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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.


Lee N.V.,Pfizer | Lira M.E.,Pfizer | Pavlicek A.,Pfizer | Ye J.,Pfizer | And 10 more authors.
PLoS ONE | Year: 2012

Targeting cancers with amplified or abnormally activated c-Met (hepatocyte growth factor receptor) may have therapeutic benefit based on nonclinical and emerging clinical findings. However, the eventual emergence of drug resistant tumors motivates the pre-emptive identification of potential mechanisms of clinical resistance. We rendered a MET amplified gastric cancer cell line, GTL16, resistant to c-Met inhibition with prolonged exposure to a c-Met inhibitor, PF-04217903 (METi). Characterization of surviving cells identified an amplified chromosomal rearrangement between 7q32 and 7q34 which overexpresses a constitutively active SND1-BRAF fusion protein. In the resistant clones, hyperactivation of the downstream MAPK pathway via SND1-BRAF conferred resistance to c-Met receptor tyrosine kinase inhibition. Combination treatment with METi and a RAF inhibitor, PF-04880594 (RAFi) inhibited ERK activation and circumvented resistance to either single agent. Alternatively, treatment with a MEK inhibitor, PD-0325901 (MEKi) alone effectively blocked ERK phosphorylation and inhibited cell growth. Our results suggest that combination of a c-Met tyrosine kinase inhibitor with a BRAF or a MEK inhibitor may be effective in treating resistant tumors that use activated BRAF to escape suppression of c-Met signaling. © 2012 Lee et al.


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.


Laurie J.D.,Helmholtz Center for Environmental Research | Laurie J.D.,University of Arizona | Ali S.,Agriculture and Agri Food Canada | Ali S.,Université 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.


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.


Khodabakhshi A.H.,Michael Smith Genome science Center | Morin R.D.,Michael Smith Genome science Center | Fejes A.P.,Michael Smith Genome science Center | Mungall A.J.,Michael Smith Genome science Center | And 12 more authors.
Oncotarget | Year: 2012

Somatic hypermutation (SHM) in the variable region of immunoglobulin genes (IGV) naturally occurs in a narrow window of B cell development to provide high-affinity antibodies. However, SHM can also aberrantly target proto-oncogenes and cause genome instability. The role of aberrant SHM (aSHM) has been widely studied in various non-Hodgkin's lymphoma particularly in diffuse large B-cell lymphoma (DLBCL). Although, it has been speculated that aSHM targets a wide range of genome loci so far only twelve genes have been identified as targets of aSHM through the targeted sequencing of selected genes. A genome-wide study aiming at identifying a comprehensive set of aSHM targets recurrently occurring in DLBCL has not been previously undertaken. Here, we present a comprehensive assessment of the somatic hypermutated genes in DLBCL identified through an analysis of genomic and transcriptome data derived from 40 DLBCL patients. Our analysis verifies that there are indeed many genes that are recurrently affected by aSHM. In particular, we have identified 32 novel targets that show same or higher level of aSHM activity than genes previously reported. Amongst these novel targets, 22 genes showed a significant correlation between mRNA abundance and aSHM. © Khodabakhshi et al.


Johner A.,University of British Columbia | Griffith O.L.,University of British Columbia | Griffith O.L.,Michael Smith Genome science Center | Walker B.,University of British Columbia | And 7 more authors.
Annals of Surgical Oncology | Year: 2011

Background: The objectives of this study were to determine: (1) the incidence permanent hypothyroidism after thyroid lobectomy (TL), (2) whether asymptomatic patients with mildly elevated thyrotropin (TSH) levels can be managed without thyroid hormone replacement, and (3) if the degree of lymphocytic infiltration (LI) and germinal center (GC) formation in the resected thyroid lobe correlates with the development of post-TL hypothyroidism. Methods: Subjects undergoing TL between January 2006 and January 2008 at 2 centers were enrolled in the study and thyroid function was followed prospectively based on a previously published algorithm. The histology of each resected thyroid lobe was examined, and the degree of LI and GC was quantified. Results: The study cohort consisted of 117 patients. Early postoperative TSH levels were significantly increased over preoperative levels (P < .001). TSH measured at 6 months to 1 year postoperatively, while still significantly increased over preoperative levels (P < .001), was also significantly reduced (P = .006) compared with early postoperative levels. Of the patients who presented with early postoperative hypothyroidism, 69.2% recovered to normal levels without intervention. The overall incidence of early postoperative hypothyroidism was 21.6%, and permanent hypothyroidism was 7.8%. A high degree of LI and GC correlated with a significantly higher mean TSH level (P = .003). Conclusions: The incidence of hypothyroidism following TL is low, and a significant proportion of individuals who become biochemically hypothyroid will demonstrate only a transient elevation in their TSH levels. As well, individuals with LI, or GC formation, within their resected thyroid lobe may be at increased risk for post-TL hypothyroidism. © 2011 Society of Surgical Oncology.


Stirling P.C.,University of British Columbia | Stirling P.C.,British Columbia Cancer Agency | Shen Y.,Michael Smith Genome science Center | Corbett R.,Michael Smith Genome science Center | And 2 more authors.
Genetics | Year: 2014

In addition to environmental factors and intrinsic variations in base substitution rates, specific genome-destabilizing mutations can shape the mutational trajectory of genomes. How specific alleles influence the nature and position of accumulated mutations in a genomic context is largely unknown. Understanding the impact of genome-destabilizing alleles is particularly relevant to cancer genomes where biased mutational signatures are identifiable. We first created a more complete picture of cellular pathways that impact mutation rate using a primary screen to identify essential Saccharomyces cerevisiae gene mutations that cause mutator phenotypes. Drawing primarily on new alleles identified in this resource, we measure the impact of diverse mutator alleles on mutation patterns directly by whole-genome sequencing of 68 mutation-accumulation strains derived from wild-type and 11 parental mutator genotypes. The accumulated mutations differ across mutator strains, displaying base-substitution biases, allele-specific mutation hotspots, and break-associated mutation clustering. For example, in mutants of POLα and the Cdc13-Stn1-Ten1 complex, we find a distinct subtelomeric bias for mutations that we show is independent of the target sequence. Together our data suggest that specific genome-instability mutations are sufficient to drive discrete mutational signatures, some of which share properties with mutation patterns seen in tumors. Thus, in a population of cells, genome-instability mutations could influence clonal evolution by establishing discrete mutational trajectories for genomes. © 2014 by the Genetics Society of America.


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.


Mead C.-L.R.,Michael Smith Genome science Center | Kuzyk M.A.,GenoLogics | Moradian A.,Michael Smith Genome science Center | Wilson G.M.,Michael Smith Genome science Center | And 5 more authors.
Journal of Neurochemistry | Year: 2010

Using immunoprecipitation, mass spectrometry, and western blot analysis we investigated cytosolic protein interactions of the schizophrenia susceptibility gene dysbindin in mammalian cells. We identified novel interactions with members of the exocyst, dynactin and chaperonin containing T-complex protein complexes, and we confirmed interactions reported previously with all members of the biogenesis of lysosome-related organelles complex-1 and the adaptor-related protein complex 3. We report interactions between dysbindin and the exocyst and dynactin complex that confirm a link between two important schizophrenia susceptibility genes: dysbindin and disrupted-in-schizophrenia-1. To expand upon this network of interacting proteins we also investigated protein interactions for members of the exocyst and dynactin complexes in mammalian cells. Our results are consistent with the notion that impairment of aspects of the synaptic vesicle life cycle may be a pathogenic mechanism in schizophrenia. © 2010 International Society for Neurochemistry.

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