The hydrophobic region of the Leishmania peroxin 14 : requirements for association with a glycosome mimetic membrane

This work was funded by operating grants from the Canadian Institutes of Health Research (CIHR) and a Natural Sciences Engineering Research Council of Canada (NSERC) Discovery grant [Fonds de recherche du Québec — Nature et technologies (FRQNT) Regroupement Stratégique grant to the Centre for Host-Parasite Interactions (A.J.)]. N.C. was supported by a doctoral research scholarship from FRQNT. E.B. was supported by a Banting postdoctoral fellowship from CIHR. This work was also supported in part by Wellcome Trust grants [086658 and 093228] to T.K.S. C.S. recognizes the financial support from the Natural Sciences and Engineering Research Council of Canada and a Canada Foundation for Innovation grant [16299].Protein import into the Leishmania glycosome requires docking of the cargo-loaded peroxin 5 (PEX5) receptor to the peroxin 14 (PEX14) bound to the glycosome surface. To examine the LdPEX14-membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol (PG) modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence-activated cell sorting technique, we established that the LdPEX14 membrane-binding activity was dependent on a predicted transmembrane helix found within residues 149-179. Monolayer experiments showed that the incorporation of PG and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5-PTS1 receptor-cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs.PostprintPeer reviewe

Comprehensive molecular characterization of gastric adenocarcinoma

Gastric cancer is a leading cause of cancer deaths, but analysis of its molecular and clinical characteristics has been complicated by histological and aetiological heterogeneity. Here we describe a comprehensive molecular evaluation of 295 primary gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) project. We propose a molecular classification dividing gastric cancer into four subtypes: tumours positive for Epstein–Barr virus, which display recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, CD274 (also known as PD-L1) and PDCD1LG2 (also knownasPD-L2); microsatellite unstable tumours, which show elevated mutation rates, including mutations of genes encoding targetable oncogenic signalling proteins; genomically stable tumours, which are enriched for the diffuse histological variant and mutations of RHOA or fusions involving RHO-family GTPase-activating proteins; and tumours with chromosomal instability, which show marked aneuploidy and focal amplification of receptor tyrosine kinases. Identification of these subtypes provides a roadmap for patient stratification and trials of targeted therapies

The Acute Satellite Cell Response and Skeletal Muscle Hypertrophy following Resistance Training

The extent of skeletal muscle hypertrophy in response to resistance training is highly variable in humans. The main objective of this study was to explain the nature of this variability. More specifically, we focused on the myogenic stem cell population, the satellite cell (SC) as a potential mediator of hypertrophy. Twenty-three males (aged 18–35 yrs) participated in 16 wk of progressive, whole body resistance training, resulting in changes of 7.9±1.6% (range of −1.9–24.7%) and 21.0±4.0% (range of −7.0 to 51.7%) in quadriceps volume and myofibre cross-sectional area (CSA), respectively. The SC response to a single bout of resistance exercise (80% 1RM), analyzed via immunofluorescent staining resulted in an expansion of type II fibre associated SC 72 h following exercise (pre: 11.3±0.9; 72 h: 14.8±1.4 SC/type II fibre; p<0.05). Training resulted in an expansion of the SC pool associated with type I (pre: 10.7±1.1; post: 12.1±1.2 SC/type I fibre; p<0.05) and type II fibres (pre: 11.3±0.9; post: 13.0±1.2 SC/type II fibre; p<0.05). Analysis of individual SC responses revealed a correlation between the relative change in type I associated SC 24 to 72 hours following an acute bout of resistance exercise and the percentage increase in quadriceps lean tissue mass assessed by MRI (r2 = 0.566, p = 0.012) and the relative change in type II associated SC following 16 weeks of resistance training and the percentage increase in quadriceps lean tissue mass assessed by MRI (r2 = 0.493, p = 0.027). Our results suggest that the SC response to resistance exercise is related to the extent of muscular hypertrophy induced by training

The Somatic Genomic Landscape of Chromophobe Renal Cell Carcinoma

We describe the landscape of somatic genomic alterations of 66 chromophobe renal cell carcinomas (ChRCCs) based on multidimensional and comprehensive characterization, including mitochondrial DNA (mtDNA) and whole genome sequencing. The result is consistent that ChRCC originates from the distal nephron compared to other kidney cancers with more proximal origins. Combined mtDNA and gene expression analysis implicates changes in mitochondrial function as a component of the disease biology, while suggesting alternative roles for mtDNA mutations in cancers relying on oxidative phosphorylation. Genomic rearrangements lead to recurrent structural breakpoints within TERT promoter region, which correlates with highly elevated TERT expression and manifestation of kataegis, representing a mechanism of TERT up-regulation in cancer distinct from previously-observed amplifications and point mutations

Biophysical investigations of structural features and interactions of «Leishmania donovani» Peroxin 5

Parasites of the genus Leishmania cause a broad array of diseases, collectively termed leishmaniasis. These diseases range in morbidity; the cutaneous form is typically self healing, while the mucocutaneous and visceral manifestations require chemotherapeutic intervention to avoid lethality. At present there is no vaccine, and current methods of chemotherapeutic intervention have severe drawbacks, together creating a dire need for new options to combat these destructive diseases. An organelle within the parasite, the glycosome, has been identified as an attractive drug target. The glycosome compartmentalizes several enzymes from important biosynthetic and metabolic pathways, which has been shown to be necessary for the viability of the parasite. Although the organelle is structurally and evolutionarily related to peroxisomes of higher eukaryotes, the import machinery of the organelles differs significantly. The majority of proteins entering the glycosome contain a C-terminal PTS1 tri-peptide sequence, which is readily recognized and bound by the soluble cytosolic receptor LPEX5. The receptor binds PTS1 cargo in the cytosol, shuttling it to the glycosomal membrane where the protein interacts with LPEX14, a peripherally membrane bound protein. The interaction with LPEX14 at the glycosomal membrane, facilitated by several other biogenesis proteins, initiates the formation of a transient import pore. In this thesis research project the role of Leishmania donovani PEX5 (LdPEX5) in formation of this crucial import pore was analyzed. Using biophysical techniques, it was found that interaction of the receptor with a PTS1 did not cause major changes in secondary structure, although did provoke a conformational change in the protein, preceding and possibly facilitating its interactions with LdPEX14 at a glycosomal membrane. Using large unilamellar vesicles mimicking the glycosomal lipid composition, the domain of LdPEX5 necessary to interact with LdPEX14 was then narrowed to 268-302. Furthermore, using serial carbonate-urea extractions, the domain identified to be necessary for interaction with LdPEX14 at a glycosomal mimetic was also found to insert into the liposomal membrane, implying that the insertion of LdPEX14 into the glycosomal membrane could be drawing LdPEX5 into the membrane as part of pore formation. In conclusion, this study has implicated LdPEX5 in having a central role in formation of the transient glycosomal import pore.Les parasites du genre Leishmania provoquent un large éventail de maladies, appelées collectivement leishmanioses. Ces maladies varient en termes de morbidité ; la forme cutanée se conclut généralement par une auto-guérison, alors que les manifestations cutanéo-muqueuses et viscérales nécessitent une intervention chimiothérapeutique pour éviter le décès. À l'heure actuelle, il n'existe aucun vaccin, et les méthodes actuelles d'intervention chimiothérapeutique présentent de graves conséquences. Il existe aujourd'hui un besoin urgent de trouver de nouvelles options pour lutter contre ces maladies destructrices. Un organite dans le parasite, le glycosome, a été identifié comme une cible thérapeutique intéressante. Le glycosome compartimente plusieurs enzymes de biosynthèses et voies métaboliques importantes; il a été prouvé que cet organite est nécessaire pour assurer la viabilité du parasite. Bien que l'organite soit structurellement et évolutivement lié aux peroxysomes des eucaryotes supérieurs, le mécanisme d'importation des organites diffère sensiblement. La majorité des protéines entrant dans le glycosome contient une séquence tri-peptide PTS1 C-terminal, qui est facilement reconnue et liée par le récepteur cytosolique soluble LPEX5. Le récepteur se lie au cargo PTS1 dans le cytosol, le conduisant vers la membrane glycosomale où la protéine interagit avec LPEX14, une protéine liée à la membrane périphérique. L'interaction avec LPEX14 au niveau de la membrane glycosomale, facilitée par plusieurs autres protéines de biogenèse, initie la formation d'un pore d'importation transitoire. Dans ce projet de thèse, le rôle de PEX5 dans la formation de ce pore d'importations essentiel a été analysé chez Leishmania donovani. En utilisant des techniques biophysiques, il a été constaté que l'interaction du récepteur avec un PTS1 n'a pas causé de changements majeurs dans la structure secondaire, bien qu'elle ait provoqué un changement de conformation de la protéine, précédant et éventuellement facilitant ses interactions avec LdPEX14 à une membrane glycosomale. Grâce à l'utilisation de grandes vésicules unilamellaires mimant la composition lipidique glycosomale, le domaine de LdPEX5 nécessaire pour interagir avec LdPEX14 fut ramené à 268-302. En outre, en utilisant des extractions carbonate-urée en série, il a été prouvé que le domaine identifié comme étant nécessaire pour l'interaction avec LdPEX14 au mimétique glycosomal, s'insère dans la membrane liposomale. De ce fait, l'insertion de LdPEX14 dans la membrane glycosomale pourrait tirer LdPEX5 dans la membrane dans le contexte de la formation de pores. En conclusion, cette étude a démontré que LdPEX5 possède un rôle central dans la formation du pore d'importation glycosomale transitoire

Proteome and Dihydrorhodamine Profiling of Bronchoalveolar Lavage in Patients with Chronic Pulmonary Aspergillosis

Neutrophil and (alveolar) macrophage immunity is considered crucial for eliminating Aspergillus fumigatus. Data derived from bronchoalveloar lavage (BAL) characterizing the human immuno-pulmonary response to Aspergillus fumigatus are non-existent. To obtain a comprehensive picture of the immune pathways involved in chronic pulmonary aspergillosis (CPA), we performed proteome analysis on AL of 9 CPA patients and 17 patients with interstitial lung disease (ILD). The dihydrorhodamine (DHR) test was also performed on BAL and blood neutrophils from CPA patients and compared to blood neutrophils from healthy controls (HCs). BAL from CPA patients primarily contained neutrophils, while ILD BAL was also characterized by a large fraction of lymphocytes; these differences likely reflecting the different immunological etiologies underlying the two disorders. BAL and blood neutrophils from CPA patients displayed the same oxidative burst capacity as HC blood neutrophils. Hence, immune evasion by Aspergillus involves other mechanisms than impaired neutrophil oxidative burst capacity per se. CPA BAL was enriched by proteins associated with innate immunity, as well as, more specifically, with neutrophil degranulation, Toll-like receptor 4 signaling, and neutrophil-mediated iron chelation. Our data provide the first comprehensive target organ-derived immune data on the human pulmonary immune response to Aspergillus fumigatus

The hydrophobic region of the <i>Leishmania peroxin</i> 14:requirements for association with a glycosome mimetic membrane

Protein import into the Leishmania glycosome requires docking of the cargo-loaded peroxin 5 (PEX5) receptor to the peroxin 14 (PEX14) bound to the glycosome surface. To examine the LdPEX14-membrane interaction, we purified L. donovani promastigote glycosomes and determined the phospholipid and fatty acid composition. These membranes contained predominately phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol (PG) modified primarily with C18 and C22 unsaturated fatty acid. Using large unilamellar vesicles (LUVs) with a lipid composition mimicking the glycosomal membrane in combination with sucrose density centrifugation and fluorescence-activated cell sorting technique, we established that the LdPEX14 membrane-binding activity was dependent on a predicted transmembrane helix found within residues 149-179. Monolayer experiments showed that the incorporation of PG and phospholipids with unsaturated fatty acids, which increase membrane fluidity and favor a liquid expanded phase, facilitated the penetration of LdPEX14 into biological membranes. Moreover, we demonstrated that the binding of LdPEX5 receptor or LdPEX5-PTS1 receptor-cargo complex was contingent on the presence of LdPEX14 at the surface of LUVs.</p

Genomics of sablefish (Anoplopoma fimbria): expressed genes, mitochondrial phylogeny, linkage map and identification of a putative sex gene

Abstract Background The sablefish (order: Scorpaeniformes) is an economically important species in commercial fisheries of the North Pacific and an emerging species in aquaculture. Aside from a handful of sequences in NCBI and a few published microsatellite markers, little is known about the genetics of this species. The development of genetic tools, including polymorphic markers and a linkage map will allow for the successful development of future broodstock and mapping of phenotypes of interest. The significant sexual dimorphism between females and males makes a genetic test for early identification of sex desirable. Results A full mitochondrial genome is presented and the resulting phylogenetic analysis verifies the placement of the sablefish within the Scorpaeniformes. Nearly 35,000 assembled transcript sequences are used to identify genes and obtain polymorphic SNP and microsatellite markers. 360 transcribed polymorphic loci from two sablefish families produce a map of 24 linkage groups. The sex phenotype maps to sablefish LG14 of the male map. We show significant conserved synteny and conservation of gene-order between the threespine stickleback Gasterosteus aculeatus and sablefish. An additional 1843 polymorphic SNP markers are identified through next-generation sequencing techniques. Sex-specific markers and sequence insertions are identified immediately upstream of the gene gonadal-soma derived factor (gsdf), the master sex determinant locus in the medaka species Oryzias luzonensis. Conclusions The first genomic resources for sablefish provide a foundation for further studies. Over 35,000 transcripts are presented, and the genetic map represents, as far as we can determine, the first linkage map for a member of the Scorpaeniformes. The observed level of conserved synteny and comparative mapping will allow the use of the stickleback genome in future genetic studies on sablefish and other related fish, particularly as a guide to whole-genome assembly. The identification of sex-specific insertions immediately upstream of a known master sex determinant implicates gsdf as an excellent candidate for the master sex determinant for sablefish