Paul August

Cloning the Soil Metagenome: a Strategy for Accessing the Genetic and Functional Diversity of Uncultured Microorganisms

ABSTRACT Recent progress in molecular microbial ecology has revealed that traditional culturing methods fail to represent the scope of microbial diversity in nature, since only a small proportion of viable microorganisms in a sample are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromosome (BAC) vector to construct libraries of genomic DNA isolated directly from soil (termed metagenomic libraries). To date, we have constructed two such libraries, which contain more than 1 Gbp of DNA. Phylogenetic analysis of 16S rRNA gene sequences recovered from one of the libraries indicates that the BAC libraries contain DNA from a wide diversity of microbial phyla, including sequences from diverse taxa such as the low-G+C, gram-positive Acidobacterium,Cytophagales, and Proteobacteria. Initial screening of the libraries in Escherichia coli identified several clones that express heterologous genes from the inserts, confirming that the BAC vector can be used to maintain, express, and analyze environmental DNA. The phenotypes expressed by these clones include antibacterial, lipase, amylase, nuclease, and hemolytic activities. Metagenomic libraries are a powerful tool for exploring soil microbial diversity, providing access to the genetic information of uncultured soil microorganisms. Such libraries will be the basis of new initiatives to conduct genomic studies that link phylogenetic and functional information about the microbiota of environments dominated by microorganisms that are refractory to cultivation.

Recombinant environmental libraries provide access to microbial diversity for drug discovery from natural products

ABSTRACT To further explore possible avenues for accessing microbial biodiversity for drug discovery from natural products, we constructed and screened a 5,000-clone “shotgun” environmental DNA library by using an Escherichia coli-Streptomyces lividans shuttle cosmid vector and DNA inserts from microbes derived directly (without cultivation) from soil. The library was analyzed by several means to assess diversity, genetic content, and expression of heterologous genes in both expression hosts. We found that the phylogenetic content of the DNA library was extremely diverse, representing mostly microorganisms that have not been described previously. The library was screened by PCR for sequences similar to parts of type I polyketide synthase genes and tested for the expression of new molecules by screening of live colonies and cell extracts. The results revealed new polyketide synthase genes in at least eight clones. In addition, at least five additional clones were confirmed by high-pressure liquid chromatography analysis and/or biological activity to produce heterologous molecules. These data reinforce the idea that exploiting previously unknown or uncultivated microorganisms for the discovery of novel natural products has potential value and, most importantly, suggest a strategy for developing this technology into a realistic and effective drug discovery tool.

A Functional Screen in Human Cells Identifies UBF2 as an RNA Polymerase II Transcription Factor That Enhances the β-Catenin Signaling Pathway

ABSTRACT β-Catenin signaling plays an important role in the development of many organisms and has a key part in driving the malignant transformation of epithelial cells comprising a variety of cancers. β-Catenin can activate gene expression through its association with transcription factors of the lymphoid enhancer factor 1 (LEF-1)/T-cell factor (TCF) family. We designed a screen in human cells to identify novel genes that activate a β-catenin-LEF/TCF-responsive promoter and isolated the high-mobility group box transcription factor, UBF2. UBF1 and UBF2 are splice variants of a common precursor RNA. Although UBF1 has been shown to activate RNA polymerase I-regulated genes, the function of UBF2 has remained obscure. Here, we show for the first time that both UBF1 and UBF2 activate RNA polymerase II-regulated promoters. UBF2 associates with LEF-1, as shown by coimmunoprecipitation experiments, and potentiates transcriptional activation stimulated by LEF-1/β-catenin from a synthetic promoter with multimerized LEF/TCF binding sites and a natural cyclin D1 promoter with consensus LEF/TCF binding sites. Downregulation of endogenous UBF expression using an RNA interference approach reduces transcriptional activation of a β-catenin-LEF/TCF-responsive promoter by means of overexpressed β-catenin, further implicating UBF as a transcriptional enhancer of the β-catenin pathway.

Identification of Novel Tumor-Associated Cell Surface Sialoglycoproteins in Human Glioblastoma Tumors Using Quantitative Proteomics

Glioblastoma multiform (GBM) remains clinical indication with significant “unmet medical need”. Innovative new therapy to eliminate residual tumor cells and prevent tumor recurrences is critically needed for this deadly disease. A major challenge of GBM research has been the identification of novel molecular therapeutic targets and accurate diagnostic/prognostic biomarkers. Many of the current clinical therapeutic targets of immunotoxins and ligand-directed toxins for high-grade glioma (HGG) cells are surface sialylated glycoproteins. Therefore, methods that systematically and quantitatively analyze cell surface sialoglycoproteins in human clinical tumor samples would be useful for the identification of potential diagnostic markers and therapeutic targets for malignant gliomas. In this study, we used the bioorthogonal chemical reporter strategy (BOCR) in combination with label-free quantitative mass spectrometry (LFQ-MS) to characterize and accurately quantify the individual cell surface sialoproteome in human GBM tissues, in fetal, adult human astrocytes, and in human neural progenitor cells (NPCs). We identified and quantified a total of 843 proteins, including 801 glycoproteins. Among the 843 proteins, 606 (72%) are known cell surface or secreted glycoproteins, including 156 CD-antigens, all major classes of cell surface receptor proteins, transporters, and adhesion proteins. Our findings identified several known as well as new cell surface antigens whose expression is predominantly restricted to human GBM tumors as confirmed by microarray transcription profiling, quantitative RT-PCR and immunohistochemical staining. This report presents the comprehensive identification of new biomarkers and therapeutic targets for the treatment of malignant gliomas using quantitative sialoglycoproteomics with clinically relevant, patient derived primary glioma cells.

Laminin 521 maintains differentiation potential of mouse and human satellite cell-derived myoblasts during long-term culture expansion

BackgroundLarge-scale expansion of myogenic progenitors is necessary to support the development of high-throughput cellular assays in vitro and to advance genetic engineering approaches necessary to develop cellular therapies for rare muscle diseases. However, optimization has not been performed in order to maintain the differentiation capacity of myogenic cells undergoing long-term cell culture. Multiple extracellular matrices have been utilized for myogenic cell studies, but it remains unclear how different matrices influence long-term myogenic activity in culture. To address this challenge, we have evaluated multiple extracellular matrices in myogenic studies over long-term expansion.MethodsWe evaluated the consequence of propagating mouse and human myogenic stem cell progenitors on various extracellular matrices to determine if they could enhance long-term myogenic potential. For the first time reported, we comprehensively examine the effect of physiologically relevant laminins, laminin 211 and laminin 521, compared to traditionally utilized ECMs (e.g., laminin 111, gelatin, and Matrigel) to assess their capacity to preserve myogenic differentiation potential.ResultsLaminin 521 supported increased proliferation in early phases of expansion and was the only substrate facilitating high-level fusion following eight passages in mouse myoblast cell cultures. In human myoblast cell cultures, laminin 521 supported increased proliferation during expansion and superior differentiation with myotube hypertrophy. Counterintuitively however, laminin 211, the native laminin isoform in resting skeletal muscle, resulted in low proliferation and poor differentiation in mouse and human cultures. Matrigel performed excellent in short-term mouse studies but showed high amounts of variability following long-term expansion.ConclusionsThese results demonstrate laminin 521 is a superior substrate for both short-term and long-term myogenic cell culture applications compared to other commonly utilized substrates. Since Matrigel cannot be used for clinical applications, we propose that laminin 521 could possibly be employed in the future to provide myoblasts for cellular therapy directed clinical studies.

Abstract 2201: The molecular signature of tumor cells that are sensitives to a new tight binder NAMPT inhibitor.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC SAR154782 is a tight binder, low reversible NAMPT inhibitor that has been identified by a phenotypic screen and chemogenomic approaches. This class of NAMPT inhibitor induces a profound and time dependent modulation of tumor metabolism which in turn induces the reprogrammation of tumor cell transcriptome. The transcriptional profile revealed that different metabolic and oncogenic signaling pathways are modulated by SAR154782. In order to define the context of tumor cell sensitivity to NAMPT inhibition, the effects of this compound on tumor cell growth and viability have been assessed against a panel of about 240 cell lines from different tissue origin. Based on the difference of tumor cell lines sensitivity to NAMPT inhibitor, a panel of 60 genes that are differentially expressed in the sensitive versus non-sensitive cell lines has been identified. Interestingly, the low expression of NAMPT concomitant with the high expression of Sirtuin1 and PARP1 are the main outstanding features in the panel of genes identified for the most sensitive cell lines. This finding is in agreement with high NAD turnover. The relevance of the preceding biomarkers to determine the potential cancer target population that could benefit upon treatment with a NAMPT inhibitor has been investigated in in vivo settings by evaluating the antitumor activity of an advanced new class of NAMPT inhibitors in several mouse tumor xenografts and human primary tumor models. The results obtained from these studies highlighted the modulation upon NAMPT inhibition of important tumor growth pathways and revealed for the first time a potential molecular signature of tumor sensitivity and support further investigation into use of the signature for patient stratification in the clinical setting Citation Format: Sandrine Hilairet, Jerome Arigon, Frederico Nardi, Samir Jegham, Annick Peleraux, Sylvie Cosnier, Regine Floutard, Omar Jbilo, Paul August, Maysoun Shomali, Francois Autelitano, Rosalia Arrebola, Stephan Reiling, Jack Pollard, Francisco Adrian, Jason Sager, Christoph Lengauer, Marion Dorsh, Carlos Garcia-Echeverria, Monsif Bouaboula. The molecular signature of tumor cells that are sensitives to a new tight binder NAMPT inhibitor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2201. doi:10.1158/1538-7445.AM2013-2201