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Leung A.K.L.,Massachusetts Institute of Technology | Young A.G.,Massachusetts Institute of Technology | Young A.G.,Salk Institute for Biological Studies | Bhutkar A.,Massachusetts Institute of Technology | And 8 more authors.
Nature Structural and Molecular Biology | Year: 2011

MicroRNAs (miRNAs) are 19-22-nucleotide noncoding RNAs that post-transcriptionally regulate mRNA targets. We have identified endogenous miRNA binding sites in mouse embryonic stem cells (mESCs), by performing photo-cross-linking immunoprecipitation using antibodies to Argonaute (Ago2) followed by deep sequencing of RNAs (CLIP-seq). We also performed CLIP-seq in Dicer -' mESCs that lack mature miRNAs, allowing us to define whether the association of Ago2 with the identified sites was miRNA dependent. A significantly enriched motif, GCACUU, was identified only in wild-type mESCs in 3-2 untranslated and coding regions. This motif matches the seed of a miRNA family that constitutes ∼68% of the mESC miRNA population. Unexpectedly, a G-rich motif was enriched in sequences cross-linked to Ago2 in both the presence and absence of miRNAs. Expression analysis and reporter assays confirmed that the seed-related motif confers miRNA-directed regulation on host mRNAs and that the G-rich motif can modulate this regulation. © 2011 Nature America, Inc. All rights reserved.


Choi H.,University of British Columbia | Kasaian K.,Michael Smith Genome science Center | Melck A.,University of British Columbia | Ong K.,University of British Columbia | And 3 more authors.
American Journal of Surgery | Year: 2015

Background The objective of this study was to evaluate whether the clinical presentation of papillary thyroid carcinoma (PTC) has prognostic significance. Methods Retrospective evaluation was carried out of sequential, primary presentation, >1 cm diameter, PTC cases treated at a single center. PTC cases were grouped into 3 groups: (1) incidental detection by imaging, (2) incidental detection by physical examination, and (3) detection because of complaints related to a thyroid mass. The MACIS (metastasis, age, completeness of resection, invasion, and size) system was used to determine cancer prognosis for each group. Results Of the 168 PTC cases, 28 patients (17%) were in group 1, 60 patients (36%) were in group 2, and 80 patients (47%) were in group 3. Overall, 53% of differentiated thyroid cancers were detected incidentally. The difference in the proportion of patients in each MACIS score groups among the 3 clinical presentation categories, and for each component of the MACIS score, was not statistically significant (P =.36). Conclusion The manner in which PTC initially clinically presents has no relationship with cancer prognosis. © 2015 Elsevier Inc. All rights reserved.


Li S.,University of Melbourne | Li S.,Burnet Institute | Li S.,Monash University | Lefranc M.-P.,Montpellier University | And 20 more authors.
Nature Communications | Year: 2013

T cell repertoire diversity and clonotype follow-up in vaccination, cancer, infectious and immune diseases represent a major challenge owing to the enormous complexity of the data generated. Here we describe a next generation methodology, which combines 5′RACE PCR, 454 sequencing and, for analysis, IMGT, the international ImMunoGeneTics information system (IMGT), IMGT/HighV-QUEST web portal and IMGT-ONTOLOGY concepts. The approach is validated in a human case study of T cell receptor beta (TRB) repertoire, by chronologically tracking the effects of influenza vaccination on conventional and regulatory T cell subpopulations. The IMGT/HighV-QUEST paradigm defines standards for genotype/haplotype analysis and characterization of IMGT clonotypes for clonal diversity and expression and achieves a degree of resolution for next generation sequencing verifiable by the user at the sequence level, while providing a normalized reference immunoprofile for human TRB. © 2013 Macmillan Publishers Limited.


Jardon M.A.,University of British Columbia | Braasch K.,University of Manitoba | Leung A.O.,Michael Smith Genome science Center | Cote H.C.,University of Manitoba | And 4 more authors.
Biotechnology and Bioengineering | Year: 2012

Understanding the cellular responses caused by metabolic stress is crucial for the design of robust fed-batch bioprocesses that maximize the expression of recombinant proteins. Chinese hamster ovary cells were investigated in chemically defined, serum-free cultures yielding 10 7cells/mL and up to 500mg/L recombinant tissue-plasminogen activator (t-PA). Upon glutamine depletion increased autophagosome formation and autophagic flux were observed, along with decreased proliferation and high viability. Higher lysosomal levels correlated with decreased productivity. Chemical inhibition of autophagy with 3-methyl adenine (3-MA) increased the t-PA yield by 2.8-fold. Autophagy-related MAP1LC3 and LAMP2 mRNA levels increased continuously in all cultures. Analysis of protein quality revealed that 3-MA treatment did not alter glycan antennarity while increasing fucosylation, galactosylation, and sialylation. Taken together, these findings indicate that inhibition of autophagy can considerably increase the yield of biotechnology fed-batch processes, without compromising the glycosylation capacity of cells. Monitoring or genetic engineering of autophagy provides novel avenues to improve the performance of cell culture-based recombinant protein production. © 2011 Wiley Periodicals, Inc.


Laurie J.D.,Helmholtz Center for Environmental Research | Laurie J.D.,University of Arizona | Ali S.,Agriculture and Agri Food Canada | Ali S.,Universite de Sherbrooke | And 13 more authors.
Plant Cell | Year: 2012

Ustilago hordei is a biotrophic parasite of barley (Hordeum vulgare). After seedling infection, the fungus persists in the plant until head emergence when fungal spores develop and are released from sori formed at kernel positions. The 26.1-Mb U. hordei genome contains 7113 protein encoding genes with high synteny to the smaller genomes of the related, maizeinfecting smut fungi Ustilago maydis and Sporisorium reilianum but has a larger repeat content that affected genome evolution at important loci, including mating-type and effector loci. The U. hordei genome encodes components involved in RNA interference and heterochromatin formation, normally involved in genome defense, that are lacking in the U. maydis genome due to clean excision events. These excision events were possibly a result of former presence of repetitive DNA and of an efficient homologous recombination system in U. maydis. We found evidence of repeat-induced point mutations in the genome of U. hordei, indicating that smut fungi use different strategies to counteract the deleterious effects of repetitive DNA. The complement of U. hordei effector genes is comparable to the other two smuts but reveals differences in family expansion and clustering. The availability of the genome sequence will facilitate the identification of genes responsible for virulence and evolution of smut fungi on their respective hosts. © 2012 American Society of Plant Biologists. All rights reserved.

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