SigWinR; the SigWin-detector updated and ported to R

<p>Abstract</p> <p>Background</p> <p>Our SigWin-detector discovers significantly enriched windows of (genomic) elements in any sequence of values (genes or other genomic elements in a DNA sequence) in a fast and reproducible way. However, since it is grid based, only (life) scientists with access to the grid can use this tool. Therefore and on request, we have developed the SigWinR package which makes the SigWin-detector available to a much wider audience. At the same time, we have introduced several improvements to its algorithm as well as its functionality, based on the feedback of SigWin-detector end users.</p> <p>Findings</p> <p>To allow usage of the SigWin-detector on a desktop computer, we have rewritten it as a package for R: SigWinR. R is a free and widely used multi platform software environment for statistical computing and graphics. The package can be installed and used on all platforms for which R is available. The improvements involve: a visualization of the input-sequence values supporting the interpretation of Ridgeograms; a visualization allowing for an easy interpretation of enriched or depleted regions in the sequence using windows of pre-defined size; an option that allows the analysis of circular sequences, which results in rectangular Ridgeograms; an application to identify regions of co-altered gene expression (ROCAGEs) with a real-life biological use-case; adaptation of the algorithm to allow analysis of non-regularly sampled data using a constant window size in physical space without resampling the data. To achieve this, support for analysis of windows with an even number of elements was added.</p> <p>Conclusion</p> <p>By porting the SigWin-detector as an R package, SigWinR, improving its algorithm and functionality combined with adequate performance, we have made SigWin-detector more useful as well as more easily accessible to scientists without a grid infrastructure.</p

Single cell transcriptomics of neighboring hyphae of Aspergillus niger

Single cell profiling was performed to assess differences in RNA accumulation in neighboring hyphae of the fungus Aspergillus niger. A protocol was developed to isolate and amplify RNA from single hyphae or parts thereof. Microarray analysis resulted in a present call for 4 to 7% of the A. niger genes, of which 12% showed heterogeneous RNA levels. These genes belonged to a wide range of gene categories

RNA isolation method for single embryo transcriptome analysis in zebrafish

Background: Transcriptome analysis during embryogenesis usually requires pooling of embryos to obtain sufficient RNA. Hence, the measured levels of gene-expression represent the average mRNA levels of pooled samples and the biological variation among individuals is confounded. This can irreversibly reduce the robustness, resolution, or expressiveness of the experiment. Therefore, we developed a robust method to isolate abundant high-quality RNA from individual embryos to perform single embryo transcriptome analyses using zebrafish as a model organism. Available methods for embryonic zebrafish RNA isolation minimally utilize ten embryos. Further downscaling of these methods to one embryo is practically not feasible. Findings: We developed a single embryo RNA extraction method based on sample homogenization in liquid nitrogen, RNA extraction with phenol and column purification. Evaluation of this method showed that: the quality of the RNA was very good with an average RIN value of 8.3-8.9; the yield was always ≥ 200 ng RNA per embryo; the method was applicable to all stages of zebrafish embryogenesis; the success rate was almost 100%; and the extracted RNA performed excellent in microarray experiments in that the technical variation was much lower than the biological variation. Conclusions: Presented is a high-quality, robust RNA isolation method. Obtaining sufficient RNA from single embryos eliminates the necessity of sample pooling and its associated drawbacks. Although our RNA isolation method has been setup for transcriptome analysis in zebrafish, it can also be used for other model systems and other applications like (q)PCR and transcriptome sequencing

RNA isolation for transcriptomics of human and mouse small skin biopsies

<p>Abstract</p> <p>Background</p> <p>Isolation of RNA from skin biopsies presents a challenge, due to the tough nature of skin tissue and a high presence of RNases. As we lacked the dedicated equipment, i.e. homogenizer or bead-beater, needed for the available RNA from skin isolation methods, we adapted and tested our zebrafish single-embryo RNA-isolation protocol for RNA isolation from skin punch biopsies.</p> <p>Findings</p> <p>We tested our new RNA-isolation protocol in two experiments: a large-scale study with 97 human skin samples, and a small study with 16 mouse skin samples. Human skin was sampled with 4.0 mm biopsy punches and for the mouse skin different punch diameter sizes were tested; 1.0, 1.5, 2.0, and 2.5 mm. The average RNA yield in human samples was 1.5 μg with an average RNA quality RIN value of 8.1. For the mouse biopsies, the average RNA yield was 2.4 μg with an average RIN value of 7.5. For 96% of the human biopsies and 100% of the mouse biopsies we obtained enough high-quality RNA. The RNA samples were successfully tested in a transcriptomics analysis using the Affymetrix and Roche NimbleGen platforms.</p> <p>Conclusions</p> <p>Using our new RNA-isolation protocol, we were able to consistently isolate high-quality RNA, which is apt for further transcriptomics analysis. Furthermore, this method is already useable on biopsy material obtained with a punch diameter as small as 1.5 mm.</p

Concepción de un plan de negocios para la creación de una agencia de representación de nuevos músicos: modelo de aplicación basado en el género pop- rock

Al realizar un plan de negocio a partir de la idea de creación de una agencia de representación de artistas musicales, se analizaron los distintos factores que intervienen en el mismo, para de esta manera evaluar si la idea de negocio es factible o no y así mismo aplicar los conocimiento adquiridos en la maestría al emplearlos en un caso aplicativo que es el de IOSSA, un músico italiano con gran potencial. En el capítulo II, se realizó un análisis general del mercado en el cuál se determinó la oferta y la demanda de la música grabada y en streaming, para así poder determinar el tipo de estrategias a realizar. En el capítulo III, se realizó un estudio de la competencia y de acuerdo al análisis, realizó también un plan de marketing con las estrategias a realizar, seguido por una descripción de la estructura organizacional de la Agencia de Representación. Adicionalmente, se realizó un estudio de factibilidad económica y financiera para medir la factibilidad de la idea de negocio. Por último se realizó un caso aplicativo en el cuál se muestra cómo trabajará la Agencia de representación de artistas musicales. Líneas de investigación y desarrollo futuras: La información que se ha considerado necesaria presentar va desde la situación general del mercado, para luego realizar un estudio de la competencia y finalmente realizar un análisis de factibilidad del plan de negocios internacional. Así el proyecto pueda ser entendido, aceptado y también podría ser útil para solicitar créditos o buscar inversores o socios si el caso lo amerite.Facultad de Ciencias Económica

Operon structure of Staphylococcus aureus

In bacteria, gene regulation is one of the fundamental characteristics of survival, colonization and pathogenesis. Operons play a key role in regulating expression of diverse genes involved in metabolism and virulence. However, operon structures in pathogenic bacteria have been determined only by in silico approaches that are dependent on factors such as intergenic distances and terminator/promoter sequences. Knowledge of operon structures is crucial to fully understand the pathophysiology of infections. Presently, transcriptome data obtained from growth curves in a defined medium were used to predict operons in Staphylococcus aureus. This unbiased approach and the use of five highly reproducible biological replicates resulted in 93.5% significantly regulated genes. These data, combined with Pearson’s correlation coefficients of the transcriptional profiles, enabled us to accurately compile 93% of the genome in operon structures. A total of 1640 genes of different functional classes were identified in operons. Interestingly, we found several operons containing virulence genes and showed synergistic effects for two complement convertase inhibitors transcribed in one operon. This is the first experimental approach to fully identify operon structures in S. aureus. It forms the basis for further in vitro regulation studies that will profoundly advance the understanding of bacterial pathophysiology in vivo

Serious complications in gene-expression studies with stress perturbation: An example of UV-exposed p53-mutant mouse embryonic fibroblasts

Reanalysis of our UV study of p53-mutant mouse embryonic fibroblasts revealed an intriguing orchestration of massive transcriptome responses. However, close scrutiny of the data uncovered an affected mRNA/rRNA ratio, effectively inhibiting valid data analysis. UV-dose range-finding showed low-dose UV specific- and high-dose stress-related responses, which represent a plea for UV dose range-finding in experimental design