Modulation of glucan-enzyme interactions by domain V in GTF-SI from Streptococcus mutans

Glucansucrase GTF-SI from Streptococcus mutans is a multidomain enzyme that catalyzes the synthesis of glucan polymers. Domain V locates 100 Å from the catalytic site and is required for an optimal activity. Nevertheless, the mechanism governing its functional role remains elusive. In this work, homology modeling and molecular dynamics simulations were employed to examine the effect of domain V in the structure and glucan-binding ability of GTF-SI in full and truncated enzyme models. Our results showed that domain V increases the flexibility of the α4′-loop-α4″ motif near the catalytic site resulting in a higher surface for glucan association, and modulates the orientation of a growing oligosaccharide (N=8-23) in glucan-enzyme complexes towards engaging in favorable contacts throughout the protein, whereas in the truncated model the glucan protrudes randomly from domain B towards the solvent. These results are valuable to increase understanding about the functional role of domain V in GH70 glucansucrases.     

Northern areas as refugia for temperate species under current climate warming: Atlantic salmon (Salmo salar L.) as a model in northern Europe

In this work, patterns of geographical genetic diversity in Atlantic salmon Salmo salar were studied across the whole Atlantic Arc; whether these patterns (and thus genetic population structure) were affected by water temperatures was also evaluated. Salmo salar populations were characterized using microsatellite loci and then analysed with reference to ocean surface temperature data from across the region. Analysis showed the presence of a latitudinal cline of genetic variability (higher in northern areas) and water temperatures (sea surface temperatures) determining genetic population structure (the latter in combination with genetic drift in southern populations). Under the current global change scenario, northern areas of Europe would constitute refugia for diversity in the future. This is effectively the inverse of what appears to have happened in glacial refugia during the last glacial maximum. From this perspective, the still abundant and large northern populations S. salar should be considered as precious as the small almost relict southern ones and given appropriate protection. Careful management of the species, coordinated across countries and latitudes, is needed in order to avoid its extinction in Europe.     

Pronounced genetic differentiation of small,isolated and fragmented tilapia populations inhabiting the Magadi Soda Lake in Kenya

Lake Magadi, an alkaline hypersaline lake in Kenya, is one of the most extreme water bodies known. Although its water temperatures often exceed 40°C, a particular lineage of ‘dwarf’ tilapia, Alcolapia grahami, has evolved remarkable adaptations to survive in this hostile environment. Magadi tilapia exists in small fragmented populations in isolated lagoons within Lake Magadi and its satellite Lake, Little Magadi. In spite of the potential this tilapia holds for understanding evolutionary processes in stressful environments, few genetic studies have focused on this species. We examined the genetic diversity and spatial genetic relationships of Magadi tilapia populations using microsatellite and mitochondrial markers. High levels of genetic variation were found to be supporting the hypothesis that A. grahami populations represent remnants of a much larger fish population that inhabited paleo-lake Orolonga. In contrast to previous studies, we found a well-supported genetic structure of A. grahami consisting of three differentiated genetic clusters (a) Little Magadi, (b) Fish Spring Lagoon and (c) Rest of Magadi. Given the importance of this species to the Magadi ecosystem and its potential evolutionary significance, the three genetic clusters should be considered as separate gene pools and conservation strategies aimed at protecting the species based on these clusters are recommended.     

A fast,low cost,and highly efficient fluorescent DNA labeling method using methyl green

The increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains.     

Temporal Genetic Variance and Propagule-Driven Genetic Structure Characterize Naturalized Rainbow Trout (Oncorhynchus mykiss) from a Patagonian Lake Impacted by Trout Farming

Knowledge about the genetic underpinnings of invasions—a theme addressed by invasion genetics as a discipline—is still scarce amid well documented ecological impacts of non-native species on ecosystems of Patagonia in South America. One of the most invasive species in Patagonia’s freshwater systems and elsewhere is rainbow trout (Oncorhynchus mykiss). This species was introduced to Chile during the early twentieth century for stocking and promoting recreational fishing; during the late twentieth century was reintroduced for farming purposes and is now naturalized. We used population- and individual-based inference from single nucleotide polymorphisms (SNPs) to illuminate three objectives related to the establishment and naturalization of Rainbow Trout in Lake Llanquihue. This lake has been intensively used for trout farming during the last three decades. Our results emanate from samples collected from five inlet streams over two seasons, winter and spring. First, we found that significant intra- population (temporal) genetic variance was greater than inter-population (spatial) genetic variance, downplaying the importance of spatial divergence during the process of naturalization. Allele frequency differences between cohorts, consistent with variation in fish length between spring and winter collections, might explain temporal genetic differences. Second, individual-based Bayesian clustering suggested that genetic structure within Lake Llanquihue was largely driven by putative farm propagules found at one single stream during spring, but not in winter. This suggests that farm broodstock might migrate upstream to breed during spring at that particular stream. It is unclear whether interbreeding has occurred between “pure” naturalized and farm trout in this and other streams. Third, estimates of the annual number of breeders (N _b) were below 73 in half of the collections, suggestive of genetically small and recently founded populations that might experience substantial genetic drift. Our results reinforce the notion that naturalized trout originated recently from a small yet genetically diverse source and that farm propagules might have played a significant role in the invasion of Rainbow Trout within a single lake with intensive trout farming. Our results also argue for proficient mitigation measures that include management of escapes and strategies to minimize unintentional releases from farm facilities.     

Release of 50 new,drug-like compounds and their computational target predictions for open source anti-tubercular drug discovery

As a follow up to the antimycobacterial screening exercise and the release of GSK´s first Tres Cantos Antimycobacterial Set (TCAMS-TB), this paper presents the results of a second antitubercular screening effort of two hundred and fifty thousand compounds recently added to the GSK collection. The compounds were further prioritized based on not only antitubercular potency but also on physicochemical characteristics. The 50 most attractive compounds were then progressed for evaluation in three different predictive computational biology algorithms based on structural similarity or GSK historical biological assay data in order to determine their possible mechanisms of action. This effort has resulted in the identification of novel compounds and their hypothesized targets that will hopefully fuel future TB drug discovery and target validation programs alike.     

Actin remodeling by Nck regulates endothelial lumen formation

Multiple angiogenic cues modulate phosphotyrosine signaling to promote vasculogenesis and angiogenesis. Despite its functional and clinical importance, how vascular cells integrate phosphotyrosine-dependent signaling to elicit cytoskeletal changes required for endothelial morphogenesis remains poorly understood. The family of Nck adaptors couples phosphotyrosine signals with actin dynamics and therefore is well positioned to orchestrate cellular processes required in vascular formation and remodeling. Culture of endothelial cells in three-dimensional collagen matrices in the presence of VEGF stimulation was combined with molecular genetics, optical imaging, and biochemistry to show that Nck-dependent actin remodeling promotes endothelial cell elongation and proper organization of VE-cadherin intercellular junctions. Major morphogenetic defects caused by abrogation of Nck signaling included loss of endothelial apical-basal polarity and impaired lumenization. Time-lapse imaging using a Förster resonance energy transfer biosensor, immunostaining with phospho-specific antibodies, and GST pull-down assays showed that Nck determines spatiotemporal patterns of Cdc42/aPKC activation during endothelial morphogenesis. Our results demonstrate that Nck acts as an important hub integrating angiogenic cues with cytoskeletal changes that enable endothelial apical-basal polarization and lumen formation. These findings point to Nck as an emergent target for effective antiangiogenic therapy.     

Exploring the molecular diversity of terrestrial nemerteans (Hoplonemertea, Monostilifera, Acteonemertidae) in a continental landmass

We report the finding of terrestrial nemerteans of the family Acteonemertidae from the northern part of Spain. The specimens were studied using molecular data from the nuclear ribosomal 18S rRNA and the mitochondrial cytochrome c oxidase subunit I genes. The 18S rRNA data strongly suggest the presence of three species of terrestrial nemerteans in the Iberian Peninsula, an old landmass where terrestrial nemerteans had not previously been reported. These specimens originate from primarily undisturbed forests across a distance of c . 1000 km. Cytochrome c oxidase subunit I data also indicate the presence of multiple lineages of Iberian terrestrial nemerteans. Nonetheless, the pattern obtained from this marker is obscured most probably by deep genetic divergences. This molecular diversity, at least in some of the clades, suggests that the Iberian species are not the result of recent introductions, as proposed for other terrestrial nemerteans found in Europe. Our data also touch upon the question of a single origin of terrestriality in nemerteans, a hypothesis rejected by both data sets. Nevertheless, terrestriality seems to have had a single origin in the family Acteonemertidae.