As most biologists are probably aware, technological advances in molecular biology during the last few years have opened up possibilities to rapidly generate large-scale sequencing data from non-model organisms at a reasonable cost. In an era when virtually any study organism can 'go genomic', it is worthwhile to review how this may impact molecular ecology. The first studies to put the next generation sequencing (NGS) to the test in ecologically well-characterized species without previous genome information were published in 2007 and the beginning of 2008. Since then several studies have followed in their footsteps, and a large number are undoubtedly under way. This review focuses on how NGS has been, and can be, applied to ecological, population genetic and conservation genetic studies of non-model species, in which there is no (or very limited) genomic resources. Our aim is to draw attention to the various possibilities that are opening up using the new technologies, but we also highlight some of the pitfalls and drawbacks with these methods. We will try to provide a snapshot of the current state of the art for this rapidly advancing and expanding field of research and give some likely directions for future developments.

/pdf/applications-of-next-generation-sequencing-in-molecular-3x2xhuh15b.pdf

Applications of next generation sequencing in molecular ecology of non-model organisms

The present invention provides compounds, pharmaceutically acceptable compositions thereof, and methods of using the same.

Heteroaryl compounds and uses thereof

We have generated a database of expression profiles carrying the transcriptional responses of the model organism Bacillus subtilis following treatment with 37 well-characterized antibacterial compounds of different classes. The database was used to build a predictor for the assignment of the mechanisms of action (MoAs) of antibacterial compounds by the use of support vector machines. This predictor was able to correctly classify the MoA class for most compounds tested. Furthermore, we provide evidence that the in vivo MoA of hexachlorophene does not match the MoA predicted from in vitro data, a situation frequently faced in drug discovery. A database of this kind may facilitate the prioritization of novel antibacterial entities in drug discovery programs. Potential applications and limitations are discussed.

Prediction of Mechanisms of Action of Antibacterial Compounds by Gene Expression Profiling

Actinobacteria within the acI lineage are often numerically dominating in freshwater ecosystems, where they can account for >50% of total bacteria in the surface water. However, they remain uncultured to date. We thus set out to use single-cell genomics to gain insights into their genetic make-up, with the aim of learning about their physiology and ecological niche. A representative from the highly abundant acI-B1 group was selected for shotgun genomic sequencing. We obtained a draft genomic sequence in 75 larger contigs (sum=1.16 Mb), with an unusually low genomic G+C mol% (∼42%). Actinobacteria core gene analysis suggests an almost complete genome recovery. We found that the acI-B1 cell had a small genome, with a rather low percentage of genes having no predicted functions (∼15%) as compared with other cultured and genome-sequenced microbial species. Our metabolic reconstruction hints at a facultative aerobe microorganism with many transporters and enzymes for pentoses utilization (for example, xylose). We also found an actinorhodopsin gene that may contribute to energy conservation under unfavorable conditions. This project reveals the metabolic potential of a member of the global abundant freshwater Actinobacteria.

/pdf/metabolic-potential-of-a-single-cell-belonging-to-one-of-the-2blf41nklt.pdf

Metabolic potential of a single cell belonging to one of the most abundant lineages in freshwater bacterioplankton

In this study, curcumin, a potential anticancer agent, was found to dampen the dynamic instability of individual microtubules in living MCF-7 cells. It strongly reduced the rate and extent of shortening states, and modestly reduced the rate and extent of growing states. In addition, curcumin decreased the fraction of time microtubules spent in the growing state and strongly increased the time microtubules spent in the pause state. Brief treatment with curcumin depolymerized mitotic microtubules, perturbed microtubule-kinetochore attachment and disturbed the mitotic spindle structure. Curcumin also perturbed the localization of the kinesin protein Eg5 and induced monopolar spindle formation. Further, curcumin increased the accumulation of Mad2 and BubR1 at the kinetochores, indicating that it activated the mitotic checkpoint. In addition, curcumin treatment increased the metaphase/anaphase ratio, indicating that it can delay mitotic progression from the metaphase to anaphase. We provide evidence suggesting that the affected cells underwent apoptosis via the p53-dependent apoptotic pathway. The results support the idea that kinetic stabilization of microtubule dynamics assists in the nuclear translocation of p53. Curcumin exerted additive effects when combined with vinblastine, a microtubule depolymerizing drug, whereas the combination of curcumin with paclitaxel, a microtubule-stabilizing drug, produced an antagonistic effect on the inhibition of MCF-7 cell proliferation. The results together suggested that curcumin inhibited MCF-7 cell proliferation by inhibiting the assembly dynamics of microtubules.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1742-4658.2010.07750.x

Curcumin suppresses the dynamic instability of microtubules, activates the mitotic checkpoint and induces apoptosis in MCF-7 cells

A novel DSN-depletion method allows elimination of selected sequences from full-length-enriched cDNA libraries. Depleted cDNA can be applied for subsequent EST sequencing, expression cloning, and functional screening approaches. The method employs specific features of the kamchatka crab duplex-specific nuclease (DSN). This thermostable enzyme is specific for double-stranded (ds) DNA, and is thus used for selective degradation of ds DNA in complex nucleic acids. DSN depletion is performed prior to library cloning, and includes the following steps: target cDNA is mixed with excess driver DNA (representing fragments of the genes to be eliminated), denatured, and allowed to hybridize. During hybridization, driver molecules form hybrids with the target sequences, leading to their removal from the ss DNA fraction. Next, the ds DNA fraction is hydrolyzed by DSN, and the ss fraction is amplified using long-distance PCR. DSN depletion has been tested in model experiments.

DSN depletion is a simple method to remove selected transcripts from cDNA populations.

Genomic DNA labeling for hybridization with DNA arrays.

The present invention provides derivatives of pyrido[2,3-d]pyrimidin-7-one. These compounds are inhibitors or kinases such as Raf, including compounds that show anti-proliferative activity, including against tumor cells, and are useful in the treatment of diseases including cancer.

Improved raf inhibitors

4458 The identification of cellular targets of small molecules is of fundamental importance in elucidating the molecular basis of drug action We have recently described the development and application of a yeast three-hybrid (Y3H) method for large-scale identification of small molecule-protein interactions (Becker et al (2004) ChemBiol , 11, pp211–223) In this system, a small molecule-protein interaction promotes the clonal growth of yeast cells that express a cDNA encoding the interacting target protein The identity of the protein is then determined by individual amplification of the respective yeast cells, plasmid rescue and cDNA sequencing Here we describe an alternative, faster, higher throughput and larger-scale approach to the analysis of candidate interactors in Y3H cDNA library screening This approach leverages whole genome microarray analysis to characterize cDNAs that are significantly enriched in Y3H cDNA library screens with a test compound as compared to a control compound We used the Whole Human Genome Oligo Microarray (Agilent, Palo Alto, USA) containing 41,000 human genes and transripts We applied this technique to screen cDNA libraries for targets of a number of small molecule kinase inhibitors, including various CDK inhibitors (eg purvalanol B, roscovitine and pyrazolopyrimidones (PPs) - for reference, see M Caligiuri et al (2005) ChemBiol , 12, pp1–13), the Raf inhibitor BAY43-9006 and tyrosine kinase inhibitors (eg, IRESSA and PD-173955) Known targets for all these compounds could be identified The whole genome microarray analysis also indicates that roscovitine and pyrazolopyrimidones have a very limited target spectrum, whereas some of the other kinase inhibitors appeared very promiscuous Our results suggest that this new approach to Y3H interaction analysis enables a rapid and in-depth proteome scan of candidate targets of organic small molecules

A whole genome approach to identifying small molecule - protein interactions

Understanding of molecular markers responsible for activity/resistance profiles of anti‐cancer agents is essential for successful drug development Here we demonstrate an approach to identify genes selectively regulated in either sensitive or resistant cell lines following treatment with RGB‐286638, a multi‐targeted protein kinase inhibitor currently in Phase I development In order to assess whether the experimental conditions for in‐vitro treatment of cancer cell lines with RGB‐286638 used in this study resembles the mouse in‐vivo situation, we adopted an in vitro assay based on the time growth curve rather than dose response curve We found that the time growth curves were very similar to “standard” tumor growth curves observed in the mouse xenograft models Next, a range of concentrations (25–100 nM of RGB‐286638) was selected in which the shape of the in vitro growth curves closely resembled the shape of the in vivo growth curves In addition to the similarity of growth curves, the pharmacodynamic markers PARP cleavage and RB‐phosphorylation were used to confirm similarity of responses observed in xenograft models and in vitro cell culture Using eight concentrations within the selected range and 6 cell lines with different responsiveness to RGB‐286638, we analyzed gene expression profiles following RGB‐286638 treatment for 24 hours We could identify genes that were specifically deregulated in sensitive or resistant cells In addition, some genes were down‐regulated upon treatment independent of responsiveness of the cell lines to RGB‐286638 treatment Surprisingly, some of these genes are involved in cell cycle, eg regulation of cyclin B1, and were previously considered as pharmacodynamic markers but now appear to be nonspecific for the action of RGB‐286638 These results facilitate the selection of relevant biomarkers and can provide a rational for future combination studies Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B43

Abstract B43: Determination of biomarkers responsible for sensitivity and resistance of RGB‐286638 action in vitro

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