A note on the incidence of reverse complementary fungal ITS sequences in the public sequence databases and a software tool for their detection and reorientation

Reverse complementary DNA sequences––sequences that are inadvertently cast backward and in which all purines and pyrimidines are transposed––are not uncommon in sequence databases, where they may introduce noise into sequence-based research. We show that about 1% of the public fungal ITS sequences, the most commonly sequenced genetic marker in mycology, are reverse complementary, and we introduce an open source software solution to automate their detection and reorientation. The MacOSX/Linux/UNIX software operates on public or private datasets of any size, although some 50 base pairs of the 5.8S gene of the ITS region are needed for the analysis.     

A promiscuous DNA packaging machine from bacteriophage T4

Complex viruses are assembled from simple protein subunits by sequential and irreversible assembly. During genome packaging in bacteriophages, a powerful molecular motor assembles at the special portal vertex of an empty prohead to initiate packaging. The capsid expands after about 10%-25% of the genome is packaged. When the head is full, the motor cuts the concatemeric DNA and dissociates from the head. Conformational changes, particularly in the portal, are thought to drive these sequential transitions. We found that the phage T4 packaging machine is highly promiscuous, translocating DNA into finished phage heads as well as into proheads. Optical tweezers experiments show that single motors can force exogenous DNA into phage heads at the same rate as into proheads. Single molecule fluorescence measurements demonstrate that phage heads undergo repeated initiations, packaging multiple DNA molecules into the same head. These results suggest that the phage DNA packaging machine has unusual conformational plasticity, powering DNA into an apparently passive capsid receptacle, including the highly stable virus shell, until it is full. These features probably led to the evolution of viral genomes that fit capsid volume, a strikingly common phenomenon in double-stranded DNA viruses, and will potentially allow design of a novel class of nanocapsid delivery vehicles.     

Incidental findings in data-intensive postgenomics science and legal liability of clinician-researchers: ready for vaccinomics?

Vaccinomics encompasses a host of multiomics approaches to characterize variability in host-environment (including pathogens) interactions, with a view to a more directed or personalized use of vaccine-based health interventions. Although vaccinomics has the potential to reduce adverse effects and increase efficacy of vaccines, the use of high-throughput, data-intensive technologies may also lead to unanticipated discoveries beyond the initial aims of a vaccinomics study--discoveries that could be highly significant to the health of the research participants. How do clinician-researchers faced with such information have to act? What are the attendant legal duties in such circumstances and how do they differ from the duties of non-clinician researchers? Together with a critical analysis of the international laws and policies framing researchers' duties with regard to incidental findings, this article also draws from Quebec's civil law--with its rich jurisprudence on clinician and researcher liability--as a case study to evaluate the potential legal implications associated with vaccinomics investigations. Given previous lessons learned from other data-intensive sciences, the education of clinician-researchers with regard to their roles, limitations, and legal obligations remains an important strategy to prevent potential legal complications and civil liability in vaccinomics research in the postgenomics era.     

Evolving Teeth Within a Stable Masticatory Apparatus in Orkney Mice

Mice from the Orkney archipelago exhibit an important diversity regarding molar shape. While on some islands mice display a usual dental pattern, teeth from other islands display additional cusplets and unusual phenotypes that may constitute case studies for evaluating the potential functional relevance of dental changes. We developed a multifaceted approach combining 2D and 3D geometric morphometrics, dental topography, dental wear, biomechanics, estimations of masticatory muscles force, and in vivo bite force on wild-derived lab descendants exemplifying the two extreme dental morphologies. The two strains differed in the geometry of the upper and lower tooth rows, and in the topography of the upper row only. Surprisingly, the most unusual tooth morphology appeared as the least complex because tooth simplification overwhelmed the signal provided by the occurrence of additional cusplets. No difference in bite force nor muscle force was evidenced, showing that the important change in dental morphology was accommodated without major changes in the rest of the masticatory apparatus. The evolution of unusual dental phenotypes was possibly fueled by drift and inbreeding in small and isolated populations on remote islands of the archipelago. No functional counter-selection impeded this diversification, since the unusual dental phenotypes did not disrupt occlusion and mastication.     

Relatedness,parentage, and philopatry within a Natterer’s bat (<Emphasis Type="Italic">Myotis nattereri</Emphasis>) maternity colony

Given their cryptic behaviour, it is often difficult to establish kinship within microchiropteran maternity colonies. This limits understanding of group formation within this highly social group. Following a concerted effort to comprehensively sample a Natterer’s bat (Myotis nattereri) maternity colony over two consecutive summers, we employed microsatellite DNA profiling to examine genetic relatedness among individuals. Resulting data were used to ascertain female kinship, parentage, mating strategies, and philopatry. Overall, despite evidence of female philopatry, relatedness was low both for adult females and juveniles of both sexes. The majority of individuals within the colony were found to be unrelated or distantly related. However, parentage analysis indicates the existence of a number of maternal lineages (e.g., grandmother, mother, or daughter). There was no evidence suggesting that males born within the colony are mating with females of the same colony. Thus, in this species, males appear to be the dispersive sex. In the Natterer’s bat, colony formation is likely to be based on the benefits of group living, rather than kin selection.     

An aminoacylase activity from Streptomyces ambofaciens catalyzes the acylation of lysine on α‐position and peptides on N‐terminal position

The presence of aminoacylase activities was investigated in a crude extract of Streptomyces ambofaciens ATCC23877. First activities catalyzing the hydrolysis of N‐α or ε‐acetyl‐L‐lysine were identified. Furthermore, the acylation of lysine and different peptides was studied and compared with results obtained with lipase B of Candida antarctica (CALB). Different regioselectivities were demonstrated for the two classes of enzymes. CALB was able to catalyze acylation only on the ε‐position whereas the crude extract from S. ambofaciens possessed the rare ability to catalyze the N‐acylation on the α‐position of the lysine or of the amino‐acid in N‐terminal position of peptides. Two genes, SAM23877_1485 and SAM23877_1734, were identified in the genome of Streptomyces ambofaciens ATCC23877 whose products show similarities with the previously identified aminoacylases from Streptomyces mobaraensis. The proteins encoded by these two genes were responsible for the major aminoacylase hydrolytic activities. Furthermore, we show that the hydrolysis of N‐α‐acetyl‐L‐lysine could be attributed to the product of SAM23877_1734 gene.     

CMA restricted to mammals and birds: myth or reality?

Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis essential for the control of intermediary metabolism. So far, the absence of any identifiable LAMP2A – a necessary and limiting protein for CMA – outside of the tetrapod clade, led to the paradigm that this cellular function was (presumably) restricted to mammals and birds. However, after we identified expressed sequences displaying high sequence homology with the mammalian LAMP2A in several fish species, our findings challenge that view and suggest that CMA likely appeared much earlier during evolution than initially thought. Hence, our results do not only shed an entirely new light on the evolution of CMA, but also bring new perspectives on the possible use of complementary genetic models, such as zebrafish or medaka for studying CMA function from a comparative angle/view.     

Pathological mutations differentially affect the self-assembly and polymerisation of the innate immune system signalling adaptor molecule MyD88

Background

Higher-order self-assembly of proteins, or “prion-like” polymerisation, is now emerging as a simple and robust mechanism for signal amplification, in particular within the innate immune system, where the recognition of pathogens or danger-associated molecular patterns needs to trigger a strong, binary response within cells. MyD88, an important adaptor protein downstream of TLRs, is one of the most recent candidates for involvement in signalling by higher order self-assembly. In this new light, we set out to re-interpret the role of polymerisation in MyD88-related diseases and study the impact of disease-associated point mutations L93P, R196C, and L252P/L265P at the molecular level.

Results

We first developed new in vitro strategies to characterise the behaviour of polymerising, full-length MyD88 at physiological levels. To this end, we used single-molecule fluorescence fluctuation spectroscopy coupled to a eukaryotic cell-free protein expression system. We were then able to explore the polymerisation propensity of full-length MyD88, at low protein concentration and without purification, and compare it to the behaviours of the isolated TIR domain and death domain that have been shown to have self-assembly properties on their own. These experiments demonstrate that the presence of both domains is required to cooperatively lead to efficient polymerisation of the protein. We then characterised three pathological mutants of MyD88.

Conclusion

We discovered that all mutations block the ability of MyD88 to polymerise fully. Interestingly, we show that, in contrast to L93P and R196C, L252P is a gain-of-function mutation, which allows the MyD88 mutant to form extremely stable oligomers, even at low nanomolar concentrations. Thus, our results shed new light on the digital “all-or-none” responses by the myddosomes and the behaviour of the oncogenic mutations of MyD88.