Mutation discovery in mice by whole exome sequencing

We report the development and optimization of reagents for in-solution, hybridization-based capture of the mouse exome. By validating this approach in a multiple inbred strains and in novel mutant strains, we show that whole exome sequencing is a robust approach for discovery of putative mutations, irrespective of strain background. We found strong candidate mutations for the majority of mutant exomes sequenced, including new models of orofacial clefting, urogenital dysmorphology, kyphosis and autoimmune hepatitis.     

Mycolactone diffuses into the peripheral blood of Buruli ulcer patients--implications for diagnosis and disease monitoring

Background

Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), is unique among human pathogens in its capacity to produce a polyketide-derived macrolide called mycolactone, making this molecule an attractive candidate target for diagnosis and disease monitoring. Whether mycolactone diffuses from ulcerated lesions in clinically accessible samples and is modulated by antibiotic therapy remained to be established.

Methodology/Principal Finding

Peripheral blood and ulcer exudates were sampled from patients at various stages of antibiotic therapy in Ghana and Ivory Coast. Total lipids were extracted from serum, white cell pellets and ulcer exudates with organic solvents. The presence of mycolactone in these extracts was then analyzed by a recently published, field-friendly method using thin layer chromatography and fluorescence detection. This approach did not allow us to detect mycolactone accurately, because of a high background due to co-extracted human lipids. We thus used a previously established approach based on high performance liquid chromatography coupled to mass spectrometry. By this means, we could identify structurally intact mycolactone in ulcer exudates and serum of patients, and evaluate the impact of antibiotic treatment on the concentration of mycolactone.

Conclusions/Significance

Our study provides the proof of concept that assays based on mycolactone detection in serum and ulcer exudates can form the basis of BU diagnostic tests. However, the identification of mycolactone required a technology that is not compatible with field conditions and point-of-care assays for mycolactone detection remain to be worked out. Notably, we found mycolactone in ulcer exudates harvested at the end of antibiotic therapy, suggesting that the toxin is eliminated by BU patients at a slow rate. Our results also indicated that mycolactone titres in the serum may reflect a positive response to antibiotics, a possibility that it will be interesting to examine further through longitudinal studies.     

Analysis of Jak2 catalytic function by peptide microarrays: the role of the JH2 domain and V617F mutation

Janus kinase 2 (JAK2) initiates signaling from several cytokine receptors and is required for biological responses such as erythropoiesis. JAK2 activity is controlled by regulatory proteins such as Suppressor of Cytokine Signaling (SOCS) proteins and protein tyrosine phosphatases. JAK2 activity is also intrinsically controlled by regulatory domains, where the pseudokinase (JAK homology 2, JH2) domain has been shown to play an essential role. The physiological role of the JH2 domain in the regulation of JAK2 activity was highlighted by the discovery of the acquired missense point mutation V617F in myeloproliferative neoplasms (MPN). Hence, determining the precise role of this domain is critical for understanding disease pathogenesis and design of new treatment modalities. Here, we have evaluated the effect of inter-domain interactions in kinase activity and substrate specificity. By using for the first time purified recombinant JAK2 proteins and a novel peptide micro-array platform, we have determined initial phosphorylation rates and peptide substrate preference for the recombinant kinase domain (JH1) of JAK2, and two constructs comprising both the kinase and pseudokinase domains (JH1-JH2) of JAK2. The data demonstrate that (i) JH2 drastically decreases the activity of the JAK2 JH1 domain, (ii) JH2 increased the K(m) for ATP (iii) JH2 modulates the peptide preference of JAK2 (iv) the V617F mutation partially releases this inhibitory mechanism but does not significantly affect substrate preference or K(m) for ATP. These results provide the biochemical basis for understanding the interaction between the kinase and the pseudokinase domain of JAK2 and identify a novel regulatory role for the JAK2 pseudokinase domain. Additionally, this method can be used to identify new regulatory mechanisms for protein kinases that provide a better platform for designing specific strategies for therapeutic approaches.     

Cryptic speciation in Brazilian Epiperipatus (Onychophora: Peripatidae) reveals an underestimated diversity among the peripatid velvet worms

Background

Taxonomical studies of the neotropical Peripatidae (Onychophora, velvet worms) have proven difficult, due to intraspecific variation and uniformity of morphological characters across this onychophoran subgroup. We therefore used molecular approaches, in addition to morphological methods, to explore the diversity of Epiperipatus from the Minas Gerais State of Brazil.

Methodology/Principal Findings

Our analyses revealed three new species. While Epiperipatus diadenoproctus sp. nov. can be distinguished from E. adenocryptus sp. nov. and E. paurognostus sp. nov. based on morphology and specific nucleotide positions in the mitochondrial cytochrome c oxidase subunit I (COI) and small ribosomal subunit RNA gene sequences (12S rRNA), anatomical differences between the two latter species are not evident. However, our phylogenetic analyses of molecular data suggest that they are cryptic species, with high Bayesian posterior probabilities and bootstrap and Bremer support values for each species clade. The sister group relationship of E. adenocryptus sp. nov. and E. paurognostus sp. nov. in our analyses correlates with the remarkable morphological similarity of these two species. To assess the species status of the new species, we performed a statistical parsimony network analysis based on 582 base pairs of the COI gene in our specimens, with the connection probability set to 95%. Our findings revealed no connections between groups of haplotypes, which have been recognized as allopatric lineages in our phylogenetic analyses, thus supporting our suggestion that they are separate species.

Conclusions/Significance

Our findings suggest high cryptic species diversity and endemism among the neotropical Peripatidae and demonstrate that the combination of morphological and molecular approaches is helpful for clarifying the taxonomy and species diversity of this apparently large and diverse onychophoran group.     

Habitat selection by large mammals in a southern Brazilian Atlantic Forest

Habitat selection, which is mainly a consequence of competition and predation, allows species to coexist. The present study was conducted in two reserves in an Atlantic Forest area in Santa Catarina State, southern Brazil, and provided information on several large mammal species through photographic records. Records were related to certain environmental parameters, such as width of passages (trails and roads), vegetation density and proximity to water, in order to assess the relationship between each mammal species and its microhabitat. Thirty-two camera trap stations were placed during 17 months for 150.8 (±62.2) days on average. Terrestrial mammals tended to use different habitats: Puma concolor used mainly dirt roads and open areas; Leopardus pardalis, Cerdocyon thous and Nasua nasua used more large trails and intermediate-forested sites; and Cuniculus paca, Dasypus novemcinctus, Leopardus tigrinus, Eira barbara and Leopardus wiedii were recorded more often on narrow trails and in densely forested sites. Some of these forest species, such as D. novemcinctus, C. paca and L. pardalis, also showed relationships with watercourses. Information on habitat selection allows more effort to be addressed to the habitat associated with focal species, and indicates the significance of environmental heterogeneity, which makes it possible for species to coexist.     

The G protein-coupled receptor kinase-2 is a TGFbeta-inducible antagonist of TGFbeta signal transduction

Signaling from the activin/transforming growth factor beta (TGFbeta) family of cytokines is a tightly regulated process. Disregulation of TGFbeta signaling is often the underlying basis for various cancers, tumor metastasis, inflammatory and autoimmune diseases. In this study, we identify the protein G-coupled receptor kinase 2 (GRK2), a kinase involved in the desensitization of G protein-coupled receptors (GPCR), as a downstream target and regulator of the TGFbeta-signaling cascade. TGFbeta-induced expression of GRK2 acts in a negative feedback loop to control TGFbeta biological responses. Upon TGFbeta stimulation, GRK2 associates with the receptor-regulated Smads (R-Smads) through their MH1 and MH2 domains and phosphorylates their linker region. GRK2 phosphorylation of the R-Smads inhibits their carboxyl-terminal, activating phosphorylation by the type I receptor kinase, thus preventing nuclear translocation of the Smad complex, leading to the inhibition of TGFbeta-mediated target gene expression, cell growth inhibition and apoptosis. Furthermore, we demonstrate that GRK2 antagonizes TGFbeta-induced target gene expression and apoptosis ex vivo in primary hepatocytes, establishing a new role for GRK2 in modulating single-transmembrane serine/threonine kinase receptor-mediated signal transduction.     

The Bud14p-Glc7p complex functions as a cortical regulator of dynein in budding yeast

Regulated interactions between microtubules (MTs) and the cell cortex control MT dynamics and position the mitotic spindle. In eukaryotic cells, the adenomatous polyposis coli/Kar9p and dynein/dynactin pathways are involved in guiding MT plus ends and MT sliding along the cortex, respectively. Here we identify Bud14p as a novel cortical activator of the dynein/dynactin complex in budding yeast. Bud14p accumulates at sites of polarized growth and the mother-bud neck during cytokinesis. The localization to bud and shmoo tips requires an intact actin cytoskeleton and the kelch-domain-containing proteins Kel1p and Kel2p. While cells lacking Bud14p function fail to stabilize the pre-anaphase spindle at the mother-bud neck, overexpression of Bud14p is toxic and leads to elongated astral MTs and increased dynein-dependent sliding along the cell cortex. Bud14p physically interacts with the type-I phosphatase Glc7p, and localizes Glc7p to the bud cortex. Importantly, the formation of Bud14p-Glc7p complexes is necessary to regulate MT dynamics at the cortex. Taken together, our results suggest that Bud14p functions as a regulatory subunit of the Glc7p type-I phosphatase to stabilize MT interactions specifically at sites of polarized growth.     

Breast pathology and reduction mammaplasty

Breast cancer is the tumor with the highest prevalence and incidence in women. Reduction mammaplasty is one of the most common procedures performed in Brazil by the plastic surgeon, and it is not uncommon for the surgeon to find a breast tumor during the operation or afterward, when the histopathological report is received. In this study, 2488 patient files were reviewed retrospectively. All patients had undergone reduction mammaplasty at the senior author's private clinic (the Ivo Pitanguy Clinic) between January of 1957 and December of 2002. Resected breast tissue was examined histopathologically. The objective of this study was to verify the occurrence of breast carcinoma found accidentally postoperatively. The senior author's team performed all of the operations and the same pathologist performed every histopathological examination. The histopathological test results were divided into two groups: benign lesions and tumors. The highest frequency of breast pathology was benign lesions, and of them, 80.8 percent involved fibrocystic changes and fibroadiposity. The tumor group was subdivided into benign tumors and malignant tumors. Among the benign tumors, fibroadenoma was the one most common, in 2.2 percent. The frequency of malignant tumors was 0.5 percent of all patients. Most of the histopathological lesions were found in patients between 30 and 50 years of age. A reduced number of patients had no lesions (3.7 percent). Lack of a pathological investigation or a cursory or hurried examination of any mammary tissue by the pathologist may overlook important lesions. In the analysis of these statistics, the concept of normal breast tissue was questioned.