Darwin's finches: a model of landscape effects on metacommunity dynamics in the Galápagos Archipelago

Darwin's finches represent a dynamic radiation of birds within the Galápagos Archipelago. Unlike classic island radiations dominated by island endemics and intuitive ‘conveyer belt’ colonization with little subsequent dispersal, species of Darwin's finches have populations distributed across many islands and each island contains complex metacommunities of closely related birds. Understanding the role of metacommunity and structured population dynamics in speciation within this heterogeneous island system would provide insights into the roles of fragmentation and dispersal in evolution. In this study, a large multi‐species dataset and a comparative ground finch dataset (two co‐distributed lineages) were used to show how landscape features influence patterns of gene flow across the archipelago. Factors expected to regulate migration including distance and movement from large, central islands to small, peripheral islands were rejected in the multi‐species dataset. Instead, the harsh northeast islands contributed individuals to the larger central islands. Successful immigration relies on three factors: arriving, surviving and reproducing, thus the dispersal towards the central islands may be either be due to more migrants orienting towards these land masses due to their large size and high elevation, or may reflect a higher likelihood of survival and successful reproduction due to the larger diversity of habitats and more environmentally stable ecosystems that these islands possess. Further, the overall directionality of migration was south‐southwest against the dominant winds and currents. In comparing dispersal between the common cactus finch and medium ground finch, both species had similar migration rates but the cactus finch had approximately half the numbers of migrants due to lower effective populations sizes. Significant population structure in the cactus finch indicates potential for further speciation, while the medium ground finch maintains cohesive gene flow across islands. These patterns shed light on the macroevolutionary patterns that drive diversification and speciation within a radiation of highly‐volant taxa.     

Analytical approaches to determine cytochrome P450 inhibitory potential of new chemical entities in drug discovery

The use of a cassette incubation of probe substrates with human liver microsomes (HLM) - also known as the 'cocktail' approach - is becoming a widely accepted approach to determine the interaction of new chemical entities (NCEs) with cytochrome P450 enzymes (CYP450) in early drug discovery. This article describes two LC-MS/MS-based analytical methods used at the high-throughput (HT) stage and late discovery (LD) stage for analysis of 'cocktail' incubates to analyze the probe metabolites 1'-hydroxymidazolam (CYP3A4), 4'-hydroxydiclofenac (CYP2C9), dextrorphan (CYP2D6), 1'-hydroxytacrine (CYP1A2) and 4'-hydroxymephenytoin (CYP2C19). The analytical methods are advantageous over currently reported methods due to their sensitivity, shorter analyses times (<2 min/sample for the HT method and 4 min/sample for the LD method) and their ability to monitor a unique set of clinically relevant probe metabolites from a biological incubate containing low microsomal protein (0.1mg/mL). The analytical methods employ the same mobile phase, acetonitrile and 0.1% formic acid, under similar LC-MS/MS conditions. In the HT method, the chromatographic method consists of a short robust step-gradient where the probe metabolites are simultaneously and quickly eluted to enhance throughput. The probe metabolites are chromatographically resolved in the LD stage by utilizing a true linear gradient to obtain optimal peak separation. The IC50 data generated by both analytical methods using single incubations versus cocktail incubations for various test compounds are in good agreement (correlation coefficient (r2)>or=0.98). The scientist conducting the analysis is provided with a choice of method selection depending on the stage of the test compound and on whether throughput or minimizing interference from other co-eluting metabolites is the most important criterion.     

A simple DNA stretching method for fluorescence imaging of single DNA molecules

Stretching or aligning DNA molecules onto a surface by means of molecular combing techniques is one of the critical steps in single DNA molecule analysis. However, many of the current studies have focused on λ-DNA, or other large DNA molecules. There are very few studies on stretching methodologies for DNA molecules generated via PCR (typically smaller than 20 kb). Here we describe a simple method of stretching DNA molecules up to 18 kb in size on a modified glass surface. The very low background fluorescence allows efficient detection of single fluorescent dye labels incorporated into the stretched DNA molecules.     

Histocytological changes and reserve accumulation during somatic embryogenesis in Eucalyptus globulus

We recently described a protocol for Eucalyptus globulus somatic embryogenesis (SE). For its immediate use at industrial levels, some stages of the process require better control. In particular, SE germination rates are variable, decreasing SE efficacy. As reserves may play a central role in embryogenic processes, we followed histocytological changes and reserve fluctuations, during SE. For SE induction, explants of mature zygotic embryos were grown on Murashige and Skoog (MS) medium with 3 mg l⁻¹ α-naphthalene acetic acid and later transferred to MS without growth regulators (MS_WH). Samples of zygotic embryo cotyledons (explants), of globular and dicotyledonar somatic embryos, and of embling leaves were analysed for reserve accumulation and histocytological profiles. Cotyledon cells of zygotic embryos were rich in lipid and protein bodies, having almost no starch. After 3 weeks of induction, starch grain density increased in differentiated mesophyll regions, while in meristematic regions their occurrence was diffuse. In globular somatic embryos, starch accumulation increased with time (in amyloplasts), but protein bodies were absent. Cotyledonary somatic embryos had lower density of starch grains and absence of lipid and protein bodies. Embling leaves showed typical histological organisation. This is the first comprehensive study on histological and cytological changes during Eucalyptus SE with emphasis in reserve accumulation. With this work we demonstrate that the presently available SE protocol for E. globulus leads to reserve fluctuations during the process. Moreover, the reserves of somatic embryo cotyledons differ from those of their zygotic embryo counterparts, which reinforce the importance of reserves in the embryogenic process and suggests that manipulating external conditions, SE may be optimised giving suitable emblings production for industrial purposes.     

Rigid linkers for bioactive peptides

Rigid linkers of variable length were used to connect two high-affinity Nle4-D-Phe7-alpha-melanocyte stimulating hormone (NDP-alpha-MSH) or two low-affinity MSH(4) ligands. The linked peptides were synthesized by solid-phase methods. Control experiments indicate there is little or no effect of these linkers on NDP-alpha-MSH or MSH(4) binding to the human melanocortin 4 receptor (hMC4R). Tethering two high-affinity ligands gave no binding enhancement, while tethering two low-affinity ligands resulted in binding enhancement that decreased with increased linker length. Furthermore, for the low-affinity ligands, the enhancement of affinity is inversely proportional to the estimated molecular moments of inertia. These results are consistent with a model wherein binding is enhanced when the rate of ligand reattachment to the receptor is fast relative to the rate of ligand diffusion.     

Genomics of Helicobacter pylori

During this review period, we have definitely entered into the genomic era. The Helicobacter pylori studies reported here illustrate the use of most of the technologies currently available to globally interrogate the genome of a pathogen. Global analysis of the gene content of H. pylori strains gives insight into the extent of its genetic diversity and its in vivo evolution. Our understanding of the particularities of H. pylori as a gastric pathogen colonizing a unique niche has been improved by studies aimed at: (i) the identification of H. pylori-specific genes; (ii) the establishment of correlations between the presence of one or a group of genes (or proteins) with clinical outcome; and (iii) the analysis of global regulatory circuits or responses to the extracellular signals. The response of host cells to H. pylori infection will be developed in the chapter 'H. pylori and gastric malignancies' by Sepulveda and Coehlo. Despite our knowledge of the H. pylori genome, the function of about one third of its total proteins is still unknown. Functional genomics are straightforward approaches for the identification of new gene functions or metabolic pathways as well as for the understanding of cellular processes and the detection of new virulence factors. In silico studies combined with experimental work will undoubtedly continue to develop. To date, the expansion of proteomics with refinements in mass spectrometry technology has illustrated that through immunoproteomics and comparative studies, relevant novel antigens can be identified. Genomics not only provides invaluable information on H. pylori but also opens new perspectives for diagnostic or therapeutic applications.     

HYBRIDS AND PHYLOGENETIC SYSTEMATICS I. PATTERNS OF CHARACTER EXPRESSION IN HYBRIDS AND THEIR IMPLICATIONS FOR CLADISTIC ANALYSIS

Interspecific hybridization is considered common among plants, but the methods of cladistic systematics produce only divergently branching phylogenetic hypotheses and thus cannot give the correct phylogeny if an analysis includes hybrids. Empirical studies of the impact of known hybrids on phylogenetic analysis are lacking, and are necessary to begin to understand the problems that we face if hybrids are often included in cladistic analysis. Examination of the implications of hybrids for cladistics must begin with patterns of character expression in hybrids. This study includes 17 hybrids and their nine parental taxa that are Central American species of Aphelandra (Acanthaceae), analyzed using a set of 50 morphological characters. The hybrids are overwhelmingly intermediate as quantitatively scored for phylogenetic analysis. They express maternal and paternal, and primitive and derived characters in equal frequencies, showing no evidence of predominant inheritance of derived character states as has been assumed by most cladists who have considered hybrids theoretically. Because of their known genetic constitution, hybrids were useful in homology assessment and ordering character states. The parental character set was generally robust, but some changes were made to reflect the special evidence offered by the hybrids. These hybrids suggest that the inclusion of hybrids in phylogenetic analysis will not lead to unresolved cladograms with rampant homoplasy, as has been predicted by other authors. Instead, the patterns of character inheritance in these hybrids lead to the prediction that a hybrid will be placed by phylogenetic analysis as a basal lineage to the clade that includes its most derived parent, with relatively little effect on homoplasy. These predictions will be evaluated by incorporation of the hybrids in phylogenetic analyses, to be reported in a subsequent paper.