Papers

Feasibility of real time next generation sequencing of cancer genes linked to drug response: results from a clinical trial.

Int J Cancer. 2013 Apr 1;132(7):1547-55

Authors: Tran B, Brown AM, Bedard PL, Winquist E, Goss GD, Hotte SJ, Welch SA, Hirte HW, Zhang T, Stein LD, Ferretti V, Watt S, Jiao W, Ng K, Ghai S, Shaw P, Petrocelli T, Hudson TJ, Neel BG, Onetto N, Siu LL, McPherson JD, Kamel-Reid S, Dancey JE

Abstract
The successes of targeted drugs with companion predictive biomarkers and the technological advances in gene sequencing have generated enthusiasm for evaluating personalized cancer medicine strategies using genomic profiling. We assessed the feasibility of incorporating real-time analysis of somatic mutations within exons of 19 genes into patient management. Blood, tumor biopsy and archived tumor samples were collected from 50 patients recruited from four cancer centers. Samples were analyzed using three technologies: targeted exon sequencing using Pacific Biosciences PacBio RS, multiplex somatic mutation genotyping using Sequenom MassARRAY and Sanger sequencing. An expert panel reviewed results prior to reporting to clinicians. A clinical laboratory verified actionable mutations. Fifty patients were recruited. Nineteen actionable mutations were identified in 16 (32%) patients. Across technologies, results were in agreement in 100% of biopsy specimens and 95% of archival specimens. Profiling results from paired archival/biopsy specimens were concordant in 30/34 (88%) patients. We demonstrated that the use of next generation sequencing for real-time genomic profiling in advanced cancer patients is feasible. Additionally, actionable mutations identified in this study were relatively stable between archival and biopsy samples, implying that cancer mutations that are good predictors of drug response may remain constant across clinical stages.

PMID: 22948899 [PubMed - indexed for MEDLINE]

Altered DNA methylation landscapes of polycomb-repressed loci are associated with Gleason score and ERG oncogene expression in prostate cancer.

Clin Cancer Res. 2013 Apr 2;

Authors: Kron K, Trudel D, Pethe V, Briollais L, Fleshner N, van der Kwast T, Bapat B

Abstract
PURPOSE: To assess differentially methylated 'landscapes' according to prostate cancer (PCa) Gleason score (GS) and ERG oncogene expression status, and to determine the extent of polycomb group (PcG) target gene involvement, we sought to assess the genome-wide DNA methylation profile of PCa according to GS and ERG expression. EXPERIMENTAL DESIGN: Genomic DNA from 39 PCa specimens was hybridized to CpG island microarrays through differential methylation hybridization. We compared methylation profiles between GS and ERG expression status as well as GS stratified by ERG expression status. In addition, we compared results from our dataset to publicly available datasets of histone modifications in benign prostate cells. RESULTS: We discovered hundreds of distinct differentially methylated regions (DMRs) associated with increasing GS and ERG. Furthermore, the number of DMRs associated with GS was greatly expanded by stratifying samples into ERG positive versus ERG negative, with ERG positive/GS associated DMRs being primarily hypermethylated as opposed to hypomethylated. Finally, we found that there was a significant overlap between either GS-related or ERG hypermethylated DMRs and distinct regions in benign epithelial cells that have PcG signatures (H3K27me3, SUZ12) and lack active gene expression signatures (H3K4me3, RNA pol II). CONCLUSIONS: This work defines methylation landscapes of PCa according to GS, and suggests that initiating genetic events may influence the PCa epigenome which is further perturbed as PCa progresses. Moreover, CpG islands with silent chromatin signatures in benign cells are particularly susceptible to PCa related hypermethylation.

PMID: 23549870 [PubMed - as supplied by publisher]

SH3 interactome conserves general function over specific form.

Mol Syst Biol. 2013 Apr 2;9:652

Authors: Xin X, Gfeller D, Cheng J, Tonikian R, Sun L, Guo A, Lopez L, Pavlenco A, Akintobi A, Zhang Y, Rual JF, Currell B, Seshagiri S, Hao T, Yang X, Shen YA, Salehi-Ashtiani K, Li J, Cheng AT, Bouamalay D, Lugari A, Hill DE, Grimes ML, Drubin DG, Grant BD, Vidal M, Boone C, Sidhu SS, Bader GD

Abstract
Src homology 3 (SH3) domains bind peptides to mediate protein-protein interactions that assemble and regulate dynamic biological processes. We surveyed the repertoire of SH3 binding specificity using peptide phage display in a metazoan, the worm Caenorhabditis elegans, and discovered that it structurally mirrors that of the budding yeast Saccharomyces cerevisiae. We then mapped the worm SH3 interactome using stringent yeast two-hybrid and compared it with the equivalent map for yeast. We found that the worm SH3 interactome resembles the analogous yeast network because it is significantly enriched for proteins with roles in endocytosis. Nevertheless, orthologous SH3 domain-mediated interactions are highly rewired. Our results suggest a model of network evolution where general function of the SH3 domain network is conserved over its specific form.

PMID: 23549480 [PubMed - in process]

Integrated bioinformatic and targeted deletion analyses of the SRS gene superfamily identify SRS29C as a negative regulator of Toxoplasma virulence.

MBio. 2012;3(6)

Authors: Wasmuth JD, Pszenny V, Haile S, Jansen EM, Gast AT, Sher A, Boyle JP, Boulanger MJ, Parkinson J, Grigg ME

Abstract
The Toxoplasma gondii SRS gene superfamily is structurally related to SRS29B (formerly SAG1), a surface adhesin that binds host cells and stimulates host immunity. Comparative genomic analyses of three Toxoplasma strains identified 182 SRS genes distributed across 14 chromosomes at 57 genomic loci. Eight distinct SRS subfamilies were resolved. A core 69 functional gene orthologs were identified, and strain-specific expansions and pseudogenization were common. Gene expression profiling demonstrated differential expression of SRS genes in a developmental-stage- and strain-specific fashion and identified nine SRS genes as priority targets for gene deletion among the tissue-encysting coccidia. A Δsag1 sag2A mutant was significantly attenuated in murine acute virulence and showed upregulated SRS29C (formerly SRS2) expression. Transgenic overexpression of SRS29C in the virulent RH parent was similarly attenuated. Together, these findings reveal SRS29C to be an important regulator of acute virulence in mice and demonstrate the power of integrated genomic analysis to guide experimental investigations. IMPORTANCE: Parasitic species employ large gene families to subvert host immunity to enable pathogen colonization and cause disease. Toxoplasma gondii contains a large surface coat gene superfamily that encodes adhesins and virulence factors that facilitate infection in susceptible hosts. We generated an integrated bioinformatic resource to predict which genes from within this 182-gene superfamily of adhesin-encoding genes play an essential role in the host-pathogen interaction. Targeted gene deletion experiments with predicted candidate surface antigens identified SRS29C as an important negative regulator of acute virulence in murine models of Toxoplasma infection. Our integrated computational and experimental approach provides a comprehensive framework, or road map, for the assembly and discovery of additional key pathogenesis genes contained within other large surface coat gene superfamilies from a broad array of eukaryotic pathogens.

PMID: 23149485 [PubMed - indexed for MEDLINE]

Microarray meta-analysis identifies acute lung injury biomarkers in donor lungs that predict development of primary graft failure in recipients.

PLoS One. 2012;7(10):e45506

Authors: Hu P, Wang X, Haitsma JJ, Furmli S, Masoom H, Liu M, Imai Y, Slutsky AS, Beyene J, Greenwood CM, dos Santos C

Abstract
OBJECTIVES: To perform a meta-analysis of gene expression microarray data from animal studies of lung injury, and to identify an injury-specific gene expression signature capable of predicting the development of lung injury in humans.
METHODS: We performed a microarray meta-analysis using 77 microarray chips across six platforms, two species and different animal lung injury models exposed to lung injury with or/and without mechanical ventilation. Individual gene chips were classified and grouped based on the strategy used to induce lung injury. Effect size (change in gene expression) was calculated between non-injurious and injurious conditions comparing two main strategies to pool chips: (1) one-hit and (2) two-hit lung injury models. A random effects model was used to integrate individual effect sizes calculated from each experiment. Classification models were built using the gene expression signatures generated by the meta-analysis to predict the development of lung injury in human lung transplant recipients.
RESULTS: Two injury-specific lists of differentially expressed genes generated from our meta-analysis of lung injury models were validated using external data sets and prospective data from animal models of ventilator-induced lung injury (VILI). Pathway analysis of gene sets revealed that both new and previously implicated VILI-related pathways are enriched with differentially regulated genes. Classification model based on gene expression signatures identified in animal models of lung injury predicted development of primary graft failure (PGF) in lung transplant recipients with larger than 80% accuracy based upon injury profiles from transplant donors. We also found that better classifier performance can be achieved by using meta-analysis to identify differentially-expressed genes than using single study-based differential analysis.
CONCLUSION: Taken together, our data suggests that microarray analysis of gene expression data allows for the detection of "injury" gene predictors that can classify lung injury samples and identify patients at risk for clinically relevant lung injury complications.

PMID: 23071521 [PubMed - indexed for MEDLINE]

A systematic review and meta-analysis of anti-pseudomonal penicillins and carbapenems in pediatric febrile neutropenia.

Support Care Cancer. 2012 Oct;20(10):2295-304

Authors: Manji A, Lehrnbecher T, Dupuis LL, Beyene J, Sung L

Abstract
PURPOSE: Carbapenems represent a broad-spectrum alternative to anti-pseudomonal penicillin (APP) combination or single-agent therapy for the management of pediatric febrile neutropenia (FN). Our primary objective was to describe the risk of treatment failure in children treated with an APP or carbapenem as initial empiric treatment for FN. Our secondary objective was to compare outcomes of APP versus carbapenem therapy in this population.
METHODS: An electronic search of Ovid Medline, EMBASE, and the Cochrane Central Register of Controlled Trials was performed. We limited studies to prospective pediatric trials of FN in which at least one treatment arm consisted of an APP (with or without an aminoglycoside) or a carbapenem.
RESULTS: Of 7,281 articles reviewed, 27 studies comprising 30 treatment regimens were included for meta-analysis. Treatment failure, including antibiotic modification, occurred in 41% (95% confidence interval (CI) 32-50%), 34% (95% CI 27-41%), and 35% (95% CI 24-45%) of patients treated with APP-aminoglycoside, APP monotherapy, and carbapenem monotherapy regimens, respectively. There was no significant difference in treatment failure including antibiotic modification, infection-related mortality, or adverse events when comparing either APP regimen with carbapenem monotherapy. Although a limited number of studies were available, when stratified by FN risk group, no differences were seen in any outcome.
CONCLUSIONS: Our meta-analysis suggests that APP-aminoglycoside, APP monotherapy, and carbapenem monotherapy are all efficacious therapeutic options for the empiric management of pediatric FN.

PMID: 22138849 [PubMed - indexed for MEDLINE]

Ultrasound imaging for lumbar punctures and epidural catheterisations: systematic review and meta-analysis.

BMJ. 2013;346:f1720

Authors: Shaikh F, Brzezinski J, Alexander S, Arzola C, Carvalho JC, Beyene J, Sung L

Abstract
OBJECTIVE: To determine whether ultrasound imaging can reduce the risk of failed lumbar punctures or epidural catheterisations, when compared with standard palpation methods, and whether ultrasound imaging can reduce traumatic procedures, insertion attempts, and needle redirections.
DESIGN: Systematic review and meta-analysis of randomised controlled trials.
DATA SOURCES: Ovid Medline, Embase, and Cochrane Central Register of Controlled Trials up to May 2012, without restriction by language or publication status.
REVIEW METHODS: Randomised trials that compared ultrasound imaging with standard methods (no imaging) in the performance of a lumbar puncture or epidural catheterisation were identified.
RESULTS: 14 studies with a total of 1334 patients were included (674 patients assigned to the ultrasound group, 660 to the control group). Five studies evaluated lumbar punctures and nine evaluated epidural catheterisations. Six of 624 procedures conducted in the ultrasound group failed; 44 of 610 procedures in the control group failed. Ultrasound imaging reduced the risk of failed procedures (risk ratio 0.21 (95% confidence interval 0.10 to 0.43), P<0.001). Risk reduction was similar when subgroup analysis was performed for lumbar punctures (risk ratio 0.19 (0.07 to 0.56), P=0.002) or epidural catheterisations (0.23 (0.09 to 0.60), P=0.003). Ultrasound imaging also significantly reduced the risk of traumatic procedures (risk ratio 0.27 (0.11 to 0.67), P=0.005), the number of insertion attempts (mean difference -0.44 (-0.64 to -0.24), P<0.001), and the number of needle redirections (mean difference -1.00 (-1.24 to -0.75), P<0.001).
CONCLUSIONS: Ultrasound imaging can reduce the risk of failed or traumatic lumbar punctures and epidural catheterisations, as well as the number of needle insertions and redirections. Ultrasound may be a useful adjunct for these procedures.

PMID: 23532866 [PubMed - in process]

Vitamin D intake is negatively associated with promoter methylation of the Wnt antagonist gene DKK1 in a large group of colorectal cancer patients.

Nutr Cancer. 2012;64(7):919-28

Authors: Rawson JB, Sun Z, Dicks E, Daftary D, Parfrey PS, Green RC, Gallinger S, McLaughlin JR, Wang PP, Knight JA, Bapat B

Abstract
Diet and lifestyle influence colorectal cancer (CRC) risk but the molecular events that mediate these effects are poorly characterized. Several dietary and lifestyle factors can modulate DNA methylation suggesting that they may influence CRC risk through epigenetic regulation of cancer-related genes. The Wnt regulatory genes DKK1 and Wnt5a are important contributors to colonic carcinogenesis and are often silenced by promoter hypermethylation in CRC; however, the dietary contributions to these events have not been explored. To investigate the link between dietary/lifestyle factors and epigenetic regulation of these Wnt signaling genes, we assessed promoter methylation of these genes in a large cohort of Canadian CRC patients from Ontario (n = 549) and Newfoundland (n = 443) and examined associations to dietary/lifestyle factors implicated in CRC risk and/or DNA methylation including intake of vitamins, fats, cholesterol, fiber, and alcohol as well as body mass index (BMI), and smoking status. Several factors were associated with methylation status including alcohol intake, BMI, and cigarette smoking. Most significantly, however, dietary vitamin D intake was strongly negatively associated with DKK1 methylation in Newfoundland (P = 0.001) and a similar trend was observed in Ontario. These results suggest that vitamin D and other dietary/lifestyle factors may alter CRC risk by mediating extracellular Wnt inhibition.

PMID: 22966878 [PubMed - indexed for MEDLINE]

Construction and validation of a homology model of the human voltage-gated proton channel hHV1.

J Gen Physiol. 2013 Apr;141(4):445-65

Authors: Kulleperuma K, Smith SM, Morgan D, Musset B, Holyoake J, Chakrabarti N, Cherny VV, Decoursey TE, Pomès R

Abstract
The topological similarity of voltage-gated proton channels (HV1s) to the voltage-sensing domain (VSD) of other voltage-gated ion channels raises the central question of whether HV1s have a similar structure. We present the construction and validation of a homology model of the human HV1 (hHV1). Multiple structural alignment was used to construct structural models of the open (proton-conducting) state of hHV1 by exploiting the homology of hHV1 with VSDs of K(+) and Na(+) channels of known three-dimensional structure. The comparative assessment of structural stability of the homology models and their VSD templates was performed using massively repeated molecular dynamics simulations in which the proteins were allowed to relax from their initial conformation in an explicit membrane mimetic. The analysis of structural deviations from the initial conformation based on up to 125 repeats of 100-ns simulations for each system reveals structural features consistently retained in the homology models and leads to a consensus structural model for hHV1 in which well-defined external and internal salt-bridge networks stabilize the open state. The structural and electrostatic properties of this open-state model are compatible with proton translocation and offer an explanation for the reversal of charge selectivity in neutral mutants of Asp(112). Furthermore, these structural properties are consistent with experimental accessibility data, providing a valuable basis for further structural and functional studies of hHV1. Each Arg residue in the S4 helix of hHV1 was replaced by His to test accessibility using Zn(2+) as a probe. The two outermost Arg residues in S4 were accessible to external solution, whereas the innermost one was accessible only to the internal solution. Both modeling and experimental data indicate that in the open state, Arg(211), the third Arg residue in the S4 helix in hHV1, remains accessible to the internal solution and is located near the charge transfer center, Phe(150).

PMID: 23530137 [PubMed - in process]

Intertwined Associations in Structures of Homooligomeric Proteins.

Structure. 2013 Mar 20;

Authors: Mackinnon S, Malevanets A, Wodak SJ

Abstract
Intertwined homo-oligomers are complexes comprising identical protein subunits, where small segments or compact protein substructures (domains) are exchanged between the subunits. Using a formal definition of intertwined homo-oligomers, we survey the Protein Data Bank for all such complexes. Results show that intertwining occurs in 13,442 (24%) of all surveyed structures. A majority (∼72%) exchanges one contiguous chain segment of varying length. Another ∼10%, exchange structural domains, and the remaining ∼20% display complex intertwining topologies. Smaller proteins are more often intertwined, and intertwining is dominant in solution homodimers. These findings and analyses of various properties of the major category of intertwined complexes, their interfaces and quaternary context, support the physiological role of intertwining in promoting homooligomer stability. Furthermore, the number of different intertwining modes observed in families of related proteins is limited, and likely specific to the protein fold. These findings yield unique insights into the role of intertwining in homomeric association.

PMID: 23523426 [PubMed - as supplied by publisher]

Response to commentaries on the Canadian Network for Mood and Anxiety Treatments/International Society for Bipolar Disorders 2013 updated Bipolar Disorder Guidelines.

Bipolar Disord. 2013 Mar 25;

Authors: Schaffer A, Parikh SV, Kennedy SH, Milev R, Frey BN, Goldstein BI, Beaulieu S, Alda M, O'Donovan C, Macqueen G, McIntyre RS, Sharma V, Ravindran A, Young LT, Bond DJ, Lam RW, Yatham LN

PMID: 23521579 [PubMed - as supplied by publisher]

Metabolic adaptation to chronic inhibition of mitochondrial protein synthesis in acute myeloid leukemia cells.

PLoS One. 2013;8(3):e58367

Authors: Jhas B, Sriskanthadevan S, Skrtic M, Sukhai MA, Voisin V, Jitkova Y, Gronda M, Hurren R, Laister RC, Bader GD, Minden MD, Schimmer AD

Abstract
Recently, we demonstrated that the anti-bacterial agent tigecycline preferentially induces death in leukemia cells through the inhibition of mitochondrial protein synthesis. Here, we sought to understand mechanisms of resistance to tigecycline by establishing a leukemia cell line resistant to the drug. TEX leukemia cells were treated with increasing concentrations of tigecycline over 4 months and a population of cells resistant to tigecycline (RTEX+TIG) was selected. Compared to wild type cells, RTEX+TIG cells had undetectable levels of mitochondrially translated proteins Cox-1 and Cox-2, reduced oxygen consumption and increased rates of glycolysis. Moreover, RTEX+TIG cells were more sensitive to inhibitors of glycolysis and more resistant to hypoxia. By electron microscopy, RTEX+TIG cells had abnormally swollen mitochondria with irregular cristae structures. RNA sequencing demonstrated a significant over-representation of genes with binding sites for the HIF1α:HIF1β transcription factor complex in their promoters. Upregulation of HIF1α mRNA and protein in RTEX+TIG cells was confirmed by Q-RTPCR and immunoblotting. Strikingly, upon removal of tigecycline from RTEX+TIG cells, the cells re-established aerobic metabolism. Levels of Cox-1 and Cox-2, oxygen consumption, glycolysis, mitochondrial mass and mitochondrial membrane potential returned to wild type levels, but HIF1α remained elevated. However, upon re-treatment with tigecycline for 72 hours, the glycolytic phenotype was re-established. Thus, we have generated cells with a reversible metabolic phenotype by chronic treatment with an inhibitor of mitochondrial protein synthesis. These cells will provide insight into cellular adaptations used to cope with metabolic stress.

PMID: 23520503 [PubMed - in process]

Molecular hyperdiversity and evolution in very large populations.

Mol Ecol. 2013 Mar 18;

Authors: Cutter AD, Jovelin R, Dey A

Abstract
The genomic density of sequence polymorphisms critically affects the sensitivity of inferences about ongoing sequence evolution, function and demographic history. Most animal and plant genomes have relatively low densities of polymorphisms, but some species are hyperdiverse with neutral nucleotide heterozygosity exceeding 5%. Eukaryotes with extremely large populations, mimicking bacterial and viral populations, present novel opportunities for studying molecular evolution in sexually reproducing taxa with complex development. In particular, hyperdiverse species can help answer controversial questions about the evolution of genome complexity, the limits of natural selection, modes of adaptation and subtleties of the mutation process. However, such systems have some inherent complications and here we identify topics in need of theoretical developments. Close relatives of the model organisms Caenorhabditis elegans and Drosophila melanogaster provide known examples of hyperdiverse eukaryotes, encouraging functional dissection of resulting molecular evolutionary patterns. We recommend how best to exploit hyperdiverse populations for analysis, for example, in quantifying the impact of noncrossover recombination in genomes and for determining the identity and micro-evolutionary selective pressures on noncoding regulatory elements.

PMID: 23506466 [PubMed - as supplied by publisher]

Pesticide use, immunologic conditions, and risk of non-Hodgkin lymphoma in Canadian men in six provinces.

Int J Cancer. 2012 Dec 1;131(11):2650-9

Authors: Pahwa M, Harris SA, Hohenadel K, McLaughlin JR, Spinelli JJ, Pahwa P, Dosman JA, Blair A

Abstract
Pesticide exposures and immune suppression have been independently associated with the risk of non-Hodgkin lymphoma (NHL), but their joint effect has not been well explored. Data from a case-control study of men from six Canadian provinces were used to evaluate the potential effect modification of asthma, allergies, or asthma and allergies and hay fever combined on NHL risk from use of: (i) any pesticide; (ii) any organochlorine insecticide; (iii) any organophosphate insecticide; (iv) any phenoxy herbicide; (v) selected individual pesticides [1,1'-(2,2,2-trichloroethylidene)bis[4-chlorobenzene]; 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (DDT), malathion, (4-chloro-2-methylphenoxy)acetic acid (MCPA), mecoprop, and (2,4-dichlorophenoxy)acetic acid (2,4-D); and (vi) from the number of potentially carcinogenic pesticides. Incident NHL cases (n = 513) diagnosed between 1991 and 1994 were recruited from provincial cancer registries and hospitalization records and compared to 1,506 controls. A stratified analysis was conducted to calculate odds ratios (ORs) adjusted for age, province, proxy respondent, and diesel oil exposure. Subjects with asthma, allergies, or hay fever had non-significantly elevated risks of NHL associated with use of MCPA (OR = 2.67, 95% confidence interval [CI]: 0.90-7.93) compared to subjects without any of these conditions (OR = 0.81, 95% CI: 0.39-1.70). Conversely, those with asthma, allergies, or hay fever who reported use of malathion had lower risks of NHL (OR = 1.25, 95% CI: 0.69-2.26) versus subjects with none of these conditions (OR = 2.44, 95% CI: 1.65-3.61). Similar effects were observed for asthma and allergies evaluated individually. Although there were some leads regarding effect modification by these immunologic conditions on the association between pesticide use and NHL, small numbers, measurement error and possible recall bias limit interpretation of these results.

PMID: 22396152 [PubMed - indexed for MEDLINE]

MSH3 Polymorphisms and Protein Levels Affect CAG Repeat Instability in Huntington's Disease Mice.

PLoS Genet. 2013 Feb;9(2):e1003280

Authors: Tomé S, Manley K, Simard JP, Clark GW, Slean MM, Swami M, Shelbourne PF, Tillier ER, Monckton DG, Messer A, Pearson CE

Abstract
Expansions of trinucleotide CAG/CTG repeats in somatic tissues are thought to contribute to ongoing disease progression through an affected individual's life with Huntington's disease or myotonic dystrophy. Broad ranges of repeat instability arise between individuals with expanded repeats, suggesting the existence of modifiers of repeat instability. Mice with expanded CAG/CTG repeats show variable levels of instability depending upon mouse strain. However, to date the genetic modifiers underlying these differences have not been identified. We show that in liver and striatum the R6/1 Huntington's disease (HD) (CAG)∼100 transgene, when present in a congenic C57BL/6J (B6) background, incurred expansion-biased repeat mutations, whereas the repeat was stable in a congenic BALB/cByJ (CBy) background. Reciprocal congenic mice revealed the gene as the determinant for the differences in repeat instability. Expansion bias was observed in congenic mice homozygous for the B6 gene on a CBy background, while the CAG tract was stabilized in congenics homozygous for the CBy gene on a B6 background. The CAG stabilization was as dramatic as genetic deficiency of . The B6 and CBy genes had identical promoters but differed in coding regions and showed strikingly different protein levels. B6 MSH3 variant protein is highly expressed and associated with CAG expansions, while the CBy MSH3 variant protein is expressed at barely detectable levels, associating with CAG stability. The DHFR protein, which is divergently transcribed from a promoter shared by the gene, did not show varied levels between mouse strains. Thus, naturally occurring MSH3 protein polymorphisms are modifiers of CAG repeat instability, likely through variable MSH3 protein stability. Since evidence supports that somatic CAG instability is a modifier and predictor of disease, our data are consistent with the hypothesis that variable levels of CAG instability associated with polymorphisms of DNA repair genes may have prognostic implications for various repeat-associated diseases.

PMID: 23468640 [PubMed - in process]

Detergent-Mediated Protein Aggregation.

Chem Phys Lipids. 2013 Mar 2;

Authors: Neale C, Ghanei H, Holyoake J, Bishop RE, Privé GG, Pomès R

Abstract
Because detergents are commonly used to solvate membrane proteins for structural evaluation, much attention has been devoted to assessing the conformational bias imparted by detergent micelles in comparison to the native environment of the lipid bilayer. Here, we conduct six 500-ns simulations of a system with >600,000 atoms to investigate the spontaneous self assembly of dodecylphosphocholine detergent around multiple molecules of the integral membrane protein PagP. This detergent formed equatorial micelles in which acyl chains surround the protein's hydrophobic belt, confirming existing models of the detergent solvation of membrane proteins. In addition, unexpectedly, the extracellular and periplasmic apical surfaces of PagP interacted with the headgroups of detergents in other micelles 85% and 60% of the time, respectively, forming complexes that were stable for hundreds of nanoseconds. In some cases, an apical surface of one molecule of PagP interacted with an equatorial micelle surrounding another molecule of PagP. In other cases, the apical surfaces of two molecules of PagP simultaneously bound a neat detergent micelle. In these ways, detergents mediated the non-specific aggregation of folded PagP. These simulation results are consistent with dynamic light scattering experiments, which show that, at detergent concentrations ≥600mM, PagP induces the formation of large scattering species that are likely to contain many copies of the PagP protein. Together, these simulation and experimental results point to a potentially generic mechanism of detergent-mediated protein aggregation.

PMID: 23466535 [PubMed - as supplied by publisher]

RIPSeeker: a statistical package for identifying protein-associated transcripts from RIP-seq experiments.

Nucleic Acids Res. 2013 Feb 28;

Authors: Li Y, Zhao DY, Greenblatt JF, Zhang Z

Abstract
RIP-seq has recently been developed to discover genome-wide RNA transcripts that interact with a protein or protein complex. RIP-seq is similar to both RNA-seq and ChIP-seq, but presents unique properties and challenges. Currently, no statistical tool is dedicated to RIP-seq analysis. We developed RIPSeeker (http://www.bioconductor.org/packages/2.12/bioc/html/RIPSeeker.html), a free open-source Bioconductor/R package for de novo RIP peak predictions based on HMM. To demonstrate the utility of the software package, we applied RIPSeeker and six other published programs to three independent RIP-seq datasets and two PAR-CLIP datasets corresponding to six distinct RNA-binding proteins. Based on receiver operating curves, RIPSeeker demonstrates superior sensitivity and specificity in discriminating high-confidence peaks that are consistently agreed on among a majority of the comparison methods, and dominated 9 of the 12 evaluations, averaging 80% area under the curve. The peaks from RIPSeeker are further confirmed based on their significant enrichment for biologically meaningful genomic elements, published sequence motifs and association with canonical transcripts known to interact with the proteins examined. While RIPSeeker is specifically tailored for RIP-seq data analysis, it also provides a suite of bioinformatics tools integrated within a self-contained software package comprehensively addressing issues ranging from post-alignments' processing to visualization and annotation.

PMID: 23455476 [PubMed - as supplied by publisher]

Long-term ovarian cancer survival associated with mutation in BRCA1 or BRCA2.

J Natl Cancer Inst. 2013 Jan 16;105(2):141-8

Authors: McLaughlin JR, Rosen B, Moody J, Pal T, Fan I, Shaw PA, Risch HA, Sellers TA, Sun P, Narod SA

Abstract
BACKGROUND: Studies have suggested that the 5-year survival of women with ovarian cancer and a BRCA1 or BRCA2 mutation is better than expected. We sought to evaluate the impact of carrying a BRCA1 or BRCA2 mutation on long-term survival of women after a diagnosis of invasive ovarian cancer.
METHODS: One thousand six hundred twenty-six unselected women diagnosed with invasive ovarian cancer in Ontario, Canada, or in Tampa, Florida, between 1995 and 2004 were followed for a mean of 6.9 years (range = 0.3 to 15.7 years). Mutation screening for BRCA1 and BRCA2 revealed mutations in 218 women (13.4%). Left-truncated survival analysis was conducted to estimate ovarian cancer-specific survival at various time points after diagnosis for women with and without mutations.
RESULTS: In the 3-year period after diagnosis, the presence of a BRCA1 or BRCA2 mutation was associated with a better prognosis (adjusted hazard ratio = 0.68, 95% confidence interval [CI] = 0.48 to 0.98; P = .03), but at 10 years after diagnosis, the hazard ratio was 1.00 (95% CI = 0.83 to 1.22; P = .90). Among women with serous ovarian cancers, 27.4% of women who were BRCA1 mutation carriers, 27.7% of women who were BRCA2 carriers, and 27.1% of women who were noncarriers were alive at 12 years past diagnosis.
CONCLUSION: For women with invasive ovarian cancer, the short-term survival advantage of carrying a BRCA1 or BRCA2 mutation does not lead to a long-term survival benefit.

PMID: 23257159 [PubMed - indexed for MEDLINE]

The VPS33B-binding protein VPS16B is required in megakaryocyte and platelet α-granule biogenesis.

Blood. 2012 Dec 13;120(25):5032-40

Authors: Urban D, Li L, Christensen H, Pluthero FG, Chen SZ, Puhacz M, Garg PM, Lanka KK, Cummings JJ, Kramer H, Wasmuth JD, Parkinson J, Kahr WH

Abstract
Patients with platelet α or dense δ-granule defects have bleeding problems. Although several proteins are known to be required for δ-granule development, less is known about α-granule biogenesis. Our previous work showed that the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B are required for α-granule biogenesis. Using a yeast two-hybrid screen, mass spectrometry, coimmunoprecipitation, and bioinformatics studies, we identified VPS16B as a VPS33B-binding protein. Immunoblotting confirmed VPS16B expression in various human tissues and cells including megakaryocytes and platelets, and also in megakaryocytic Dami cells. Characterization of platelets from a patient with arthrogryposis, renal dysfunction, and cholestasis (ARC) syndrome containing mutations in C14orf133 encoding VPS16B revealed pale-appearing platelets in blood films and electron microscopy revealed a complete absence of α-granules, whereas δ-granules were observed. Soluble and membrane-bound α-granule proteins were reduced or undetectable, suggesting that both releasable and membrane-bound α-granule constituents were absent. Immunofluorescence microscopy of Dami cells stably expressing GFP-VPS16B revealed that similar to VPS33B, GFP-VPS16B colocalized with markers of the trans-Golgi network, late endosomes and α-granules. We conclude that VPS16B, similar to its binding partner VPS33B, is essential for megakaryocyte and platelet α-granule biogenesis.

PMID: 23002115 [PubMed - indexed for MEDLINE]

A comparative transcriptomic analysis reveals conserved features of stem cell pluripotency in planarians and mammals.

Stem Cells. 2012 Aug;30(8):1734-45

Authors: Labbé RM, Irimia M, Currie KW, Lin A, Zhu SJ, Brown DD, Ross EJ, Voisin V, Bader GD, Blencowe BJ, Pearson BJ

Abstract
Many long-lived species of animals require the function of adult stem cells throughout their lives. However, the transcriptomes of stem cells in invertebrates and vertebrates have not been compared, and consequently, ancestral regulatory circuits that control stem cell populations remain poorly defined. In this study, we have used data from high-throughput RNA sequencing to compare the transcriptomes of pluripotent adult stem cells from planarians with the transcriptomes of human and mouse pluripotent embryonic stem cells. From a stringently defined set of 4,432 orthologs shared between planarians, mice and humans, we identified 123 conserved genes that are ≥5-fold differentially expressed in stem cells from all three species. Guided by this gene set, we used RNAi screening in adult planarians to discover novel stem cell regulators, which we found to affect the stem cell-associated functions of tissue homeostasis, regeneration, and stem cell maintenance. Examples of genes that disrupted these processes included the orthologs of TBL3, PSD12, TTC27, and RACK1. From these analyses, we concluded that by comparing stem cell transcriptomes from diverse species, it is possible to uncover conserved factors that function in stem cell biology. These results provide insights into which genes comprised the ancestral circuitry underlying the control of stem cell self-renewal and pluripotency.

PMID: 22696458 [PubMed - indexed for MEDLINE]