Phylogenetic comparative analysis of life-history variation among populations of the lizard Sceloporus undulatus: an example and prognosis

Over the past 15 years, phylogenetic comparative methods (PCMs) have become standard in the study of life-history evolution. To date, most studies have focused on variation among species or higher taxonomic levels, generally revealing the presence of significant phylogenetic effects as well as residual variation potentially attributable to adaptive evolution. Recently, population-level phylogenetic hypotheses have become available for many species, making it possible to apply PCMs directly to the level at which experiments are typically used to test adaptive hypotheses. In this study, we present the results of PCMs applied to life-history variation among populations of the widespread and well-studied lizard Sceloporus undulatus. Using S. undulatus (which may represent four closely related species) as an example, we explore the benefits of using PCMs at the population level, as well as consider the importance of several thorny methodological problems including but not limited to nonindependence of populations, lack of sufficient variation in traits, and the typically small sample sizes dictated by the difficulty of collecting detailed demographic data. We show that phylogenetic effects on life-history variation among populations of S. undulatus appear to be unimportant, and that several classic trade-offs expected by theory and revealed by many interspecific comparisons are absent. Our results suggest that PCMs applied to variation in life-history traits below the species level may be of limited value, but more studies like ours are needed to draw a general conclusion. Finally, we discuss several outstanding problems that face studies seeking to apply PCMs below the species level.     

Differential arrangements of conserved building blocks among homologs of the Rad50/Mre11 DNA repair protein complex

Structural maintenance of chromosomes (SMC) proteins have diverse cellular functions including chromosome segregation, condensation and DNA repair. They are grouped based on a conserved set of distinct structural motifs. All SMC proteins are predicted to have a bipartite ATPase domain that is separated by a long region predicted to form a coiled coil. Recent structural data on a variety of SMC proteins shows them to be arranged as long intramolecular coiled coils with a globular ATPase at one end. SMC proteins function in pairs as heterodimers or as homodimers often in complexes with other proteins. We expect the arrangement of the SMC protein domains in complex assemblies to have important implications for their diverse functions. We used scanning force microscopy imaging to determine the architecture of human, Saccharomyces cerevisiae, and Pyrococcus furiosus Rad50/Mre11, Escherichia coli SbcCD, and S.cerevisiae SMC1/SMC3 cohesin SMC complexes. Two distinct architectural arrangements are described, based on the way their components were connected. The eukaryotic complexes were similar to each other and differed from their prokaryotic and archaeal homologs. These similarities and differences are discussed with respect to their diverse mechanistic roles in chromosome metabolism.     

FGF ligand family mRNA expression profile for mouse preimplantation embryos, early gestation human placenta, and mouse trophoblast stem cells

Signaling by fibroblast growth factor (FGF) is essential is for trophoblast stem (TS) cells and preimplantation embryos. FGF4 provides essential signaling, but the expression of the complete set of 23 FGF family members has not been analyzed. Here, semi-quantitative RT-PCR and microarray analyses were used to define expression of all FGF ligand mRNA. RT-PCR was done for developmentally important FGF subfamilies, FGF10/FGF22 and FGF8/FGF17/FGF18 as well as FGF11. FGF4 and FGF18 are detected at highest levels by RT-PCR and microarrays. FGF10 was detected at low levels in both assays. FGF11 was detected at moderate levels by microarray, but not by RT-PCR. FGF17 was detected at low levels by array and moderate levels by RT-PCR. FGF8 and FGF22 were detected by RT-PCR, but not by microarrays during late cleavage divisions. FGF8, FGF5, and FGF9 were detected in the oocyte by microarray. FGF2, FGF3, and FGF7 were not detected by RT-PCR or microarrays and FGF13, FGF14, and FGF23 were not detected by microarray. Since a major role of FGF is to maintain TS cells, we tested human and mouse placental cell lines and early gestation human placenta for expression of FGF ligands. Expression in mouse TS cells was compared with preimplantation embryos, and human placental cell line expression was compared with human placenta, to infer which ligands are expressed in placental lineage vs. other cell lineages. The data suggest that human and mouse placenta share FGF18 and its high expression suggests preimplantation and early placental function.     

Fragment screening: an introduction

There are clearly many different philosophies associated with adapting fragment screening into mainstream Drug Discovery Lead Generation strategies. Scientists at Astex, for instance, focus entirely on strategies involving use of X-ray crystallography and NMR. However, AstraZeneca uses a number of different fragment screening strategies. One approach is to screen a 2000 compound fragment set (with close to "lead-like" complexity) at 100 microM in parallel with every HTS such that the data are obtained on the entire screening collection at 10 microM plus the extra samples at 100 microM; this provides valuable compound potency data in a concentration range that is usually unexplored. The fragments are then screen-specific "privileged structures" that can be searched for in the rest of the HTS output and other databases as well as having synthesis follow-up. A typical workflow for a fragment screen within AstraZeneca is shown below (Figure 24) and highlights the desirability (particularly when screening >100 microM) for NMR and X-ray information to validate weak hits and give information on how to optimise them. In this chapter, we have provided an introduction to the theoretical and practical issues associated with the use of fragment methods and lead-likeness. Fragment-based approaches are still in an early stage of development and are just one of many interrelated techniques that are now used to identify novel lead compounds for drug development. Fragment based screening has some advantages, but like every other drug hunting strategy will not be universally applicable. There are in particular some practical challenges associated with fragment screening that relate to the generally lower level of potency that such compounds initially possess. Considerable synthetic effort has to be applied for post-fragment screening to build the sort of potency that would be expected to be found from a traditional HTS. However, if there are no low-hanging fruit in a screening collection to be found by HTS then the use of fragment screening can help find novelty that may lead to a target not being discarded as intractable. As such, the approach offers some significant advantages by providing less complex molecules, which may have better potential for novel drug optimisation and by enabling new chemical space to be more effectively explored. Many literature examples that cover examples of fragment screening approaches are still at the "proof of concept" stage and although delivering inhibitors or ligands, may still prove to be unsuitable when further ADMET and toxicity profiling is done. The next few years should see a maturing of the area, and as our understanding of how the concepts can be best applied, there are likely to be many more examples of attractive, small molecule hits, leads and candidate drugs derived from the approaches described.     

Effects of methylmalonyl-CoA mutase gene knockouts on erythromycin production in carbohydrate-based and oil-based fermentations of Saccharopolyspora erythraea

In carbohydrate-based fermentations of Saccharopolyspora erythraea, a polar knockout of the methylmalonyl-CoA mutase (MCM) gene, mutB, improved erythromycin production an average of 126% (within the range of 102–153% for a 0.95 confidence interval). In oil-based fermentations, where erythromycin production by the wild-type strain averages 184% higher (141–236%, 0.95 CI) than in carbohydrate-based fermentations, the same polar knockout in mutB surprisingly reduced erythromycin production by 66% (53–76%, 0.95 CI). A metabolic model is proposed where in carbohydrate-based fermentations MCM acts as a drain on the methylmalonyl-CoA metabolite pool, and in oil-based fermentations, MCM acts in the reverse direction to fill the methylmalonyl-CoA pool. Therefore, the model explains, in part, how the well-known oil-based process improvement for erythromycin production operates at the biochemical level; furthermore, it illustrates how the mutB erythromycin strain improvement mutation operates at the genetic level in carbohydrate-based fermentations.     

Triumphalone, a diketone from the volatile oil of the leaves of Melaleuca triumphalis, and its spontaneous conversion into isotriumphalone

The major component (35-65%) of the volatile oil obtained by steam distillation of the leaves of Melaleuca triumphalis has been identified as (rel)-1beta-pentyl-1alpha,6alpha-dihydroxy-3,3,5,5-tetramethylcyclohexa-2,4-dione (trivial name triumphalone). Relative stereochemistry was established by nuclear Overhauser experiments and X-ray studies on the 2-(3,5-dinitrobenzoic acid) derivative. The remainder of the oil was composed of mono- and sesquiterpene hydrocarbons and alcohols. On prolonged standing the presence of a rearrangement product of triumphalone was observed which was characterized as (rel)-1beta-pentyl-1alpha,3alpha-dihydroxy-4,4,6,6-tetramethylcyclohexa-2,5-dione (trivial name isotriumphalone), presumably arising from an acid catalyzed shift of the pentyl group from C-1 to C-2.     

2-(Alkylthio)pyrimidin-4-ones as novel, reversible inhibitors of lipoprotein-associated phospholipase A2

Starting from two weakly active hits from high throughput screening, a novel series of 2-(alkylthio)-pyrimidin-4-ones with high potency and selectivity for lipoprotein-associated phospholipase A2 has been designed. In contrast to previously known inhibitors, these have been shown to act by a non-covalent and substrate competitive mechanism.     

Bioluminescence assay platform for selective and sensitive detection of Ub/Ubl proteases

As the importance of ubiquitylation in certain disease states becomes increasingly apparent, the enzymes responsible for removal of ubiquitin (Ub) from target proteins, deubiquitylases (DUBs), are becoming attractive targets for drug discovery. For rapid identification of compounds that alter DUB function, in vitro assays must be able to provide statistically robust data over a wide dynamic range of both substrate and enzyme concentrations during high throughput screening (HTS). The most established reagents for HTS are Ubs with a quenched fluorophore conjugated to the C-terminus; however, a luciferase-based strategy for detecting DUB activity (DUB-Glo?, Promega) provides a wider dynamic range than traditional fluorogenic reagents. Unfortunately, this assay requires high enzyme concentrations and lacks specificity for DUBs over other isopeptidases (e.g. desumoylases), as it is based on an aminoluciferin (AML) derivative of a peptide derived from the C-terminus of Ub (Z-RLRGG-). Conjugation of aminoluciferin to a full-length Ub (Ub-AML) yields a substrate that has a wide dynamic range, yet displays detection limits for DUBs 100- to 1000-fold lower than observed with DUB-Glo?. Ub-AML was even a sensitive substrate for DUBs (e.g. JosD1 and USP14) that do not show appreciable activity with DUB-Glo?. Aminoluciferin derivatives of hSUMO2 and NEDD8 were also shown to be sensitive substrates for desumoylases and deneddylases, respectively. Ub/Ubl-AML substrates are amenable to HTS (Z'=0.67) yielding robust signal, and providing an alternative drug discovery platform for Ub/Ubl isopeptidases. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

Analysis of ubiquitin E3 ligase activity using selective polyubiquitin binding proteins

The ubiquitin proteasome pathway controls the cellular degradation of ~80-90% of the proteome in a highly regulated manner. In this pathway, E3 ligases are responsible for the conjugation of ubiquitin to protein substrates which can lead to their destruction by the 26S proteasome. Aberrant E3 ligases have been implicated in several diseases and are widely recognized as attractive targets for drug discovery. As researchers continue to characterize E3 ligases, additional associations with various disease states are being exposed. The availability of assays that allow rapid analysis of E3 ligase activity is paramount to both biochemical studies and drug discovery efforts aimed at E3 ligases. To address this need, we have developed a homogenous assay for monitoring ubiquitin chain formation using Tandem Ubiquitin Binding Entities (TUBEs). TUBEs bind selectively to polyubiquitin chains versus mono-ubiquitin thus enabling the detection of polyubiquitin chains in the presence of mono-ubiquitin. This assay reports on the proximity between the protein substrate and TUBEs as a result of polyubiquitin chain formation by an E3 ligase. This homogenous assay is a step forward in streamlining an approach for characterizing and quantitating E3 ligase activity in a rapid and cost effective manner. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

Fluorescent silica particles for monitoring oxygen levels in three-dimensional heterogeneous cellular structures

Bacterial biofilms are a major obstacle challenging the development of more effective therapies to treat implant infections. Oxygen availability to bacterial cells has been implicated in biofilm formation and planktonic cell detachment; however, there are insufficient tools available to measure oxygen concentrations within complex three‐dimensional structures with ～1 µm resolution. Such measurements may complement measures of biofilm structure and cell activity to provide a more comprehensive understanding of biofilm biology. Thus, we developed oxygen‐sensing microparticles specifically designed to characterize oxygen transport through the volume of bacterial biofilms. The Stöber method was used to synthesize monodisperse silica microparticles of approximately the same size as a bacterium (～1 µm). Two fluorophores, oxygen‐sensitive Ru(Ph₂phen₃)Cl₂, and the reference fluorophore Nile blue chloride were immobilized on the surface of the particles. We demonstrate application of the microparticles toward measuring the oxygen concentration profiles within a live Staphylococcus aureus biofilm. Biotechnol. Bioeng. 2012; 109: 2663–2670. © 2012 Wiley Periodicals, Inc.