Validation of a modified fluorimetric assay for the screening of trichomonacidal drugs

A fluorimetric microassay that uses a redox dye to determine the viability of the flagellate Trichomonas vaginalis has been optimised to provide a more sensitive method to evaluate potential trichomonacidal compounds. Resazurin has been used in recent years to test drugs against different parasites, including trichomonadid protozoa; however, the reproducibility of these resazurin-based methods in our laboratory has been limited because the flagellate culture medium spontaneously reduces the resazurin. The objective of this work was to refine the fluorimetric microassay method previously developed by other research groups to reduce the fluorescence background generated by the media and increase the sensitivity of the screening assay. The experimental conditions, time of incubation, resazurin concentration and media used in the microtitre plates were adjusted. Different drug sensitivity studies against T. vaginalis were developed using the 5-nitroimidazole reference drugs, new 5-nitroindazolinones and 5-nitroindazole synthetic derivatives. Haemocytometer count results were compared with the resazurin assay using a 10% solution of 3 mM resazurin dissolved in phosphate buffered saline with glucose (1 mg/mL). The fluorimetric assay and the haemocytometer counts resulted in similar percentages of trichomonacidal activity in all the experiments, demonstrating that the fluorimetric microtitre assay has the necessary accuracy for high-throughput screening of new drugs against T. vaginalis.     

Forecasting phenology: from species variability to community patterns

Shifts in species' phenology in response to climate change have wide-ranging consequences for ecological systems. However, significant variability in species' responses, together with limited data, frustrates efforts to forecast the consequences of ongoing phenological changes. Herein, we use a case study of three North American plant communities to explore the implications of variability across levels of organisation (within and among species, and among communities) for forecasting responses to climate change. We show how despite significant variation among species in sensitivities to climate, comparable patterns emerge at the community level once regional climate drivers are accounted for. However, communities differ with respect to projected patterns of divergence and overlap among their species' phenological distributions in response to climate change. These analyses and a review of hypotheses suggest how explicit consideration of spatial scale and levels of biological organisation may help to understand and forecast phenological responses to climate change.     

Comparative study of the peptide composition of Complex III (quinol-cytochromec reductase)

A comparative study has been made on the subunits of Complex III from beef heart, rat liver, Neurospora, and baker's yeast mitochondria. All of the subunits of the beef heart enzyme were similar to the counterpart subunit in rat liver Complex III, both with respect to their apparent molecular weights on SDS-polyacrylamide gels and their proteolytic digestion maps obtained in the presence of S. subtilus V8 protease. In contrast, the subunits of Neurospora and yeast Complex III varied considerably from the mammalian enzyme, as well as between themselves, the only exception being cytochrome b (subunit III). Less variation was observed in the electron transport peptides (IV-V) of higher and lower eukaryotes than in those subunits (I, II, VI-VIII) for which no functions are known. However, the data imply that subunits I, II, and VI-VIII are bona fide members of the complex, and that their functions within the complex, although unknown, are also somewhat conserved. Finally, the low-molecular-weight subunits of rat liver cytochrome oxidase and Complex III were compared. They appear to contain no subunits in common, implying different roles for these peptides in the two complexes.     

Phylogeny and biogeography of Muusoctopus (Cephalopoda: Enteroctopodidae)

Deep‐sea octopuses of the genus Muusoctopus are thought to have originated in the Pacific Northern Hemisphere and then diversified throughout the Pacific and into the rest of the World Ocean. However, this hypothesis was inferred only from molecular divergence times. Here, the ancestral distribution and dispersal routes are estimated by Bayesian analysis based on a new phylogeny including 38 specimens from the south‐eastern Pacific Ocean. Morphological data and molecular sequences of three mitochondrial genes (16S rRNA, COI and COIII) are presented. The morphological data confirm that specimens newly acquired from off the coast of Chile comprise two species: Muusoctopus longibrachus and the poorly described species, Muusoctopus eicomar. The latter is here redescribed and is clearly distinguished from M. longibrachus and other closely related species in the region. A gene tree was built using Bayesian analysis to infer the phylogenetic position of these species within the species group, revealing that a large genetic distance separates the two sympatric Chilean species. M. longibrachus is confirmed as the sister species of Muusooctopus eureka from the Falkland Islands; while M. eicomar is a sister species of Muusoctopus yaquinae from the North Pacific, most closely related to the amphi‐Atlantic species Muusoctopus januarii. Molecular divergence times and ancestral distribution analyses suggest that genus Muusoctopus may have originated in the North Atlantic: one lineage dispersed directly southward to the Magellan region and another dispersed southward along the Eastern Pacific to the Southern Ocean and Antarctica. The Muusoctopus species in the Southern Hemisphere have different phylogenetic origins and represent independent invasions of this region.     

Synergistic interaction between Gdf1 and Nodal during anterior axis development

Growth and Differentiation Factor 1 (GDF-1) has been implicated in left-right patterning of the mouse embryo but has no other known function. Here, we demonstrate a genetic interaction between Gdf1 and Nodal during anterior axis development. Gdf1-/-;Nodal+/- mutants displayed several abnormalities that were not present in either Gdf1-/- or Nodal+/- single mutants, including absence of notochord and prechordal plate, and malformation of the foregut; organizing centers implicated in the development of the anterior head and branchial arches, respectively. Consistent with these deficits, Gdf1-/-;Nodal+/- mutant embryos displayed a number of axial midline abnormalities, including holoprosencephaly, anterior head truncation, cleft lip, fused nasal cavity, and lack of jaws and tongue. The absence of these defects in single mutants indicated a synergistic interaction between Nodal and GDF-1 in the node, from which the axial mesendoderm that gives rise to the notochord, prechordal plate, and foregut endoderm originates, and where the two factors are co-expressed. This notion was supported by a severe downregulation of FoxA2 and goosecoid in the anterior primitive streak of double mutant embryos. Unlike that in the lateral plate mesoderm, Nodal expression in the node was independent of GDF-1, indicating that both factors act in parallel to control the development of mesendodermal precursors. Receptor reconstitution experiments indicated that GDF-1, like Nodal, can signal through the type I receptors ALK4 and ALK7. However, analysis of compound mutants indicated that ALK4, but not ALK7, was responsible for the effects of GDF-1 and Nodal during anterior axis development. These results indicate that GDF-1 and Nodal converge on ALK4 in the anterior primitive streak to control the formation of organizing centers that are necessary for normal forebrain and branchial arch development.