Are Drosophila preferences for yeasts stable or contextual?

Whether there are general mechanisms, driving interspecific chemical communication is uncertain. Saccharomycetaceae yeast and Drosophila fruit flies, both extensively studied research models, share the same fruit habitat, and it has been suggested their interaction comprises a facultative mutualism that is instigated and maintained by yeast volatiles. Using choice tests, experimental evolution, and volatile analyses, we investigate the maintenance of this relationship and reveal little consistency between behavioral responses of two isolates of sympatric Drosophila species. While D. melanogaster was attracted to a range of different Saccharomycetaceae yeasts and this was independent of fruit type, D. simulans preference appeared specific to a particular S. cerevisiae genotype isolated from a vineyard fly population. This response, however, was not consistent across fruit types and is therefore context‐dependent. In addition, D. simulans attraction to an individual S. cerevisiae isolate was pliable over ecological timescales. Volatile candidates were analyzed to identify a common signal for yeast attraction, and while D. melanogaster generally responded to fermentation profiles, D. simulans preference was more discerning and likely threshold‐dependent. Overall, there is no strong evidence to support the idea of bespoke interactions with specific yeasts for either of these Drosophila genotypes. Rather the data support the idea Drosophila are generally adapted to sense and locate fruits infested by a range of fungal microbes and/or that yeast–Drosophila interactions may evolve rapidly.     

A chromosome-scale assembly of the bilberry genome identifies a complex locus controlling berry anthocyanin composition

Bilberry (Vaccinium myrtillus L.) belongs to the Vaccinium genus, which includes blueberries (Vaccinium spp.) and cranberry (V. macrocarpon). Unlike its cultivated relatives, bilberry remains largely undomesticated, with berry harvesting almost entirely from the wild. As such, it represents an ideal target for genomic analysis, providing comparisons with the domesticated Vaccinium species. Bilberry is prized for its taste and health properties and has provided essential nutrition for Northern European indigenous populations. It contains high concentrations of phytonutrients, with perhaps the most important being the purple colored anthocyanins, found in both skin and flesh. Here, we present the first bilberry genome assembly, comprising 12 pseudochromosomes assembled using Oxford Nanopore (ONT) and Hi-C Technologies. The pseudochromosomes represent 96.6% complete BUSCO genes with an assessed LAI score of 16.3, showing a high conservation of synteny against the blueberry genome. Kmer analysis showed an unusual third peak, indicating the sequenced samples may have been from two individuals. The alternate alleles were purged so that the final assembly represents only one haplotype. A total of 36,404 genes were annotated after nearly 48% of the assembly was masked to remove repeats. To illustrate the genome quality, we describe the complex MYBA locus, and identify the key regulating MYB genes that determine anthocyanin production. The new bilberry genome builds on the genomic resources and knowledge of Vaccinium species, to help understand the genetics underpinning some of the quality attributes that breeding programs aspire to improve. The high conservation of synteny between bilberry and blueberry genomes means that comparative genome mapping can be applied to transfer knowledge about marker-trait association between these two species, as the loci involved in key characters are orthologous.     

Multiple treatment comparisons in epilepsy monotherapy trials

Background

Previous trials of the RTS, S malaria candidate vaccine have shown that this vaccine is safe, tolerated and immunogenic. The development plan for this vaccine aims at administering it in the first year of life through the Expanded Program on Immunization (EPI). The objective was to evaluate the safety and reactogenicity of RTS, S/AS02D (0.5 ml dose), a pediatric formulation of GlaxoSmithKline Biologicals' current malaria candidate vaccine RTS, S/AS02A (0.25 ml dose). A 0.5 ml dose of AS02D is composed of the same active ingredients in the same quantities as in a 0.25 ml dose of AS02A and has been developed to be easily introduced into routine EPI practices.

Methods

We performed a phase I/IIb randomized double-blind bridging study in a malaria-endemic region of Mozambique, to compare the safety and immunogenicity of both candidate vaccines with the aim of replacing RTS, S/AS02A with RTS, S/AS02D as the candidate pediatric vaccine. 200 Mozambican children aged 3 to 5 years were randomized 1:1 to receive one of the 2 vaccines according to a 0, 1, 2 month schedule.

Results

Both vaccines were safe and had similar reactogenicity profiles. All subjects with paired pre and post-vaccination samples showed a vaccine response with respect to anti-circumsporozoite (CS) antibodies irrespective of initial anti-CS serostatus. Geometric mean titers (GMTs) were 191 EU/ml (95% CI 150–242) in recipients of RTS, S/AS02D compared to 180 EU/ml (95% CI 146–221) in recipients of RTS, S/AS02A. For the anti-hepatitis B surface antigen (HBsAg), all subjects were seroprotected at day 90, and the GMTs were 23978 mIU/ml (95% CI 17896–32127) in RTS, S/AS02D recipients and 17410 mIU/ml (95% CI 13322–22752) in RTS, S/AS02A recipients. There was a decrease in anti-CS GMTs between months 3 and 14 in both groups (191 vs 22 EU/mL in RTS, S/AS02D group and 180 vs 29 EU/mL in RTS, S/AS02A group).

Conclusion

Our data show that the RTS, S/AS02D is safe, well tolerated, and demonstrates non-inferiority (defined as upper limit of the 95% confidence interval of the anti-CS GMT ratio of RTS, S/AS02A to RTS, S/AS02D below 3.0) of the antibody responses to circumsporozoite and HBsAg induced by the RTS, S/AS02D as compared to the RTS, S/AS02A.     

B-Raf acts via the ROCKII/LIMK/cofilin pathway to maintain actin stress fibers in fibroblasts

Recent data have shown that the BRAF gene is mutated at a high frequency in human malignancies. We have analyzed the migratory characteristics of B-raf(-/-) mouse embryonic fibroblasts (MEFs) and compared these with the organization of the actin cytoskeleton and the activity of signaling pathways that are known to influence this organization. Disruption of B-raf significantly reduced the levels of phospho-ERK1/2 and, surprisingly, induced an approximately 1.5-fold increase in cell migration. Consistent with these findings, the high level of actin stress fibers normally present in MEFs was considerably reduced following disruption of B-raf, and the F-actin content of B-raf(-/-) cells was less than half that of B-raf(+/+) cells. Phosphorylation of the myosin light chain on Thr18/Ser19 residues was not reduced in B-raf(-/-) cells. Rather, reduced ROCKII expression and attenuated phosphorylation of ADF/cofilin on serine 3 occurred. Normal stress fiber and phosphocofilin levels were restored by the expression of human B-Raf and catalytically active MEK and by the overexpression of LIM kinase (LIMK). These results have important implications for the role of the B-Raf/ERK signaling pathway in regulating cell motility in normal and malignant cells. They suggest that B-Raf is involved in invasiveness by regulating the proper assembly of actin stress fibers and contractility through a ROCKII/LIMK/cofilin signaling pathway.     

TGFβ3 and loading increases osteocyte survival in human cancellous bone cultured ex vivo

The goal of this study was to assess the effect of the addition of TGFβ₃, alone or in combination with loading, on the survival of osteocytes in 3D human explant cancellous bone during long-term culture in an ex vivo loading bioreactor. Human cancellous bone explants were cultured for up to 14 days with or without TGFβ₃ (15 ng ml⁻¹) and with or without loading (300 cycles, at 1 Hz, producing 4000 microstrain). Bone core response was visualized using undecalcified histology with morphological methods after embedding with Technovit 9100 New® resin. Histological examination revealed normal gross level bone structure with or without the application of load or the addition of TGFβ₃. The viability of the osteocytes within the bone was assessed by lactate dehydrogenase (LDH) activity. We demonstrate that this ex vivo loading bioreactor is able to maintain a high percentage (over 50%) of viable osteocytes throughout the bone explants after 14 days in ex vivo culture. Further to this, the combination of daily loading and TGFβ₃ administration produced superior osteocyte survival at the core centres when compared to loading or TGFβ alone. Copyright © 2008 John Wiley & Sons, Ltd.     

Association of protein kinase C-delta with the B cell antigen receptor complex

Protein kinase C (PKC)-delta is a diacylglycerol-dependent, calcium-independent novel PKC isoform and has been demonstrated to exert negative regulatory functions in B lymphocytes as well as in mast cells. Whereas in mast cells PKC-delta functionally interacts with the high-affinity receptor for IgE, FcepsilonR1, no such association has been described for the B cell antigen receptor (BCR). In this report, for the first time, we demonstrate the interaction of PKC-delta with different classes of BCR by means of affinity purification and native protein complex analysis. Using a C-terminally truncated Ig-alpha as well as non-phosphorylated and phosphorylated peptides representing C-terminal regions of Ig-alpha, the dependence of this BCR/PKC-delta interaction on tyrosine-phosphorylated Ig-alpha is shown. Finally, splenocytes from PKC-delta-deficient mice are found to exert reduced phosphorylation of PKD (a.k.a. PKC-mu) in response to BCR engagement, suggesting the early, membrane-proximal activation of an attenuating kinase complex including PKC-delta and PKD.     

Knockdown of embryonic myosin heavy chain reveals an essential role in the morphology and function of the developing heart

The expression and function of embryonic myosin heavy chain (eMYH) has not been investigated within the early developing heart. This is despite the knowledge that other structural proteins, such as alpha and beta myosin heavy chains and cardiac alpha actin, play crucial roles in atrial septal development and cardiac function. Most cases of atrial septal defects and cardiomyopathy are not associated with a known causative gene, suggesting that further analysis into candidate genes is required. Expression studies localised eMYH in the developing chick heart. eMYH knockdown was achieved using morpholinos in a temporal manner and functional studies were carried out using electrical and calcium signalling methodologies. Knockdown in the early embryo led to abnormal atrial septal development and heart enlargement. Intriguingly, action potentials of the eMYH knockdown hearts were abnormal in comparison with the alpha and beta myosin heavy chain knockdowns and controls. Although myofibrillogenesis appeared normal, in knockdown hearts the tissue integrity was affected owing to apparent focal points of myocyte loss and an increase in cell death. An expression profile of human skeletal myosin heavy chain genes suggests that human myosin heavy chain 3 is the functional homologue of the chick eMYH gene. These data provide compelling evidence that eMYH plays a crucial role in important processes in the early developing heart and, hence, is a candidate causative gene for atrial septal defects and cardiomyopathy.     

Assessing bee species richness in two Mediterranean communities: importance of habitat type and sampling techniques

The decline of bees has raised concerns regarding their conservation and the maintenance of ecosystem services they provide to bee-pollinated wild flowers and crops. Although the Mediterranean region is a hotspot for bee species richness, their status remains poorly studied. There is an urgent need for cost-effective, reliable, and unbiased sampling methods that give good bee species richness estimates. This study aims: (a) to assess bee species richness in two common Mediterranean habitat types: semi-natural scrub (phrygana) and managed olive groves; (b) to compare species richness in those systems to that of other biogeographic regions, and (c) to assess whether six different sampling methods (pan traps, variable and standardized transect walks, observation plots and trap nests), previously tested in other European biogeographic regions, are suitable in Mediterranean communities. Eight study sites, four per habitat type, were selected on the island of Lesvos, Greece. The species richness observed was high compared to other habitat types worldwide for which comparable data exist. Pan traps collected the highest proportion of the total bee species richness across all methods at the scale of a study site. Variable and standardized transect walks detected the highest total richness over all eight study sites. Trap nests and observation plots detected only a limited fraction of the bee species richness. To assess the total bee species richness in bee diversity hotspots, such as the studied habitats, we suggest a combination of transect walks conducted by trained bee collectors and pan trap sampling.