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Coxsackie B (CVB) viruses have been associated with type 1 diabetes. We have recently observed that CVB1 was linked to the initiation of the autoimmune process leading to type 1 diabetes in Finnish children. Viral persistency in the pancreas is currently considered as one possible mechanism. In the current study persistent infection was established in pancreatic ductal and beta cell lines (PANC-1 and 1.1B4) using four different CVB1 strains, including the prototype strain and three clinical isolates. We sequenced 5&prime, untranslated region (UTR) and regions coding for structural and non-structural proteins and the second single open reading frame (ORF) protein of all persisting CVB1 strains using next generation sequencing to identify mutations that are common for all of these strains. One mutation, K257R in VP1, was found from all persisting CVB1 strains. The mutations were mainly accumulated in viral structural proteins, especially at BC, DE, EF loops and C-terminus of viral capsid protein 1 (VP1), the puff region of VP2, the knob region of VP3 and infection-enhancing epitope of VP4. This showed that the capsid region of the viruses sustains various changes during persistency some of which could be hallmark(s) of persistency.

Chemical control strategies are driving the evolution of pesticide resistance in pest populations. Understanding the genetic mechanisms of these evolutionary processes is of crucial importance to develop sustainable resistance management strategies. The acaricide pyflubumide is one of the most recently developed mitochondrial complex II inhibitors with a new mode of action that specifically targets spider mite pests. In this study, we characterize the molecular basis of pyflubumide resistance in a highly resistant population of the spider mite Tetranychus urticae. Classical genetic crosses indicated that pyflubumide resistance was incompletely recessive and controlled by more than one gene. To identify resistance loci, we crossed the resistant population to a highly susceptible T. urticae inbred strain and propagated resulting populations with and without pyflubumide exposure for multiple generations in an experimental evolution set-up. High-resolution genetic mapping by a bulked segregant analysis approach led to the identification of three quantitative trait loci (QTL) linked to pyflubumide resistance. Two QTLs were found on the first chromosome and centered on the cytochrome P450 CYP392A16 and a cluster of CYP392E6-8 genes. Comparative transcriptomics revealed a consistent overexpression of CYP392A16 and CYP392E8 in the experimental populations that were selected for pyflubumide resistance. We further corroborated the involvement of CYP392A16 in resistance by in vitro functional expression and metabolism studies. Collectively, these experiments uncovered that CYP392A16 N-demethylates the toxic carboxamide form of pyflubumide to a non-toxic compound. A third QTL coincided with cytochrome P450 reductase (CPR), a vital component of cytochrome P450 metabolism. We show here that the resistant population harbors three gene copies of CPR and that this copy number variation is associated with higher mRNA abundance. Together, we provide evidence for detoxification of pyflubumide by cytochrome P450s that is likely synergized by gene amplification of CPR., Author summary Our understanding of the causal genetic variants that drive the evolution of quantitative traits, such as polygenic pesticide resistance, remains very limited. Here, we followed a high-resolution genetic mapping approach to localize the genetic variants that cause pyflubumide resistance in the two-spotted spider mite Tetranychus urticae. Three well-supported QTL were uncovered and pointed towards a major role for cytochrome P450-mediated detoxification. Cis-regulatory variation for cytochrome P450s was observed, and in vitro cytochrome P450 experiments showed that pyflubumide was metabolized into a non-toxic derivate. A third QTL centered on cytochrome P450 reductase (CPR), which is required for cytochrome P450 activity, and is amplified in pyflubumide resistant populations. Our results indicate that pyflubumide resistance is mediated by cytochrome P450 detoxification that is enhanced by gene amplification at the CPR locus.

Emerging data highlight different clinical behaviors according to KRAS amino acid substitutions (AASs) in patients with non-small-cell lung cancer (NSCLC). We aimed to evaluate whether different KRAS AASs were associated with different responses to chemotherapy.We retrospectively reviewed data from 1190 patients with KRAS mutations who underwent first-line platinum-based chemotherapy for stage IV NSCLC. The response to different chemotherapy regimens was evaluated using the Response Evaluation Criteria In Solid Tumors criteria (v 1.1). Overall survival and time to progression (TTP) were secondary endpoints.Taxane was associated with the best response in the entire cohort (odds ratio, 2.52; 95% confidence interval [CI], 1.82-3.48; P .001), especially in G12V patients (odds ratio, 2.15; 95% CI, 1.05-4.41; P = .036). Taxane was associated with improved TTP in the entire cohort (hazard ratio [HR], 0.31; 95% CI, 0.26-0.38; P .001), especially in G13D patients (HR, 0.47; 95% CI, 0.22-1.01; P = .054). Pemetrexed was associated with the worst TTP in the entire cohort, particularly in G12V patients, who had the worst response rates (HR, 0.55; 95% CI, 0.30-0.99; P = .049). No impact on overall survival was observed according to different chemotherapy regimens and AASs.KRAS-specific AAS appears to induce different responses to chemotherapy regimens after first-line platinum-based chemotherapy in advanced NSCLC.

Enterovirus infections have been suspected to be involved in the development of type 1 diabetes. However, the pathogenetic mechanism of enterovirus-induced type 1 diabetes is not known. Pancreatic ductal cells are closely associated with pancreatic islets. Therefore, enterovirus infections in ductal cells may also affect beta-cells and be involved in the induction of type 1 diabetes. The aim of this study was to assess the ability of different enterovirus strains to infect, replicate and produce cytopathic effect in human pancreatic ductal cells. Furthermore, the viral factors that affect these capabilities were studied. The pancreatic ductal cells were highly susceptible to enterovirus infections. Both viral growth and cytolysis were detected for several enterovirus serotypes. However, the viral growth and capability to induce cytopathic effect (cpe) did not correlate completely. Some of the virus strains replicated in ductal cells without apparent cpe. Furthermore, there were strain-specific differences in the growth kinetics and the ability to cause cpe within some serotypes. Viral adaptation experiments were carried out to study the potential genetic determinants behind these phenotypic differences. The blind-passage of non-lytic CV-B6-Schmitt strain in HPDE-cells resulted in lytic phenotype and increased progeny production. This was associated with the substitution of a single amino acid (K257E) in the virus capsid protein VP1 and the viral ability to use decay accelerating factor (DAF) as a receptor. This study demonstrates considerable plasticity in the cell tropism, receptor usage and cytolytic properties of enteroviruses and underlines the strong effect of single or few amino acid substitutions in cell tropism and lytic capabilities of a given enterovirus. Since ductal cells are anatomically close to pancreatic islets, the capability of enteroviruses to infect and destroy pancreatic ductal cells may also implicate in respect to enterovirus induced type 1 diabetes. In addition, the capability for rapid adaptation to different cell types suggests that, on occasion, enterovirus strains with different pathogenetic properties may arise from less pathogenic ancestors. (C) 2015 Elsevier B.V. All rights reserved.

Background We previously identified two phenotypes of CD4+ cells with and without reactions to anti-pig CD4 monoclonal antibodies by flow cytometry in a herd of Microminipigs. In this study, we analyzed the coding sequences of CD4 and certified the expression of CD4 molecules in order to identify the genetic sequence variants responsible for the positive and negative PBMCs reactivity to anti-pig CD4 monoclonal antibodies. Results We identified two CD4 alleles, CD4.A and CD4.B, corresponding to antibody positive and negative, respectively, by nucleotide sequencing of PCR products using CD4 specific primer pairs. In comparison with the swine CD4 amino-acid sequence [GenBank: NP_001001908], CD4.A had seven amino-acid substitutions and CD4.B had 15 amino-acid substitutions. The amino-acid sequences within domain 1 of CD4.B were identical to the swine CD4.2 [GenBank: CAA46584] sequence that had been reported previously to be a modified CD4 molecule that had lost reactivity with an anti-pig CD4 antibody in NIH miniature pigs. Homozygous and heterozygous CD4.A and CD4.B alleles in the Microminipigs herd were characterised by using the RFLP technique with the restriction endonuclease, BseRI. The anti-pig CD4 antibody recognized pig PBMCs with CD4.AA and CD4.AB, but did not recognized those with CD4.BB. We transfected HeLa cells with the FLAG-tagged CD4.A or CD4.B vectors, and certified that transfected HeLa cells expressed FLAG in both vectors. The failure of cells to react with anti-CD4 antibodies in CD4.B pigs was associated to ten amino-acid substitutions in domain 1 and/or one amino-acid substitution in joining region 3 of CD4.B. We also found exon 8 was defective in some CD4.A and CD4.B resulting in the loss of the transmembrane domain, which implies that these CD4 proteins are secreted from helper T cells into the circulation. Conclusions We identified that amino-acids substitutions of domain 1 in CD4.B gave rise to the failure of some CD4 expressing cells to react with particular anti-pig CD4 monoclonal antibodies. In addition, we developed a PCR-RFLP method that enabled us to simply identify the CD4 sequence variant and the positive and negative PBMCs reactivity to our anti-pig CD4 monoclonal antibodies without the need to use flow cytometric analysis. Electronic supplementary material The online version of this article (doi:10.1186/s12917-016-0856-8) contains supplementary material, which is available to authorized users.

We determined the complete mitochondrial genomes of five cephalopods of the Subclass Coleoidea (Suborder Oegopsida: Watasenia scintillans, Todarodes pacificus, Suborder Myopsida: Sepioteuthis lessoniana, Order Sepiida: Sepia officinalis, and Order Octopoda: Octopus ocellatus) and used them to infer phylogenetic relationships. In our Maximum Likelihood (ML) tree, sepiids (cuttlefish) are at the most basal position of all decapodiformes, and oegopsids and myopsids form a monophyletic clade, thus supporting the traditional classification of the Order Teuthida. We detected extensive gene rearrangements in the mitochondrial genomes of broad cephalopod groups. It is likely that the arrangements of mitochondrial genes in Oegopsida and Sepiida were derived from those of Octopoda, which is thought to be the ancestral order, by entire gene duplication and random gene loss. Oegopsida in particular has undergone long-range gene duplications. We also found that the mitochondrial gene arrangement of Sepioteuthis lessoniana differs from that of Loligo bleekeri, although they belong to the same family. Analysis of both the phylogenetic tree and mitochondrial gene rearrangements of coleoid Cephalopoda suggests that each mitochondrial gene arrangement was acquired after the divergence of each lineage.

The phylogenetic position of the order Spenisciformes in Aves remains unclear despite several independent analyses based on morphological and molecular data. To address this issue, we determined the complete mtDNA sequence of rockhopper penguins. The mitochondrial genome, excluding the region from the D-loop to 12SrRNA, was also sequenced for petrel, albatross, frigatebird, loon and grebe, which previous studies suggest are related to penguins. A maximum likelihood analysis of the phylogenetic placement of penguins with 23 birds, including 17 species whose mtDNA sequences were previously reported, suggested that storks are the closest extant relatives of penguins, with 78% and 56% bootstrap supports, depending on the choice of outgroup species. Thus, ciconiiform birds constitute new candidates as the closest extant relatives of penguins (previously proposed candidates were either gaviiform, podicipediform, or procellariiform birds). In addition to this new evidence, our analysis gave evidence to some of ambiguous relationships in the avian tree: our analysis supported a basal split between passerines and other neoavians within Neoaves, and rejected the monophyly of Falconiformes as well as that of loons and grebes.

Delivery of secretory IgA antibodies (sIgA) to mucosal surfaces is a promising strategy to passively prevent infectious diseases. Plants have been proposed as biofactories for such complex immunoglobulin molecules. Recently, the molecular characterization of all four monomers of chicken sIgA (IgA immunoglobulin heavy and light chains, J-chain and secretory component) has been completed, allowing recombinant, up scaled production of chicken sIgA and extension of passive immune strategies to poultry. To test the suitability of the plant cell factory for bulk production of chicken sIgA, we studied the expression of chicken IgA, dIgA and sIgA in planta. To that end, new cassettes were designed that allowed the grafting of immunoglobulin variable regions derived from combinatorial libraries into full-size chicken IgA frames ready for plant expression. Using this system, 10 individual phage display clones, which had previously been selected against Eimeria acervulina antigens, were transferred "from phage to plant". Plant-made chicken antibodies showed strong differences in expression levels, which seemed governed mainly by the stability of their respective light chains. Finally, with the co-expression of chicken IgA heavy and light chains, J-chain and secretory component in N. benthamiana leaves we showed that plant cells are suitable biofactories for the production of assembled chicken sIgA complexes.

Australidelphia is the cohort, originally named by Szalay, of all Australian marsupials and the South American Dromiciops. A lot of mitochondria and nuclear genome studies support the hypothesis of a monophyly of Australidelphia, but some familial relationships in Australidelphia are still unclear. In particular, the familial relationships among the order Diprotodontia (koala, wombat, kangaroos and possums) are ambiguous. These Diprotodontian families are largely grouped into two suborders, Vombatiformes, which contains Phascolarctidae (koala) and Vombatidae (wombat), and Phalangerida, which contains Macropodidae, Potoroidae, Phalangeridae, Petauridae, Pseudocheiridae, Acrobatidae, Tarsipedidae and Burramyidae. Morphological evidence and some molecular analyses strongly support monophyly of the two families in Vombatiformes. The monophyly of Phalangerida as well as the phylogenetic relationships of families in Phalangerida remains uncertain, however, despite searches for morphological synapomorphy and mitochondrial DNA sequence analyses. Moreover, phylogenetic relationships among possum families (Phalangeridae, Petauridae, Pseudocheiridae, Acrobatidae, Tarsipedidae and Burramyidae) as well as a sister group of Macropodoidea (Macropodidae and Potoroidae) remain unclear. To evaluate familial relationships among Dromiciops and Australian marsupials as well as the familial relationships in Diprotodontia, we determined the complete mitochondrial sequence of six Diprotodontian species. We used Maximum Likelihood analyses with concatenated amino acid and codon sequences of 12 mitochondrial protein genomes. Our analysis of mitochondria amino acid sequence supports monophyly of Australian marsupials+Dromiciops and monophyly of Phalangerida. The close relatedness between Macropodidae and Phalangeridae is also weakly supported by our analysis.

The phylogenetic relationships among baleen whales (Order: Cetacea) remain uncertain despite extensive research in cetacean molecular phylogenetics and a potential morphological sample size of over 2 million animals harvested. Questions remain regarding the number of species and the monophyly of genera, as well as higher order relationships. Here, we approach mysticete phylogeny with complete mitochondrial genome sequence analysis. We determined complete mtDNA sequences of 10 extant Mysticeti species, inferred their phylogenetic relationships, and estimated node divergence times. The mtDNA sequence analysis concurs with previous molecular studies in the ordering of the principal branches, with Balaenidae (right whales) as sister to all other mysticetes base, followed by Neobalaenidae (pygmy right whale), Eschrichtiidae (gray whale), and finally Balaenopteridae (rorquals + humpback whale). The mtDNA analysis further suggests that four lineages exist within the clade of Eschrichtiidae + Balaenopteridae, including a sister relationship between the humpback and fin whales, and a monophyletic group formed by the blue, sei, and Bryde's whales, each of which represents a newly recognized phylogenetic relationship in Mysticeti. We also estimated the divergence times of all extant mysticete species, accounting for evolutionary rate heterogeneity among lineages. When the mtDNA divergence estimates are compared with the mysticete fossil record, several lineages have molecular divergence estimates strikingly older than indicated by paleontological data. We suggest this discrepancy reflects both a large amount of ancestral polymorphism and long generation times of ancestral baleen whale populations.

BACKGROUND: Simulated nucleotide or amino acid sequences are frequently used to assess the performance of phylogenetic reconstruction methods. BEAST, a Bayesian statistical framework that focuses on reconstructing time-calibrated molecular evolutionary processes, supports a wide array of evolutionary models, but lacked matching machinery for simulation of character evolution along phylogenies. RESULTS: We present a flexible Monte Carlo simulation tool, called πBUSS, that employs the BEAGLE high performance library for phylogenetic computations to rapidly generate large sequence alignments under complex evolutionary models. πBUSS sports a user-friendly graphical user interface (GUI) that allows combining a rich array of models across an arbitrary number of partitions. A command-line interface mirrors the options available through the GUI and facilitates scripting in large-scale simulation studies. πBUSS may serve as an easy-to-use, standard sequence simulation tool, but the available models and data types are particularly useful to assess the performance of complex BEAST inferences. The connection with BEAST is further strengthened through the use of a common extensible markup language (XML), allowing to specify also more advanced evolutionary models. To support simulation under the latter, as well as to support simulation and analysis in a single run, we also add the πBUSS core simulation routine to the list of BEAST XML parsers. CONCLUSIONS: πBUSS offers a unique combination of flexibility and ease-of-use for sequence simulation under realistic evolutionary scenarios. Through different interfaces, πBUSS supports simulation studies ranging from modest endeavors for illustrative purposes to complex and large-scale assessments of evolutionary inference procedures. Applications are not restricted to the BEAST framework, or even time-measured evolutionary histories, and πBUSS can be connected to various other programs using standard input and output format. 13 pages, 2 figures, 1 table ispartof: BMC bioinformatics vol:15 issue:1 pages:133- ispartof: location:England status: published

N-linked glycans attached to specific amino acids of the gp120 envelope trimer of a HIV virion can modulate the binding affinity of gp120 to CD4, influence coreceptor tropism, and play an important role in neutralising antibody responses. Because of the challenges associated with crystallising fully glycosylated proteins, most structural investigations have focused on describing the features of a non-glycosylated HIV-1 gp120 protein. Here, we use a computational approach to determine the influence of N-linked glycans on the dynamics of the HIV-1 gp120 protein and, in particular, the V3 loop. We compare the conformational dynamics of a non-glycosylated gp120 structure to that of two glycosylated gp120 structures, one with a single, and a second with five, covalently linked high-mannose glycans. Our findings provide a clear illustration of the significant effect that N-linked glycosylation has on the temporal and spatial properties of the underlying protein structure. We find that glycans surrounding the V3 loop modulate its dynamics, conferring to the loop a marked propensity towards a more narrow conformation relative to its non-glycosylated counterpart. The conformational effect on the V3 loop provides further support for the suggestion that N-linked glycosylation plays a role in determining HIV-1 coreceptor tropism.

The two-spotted spider mite Tetranychus urticae Koch is one of the economically most important pests in a wide range of outdoor and protected crops worldwide. Its control has been and still is largely based on the use of insecticides and acaricides. However, due to its short life cycle, abundant progeny and arrhenotokous reproduction, it is able to develop resistance to these compounds very rapidly. As a consequence, it has the dubious reputation to be the"most resistant species" in terms of the total number of pesticides to which populations have become resistant, and its control has become problematic in many areas worldwide. Insecticide and acaricide resistance has also been reported in the ectoparasite Sarcoptes scabiei, the causative organism of scabies, and other economically important Acari, such as the Southern cattle tick Rhipicephalus microplus, one of the biggest arthropod threats to livestock, and the parasitic mite Varroa destructor, a major economic burden for beekeepers worldwide. Although resistance research in Acari has not kept pace with that in insects, a number of studies on the molecular mechanisms responsible for the resistant phenotype has been conducted recently. In this review, state-of-the-art information on T. urticae resistance, supplemented with data on other important Acari has been brought together. Considerable attention is given to the underlying resistance mechanisms that have been elucidated at the molecular level. The incidence of bifenazate resistance in T. urticae is expanded as an insecticide resistance evolutionary paradigm in arthropods.

We have recently developed a non-cytopathic RNA replicon-based viral vector system based on the flavivirus Kunjin. Here, we illustrate the utility of the Kunjin replicon system for gene therapy. Intra-tumoral injections of Kunjin replicon virus-like particles encoding granulocyte colony-stimulating factor were able to cure >50% of established subcutaneous CT26 colon carcinoma and B16-OVA melanomas. Regression of CT26 tumours correlated with the induction of anti-cancer CD8 T cells, and treatment of subcutaneous CT26 tumours also resulted in the regression of CT26 lung metastases. Only a few immune-based strategies are able to cure these aggressive tumours once they are of a reasonable size, illustrating the potential of this vector system for intra-tumoral gene therapy applications.

We studied the association between polymorphisms of genes coding for dopamine D-2 (DRD2), dopamine D-3 (DRD3), serotonin 2(a) (HTR2A), and serotonin 2(c) (HTR2C) receptors and Antipsychotic-Induced Parkinsonism (AIP), rigidity, bradykinesia, and rest-tremor in African-Caribbeans treated with antipsychotics. Polymorphisms of DRD2 (-141CIns/Del, TaqIA, 957C > T), DRD3 (Ser9Gly), HTR2A (-1438A > G, 102T > C, His452-Tyr), and HTR2C (-759C > T, Cys23Ser) genes were determined according to standard protocols. The Unified Parkinson Disease Rating Scale was used for the measurement of AIP, rigidity, bradykinesia, and rest-tremor. Chi-squared or Fisher's exact tests were applied for the association analyses. The t-test was applied for continuous data. Ninety nine males and 27 females met the inclusion criteria (Schizophr Res 1996, 19:195). In males, but not in females, there were significant associations between -141CDel-allele carriership (DRD2) and rigidity (Fisher's Exact Test: P = 0.021) and between 23Ser-allele carriership (HTR2C) and bradykinesia (P = 0.026, chi(2) = 5.0) or AIP (P = 0.008, chi(2) = 7.1). Rest-tremor was not associated with any of the polymorphisms studied. Analyses of the age, chlorpromazine equivalents, benztropine equivalents, the number of patients using anticholinergic medication, and the utilization patterns of the antipsychotic medication did not show statistically significant differences between patients with and without AIP, rigidity, bradykinesia, rest-tremor. Conducting the analysis without gender stratification did not affect our findings considerably, except for the association between bradykinesia and 23Ser-allele which failed to reach statistical significance in the total sample (P = 0.0646, chi(2) = 3.41). Since AIPs subsymptoms (rigidity, bradykinesia, and rest-tremor) may differ pharmacogenetically, our data strongly support symptom-specific analysis of AIP. However, further research is warranted to confirm our findings. (c) 2008 Wiley-Liss, Inc.

Affiliations des auteurs : cf page 216 de l'article; International audience; Comparative analysis of multiple genomes in a phylogenetic framework dramatically improves the precision and sensitivity of evolutionary inference, producing more robust results than single-genome analyses can provide. The genomes of 12 Drosophila species, ten of which are presented here for the first time (sechellia, simulans, yakuba, erecta, ananassae, persimilis, willistoni, mojavensis, virilis and grimshawi), illustrate how rates and patterns of sequence divergence across taxa can illuminate evolutionary processes on a genomic scale. These genome sequences augment the formidable genetic tools that have made Drosophila melanogaster a pre-eminent model for animal genetics, and will further catalyse fundamental research on mechanisms of development, cell biology, genetics, disease, neurobiology, behaviour, physiology and evolution. Despite remarkable similarities among these Drosophila species, we identified many putatively non-neutral changes in protein-coding genes, non-coding RNA genes, and cis-regulatory regions. These may prove to underlie differences in the ecology and behaviour of these diverse species.

Background Genome-wide association studies of common diseases for common, low penetrance causal variants are underway. A proportion of these will alter protein sequences, the most common of which is the non-synonymous single nucleotide polymorphism (nsSNP). It would be an advantage if the functional effects of an nsSNP on protein structure and function could be predicted, both for the final identification process of a causal variant in a disease-associated chromosome region, and in further functional analyses of the nsSNP and its disease-associated protein. Results In the present report we have compared and contrasted structure- and sequence-based methods of prediction to over 5500 genes carrying nearly 24,000 nsSNPs, by employing an automatic comparative modelling procedure to build models for the genes. The nsSNP information came from two sources, the OMIM database which are rare (minor allele frequency, MAF, < 0.01) and are known to cause penetrant, monogenic diseases. Secondly, nsSNP information came from dbSNP125, for which the vast majority of nsSNPs, mostly MAF > 0.05, have no known link to a disease. For over 40% of the nsSNPs, structure-based methods predicted which of these sequence changes are likely to either disrupt the structure of the protein or interfere with the function or interactions of the protein. For the remaining 60%, we generated sequence-based predictions. Conclusion We show that, in general, the prediction tools are able distinguish disease causing mutations from those mutations which are thought to have a neutral affect. We give examples of mutations in genes that are predicted to be deleterious and may have a role in disease. Contrary to previous reports, we also show that rare mutations are consistently predicted to be deleterious as often as commonly occurring nsSNPs.

Balaenoptera omurai, formerly classified as a small form of Bryde's whale, was recently reclassified as a new baleen whale species of the family Balaenopteridae. Although researchers have investigated the evolutionary history of Balaenopteridae and their relatives using molecular phylogenetic methods, the taxonomy of the ordinary Bryde's whale (Balaenoptera brydei) and small-form Bryde's whales (Balaenoptera edeni and B. omurai) remains unclear. We have used complete mtDNA sequences and short interspersed repetitive element (SINE) insertion patterns to construct the evolutionary history of both B. omurai and the taxonomically redefined species, B. edeni. The combined results demonstrate that B. omurai forms a monophyletic lineage with B. musculus, B. brydei, B. edeni and B. borealis and that B. omurai and B. musculus successively diverged from their common ancestor. In addition, we also showed that B. edeni constitutes a sister taxon to B. brydei. Our data suggest that B. omurai evolved as an ancient independent lineage that diverged much earlier than B. borealis, B. brydei and B. edeni, which were previously believed to be closely related to B. omurai.

The complete mitochondrial genome of a lesser hedgehog tenrec Echinops telfairi was determined in this study. It is an endemic African insectivore that is found specifically in Madagascar. The tenrec's back is covered with hedgehog-like spines. Unlike other spiny mammals, such as spiny mice, spiny rats, spiny dormice and porcupines, lesser hedgehog tenrecs look amazingly like true hedgehogs (Erinaceidae). However, they are distinguished morphologically from hedgehogs by the absence of a jugal bone. We determined the complete sequence of the mitochondrial genome of a lesser hedgehog tenrec and analyzed the results phylogenetically to determine the relationships between the tenrec and other insectivores (moles, shrews and hedgehogs), as well as the relationships between the tenrec and endemic African mammals, classified as Afrotheria, that have recently been shown by molecular analysis to be close relatives of the tenrec. Our data confirmed the afrotherian status of the tenrec, and no direct relation was recovered between the tenrec and the hedgehog. Comparing our data with those of others, we found that within-species variations in the mitochondrial DNA of lesser hedgehog tenrecs appear to be the largest recognized to date among mammals, apart from orangutans, which might be interesting from the view point of evolutionary history of tenrecs on Madagascar.

Sso7d is a basic 7-kDa DNA-binding protein from Sulfolobus solfataricus, also endowed with ribonuclease activity. The protein consists of a double-stranded antiparallel beta-sheet, onto which an orthogonal triple-stranded antiparallel beta-sheet is packed, and of a small helical stretch at the C-terminus. Furthermore, the two beta-sheets enclose an aromatic cluster displaying a fishbone geometry. We previously cloned the Sso7d-encoding gene, expressed it in Escherichia coli, and produced several single-point mutants, either of residues located in the hydrophobic core or of Trp23, which is exposed to the solvent and plays a major role in DNA binding. The mutation F31A was dramatically destabilizing, with a loss in thermo- and piezostabilities by at least 27 K and 10 kbar, respectively. Here, we report the solution structure of the F31A mutant, which was determined by NMR spectroscopy using 744 distance constraints obtained from analysis of multidimensional spectra in conjunction with simulated annealing protocols. The most remarkable finding is the change in orientation of the Trp23 side chain, which in the wild type is completely exposed to the solvent, whereas in the mutant is largely buried in the aromatic cluster. This prevents the formation of a cavity in the hydrophobic core of the mutant, which would arise in the absence of structural rearrangements. We found additional changes produced by the mutation, notably a strong distortion in the beta-sheets with loss in several hydrogen bonds, increased flexibility of some stretches of the backbone, and some local strains. On one hand, these features may justify the dramatic destabilization provoked by the mutation; on the other hand, they highlight the crucial role of the hydrophobic core in protein stability. To the best of our knowledge, no similar rearrangement has been so far described as a result of a single-point mutation.