Identification of cancer genes using a statistical framework for multiexperiment analysis of nondiscretized array CGH data

Tumor formation is in part driven by DNA copy number alterations (CNAs), which can be measured using microarray-based Comparative Genomic Hybridization (aCGH). Multiexperiment analysis of aCGH data from tumors allows discovery of recurrent CNAs that are potentially causal to cancer development. Until now, multiexperiment aCGH data analysis has been dependent on discretization of measurement data to a gain, loss or no-change state. Valuable biological information is lost when a heterogeneous system such as a solid tumor is reduced to these states. We have developed a new approach which inputs nondiscretized aCGH data to identify regions that are significantly aberrant across an entire tumor set. Our method is based on kernel regression and accounts for the strength of a probe's signal, its local genomic environment and the signal distribution across multiple tumors. In an analysis of 89 human breast tumors, our method showed enrichment for known cancer genes in the detected regions and identified aberrations that are strongly associated with breast cancer subtypes and clinical parameters. Furthermore, we identified 18 recurrent aberrant regions in a new dataset of 19 p53-deficient mouse mammary tumors. These regions, combined with gene expression microarray data, point to known cancer genes and novel candidate cancer genes.     

The effect of process conditions on the alpha-amylolytic hydrolysis of amylopectin potato starch: An experimental design approach

The hydrolysis of amylopectin potato starch with Bacillus licheniformis alpha-amylase (Maxamyl) was studied under industrially relevant conditions (i.e. high dry-weight concentrations). The following ranges of process conditions were chosen and investigated by means of an experimental design: pH [5.6-7.6]; calcium addition [0-120 microg/g]; temperature [63-97 degrees C]; dry-weight concentration [3-37% [w/w]]; enzyme dosage [27.6-372.4 microL/kg] and stirring [0-200 rpm]. The rate of hydrolysis was followed as a function of the theoretical dextrose equivalent. The highest rate (at a dextrose equivalent of 10) was observed at high temperature (90 degrees C) and low pH (6). At a higher pH (7.2), the maximum temperature of hydrolysis shifted to a lower value. Also, high levels of calcium resulted in a decrease of the maximum temperature of hydrolysis. The pH, temperature, and the amount of enzyme added showed interactive effects on the observed rate of hydrolysis. No product or substrate inhibition was observed. Stirring did not effect the rate of hydrolysis. The oligosaccharide composition after hydrolysis (at a certain dextrose equivalent) did depend on the reaction temperature. The level of maltopentaose [15-24% [w/w]], a major product of starch hydrolysis by B. licheniformis alpha-amylase, was influenced mostly by temperature.     

Differentiation and anaerobiosis in standing liquid cultures of Streptomyces coelicolor

Streptomyces coelicolor differentiates on solid agar media by forming aerial hyphae that septate into spores. We here show that differentiation also occurs in standing liquid minimal media. After a period of submerged growth, hyphae migrate to the air interface, where they become fixed by a rigid reflecting film. Colonies that result from these hyphae form sporulating aerial hyphae. In addition, submerged hyphae in the liquid minimal medium may attach to the surface. Liquid standing cultures easily become anoxic only 1 to 2 mm below the surface. Yet, biomass increases, implying the existence of metabolic pathways supporting anaerobic growth.     

The Effect of Sampling and Storage on the Fecal Microbiota Composition in Healthy and Diseased Subjects

Large-scale cohort studies are currently being designed to investigate the human microbiome in health and disease. Adequate sampling strategies are required to limit bias due to shifts in microbial communities during sampling and storage. Therefore, we examined the impact of different sampling and storage conditions on the stability of fecal microbial communities in healthy and diseased subjects. Fecal samples from 10 healthy controls, 10 irritable bowel syndrome and 8 inflammatory bowel disease patients were collected on site, aliquoted immediately after defecation and stored at -80°C, -20°C for 1 week, at +4°C or room temperature for 24 hours. Fecal transport swabs (FecalSwab, Copan) were collected and stored for 48-72 hours at room temperature. We used pyrosequencing of the 16S gene to investigate the stability of microbial communities. Alpha diversity did not differ between all storage methods and -80°C, except for the fecal swabs. UPGMA clustering and principal coordinate analysis showed significant clustering by test subject (p<0.001) but not by storage method. Bray-Curtis dissimilarity and (un)weighted UniFrac showed a significant higher distance between fecal swabs and -80°C versus the other methods and -80°C samples (p<0.009). The relative abundance of Ruminococcus and Enterobacteriaceae did not differ between the storage methods versus -80°C, but was higher in fecal swabs (p<0.05). Storage up to 24 hours (at +4°C or room temperature) or freezing at -20°C did not significantly alter the fecal microbial community structure compared to direct freezing of samples from healthy subjects and patients with gastrointestinal disorders.     

The Intestinal Barrier in Irritable Bowel Syndrome: Subtype-Specific Effects of the Systemic Compartment in an In Vitro Model

BackgroundIrritable bowel syndrome (IBS) is a disorder with multifactorial pathophysiology. Intestinal barrier may be altered, especially in diarrhea-predominant IBS (IBS-D). Several mediators may contribute to increased intestinal permeability in IBS.AimWe aimed to assess effects of tryptase and LPS on in vitro permeability using a 3-dimensional cell model after basolateral cell exposure. Furthermore, we assessed the extent to which these mediators in IBS plasma play a role in intestinal barrier function.ResultsTryptase (20 and 50 mU) and LPS (6.25 – 50 ng/mL) significantly increased Caco-2 permeability versus control (all P< 0.05). Plasma of IBS-D only showed significantly elevated median tryptase concentrations (7.1 [3.9 – 11.0] vs. 4.2 [2.2 – 7.0] vs. 4.2 [2.5 – 5.9] μg/mL; P<0.05) and LPS concentrations (3.65 [3.00 – 6.10] vs. 3.10 [2.60-3.80] vs. 2.65 [2.40 – 3.40] EU/ml; P< 0.05) vs. IBS-C and HC. Also, plasma of IBS-D increased Caco-2 permeability versus HC (0.14450 ± 0.00472 vs. 0.00021 ± 0.00003; P < 0.001), which was attenuated by selective inhibition of tryptase and LPS (P< 0.05).ConclusionBasolateral exposure of spheroids to plasma of IBS-D patients resulted in a significantly increased FD4 permeation, which was partially abolished by selective inhibition of tryptase and LPS. These findings point to a role of systemic tryptase and LPS in the epithelial barrier alterations observed in patients with IBS-D.     

Contribution of Chloroflexus respiration to oxygen cycling in a hypersaline microbial mat from Lake Chiprana, Spain

In dense stratified systems such as microbial mats, photosynthesis and respiration are coupled due to a tight spatial overlap between oxygen-producing and -consuming microorganisms. We combined microsensors and a membrane inlet mass spectrometer with two independent light sources emitting in the visible (VIS) and near infrared (NIR) regions to study this coupling in more detail. Using this novel approach, we separately quantified the activity of the major players in the oxygen cycle in a hypersaline microbial mat: gross photosynthesis of cyanobacteria, NIR light-dependent respiration of Chloroflexus-like bacteria (CLB) and respiration of aerobic heterotrophs. Illumination by VIS light induced oxygen production in the top approximately 1 mm of the mat. In this zone CLB were found responsible for all respiration, while the contribution of the aerobic heterotrophs was negligible. Additional illumination of the mat with saturating NIR light completely switched off CLB respiration, resulting in zero respiration in the photosynthetically active zone. We demonstrate that microsensor-based quantification of gross and net photosyntheses in dense stratified systems should carefully consider the NIR light-dependent behaviour of CLB and other anoxygenic phototrophic groups.     

Tumor-initiating cells are not enriched in cisplatin-surviving BRCA1;p53-deficient mammary tumor cells in vivo

Although many breast cancers respond to chemotherapy or hormonal therapy, lack of tumor eradication is a central clinical problem preceding the development of drug-resistant tumors. Using the K14cre;Brca1^{F5–13/F5–13};p53^{F2–10/F2–10} mouse model for hereditary breast cancer, we have previously studied responses of mammary tumors to clinically relevant anti-cancer drugs, including cisplatin. The BRCA1- and p53-deficient tumors generated in this model are hypersensitive to cisplatin and never become resistant to this agent due to the large, irreversible deletion in Brca1. We show here that even dose-dense treatment with a maximum tolerated dose of cisplatin does not result in complete tumor eradication. To explain this result we have addressed the hypothesis that the lack of eradication of drug-sensitive tumors is due to increased in vivo chemotherapy resistance of tumor-initiating cells (TICs). Using the CD24 and CD49f cell surface markers that detect normal mouse mammary stem cells, we have identified tumor-initiating cells in BRCA1- and p53-deficient tumors. In addition to the Lin⁻/CD24⁺/CD49f⁺ subpopulation, we show that a larger population of Lin⁻/CD24⁺/CD49f cells also has tumor-initiating capability in at least two serial orthotopic transplantations, suggesting that these are not more differentiated transit-amplifying cells. However, we did not find an enrichment of TICs in cisplatin-treated tumor remnants. We conclude that in this model the tolerance of the cisplatin-surviving cells cannot be attributed to special biochemical defense mechanisms of TICs.Key words: tumor-initiating cells, cisplatin, genetically-engineered mouse model, BRCA1, breast cancer     

Bmi1 regulates stem cells and proliferation and differentiation of committed cells in mammary epithelium

PolycombGroup (PcG) proteins are epigenetic silencers involved in maintaining cellular identity, and their deregulation can result in cancer [1]. Mice without the PcG gene Bmi1 are runted and suffer from progressive loss of hematopoietic and neural stem cells [2-4]. Here, we assess the effects of Bmi1 on stem cells and differentiation of an epithelial tissue in vivo. We chose the mammary gland because it allows limiting dilution transplantations [5, 6] and because Bmi1 is overexpressed in breast cancer [7, 8]. Our analyses show that Bmi1 is expressed in all cells of the mouse mammary gland and is especially high in luminal cells. Loss of Bmi1 results in a severe mammary-epithelium growth defect, which can be rescued by codeletion of the Ink4a/Arf locus or pregnancy. Even though mammary stem cells are present in the absence of Bmi1, their activity is reduced, and this is only partially due to Ink4a/Arf expression. Interestingly, loss of Bmi1 causes premature lobuloalveolar differentiation, whereas overexpression of Bmi1 inhibits lobuloalveolar differentiation induced by pregnancy hormones. Because Bmi1 affects not only mammary stem cells but also more committed cells, our data warrant a more detailed analysis of the different roles of Bmi1 in breast-cancer etiology.