Salvaging Affymetrix probes after probe-level re-annotation

Background: Affymetrix GeneChips can be re-annotated at the probe-level by breaking up the original probe-sets and recomposing new probe-sets based on up-to-date genomic knowledge, such as available in Entrez Gene. This results in custom Chip Description Files (CDF). Using these custom CDFs improves the quality of the data and thus the results of related gene expression studies. However, 44-71% of the probes on a GeneChip are lost in this re-annotation process. Although generally aimed at less known genes, losing these probes obviously means a substantial loss of expensive experiment data. Biologists are therefore very reluctant to adopt this approach. Findings: We aimed to re-introduce the non-affected Affymetrix probe-sets after these re-annotation procedures. For this, we developed an algorithm (CDF-Merger) and applied it to standard Affymetrix CDFs and custom Brainarray CDFs to obtain Hybrid CDFs. Thus, salvaging lost Affymetrix probes with our CDF-Merger restored probe content up to 94%. Because the salvaged probes (up to 54% of the probe content on the arrays) represent less-reliable probe-sets, we made the origin of all probe-set definitions traceable, so biologists can choose at any time in their analyses, which subset of probe-sets they want to use. Conclusion: The availability of up-to-date Hybrid CDFs plus R environment allows for easy implementation of our approach

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

The core genome of the anaerobic oral pathogenic bacterium Porphyromonas gingivalis

<p>Abstract</p> <p>Background</p> <p>The Gram negative anaerobic bacterium <it>Porphyromonas gingivalis </it>has long been recognized as a causative agent of periodontitis. Periodontitis is a chronic infectious disease of the tooth supporting tissues eventually leading to tooth-loss. Capsular polysaccharide (CPS) of <it>P. gingivalis </it>has been shown to be an important virulence determinant. Seven capsular serotypes have been described. Here, we used micro-array based comparative genomic hybridization analysis (CGH) to analyze a representative of each of the capsular serotypes and a non-encapsulated strain against the highly virulent and sequenced W83 strain. We defined absent calls using <it>Arabidopsis thaliana </it>negative control probes, with the aim to distinguish between aberrations due to mutations and gene gain/loss.</p> <p>Results</p> <p>Our analyses allowed us to call aberrant genes, absent genes and divergent regions in each of the test strains. A conserved core <it>P. gingivalis </it>genome was described, which consists of 80% of the analyzed genes from the sequenced W83 strain. The percentage of aberrant genes between the test strains and control strain W83 was 8.2% to 13.7%. Among the aberrant genes many CPS biosynthesis genes were found. Most other virulence related genes could be found in the conserved core genome. Comparing highly virulent strains with less virulent strains indicates that <it>hmuS, </it>a putative CobN/Mg chelatase involved in heme uptake, may be a more relevant virulence determinant than previously expected. Furthermore, the description of the 39 W83-specific genes could give more insight in why this strain is more virulent than others.</p> <p>Conclusion</p> <p>Analyses of the genetic content of the <it>P. gingivalis </it>capsular serotypes allowed the description of a <it>P. gingivalis </it>core genome. The high resolution data from three types of analysis of triplicate hybridization experiments may explain the higher divergence between <it>P. gingivalis </it>strains than previously recognized.</p

Linking toxicant physiological mode of action with induced gene expression changes in Caenorhabditis elegans

Background Physiologically based modelling using DEBtox (dynamic energy budget in toxicology) and transcriptional profiling were used in Caenorhabditis elegans to identify how physiological modes of action, as indicated by effects on system level resource allocation were associated with changes in gene expression following exposure to three toxic chemicals: cadmium, fluoranthene (FA) and atrazine (AZ). Results For Cd, the physiological mode of action as indicated by DEBtox model fitting was an effect on energy assimilation from food, suggesting that the transcriptional response to exposure should be dominated by changes in the expression of transcripts associated with energy metabolism and the mitochondria. While evidence for effect on genes associated with energy production were seen, an ontological analysis also indicated an effect of Cd exposure on DNA integrity and transcriptional activity. DEBtox modelling showed an effect of FA on costs for growth and reproduction (i.e. for production of new and differentiated biomass). The microarray analysis supported this effect, showing an effect of FA on protein integrity and turnover that would be expected to have consequences for rates of somatic growth. For AZ, the physiological mode of action predicted by DEBtox was increased cost for maintenance. The transcriptional analysis demonstrated that this increase resulted from effects on DNA integrity as indicated by changes in the expression of genes chromosomal repair. Conclusions Our results have established that outputs from process based models and transcriptomics analyses can help to link mechanisms of action of toxic chemicals with resulting demographic effects. Such complimentary analyses can assist in the categorisation of chemicals for risk assessment purposes

Relationships between renal cytoplasmic and nuclear aldosterone-receptors

Relationships between renal cytoplasmic and nuclear aldosteronereceptors.Three 3H-aldosterone receptor complexes have been recovered from rat kidneys: 1) cytosol (high speed supernatants), 2) Tris-soluble nuclear (obtained by an osmotic shock procedure), and 3) chromatin-bound (prepared by extracting post-shock nuclei with 0.4 M KCl).Glycerol density gradient analyses of cytosol labelled in vivo or in vitro with 3H-aldosterone yielded two specific peaks -4.5S and 8.5S.These peaks were sensitive to salt concentration; 0.4 M KCl shifted the 8.5S to 4.5S and the addition of Ca++ (6 mM) resulted in a further shift to 3.5S.The Tris-soluble nuclear species sedimented at 3S and the chromatin-bound species at 4S.The time-course of generation of the 3H-aldosterone-labelled cytosol and nuclear receptor species was studied in vivo and in vitro by tissue slice and reconstitution methods.The results obtained are consistent with a three-step mechanism: cytosol (8.5S or 4.5S)→ Tris-soluble nuclear (3S)→ chromatin-bound (4S).Alternatively, the 3S and 4S complexes may be attached to independent nuclear sites.The formation of the chromatin-bound species was temperature sensitive and failed to form at 0°C.Pre-treatment with DNase but not RNase impaired the generation of both the Tris-soluble nuclear and chromatin-bound species.These results imply a close association between nuclear aldosterone-receptor complexes and intact DNA

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

Free SepF interferes with recruitment of late cell division proteins

Abstract The conserved cell division protein SepF aligns polymers of FtsZ, the key cell division protein in bacteria, during synthesis of the (Fts)Z-ring at midcell, the first stage in cytokinesis. In addition, SepF acts as a membrane anchor for the Z-ring. Recently, it was shown that SepF overexpression in Mycobacterium smegmatis blocks cell division. Why this is the case is not known. Surprisingly, we found in Bacillus subtilis that SepF overproduction does not interfere with Z-ring assembly, but instead blocks assembly of late division proteins responsible for septum synthesis. Transposon mutagenesis suggested that SepF overproduction suppresses the essential WalRK two-component system, which stimulates expression of ftsZ. Indeed, it emerged that SepF overproduction impairs normal WalK localization. However, transcriptome analysis showed that the WalRK activity was in fact not reduced in SepF overexpressing cells. Further experiments indicated that SepF competes with EzrA and FtsA for binding to FtsZ, and that binding of extra SepF by FtsZ alleviates the cell division defect. This may explain why activation of WalRK in the transposon mutant, which increases ftsZ expression, counteracts the division defect. In conclusion, our data shows that an imbalance in early cell division proteins can interfere with recruitment of late cell division proteins

Absence of Kre increases xylose induced GFP-ComK expression.

<p>(A) GFP and phase contrast images of strains PG508 (<i>amyE</i>::<i>Pxyl-gfp-comK</i>, Δ<i>comK</i>, Δ<i>mecA</i>) and PG505 (<i>amyE</i>::<i>Pxyl-gfp-comK</i>, Δ<i>comK</i>, Δ<i>mecA</i>, <i>kre</i>:<i>Tn</i>) 60, 120 and 180 min after induction of GFP-ComK with 0.05% xylose. Fluorescence levels are indicated by a colour intensity scale using the same contrast settings. (B) Quantification of GFP-ComK levels after 60 min of xylose induction. (C) Induction of GFP-ComK causes a stronger reduction in cell growth when Kre is inactivated.</p