Expression profiling of drug response-from genes to pathways

Understanding individual response to a drug—what determines its efficacy and tolerability—is the major bottleneck in current drug development and clinical trials. Intracellular response and metabolism, for example through cytochrome P- 450 enzymes, may either enhance or decrease the effect of different drugs, dependent on the genetic variant. Microarrays offer the potential to screen the genetic composition of the individual patient. However, experiments are “noisy” and must be accompanied by solid and robust data analysis. Furthermore, recent research aims at the combination of highthroughput data with methods of mathematical modeling, enabling problem-oriented assistance in the drug discovery process. This article will discuss state-of-the-art DNA array technology platforms and the basic elements of data analysis and bioinformatics research in drug discovery. Enhancing single-gene analysis, we will present a new method for interpreting gene expression changes in the context of entire pathways. Furthermore, we will introduce the concept of systems biology as a new paradigm for drug development and highlight our recent research—the development of a modeling and simulation platform for biomedical applications. We discuss the potentials of systems biology for modeling the drug response of the individual patient

Hum Mutat

Reverse genetic approaches to generate mutants of model species are useful tools to assess functions of unknown genes. Recent work has demonstrated the feasibility of such strategies in several organisms, exploiting the power of chemical mutagenesis to disrupt genes randomly throughout the genome. To increase the throughput of gene-driven mutant identification, efficient mutation screening protocols are needed. Given the availability of sequence information for large numbers of unknown genes in many species, mutation detection protocols are preferably based on PCR. Using a set of defined mutations in the Hprt1 gene of mouse embryonic stem (ES) cells, we have systematically compared several PCR-based point mutation and deletion detection methods available for their ability to identify lesions in pooled samples, which is a major criterion for an efficient large-scale mutation screening assay. Results indicate that point mutations are most effectively identified by heteroduplex cleavage using CEL I endonuclease. Small deletions can most effectively be detected employing the recently described poison primer PCR technique. Further, we employed the CEL I assay followed by conventional agarose gel electrophoresis analysis for screening a library of chemically mutagenized ES cell clones. This resulted in the isolation of several clones harboring mutations in the mouse Sult1a1 locus, demonstrating the high-throughput compatibility of this approach using simple and inexpensive laboratory equipment. Hum Mutat 25:483-490, 2005. © 2005 Wiley-Liss, Inc

Bioinformatics

The analysis of gene regulatory networks (GRNs) is a central goal of bioinformatics highly accelerated by the advent of new experimental techniques, such as RNA interference. A battery of reverse engineering methods has been developed in recent years to reconstruct the underlying GRNs from these and other experimental data. However, the performance of the individual methods is poorly understood and validation of algorithmic performances is still missing to a large extent. To enable such systematic validation, we have developed the web application GeNGe (GEne Network GEnerator), a controlled framework for the automatic generation of GRNs. The theoretical model for a GRN is a non-linear differential equation system. Networks can be user-defined or constructed in a modular way with the option to introduce global and local network perturbations. Resulting data can be used, e.g. as benchmark data for evaluating GRN reconstruction methods or for predicting effects of perturbations as theoretical counterparts of biological experiment

Fibroblast growth factor 2 modulates transforming growth factor ß signaling in mouse embryonic fibroblasts and human ESCs (hESCs) to support hESC self-renewal

Fibroblast growth factor 2 (FGF2) is known to promote self-renewal of human embryonic stem cells (hESCs). In addition, it has been shown that transforming growth factor ß (TGFß) signaling is crucial in that the TGFß/Activin/Nodal branch of the pathway needs to be activated and the bone morphogenic protein (BMP)/GDF branch repressed to prevent differentiation. This holds particularly true for Serum Replacement-based medium containing BMP-like activity. We have reinvestigated a widely used protocol for conditioning hESC medium with mouse embryonic fibroblasts (MEFs). We show that FGF2 acts on MEFs to release supportive factors and reduce differentiation-inducing activity. FGF2 stimulation experiments with supportive and nonsupportive MEFs followed by genome-wide expression profiling revealed that FGF2 regulates the expression of key members of the TGFß pathway, with Inhba, Tgfb1, Grem1, and Bmp4 being the most likely candidates orchestrating the above activities. In addition, restimulation experiments in hESCs combined with global expression analysis revealed downstream targets of FGF2 signaling in these cells. Among these were the same factors previously identified in MEFs, thus suggesting that FGF2, at least in part, promotes self-renewal of hESCs by modulating the expression of TGFß ligands, which, in turn, act on hESCs in a concerted and autocrine manner

A Method to Polarize Stored Antiprotons to a High Degree

Polarized antiprotons can be produced in a storage ring by spin--dependent interaction in a purely electron--polarized hydrogen gas target. The polarizing process is based on spin transfer from the polarized electrons of the target atoms to the orbiting antiprotons. After spin filtering for about two beam lifetimes at energies $T\approx 40-170$ MeV using a dedicated large acceptance ring, the antiproton beam polarization would reach $P=0.2-0.4$. Polarized antiprotons would open new and unique research opportunities for spin--physics experiments in $\bar{p}p$ interactions

Trends Biochem Sci

The metabolic network has a modular architecture, is robust to perturbations, and responds to biological stimuli and environmental conditions. Through monitoring by metabolite responsive macromolecules, metabolic pathways interact with the transcriptome and proteome. Whereas pathway interconnecting cofactors and substrates report on the overall state of the network, specialised intermediates measure the activity of individual functional units. Transitions in the network affect many of these regulatory metabolites, facilitating the parallel regulation of the timing and control of diverse biological processes. The metabolic network controls its own balance, chromatin structure and the biosynthesis of molecular cofactors; moreover, metabolic shifts are crucial in the response to oxidative stress and play a regulatory role in cancer

J Theor Biol

One of the most important antioxidant enzymes is superoxide dismutase (SOD), which catalyses the dismutation of superoxide radicals to hydrogen peroxide. The enzyme plays an important role in diseases like trisomy 21 and also in theories of the mechanisms of aging. But instead of being beneficial, intensified oxidative stress is associated with the increased expression of SOD and also studies on bacteria and transgenic animals show that high levels of SOD actually lead to increased lipid peroxidation and hypersensitivity to oxidative stress. Using mathematical models we investigate the question how overexpression of SOD can lead to increased oxidative stress, although it is an antioxidant enzyme. We consider the following possibilities that have been proposed in the literature: (i) Reaction of H2O2 with CuZnSOD leading to hydroxyl radical formation. (ii) Superoxide radicals might reduce membrane damage by acting as radical chain breaker. (iii) While detoxifying superoxide radicals SOD cycles between a reduced and oxidized state. At low superoxide levels the intermediates might interact with other redox partners and increase the superoxide reductase (SOR) activity of SOD. This short-circuiting of the SOD cycle could lead to an increased hydrogen peroxide production. We find that only one of the proposed mechanisms is under certain circumstances able to explain the increased oxidative stress caused by SOD. But furthermore we identified an additional mechanism that is of more general nature and might be a common basis for the experimental findings. We call it the alternative pathway mechanism

In vitro recombination cloning of entire cDNA libraries in Arabidopsis thaliana and its application to the yeast two-hybrid system

In the postgenomic era many experiments rely on the availability of transcript sequence for cloning. As these clones usually originate from cDNA libraries, the quality of these libraries is crucial. If a good library is generated it is desirable to use a versatile cloning system suitable for many different kinds of applications. The cloning systems based on in vitro recombination proves fitting for this task. However, the use of this method for shuttling entire cDNA libraries between different vectors has not yet been studied in great detail. Here we describe the construction of four cDNA libraries from different tissues of Arabidopsis thaliana, the shuttling of the libraries into expression vectors, and evaluation of this method as well as its suitability for downstream applications. Libraries were constructed from seedlings, hormone treated seedlings, flowers, developing seeds and primary leaves in the ldquoentry vectorrdquo of the Gatewaytrade cloning system. After initial characterization of the libraries, they were shuttled into an expression vector (a yeast two-hybrid prey vector). To monitor for a size bias generally assumed to be inherent to in vitro recombination methods, the libraries were characterized before and after the transfer into the expression vector. However no significant difference could be detected. The functionality of the in vitro recombination system for the shuttling of entire libraries was then further tested by protein-protein interaction screens. The results of the library characterization and of the yeast two-hybrid screens and their implications for large-scale proteomic approaches are discussed

A centrosome-independent role for {gamma}-TuRC proteins in the spindle assembly checkpoint

The spindle assembly checkpoint guards the fidelity of chromosome segregation. It requires the close cooperation of cell cycle regulatory proteins and cytoskeletal elements to sense spindle integrity. The role of the centrosome, the organizing center of the microtubule cytoskeleton, in the spindle checkpoint is unclear. We found that the molecular requirements for a functional spindle checkpoint included components of the large {gamma}-tubulin ring complex ({gamma}-TuRC). However, their localization at the centrosome and centrosome integrity were not essential for this function. Thus, the spindle checkpoint can be activated at the level of microtubule nucleation