chip containing 44,000 reporter elements. Genomic alterations (gains and losses) were readily detected in most of the samples analysed. For example, both homozygous deletions of 0.6 Mb and high level of amplifications of 0.7 Mb were identified.

Conclusions

We established a robust protocol for molecular genetic testing of dFFPE derived DNA, irrespective of fixation, decalcification or sample type used. This approach may greatly facilitate further genetic testing on rare tumour entities where archival decalcified, formalin fixed samples are the only source.     

Expression of IL-27 by Tumor Cells in InvasCutaneous and Metastatic Melanomas

Interleukin (IL)-27 is a cytokine of the IL-12 family that displays either immunostimulatory or immunosuppressive functions depending on the context. In various murine tumor models including melanoma models, ectopic expression of IL-27 has been shown to play an anti-tumoral role and to favor tumor regression. In this study, we investigated whether IL-27 might play a role in the development of melanoma in humans. We analyzed the in situ expression of IL-27 in melanocytic lesions (n = 82) representative of different stages of tumor progression. IL-27 expression was not observed in nevus (n = 8) nor in in situ melanoma (n = 9), but was detected in 28/46 (61%) cases of invasive cutaneous melanoma, notably in advanced stages (19/23 cases of stages 3 and 4). In most cases, the main source of IL-27 was tumor cells. Of note, when IL-27 was detected in primary cutaneous melanomas, its expression was maintained in metastatic lesions. These in situ data suggested that the immunosuppressive functions of IL-27 may dominate in human melanoma. Consistent with this hypothesis, we found that IL-27 could induce suppressive molecules such as PD-L1, and to a lesser extent IL-10, in melanoma cells, and that the in situ expression of IL-27 in melanoma correlated with those of PD-L1 and IL-10.