Mammalian Glutaminase Gls2 Gene Encodes Two Functional Alternative Transcripts by a Surrogate Promoter Usage Mechanism

Glutaminase is expressed in most mammalian tissues and cancer cells, but the regulation of its expression is poorly understood. An essential step to accomplish this goal is the characterization of its species- and cell-specific isoenzyme pattern of expression. Our aim was to identify and characterize transcript variants of the mammalian glutaminase Gls2 gene.We demonstrate for the first time simultaneous expression of two transcript variants from the Gls2 gene in human, rat and mouse. A combination of RT-PCR, primer-extension analysis, bioinformatics, real-time PCR, in vitro transcription and translation and immunoblot analysis was applied to investigate GLS2 transcripts in mammalian tissues. Short (LGA) and long (GAB) transcript forms were isolated in brain and liver tissue of human, rat and mouse. The short LGA transcript arises by a combination of two mechanisms of transcriptional modulation: alternative transcription initiation and alternative promoter. The LGA variant contains both the transcription start site (TSS) and the alternative promoter in the first intron of the Gls2 gene. The full human LGA transcript has two in-frame ATGs in the first exon, which are missing in orthologous rat and mouse transcripts. In vitro transcription and translation of human LGA yielded two polypeptides of the predicted size, but only the canonical full-length protein displayed catalytic activity. Relative abundance of GAB and LGA transcripts showed marked variations depending on species and tissues analyzed.This is the first report demonstrating expression of alternative transcripts of the mammalian Gls2 gene. Transcriptional mechanisms giving rise to GLS2 variants and isolation of novel GLS2 transcripts in human, rat and mouse are presented. Results were also confirmed at the protein level, where catalytic activity was demonstrated for the human LGA protein. Relative abundance of GAB and LGA transcripts was species- and tissue-specific providing evidence of a differential regulation of GLS2 transcripts in mammals

Candidate gene prioritization by network analysis of differential expression using machine learning approaches

<p>Abstract</p> <p>Background</p> <p>Discovering novel disease genes is still challenging for diseases for which no prior knowledge - such as known disease genes or disease-related pathways - is available. Performing genetic studies frequently results in large lists of candidate genes of which only few can be followed up for further investigation. We have recently developed a computational method for constitutional genetic disorders that identifies the most promising candidate genes by replacing prior knowledge by experimental data of differential gene expression between affected and healthy individuals.</p> <p>To improve the performance of our prioritization strategy, we have extended our previous work by applying different machine learning approaches that identify promising candidate genes by determining whether a gene is surrounded by highly differentially expressed genes in a functional association or protein-protein interaction network.</p> <p>Results</p> <p>We have proposed three strategies scoring disease candidate genes relying on network-based machine learning approaches, such as kernel ridge regression, heat kernel, and Arnoldi kernel approximation. For comparison purposes, a local measure based on the expression of the direct neighbors is also computed. We have benchmarked these strategies on 40 publicly available knockout experiments in mice, and performance was assessed against results obtained using a standard procedure in genetics that ranks candidate genes based solely on their differential expression levels (<it>Simple Expression Ranking</it>). Our results showed that our four strategies could outperform this standard procedure and that the best results were obtained using the <it>Heat Kernel Diffusion Ranking </it>leading to an average ranking position of 8 out of 100 genes, an AUC value of 92.3% and an error reduction of 52.8% relative to the standard procedure approach which ranked the knockout gene on average at position 17 with an AUC value of 83.7%.</p> <p>Conclusion</p> <p>In this study we could identify promising candidate genes using network based machine learning approaches even if no knowledge is available about the disease or phenotype.</p

The Heterotrimeric Laminin Coiled-Coil Domain Exerts Anti-Adhesive Effects and Induces a Pro-Invasive Phenotype

Laminins are large heterotrimeric cross-shaped extracellular matrix glycoproteins with terminal globular domains and a coiled-coil region through which the three chains are assembled and covalently linked. Laminins are key components of basement membranes, and they serve as attachment sites for cell adhesion, migration and proliferation. In this work, we produced a recombinant fragment comprising the entire laminin coiled-coil of the α1-, β1-, and γ1-chains that assemble into a stable heterotrimeric coiled-coil structure independently of the rest of the molecule. This domain was biologically active and not only failed to serve as a substrate for cell attachment, spreading and focal adhesion formation but also inhibited cell adhesion to laminin when added to cells in a soluble form at the time of seeding. Furthermore, gene array expression profiling in cells cultured in the presence of the laminin coiled-coil domain revealed up-regulation of genes involved in cell motility and invasion. These findings were confirmed by real-time quantitative PCR and zymography assays. In conclusion, this study shows for the first time that the laminin coiled-coil domain displays anti-adhesive functions and has potential implications for cell migration during matrix remodeling

Transcriptome Analysis during Human Trophectoderm Specification Suggests New Roles of Metabolic and Epigenetic Genes

In humans, successful pregnancy depends on a cascade of dynamic events during early embryonic development. Unfortunately, molecular data on these critical events is scarce. To improve our understanding of the molecular mechanisms that govern the specification/development of the trophoblast cell lineage, the transcriptome of human trophectoderm (TE) cells from day 5 blastocysts was compared to that of single day 3 embryos from our in vitro fertilization program by using Human Genome U133 Plus 2.0 microarrays. Some of the microarray data were validated by quantitative RT-PCR. The TE molecular signature included 2,196 transcripts, among which were genes already known to be TE-specific (GATA2, GATA3 and GCM1) but also genes involved in trophoblast invasion (MUC15), chromatin remodeling (specifically the DNA methyltransferase DNMT3L) and steroid metabolism (HSD3B1, HSD17B1 and FDX1). In day 3 human embryos 1,714 transcripts were specifically up-regulated. Besides stemness genes such as NANOG and DPPA2, this signature included genes belonging to the NLR family (NALP4, 5, 9, 11 and 13), Ret finger protein-like family (RFPL1, 2 and 3), Melanoma Antigen family (MAGEA1, 2, 3, 5, 6 and 12) and previously unreported transcripts, such as MBD3L2 and ZSCAN4. This study provides a comprehensive outlook of the genes that are expressed during the initial embryo-trophectoderm transition in humans. Further understanding of the biological functions of the key genes involved in steroidogenesis and epigenetic regulation of transcription that are up-regulated in TE cells may clarify their contribution to TE specification and might also provide new biomarkers for the selection of viable and competent blastocysts

The Sea Urchin Diadema antillarum (Echinodermata, Equinoidea), algal cover and juvenile coral densities in La Parguera, Puerto Rico

Grazing by the black sea urchin Diadema antillarum reduces algal cover and enhances coral recruitment. The overall goal of this project was to examine if there is a relationship between densities of D. antillarum with algal cover and abundance of juvenile corals. Population densities of the black sea urchin, juvenile coral abundances and algal cover were assessed along four 20m2 transects at two depth intervals (0-3 and 3-8m) within each of two inner shelf coral reefs off La Parguera Natural Reserve (San Cristóbal and Enrique) in southwest, Puerto Rico using a 1m2 quadrat (divided into 100 areas of 100cm2, each area encompassing 1% of the quadrat). Juvenile coral densities were counted and identified to genus or species. Algal cover and composition was measured using each 1% square of the 1m2 quadrat. Urchin population densities were significantly higher at 0-3m at the two sites studied. Population densities were higher at Enrique at 0-3m than at San Cristobal but at 3-8m Diadema was not seen at Enrique. A total of 30 juvenile corals belonging to eight different genera were found. Juvenile coral densities were higher at 3-8m at the two reef sites studied. Algal cover and composition was mainly composed by crustose coralline algae at 0-3m at both reef sites. Macroalgal cover was low at both reefs. The results of this study suggest that densities of coral recruits at inner shelf reefs in La Parguera, Puerto Rico are driven mainly by differences in habitats (depth).KEY WORDScoral recruitment, abundance, Diadema antillarum, densities, composition, correlationEl pastoreo del erizo negro, Diadema antillarum reduce la cobertura de algas y provee sustrato disponible para el reclutamiento coralino. La meta principal de nuestro trabajo era examinar si existe una relación entre las densidades de D. antillarum, la cobertura de algas y el reclutamiento de corales. La densidad poblacional del D. antillarum, la abundancia de corales juveniles y la cobertura de algas fueron evaluados a lo largo de cuatro transectos de 20m2, en dos intérvalos de profundidad (0-3m y 3-8m) en dos arrecifes de la zona interior de la Reserva Natural de La Parguera en el suroeste de Puerto Rico utilizando un cuadrante de 1m2 (dividida en 100 áreas de 100cm2, cada área abarcando 1% del cuadrante). La densidad de corales juveniles fue contada e identificada a género o especie. La cobertura y composición de algas fue medida utilizando cada cuadro pequeño representando 1% del cuadrante de 1m2. La densidad poblacional de D. antillarum fue significativamente diferente en 0-3m de profundidad en ambos lugares. Dichas densidades fueron más altas en Enrique en 0-3m de profundidad. Sin embargo en 3-8m no fueron encontrados los erizos en dicho arrecife. Un total de 30 corales juveniles pertenecientes a ocho géneros diferentes fueron encontrados. La densidad de los corales juveniles fue mayor en 3-8m en ambos lugares. La cobertura y composición de algas fue dominada por algas crustosas coralinas de 0-3m en ambos arrecifes. La cobertura de macroalgas fue baja en ambos arrecifes. Nuestros resultados sugieren que las densidades de corales juveniles en arrecifes de la zona interior de La Parguera son guiados mayormente por diferencias en hábitats (profundidad).PALABRAS CLAVEReclutamiento coralino, abundancia, Diadema antillarum, densidades, composición, correlació

Recovery of outer retinal laminations on optical coherence tomography after treatment of cancer associated retinopathy

Purpose: To report novel optical coherence tomography findings in a case of anti-α-enolase cancer associated retinopathy. Observations: An elderly female presented with bilateral decreased vision and a recent diagnosis of ovarian carcinoma. Optical coherence tomography demonstrated bilateral loss of outer retinal structures and macular edema. Serum testing found antibodies against α-enolase and 82–84 kDa proteins. Outer retinal structures showed recovery, macular edema resolved and repeat anti-retinal antibody testing became negative following cancer therapy and topical difluprednate treatment. Conclusions and importance: Cancer associated retinopathy is a paraneoplastic disease that results in damage to retinal structures through an autoimmune response. The damage is generally considered to be irreversible; however, in rare cases, such as observed here, retinal structures may demonstrate recovery after treatment