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1.  Trypanosoma brucei Translation Initiation Factor Homolog EIF4E6 Forms a Tripartite Cytosolic Complex with EIF4G5 and a Capping Enzyme Homolog 
Eukaryotic Cell  2014;13(7):896-908.
Trypanosomes lack the transcriptional control characteristic of the majority of eukaryotes that is mediated by gene-specific promoters in a one-gene–one-promoter arrangement. Rather, their genomes are transcribed in large polycistrons with no obvious functional linkage. Posttranscriptional regulation of gene expression must thus play a larger role in these organisms. The eIF4E homolog TbEIF4E6 binds mRNA cap analogs in vitro and is part of a complex in vivo that may fulfill such a role. Knockdown of TbEIF4E6 tagged with protein A-tobacco etch virus protease cleavage site-protein C to approximately 15% of the normal expression level resulted in viable cells that displayed a set of phenotypes linked to detachment of the flagellum from the length of the cell body, if not outright flagellum loss. While these cells appeared and behaved as normal under stationary liquid culture conditions, standard centrifugation resulted in a marked increase in flagellar detachment. Furthermore, the ability of TbEIF4E6-depleted cells to engage in social motility was reduced. The TbEIF4E6 protein forms a cytosolic complex containing a triad of proteins, including the eIF4G homolog TbEIF4G5 and a hypothetical protein of 70.3 kDa, referred to as TbG5-IP. The TbG5-IP analysis revealed two domains with predicted secondary structures conserved in mRNA capping enzymes: nucleoside triphosphate hydrolase and guanylyltransferase. These complex members have the potential for RNA interaction, either via the 5′ cap structure for TbEIF4E6 and TbG5-IP or through RNA-binding domains in TbEIF4G5. The associated proteins provide a signpost for future studies to determine how this complex affects capped RNA molecules.
PMCID: PMC4135740  PMID: 24839125
2.  Low Wear of a Second-generation Highly Crosslinked Polyethylene Liner: A 5-year Radiostereometric Analysis Study 
A sequentially irradiated and annealed, second-generation highly crosslinked polyethylene (XLPE) liner was introduced clinically in 2005 to reduce in vivo oxidation. This liner design has also been shown to reduce wear in vitro when compared with conventional and first-generation crosslinked liners. To date, there is only one study reporting an in vivo wear rate of this liner at 5 years’ followup. However, that study used measurements made from plain radiographs, which have limited sensitivity, particularly when monitoring very low amounts of wear.
What is the amount and direction of wear at 5 years using radiostereometric analysis (RSA) in patients who had THAs that included second-generation XLPE?
We prospectively reviewed 21 patients who underwent primary cementless THA with the same design of XLPE acetabular liner and 32-mm articulation. Tantalum markers were inserted during surgery and all patients had RSA radiographs at 1 week, 6 months, and 1, 2, and 5 years postoperatively. Femoral head penetration within the acetabular component was measured with UmRSA® software. One patient died and two had incomplete radiographs leaving 18 radiographic series for analysis.
The mean amounts of proximal, two-dimensional, and three-dimensional head penetration between 1 week and 5 years were 0.018, 0.071, and 0.149 mm, respectively. The mean proximal, two-dimensional, and three-dimensional wear rates calculated between 1 year and 5 years were all less than 0.001 mm/year with no patient recording a wear rate of more than 0.040 mm/year.
The head penetration of a second-generation XLPE liner remained low at 5 years and the wear rate calculated after the first year was low in all directions. This low level of wear remains encouraging for the future clinical performance of this material.
Level of Evidence
Level IV, therapeutic study. See Instructions for Authors for a complete description of levels of evidence.
PMCID: PMC3792282  PMID: 23893361
3.  Age modification of diabetes-related hospitalization among First Nations adults in Alberta, Canada 
We sought to determine the modifying effects of age and multimorbidity on the association between First Nations status and hospitalizations for diabetes-specific ambulatory care sensitive conditions (ACSC).
We identified 183,654 adults with diabetes from Alberta Canada, and followed them for one year for the outcome of hospitalization or emergency department (ED) visit for a diabetes-specific ACSC. We used logistic regression to determine the association between First Nations status and the outcome, assessing for effect modification by age and multimorbidity with interaction terms. In a model adjusting for age, age2, baseline A1c, duration of diabetes, and multimorbidity, First Nations people were at greater risk than non-First Nations to experience a diabetes-specific hospitalization or ED visit (unadjusted odds ratio [OR] 3.74; 95% confidence interval [CI]: 3.45-4.07). After adjustment for relevant covariates, this association varied by age (interaction: p = 0.018): adjusted OR 3.94 (95% CI: 3.11-4.99) and 5.74 (95% CI: 3.36-9.80) for First Nations compared to non-First Nations at ages 30 and 80 years, respectively.
Compared with non-First Nations, older First Nations patients with diabetes are at greater risk for diabetes-specific hospitalizations. Older First Nations patients with diabetes should be given priority access to primary care services as they are at greatest risk for requiring hospitalization for stabilization of their condition.
PMCID: PMC4192759  PMID: 25309626
American indian; First Nations; Hospitalization; Diabetes mellitus; Risk adjustment
4.  Noninvasive Image Texture Analysis Differentiates K-ras Mutation from Pan-Wildtype NSCLC and Is Prognostic 
PLoS ONE  2014;9(7):e100244.
Non-invasive characterization of a tumor's molecular features could enhance treatment management. Quantitative computed tomography (CT) based texture analysis (QTA) has been used to derive tumor heterogeneity information, and the appearance of the tumors has been shown to relate to patient outcome in non-small cell lung cancer (NSCLC) and other cancers. In this study, we examined the potential of tumoral QTA to differentiate K-ras mutant from pan-wildtype tumors and its prognostic potential using baseline pre-treatment non-contrast CT imaging in NSCLC.
Tumor DNA from patients with early-stage NSCLC was analyzed on the LungCarta Panel. Cases with a K-ras mutation or pan-wildtype for 26 oncogenes and tumor suppressor genes were selected for QTA. QTA was applied to regions of interest in the primary tumor. Non-parametric Mann Whitney test assessed the ability of the QTA, clinical and patient characteristics to differentiate between K-ras mutation from pan-wildtype. A recursive decision tree was developed to determine whether the differentiation of K-ras mutant from pan-wildtype tumors could be improved by sequential application of QTA parameters. Kaplan-Meier survival analysis assessed the ability of these markers to predict survival.
QTA was applied to 48 cases identified, 27 had a K-ras mutation and 21 cases were pan-wildtype. Positive skewness and lower kurtosis were significantly associated with the presence of a K-ras mutation. A five node decision tree had sensitivity, specificity, and accuracy values (95% CI) of 96.3% (78.1–100), 81.0% (50.5–97.4), and 89.6% (72.9–97.0); respectively. Kurtosis was a significant predictor of OS and DFS, with a lower kurtosis value linked with poorer survival.
Lower kurtosis and positive skewness are significantly associated with K-ras mutations. A QTA feature such as kurtosis is prognostic for OS and DFS. Non-invasive QTA can differentiate the presence of K-ras mutation from pan-wildtype NSCLC and is associated with patient survival.
PMCID: PMC4079229  PMID: 24987838
5.  Wear of a 5 Megarad Cross-linked Polyethylene Liner: A 6-year RSA Study 
One cross-linked polyethylene (XLPE) liner is manufactured using a lower dose of radiation, 5 Mrad, which may result in less cross-linking. The reported in vivo wear rate of this XLPE liner in patients undergoing THA has varied, and has included some patients in each reported cohort who had greater than 0.1 mm/year of wear, which is an historical threshold for osteolysis. Previous studies have measured wear on plain radiographs, an approach that has limited sensitivity.
We therefore measured the amount and direction of wear at 6 years using Radiostereometric analysis (RSA) in patients who had THAs that included a cross-linked polyethylene liner manufactured using 5 Mrad radiation.
We prospectively reviewed wear in 30 patients who underwent primary THAs with the same design of cross-linked acetabular liner and a 28-mm articulation. Tantalum markers were inserted during surgery and all patients had RSA radiographic examinations at 1 week, 6 months, 1, 2, and 6 years postoperatively.
The mean proximal, two-dimensional (2-D) and three-dimensional (3-D) wear rates calculated between 1 year and 6 years were 0.014, 0.014, and 0.018 mm/per year, respectively. The direction of the head penetration recorded between 1 week and 6 years was in a proximal direction for all patients, proximolateral for 16 of 24 patients, and proximomedial for eight of 24 patients.
The proximal, 2-D and 3-D wear of a XLPE liner produced using 5 Mrad of radiation was low but measurable by RSA after 6 years. No patients had proximal 2-D or 3-D wear rates exceeding 0.1 mm/year. Further followup is needed to evaluate the effect of XLPE wear particles on the development of long-term osteolysis.
PMCID: PMC3676600  PMID: 23334705
6.  Interplay between Polo kinase, LKB1-activated NUAK1 kinase, PP1βMYPT1 phosphatase complex and the SCFβTrCP E3 ubiquitin ligase 
Biochemical Journal  2014;461(Pt 2):233-245.
NUAK1 (NUAK family SnF1-like kinase-1) and NUAK2 protein kinases are activated by the LKB1 tumour suppressor and have been implicated in regulating multiple processes such as cell survival, senescence, adhesion and polarity. In the present paper we present evidence that expression of NUAK1 is controlled by CDK (cyclin-dependent kinase), PLK (Polo kinase) and the SCFβTrCP (Skp, Cullin and F-boxβTrCP) E3 ubiquitin ligase complex. Our data indicate that CDK phosphorylates NUAK1 at Ser445, triggering binding to PLK, which subsequently phosphorylates NUAK1 at two conserved non-catalytic serine residues (Ser476 and Ser480). This induces binding of NUAK1 to βTrCP, the substrate-recognition subunit of the SCFβTrCP E3 ligase, resulting in NUAK1 becoming ubiquitylated and degraded. We also show that NUAK1 and PLK1 are reciprocally controlled in the cell cycle. In G2–M-phase, when PLK1 is most active, NUAK1 levels are low and vice versa in S-phase, when PLK1 expression is low, NUAK1 is more highly expressed. Moreover, NUAK1 inhibitors (WZ4003 or HTH-01-015) suppress proliferation by reducing the population of cells in S-phase and mitosis, an effect that can be rescued by overexpression of a NUAK1 mutant in which Ser476 and Ser480 are mutated to alanine. Finally, previous work has suggested that NUAK1 phosphorylates and inhibits PP1βMYPT1 (where PP1 is protein phosphatase 1) and that a major role for the PP1βMYPT1 complex is to inhibit PLK1 by dephosphorylating its T-loop (Thr210). We demonstrate that activation of NUAK1 leads to a striking increase in phosphorylation of PLK1 at Thr210, an effect that is suppressed by NUAK1 inhibitors. Our data link NUAK1 to important cell-cycle signalling components (CDK, PLK and SCFβTrCP) and suggest that NUAK1 plays a role in stimulating S-phase, as well as PLK1 activity via its ability to regulate the PP1βMYPT1 phosphatase.
The present study provides insights into the biological regulation of the NUAK isoforms and highlights the remarkable interplay that exists between Polo kinase, NUAK1, PP1βMYPT1 and SCFβTrCP signalling components. It demonstrates NUAK1 inhibitors suppress cell proliferation and PLK1.
PMCID: PMC4109838  PMID: 24785407
AMP-activated protein kinase (AMPK); AMPK-related kinase 5 (ARK5); cell cycle; degron; mitosis; Polo kinase (PLK) ubiquitylation; AMPK, AMP-activated protein kinase; CDK, cyclin-dependent kinase; CK1, casein kinase 1; Cul1, cullin 1; DMEM, Dulbecco’s modified Eagle’s medium; DTB, double thymidine block; Emi1, early mitotic inhibitor 1; GST, glutathione transferase; HA, haemagglutinin; HEK, human embryonic kidney; HRP, horseradish peroxidase; IKK, inhibitor of nuclear factor κB kinase; MEF, mouse embryonic fibroblast; LKB1, liver kinase B1; NEM, N-ethylmaleimide; NUAK, NUAK family SnF1-like kinase; PEI, polyethylenimine; PI, propidium iodide; PLK1, Polo kinase 1; PP1, protein phosphatase 1; SCFβTrCP, Skp, Cullin and F-boxβTrCP; SKP1, S-phase kinase-associated protein 1; Wee1, WEE1 G2 checkpoint kinase; WT, wild-type; XIC, extracted ion chromatogram analysis
7.  Targeting AKT with the allosteric AKT inhibitor MK-2206 in non-small cell lung cancer cells with acquired resistance to cetuximab 
Cancer Biology & Therapy  2013;14(6):481-491.
The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR monoclonal antibody that has been approved for use in oncology. Despite clinical success the majority of patients do not respond to cetuximab and those who initially respond frequently acquire resistance. To understand how tumor cells acquire resistance to cetuximab we developed a model of resistance using the non-small cell lung cancer line NCI-H226. We found that cetuximab-resistant (CtxR) clones manifested strong activation of EGFR, PI3K/AKT and MAPK. To investigate the role of AKT signaling in cetuximab resistance we analyzed the activation of the AKT pathway effector molecules using a human AKT phospho-antibody array. Strong activation was observed in CtxR clones for several key AKT substrates including c-jun, GSK3β, eIF4E, rpS6, IKKα, IRS-1 and Raf1. Inhibition of AKT signaling by siAKT1/2 or by the allosteric AKT inhibitor MK-2206 resulted in robust inhibition of cell proliferation in all CtxR clones. Moreover, the combinational treatment of cetuximab and MK-2206 resulted in further decreases in proliferation than either drug alone. This combinatorial treatment resulted in decreased activity of both AKT and MAPK thus highlighting the importance of simultaneous pathway inhibition to maximally affect the growth of CtxR cells. Collectively, our findings demonstrate that AKT activation is an important pathway in acquired resistance to cetuximab and suggests that combinatorial therapy directed at both the AKT and EGFR/MAPK pathways may be beneficial in this setting.
PMCID: PMC3813564  PMID: 23760490
AKT; EGFR; MK-2206; cetuximab; acquired cetuximab-resistance; non-small cell lung cancer; MAPK
8.  The Mtb Proteome Library: A Resource of Assays to Quantify the Complete Proteome of Mycobacterium tuberculosis 
Cell host & microbe  2013;13(5):602-612.
Research advancing our understanding of Mycobacterium tuberculosis (Mtb) biology and complex host-Mtb interactions requires consistent and precise quantitative measurements of Mtb proteins. We describe the generation and validation of a compendium of assays to quantify 97% of the 4,012 annotated Mtb proteins by the targeted mass spectrometric method selected reaction monitoring (SRM). Furthermore, we estimate the absolute abundance for 55% of all Mtb proteins, revealing a dynamic range within the Mtb proteome of over four orders of magnitude, and identify previously un-annotated proteins. As an example of the assay library utility, we monitored the entire Mtb dormancy survival regulon (DosR), which is linked to anaerobic survival and Mtb persistence, and show its dynamic protein-level regulation during hypoxia. In conclusion, we present a publicly available research resource that supports the sensitive, precise, and reproducible quantification of virtually any Mtb protein by a robust and widely accessible mass spectrometric method.
PMCID: PMC3766585  PMID: 23684311
9.  Parkin is activated by PINK1-dependent phosphorylation of ubiquitin at Ser65 
Biochemical Journal  2014;460(Pt 1):127-139.
We have previously reported that the Parkinson's disease-associated kinase PINK1 (PTEN-induced putative kinase 1) is activated by mitochondrial depolarization and stimulates the Parkin E3 ligase by phosphorylating Ser65 within its Ubl (ubiquitin-like) domain. Using phosphoproteomic analysis, we identified a novel ubiquitin phosphopeptide phosphorylated at Ser65 that was enriched 14-fold in HEK (human embryonic kidney)-293 cells overexpressing wild-type PINK1 stimulated with the mitochondrial uncoupling agent CCCP (carbonyl cyanide m-chlorophenylhydrazone), to activate PINK1, compared with cells expressing kinase-inactive PINK1. Ser65 in ubiquitin lies in a similar motif to Ser65 in the Ubl domain of Parkin. Remarkably, PINK1 directly phosphorylates Ser65 of ubiquitin in vitro. We undertook a series of experiments that provide striking evidence that Ser65-phosphorylated ubiquitin (ubiquitinPhospho−Ser65) functions as a critical activator of Parkin. First, we demonstrate that a fragment of Parkin lacking the Ubl domain encompassing Ser65 (ΔUbl-Parkin) is robustly activated by ubiquitinPhospho−Ser65, but not by non-phosphorylated ubiquitin. Secondly, we find that the isolated Parkin Ubl domain phosphorylated at Ser65 (UblPhospho−Ser65) can also activate ΔUbl-Parkin similarly to ubiquitinPhospho−Ser65. Thirdly, we establish that ubiquitinPhospho−Ser65, but not non-phosphorylated ubiquitin or UblPhospho−Ser65, activates full-length wild-type Parkin as well as the non-phosphorylatable S65A Parkin mutant. Fourthly, we provide evidence that optimal activation of full-length Parkin E3 ligase is dependent on PINK1-mediated phosphorylation of both Parkin at Ser65 and ubiquitin at Ser65, since only mutation of both proteins at Ser65 completely abolishes Parkin activation. In conclusion, the findings of the present study reveal that PINK1 controls Parkin E3 ligase activity not only by phosphorylating Parkin at Ser65, but also by phosphorylating ubiquitin at Ser65. We propose that phosphorylation of Parkin at Ser65 serves to prime the E3 ligase enzyme for activation by ubiquitinPhospho−Ser65, suggesting that small molecules that mimic ubiquitinPhospho−Ser65 could hold promise as novel therapies for Parkinson's disease.
We describe a novel and unexpected mechanism by which PINK1 protein kinase activates Parkin E3 ligase. We show that PINK1 phosphorylates ubiquitin at Ser65 and that phosphorylated ubiquitin acts as a direct activator of Parkin.
PMCID: PMC4000136  PMID: 24660806
Parkin; Parkinson’s disease; phosphorylation; PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1 (PINK1); ubiquitin; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CDK2, cyclin-dependent kinase 2; GSK3β, glycogen synthase kinase-3β; HEK, human embryonic kidney; HOIL1, haem-oxidized IRP2 (iron-regulatory protein 2) ubiquitin ligase 1; HRP, horseradish peroxidase; IKK, IκB (inhibitor of nuclear factor κB) kinase; ISG15, interferon-induced 17 kDa protein; MBP, maltose-binding protein; MLK1, mixed lineage kinase 1; Nedd8, neural-precursor-cell-expressed developmentally down-regulated 8; Ni-NTA, Ni2+-nitrilotriacetate; NUAK1, NUAK family SNF1-like kinase 1; OTU1, OTU (ovarian tumour) domain-containing protein 1; PD, Parkinson’s disease; PINK1, PTEN (phosphatase and tensin homologue deleted on chromosome 10)-induced putative kinase 1; PLK1, Polo-like kinase 1; SILAC, stable isotope labelling by amino acids in cell culture; SUMO, small ubiquitin-related modifier; TCEP, tris-(2-carboxyethyl)phosphine; TcPINK1, Tribolium castaneum PINK1; Ubl, ubiquitin-like
10.  The Association of Income with Health Behavior Change and Disease Monitoring among Patients with Chronic Disease 
PLoS ONE  2014;9(4):e94007.
Management of chronic diseases requires patients to adhere to recommended health behavior change and complete tests for monitoring. While studies have shown an association between low income and lack of adherence, the reasons why people with low income may be less likely to adhere are unclear. We sought to determine the association between household income and receipt of health behavior change advice, adherence to advice, receipt of recommended monitoring tests, and self-reported reasons for non-adherence/non-receipt.
We conducted a population-weighted survey, with 1849 respondents with cardiovascular-related chronic diseases (heart disease, hypertension, diabetes, stroke) from Western Canada (n = 1849). We used log-binomial regression to examine the association between household income and the outcome variables of interest: receipt of advice for and adherence to health behavior change (sodium reduction, dietary improvement, increased physical activity, smoking cessation, weight loss), reasons for non-adherence, receipt of recommended monitoring tests (cholesterol, blood glucose, blood pressure), and reasons for non-receipt of tests.
Behavior change advice was received equally by both low and high income respondents. Low income respondents were more likely than those with high income to not adhere to recommendations regarding smoking cessation (adjusted prevalence rate ratio (PRR): 1.55, 95%CI: 1.09–2.20), and more likely to not receive measurements of blood cholesterol (PRR: 1.72, 95%CI 1.24–2.40) or glucose (PRR: 1.80, 95%CI: 1.26–2.58). Those with low income were less likely to state that non-adherence/non-receipt was due to personal choice, and more likely to state that it was due to an extrinsic factor, such as cost or lack of accessibility.
There are important income-related differences in the patterns of health behavior change and disease monitoring, as well as reasons for non-adherence or non-receipt. Among those with low income, adherence to health behavior change and monitoring may be improved by addressing modifiable barriers such as cost and access.
PMCID: PMC3983092  PMID: 24722618
11.  Association between Drug Insurance Cost Sharing Strategies and Outcomes in Patients with Chronic Diseases: A Systematic Review 
PLoS ONE  2014;9(3):e89168.
Prescription drugs are used in people with hypertension, diabetes, and cardiovascular disease to manage their illness. Patient cost sharing strategies such as copayments and deductibles are often employed to lower expenditures for prescription drug insurance plans, but the impact on health outcomes in these patients is unclear.
To determine the association between drug insurance and patient cost sharing strategies on medication adherence, clinical and economic outcomes in those with chronic diseases (defined herein as diabetes, hypertension, hypercholesterolemia, coronary artery disease, and cerebrovascular disease).
Studies were included if they examined various cost sharing strategies including copayments, coinsurance, fixed copayments, deductibles and maximum out-of-pocket expenditures. Value-based insurance design and reference based pricing studies were excluded. Two reviewers independently identified original intervention studies (randomized controlled trials, interrupted time series, and controlled before-after designs). MEDLINE, EMBASE, Cochrane Library, CINAHL, and relevant reference lists were searched until March 2013. Two reviewers independently assessed studies for inclusion, quality, and extracted data. Eleven studies, assessing the impact of seven policy changes, were included: 2 separate reports of one randomized controlled trial, 4 interrupted time series, and 5 controlled before-after studies.
Outcomes included medication adherence, clinical events (myocardial infarction, stroke, death), quality of life, healthcare utilization, or cost. The heterogeneity among the studies precluded meta-analysis. Few studies reported the impact of cost sharing strategies on mortality, clinical and economic outcomes. The association between patient copayments and medication adherence varied across studies, ranging from no difference to significantly lower adherence, depending on the amount of the copayment.
Lowering cost sharing in patients with chronic diseases may improve adherence, but the impact on clinical and economic outcomes is uncertain.
PMCID: PMC3965394  PMID: 24667163
12.  Machine-learning prediction of cancer survival: a retrospective study using electronic administrative records and a cancer registry 
BMJ Open  2014;4(3):e004007.
Using the prediction of cancer outcome as a model, we have tested the hypothesis that through analysing routinely collected digital data contained in an electronic administrative record (EAR), using machine-learning techniques, we could enhance conventional methods in predicting clinical outcomes.
A regional cancer centre in Australia.
Disease-specific data from a purpose-built cancer registry (Evaluation of Cancer Outcomes (ECO)) from 869 patients were used to predict survival at 6, 12 and 24 months. The model was validated with data from a further 94 patients, and results compared to the assessment of five specialist oncologists. Machine-learning prediction using ECO data was compared with that using EAR and a model combining ECO and EAR data.
Primary and secondary outcome measures
Survival prediction accuracy in terms of the area under the receiver operating characteristic curve (AUC).
The ECO model yielded AUCs of 0.87 (95% CI 0.848 to 0.890) at 6 months, 0.796 (95% CI 0.774 to 0.823) at 12 months and 0.764 (95% CI 0.737 to 0.789) at 24 months. Each was slightly better than the performance of the clinician panel. The model performed consistently across a range of cancers, including rare cancers. Combining ECO and EAR data yielded better prediction than the ECO-based model (AUCs ranging from 0.757 to 0.997 for 6 months, AUCs from 0.689 to 0.988 for 12 months and AUCs from 0.713 to 0.973 for 24 months). The best prediction was for genitourinary, head and neck, lung, skin, and upper gastrointestinal tumours.
Machine learning applied to information from a disease-specific (cancer) database and the EAR can be used to predict clinical outcomes. Importantly, the approach described made use of digital data that is already routinely collected but underexploited by clinical health systems.
PMCID: PMC3963101  PMID: 24643167
Cancer; Survival; Prediction; Machine Learning; Electronic Medical Record
13.  A complete mass spectrometric map for the analysis of the yeast proteome and its application to quantitative trait analysis 
Nature  2013;494(7436):266-270.
Complete reference maps or datasets, like the genomic map of an organism, are highly beneficial tools for biological and biomedical research. Attempts to generate such reference datasets for a proteome so far failed to reach complete proteome coverage, with saturation apparent at approximately two thirds of the proteomes tested, even for the most thoroughly characterized proteomes. Here, we used a strategy based on high-throughput peptide synthesis and mass spectrometry to generate a close to complete reference map (97% of the genome-predicted proteins) of the S. cerevisiae proteome. We generated two versions of this mass spectrometric map one supporting discovery- (shotgun) and the other hypothesis-driven (targeted) proteomic measurements. The two versions of the map, therefore, constitute a complete set of proteomic assays to support most studies performed with contemporary proteomic technologies. The reference libraries can be browsed via a web-based repository and associated navigation tools. To demonstrate the utility of the reference libraries we applied them to a protein quantitative trait locus (pQTL) analysis, which requires measurement of the same peptides over a large number of samples with high precision. Protein measurements over a set of 78 S. cerevisiae strains revealed a complex relationship between independent genetic loci, impacting on the levels of related proteins. Our results suggest that selective pressure favors the acquisition of sets of polymorphisms that maintain the stoichiometry of protein complexes and pathways.
PMCID: PMC3951219  PMID: 23334424
S. cerevisiae; selected reaction monitoring; SRM; MRM; spectral library; peptide library; mass spectrometric map; protein QTL
14.  The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K+–Cl− co-transporters 
Biochemical Journal  2014;458(Pt 3):559-573.
Precise homoeostasis of the intracellular concentration of Cl− is achieved via the co-ordinated activities of the Cl− influx and efflux. We demonstrate that the WNK (WNK lysine-deficient protein kinase)-activated SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) known to directly phosphorylate and stimulate the N[K]CCs (Na+–K+ ion co-transporters), also promote inhibition of the KCCs (K+–Cl− co-transporters) by directly phosphorylating a recently described C-terminal threonine residue conserved in all KCC isoforms [Site-2 (Thr1048)]. First, we demonstrate that SPAK and OSR1, in the presence of the MO25 regulatory subunit, robustly phosphorylates all KCC isoforms at Site-2 in vitro. Secondly, STOCK1S-50699, a WNK pathway inhibitor, suppresses SPAK/OSR1 activation and KCC3A Site-2 phosphorylation with similar efficiency. Thirdly, in ES (embryonic stem) cells lacking SPAK/OSR1 activity, endogenous phosphorylation of KCC isoforms at Site-2 is abolished and these cells display elevated basal activity of 86Rb+ uptake that was not markedly stimulated further by hypotonic high K+ conditions, consistent with KCC3A activation. Fourthly, a tight correlation exists between SPAK/OSR1 activity and the magnitude of KCC3A Site-2 phosphorylation. Lastly, a Site-2 alanine KCC3A mutant preventing SPAK/OSR1 phosphorylation exhibits increased activity. We also observe that KCCs are directly phosphorylated by SPAK/OSR1, at a novel Site-3 (Thr5 in KCC1/KCC3 and Thr6 in KCC2/KCC4), and a previously recognized KCC3-specific residue, Site-4 (Ser96). These data demonstrate that the WNK-regulated SPAK/OSR1 kinases directly phosphorylate the N[K]CCs and KCCs, promoting their stimulation and inhibition respectively. Given these reciprocal actions with anticipated net effects of increasing Cl− influx, we propose that the targeting of WNK–SPAK/OSR1 with kinase inhibitors might be a novel potent strategy to enhance cellular Cl− extrusion, with potential implications for the therapeutic modulation of epithelial and neuronal ion transport in human disease states.
WNK-regulated SPAK/OSR1 act as direct phosphorylators and major regulators of the KCC isoforms, which explains how activation of the WNK signalling pathway can co-ordinately regulate Cl− influx and efflux by reciprocally controlling the SLC12A family N[K]CC and KCC isoforms.
PMCID: PMC3940040  PMID: 24393035
γ-aminobutyric acid (GABA); blood pressure/hypertension; ion homoeostasis; K+–Cl− co-transporter 2 (KCC2); K+–Cl− co-transporter 3 (KCC3); Na+–Cl− co-transporter (NCC); Na+–K+–2Cl− co-transporter 1 (NKCC1); protein kinase; signal transduction; CCC, cation–Cl− co-transporter; CCT, conserved C-terminal; CTD, C-terminal cytoplasmic domain; ERK1, extracellular-signal-regulated kinase 1; ES, embryonic stem; HEK, human embryonic kidney; HRP, horseradish peroxidase; KCC, K+–Cl− co-transporter; LDS, lithium dodecyl sulfate; NCC, Na+–Cl− co-transporter; N[K]CC, Na+–K+ ion co-transporter; NKCC, Na+–K+–2Cl− co-transporter; NTD, N-terminal cytoplasmic domain; OSR1, oxidative stress-responsive kinase 1; SLC12, solute carrier family 12; SPAK, SPS1-related proline/alanine-rich kinase; TTBS, Tris-buffered saline containing Tween 20; WNK, WNK lysine-deficient protein kinase; XIC, extracted ion chromatogram
15.  The Streamlined Genome of Phytomonas spp. Relative to Human Pathogenic Kinetoplastids Reveals a Parasite Tailored for Plants 
PLoS Genetics  2014;10(2):e1004007.
Members of the family Trypanosomatidae infect many organisms, including animals, plants and humans. Plant-infecting trypanosomes are grouped under the single genus Phytomonas, failing to reflect the wide biological and pathological diversity of these protists. While some Phytomonas spp. multiply in the latex of plants, or in fruit or seeds without apparent pathogenicity, others colonize the phloem sap and afflict plants of substantial economic value, including the coffee tree, coconut and oil palms. Plant trypanosomes have not been studied extensively at the genome level, a major gap in understanding and controlling pathogenesis. We describe the genome sequences of two plant trypanosomatids, one pathogenic isolate from a Guianan coconut and one non-symptomatic isolate from Euphorbia collected in France. Although these parasites have extremely distinct pathogenic impacts, very few genes are unique to either, with the vast majority of genes shared by both isolates. Significantly, both Phytomonas spp. genomes consist essentially of single copy genes for the bulk of their metabolic enzymes, whereas other trypanosomatids e.g. Leishmania and Trypanosoma possess multiple paralogous genes or families. Indeed, comparison with other trypanosomatid genomes revealed a highly streamlined genome, encoding for a minimized metabolic system while conserving the major pathways, and with retention of a full complement of endomembrane organelles, but with no evidence for functional complexity. Identification of the metabolic genes of Phytomonas provides opportunities for establishing in vitro culturing of these fastidious parasites and new tools for the control of agricultural plant disease.
Author Summary
Some plant trypanosomes, single-celled organisms living in phloem sap, are responsible for important palm diseases, inducing frequent expensive and toxic insecticide treatments against their insect vectors. Other trypanosomes multiply in latex tubes without detriment to their host. Despite the wide range of behaviors and impacts, these trypanosomes have been rather unceremoniously lumped into a single genus: Phytomonas. A battery of molecular probes has been used for their characterization but no clear phylogeny or classification has been established. We have sequenced the genomes of a pathogenic phloem-specific Phytomonas from a diseased South American coconut palm and a latex-specific isolate collected from an apparently healthy wild euphorb in the south of France. Upon comparison with each other and with human pathogenic trypanosomes, both Phytomonas revealed distinctive compact genomes, consisting essentially of single-copy genes, with the vast majority of genes shared by both isolates irrespective of their effect on the host. A strong cohort of enzymes in the sugar metabolism pathways was consistent with the nutritional environments found in plants. The genetic nuances may reveal the basis for the behavioral differences between these two unique plant parasites, and indicate the direction of our future studies in search of effective treatment of the crop disease parasites.
PMCID: PMC3916237  PMID: 24516393
16.  Protein associated with SMAD1 (PAWS1/FAM83G) is a substrate for type I bone morphogenetic protein receptors and modulates bone morphogenetic protein signalling 
Open Biology  2014;4(2):130210.
Bone morphogenetic proteins (BMPs) control multiple cellular processes in embryos and adult tissues. BMPs signal through the activation of type I BMP receptor kinases, which then phosphorylate SMADs 1/5/8. In the canonical pathway, this triggers the association of these SMADs with SMAD4 and their translocation to the nucleus, where they regulate gene expression. BMPs can also signal independently of SMAD4, but this pathway is poorly understood. Here, we report the discovery and characterization of PAWS1/FAM83G as a novel SMAD1 interactor. PAWS1 forms a complex with SMAD1 in a SMAD4-independent manner, and BMP signalling induces the phosphorylation of PAWS1 through BMPR1A. The phosphorylation of PAWS1 in response to BMP is essential for activation of the SMAD4-independent BMP target genes NEDD9 and ASNS. Our findings identify PAWS1 as the first non-SMAD substrate for type I BMP receptor kinases and as a novel player in the BMP pathway. We also demonstrate that PAWS1 regulates the expression of several non-BMP target genes, suggesting roles for PAWS1 beyond the BMP pathway.
PMCID: PMC3938053  PMID: 24554596
bone morphogenetic protein; SMAD1; FAM83G; PAWS1; ALK3; BMPR1
17.  Access to primary care and other health care use among western Canadians with chronic conditions: a population-based survey 
CMAJ Open  2014;2(1):E27-E34.
For adults with chronic conditions, access to primary care, including multidisciplinary care, is associated with better outcomes. Few studies have assessed barriers to such care. We sought to describe barriers to primary care, including care from allied health professionals, for adults with chronic conditions.
We surveyed western Canadians aged 40 years or older who had hypertension, diabetes, heart disease or stroke about access to primary care and other use of health care. Using log binomial regression, we determined the association between sociodemographic variables and several indicators of access to primary care and care from allied health professionals.
Of the 2316 people who were approached, 1849 (79.8%) completed the survey. Most of the respondents (95.1%) had a regular medical doctor, but two-thirds (68.1%) did not have after-hours access. Only 6.1% indicated that allied health professionals were involved in their care, although most respondents (87.3%) indicated they would be willing to see a nurse practitioner if their primary care physician was not available. Respondents who were obese or less than 65 years of age were less likely to have a regular medical doctor. Individuals who had diabetes, lived in a rural area, were residents of Alberta or had poorer health were more likely to have allied health professionals involved in their care.
The survey results identified barriers to accessing primary care for people with chronic conditions. Opportunities for improving access to primary care may include greater involvement by allied health professionals, such as nurse practitioners.
PMCID: PMC3985957  PMID: 25077122
18.  A Rev-Independent gag/pol Eliminates Detectable psi-gag Recombination in Lentiviral Vectors 
BioResearch Open Access  2013;2(6):421-430.
Lentiviral vectors (LVs) are being developed for clinical use in humans for applications including gene therapy and immunotherapy. A safety concern for use of LVs in humans is the generation of replication-competent lentivirus (RCL), which may arise due to recombination between the split genomes of third-generation LVs. Although no RCL has been detected to date, design optimizations that minimize recombination events between split genome vectors would provide an added safety benefit that may further reduce the risk of RCL formation. Here we describe design elements introduced to the gag/pol plasmid with the intention of eliminating psi-gag recombination between the vector genome and gag/pol. These design changes, consisting of codon optimization of the gag/pol sequence and the deletion of the Rev-responsive element, abrogate the requirement for Rev in expression of Gag protein, thus the resulting gag/pol construct being Rev independent (RI gag/pol). We show that generating vector using the RI gag/pol construct has no effect on particle production or transduction titers. The RI and wild-type gag/pol vectors function equivalently as antigen-specific immunotherapy, potently inducing antigen-specific CD8 T cells that protect against challenge with vaccinia virus. Most importantly, the designed RI gag/pol eliminated detectable psi-gag recombination. Interestingly, we detected recombination between the vector genome and gag/pol from regions without sequence homology. Our findings imply that although unpredictable recombination events may still occur, the RI gag/pol design is sufficient to prevent psi-gag recombination.
PMCID: PMC3869434  PMID: 24380052
gene therapy; immunotherapy; retrovirus; viral vectors
19.  PASSEL: The PeptideAtlas SRM Experiment Library 
Proteomics  2012;12(8):10.1002/pmic.201100515.
Public repositories for proteomics data have accelerated proteomics research by enabling more efficient cross-analyses of datasets, supporting the creation of protein and peptide compendia of experimental results, supporting the development and testing of new software tools, and facilitating the manuscript review process. The repositories available to date have been designed to accommodate either shotgun experiments or generic proteomic data files. Here, we describe a new kind of proteomic data repository for the collection and representation of data from selected reaction monitoring (SRM) measurements. The PeptideAtlas SRM Experiment Library (PASSEL) allows researchers to easily submit proteomic data sets generated by SRM. The raw data are automatically processed in a uniform manner and the results are stored in a database, where they may be downloaded or browsed via a web interface that includes a chromatogram viewer. PASSEL enables cross-analysis of SRM data, supports optimization of SRM data collection, and facilitates the review process of SRM data. Further, PASSEL will help in the assessment of proteotypic peptide performance in a wide array of samples containing the same peptide, as well as across multiple experimental protocols.
PMCID: PMC3832291  PMID: 22318887
data repository; MRM; software; SRM; targeted proteomics
20.  Economic Rationality in Choosing between Short-Term Bad-Health Choices and Longer-Term Good-Health Choices 
Non-contagious, chronic disease has been identified as a global health risk. Poor lifestyle choices, such as smoking, alcohol, drug and solvent abuse, physical inactivity, and unhealthy diet have been identified as important factors affecting the increasing incidence of chronic disease. The following focuses on the circumstance affecting the lifestyle or behavioral choices of Aboriginal and Torres Strait Islander peoples in remote-/very remote Australia. Poor behavioral choices are the result of endogenous characteristics that are influenced by a range of stressful exogenous variables making up the psychosocial determinants including social disenfranchisement, cultural loss, insurmountable tasks, the loss of volitional control and resource constraints. It is shown that poor behavioral choices can be economically rational; especially under highly stressful conditions. Stressful circumstances erode individual capacity to commit to long-term positive health alternatives such as self-investment in education. Policies directed at removing the impediments and providing incentives to behaviors involving better health choices can lead to reductions in smoking and alcohol consumption and improved health outcomes. Multijurisdictional culturally acceptable policies directed at distal variables relating to the psychosocial determinants of health and personal mastery and control can be cost effective. While the content of this paper is focused on the conditions of colonized peoples, it has broader relevance.
PMCID: PMC3863881  PMID: 24217181
smoking; alcohol abuse; health; behavioral choice; psychosocial determinants; human capital
21.  Deprivation, timing of preschool infections and H. pylori seropositivity at age 49-51 years: the Newcastle thousand families birth cohort 
BMC Infectious Diseases  2013;13:422.
Helicobacter pylori infection is acquired in early childhood and persists for life (or until eradication treatment is taken). Seropositivity of H. pylori at age 49-51 years was assessed in relation to socio-economic deprivation in early life and the timing of other childhood infections common at that time.
Prospectively collected socio-economic and morbidity data from the Newcastle Thousand Families study, a birth cohort established in 1947. H. pylori IgG seropositivity was assessed at 49-51 years and examined in relation to both whether the individual had been diagnosed with one of measles, mumps or chicken pox, and, if so, the age at first infection. This was done in logistic regression models, allowing adjustment for socio-economic status and housing quality in childhood.
Adult H. pylori status was strongly linked to disadvantaged socio-economic status in early life (p ≤ 0.002), unlike measles, mumps and chicken pox which showed no associations. Early measles infection was independently associated with H. pylori seropositivity (p = 0.01).
Of the four infectious diseases that we have studied, it appears that H. pylori differs from the others by the strength of association with socio economic deprivation in early childhood.
Our findings further highlight the complex interaction between measles, childhood infections and other non-microbiological factors that occur within a whole population. These data suggest a strong association between H. pylori and deprivation and raise the possibility of an interaction between early measles exposure and increased risk of exposure to H. pylori infection.
PMCID: PMC3847688  PMID: 24010891
Helicobacter pylori; Socio-economic status; Measles; Chicken pox; Mumps
22.  Yes and Lyn play a role in nuclear translocation of the Epidermal Growth Factor Receptor 
Oncogene  2012;32(6):759-767.
The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR antibody that has been approved for use in oncology. Previously we investigated mechanisms of resistance to cetuximab using a model derived from the non-small cell lung cancer line NCI-H226. We demonstrated that cetuximab-resistant clones (CtxR) had increased nuclear localization of the EGFR. This process was mediated by Src family kinases (SFK), and nuclear EGFR played a role in resistance to cetuximab. To better understand SFK mediated nuclear translocation of EGFR, we investigated which SFK member(s) controlled this process as well as the EGFR tyrosine residues that are involved. Analyses of mRNA and protein expression indicated up-regulation of the SFK members Yes and Lyn in all CtxR clones. Further, immunoprecipitation analysis revealed that EGFR interacts with Yes and Lyn in CtxR clones, but not in cetuximab-sensitive (CtxS) parental cells. Using RNAi interference, we found that knockdown of either Yes or Lyn led to loss of EGFR translocation to the nucleus. Conversely, overexpression of Yes or Lyn in low nuclear EGFR expressing CtxS parental cells led to increased nuclear EGFR. Chromatin immunoprecipitation (ChIP) assays confirmed nuclear EGFR complexes associated with the promoter of the known EGFR target genes B-Myb and iNOS. Further, all CtxR clones exhibited up-regulation of B-Myb and iNOS at the mRNA and protein levels. siRNAs directed at Yes or Lyn led to decreased binding of EGFR complexes to the B-Myb and iNOS promoters based on ChIP analyses. SFKs have been shown to phosphorylate EGFR on tyrosines 845 and 1101 (Y845 and Y1101) and mutation of Y1101, but not Y845, impaired nuclear entry of the EGFR. Taken together, our findings demonstrate that Yes and Lyn phosphorylate EGFR at Y1101 which influences EGFR nuclear translocation in this model of cetuximab resistance.
PMCID: PMC3381861  PMID: 22430206
nuclear EGFR; SFK; Yes; Lyn
23.  Protein phosphatase 4 is phosphorylated and inactivated by Cdk in response to spindle toxins and interacts with γ-tubulin 
Cell Cycle  2013;12(17):2876-2887.
Many pharmaceuticals used to treat cancer target the cell cycle or mitotic spindle dynamics, such as the anti-tumor drug, paclitaxel, which stabilizes microtubules. Here we show that, in cells arrested in mitosis with the spindle toxins, nocodazole, or paclitaxel, the endogenous protein phosphatase 4 (Ppp4) complex Ppp4c-R2-R3A is phosphorylated on its regulatory (R) subunits, and its activity is inhibited. The phosphorylations are blocked by roscovitine, indicating that they may be mediated by Cdk1-cyclin B. Endogenous Ppp4c is enriched at the centrosomes in the absence and presence of paclitaxel, nocodazole, or roscovitine, and the activity of endogenous Ppp4c-R2-R3A is inhibited from G1/S to the G2/M phase of the cell cycle. Endogenous γ-tubulin and its associated protein, γ-tubulin complex protein 2, both of which are essential for nucleation of microtubules at centrosomes, interact with the Ppp4 complex. Recombinant γ-tubulin can be phosphorylated by Cdk1-cyclin B or Brsk1 and dephosphorylated by Ppp4c-R2-R3A in vitro. The data indicate that Ppp4c-R2-R3A regulates microtubule organization at centrosomes during cell division in response to stress signals such as spindle toxins, paclitaxel, and nocodazole, and that inhibition of the Ppp4 complex may be advantageous for treatment of some cancers.
PMCID: PMC3899200  PMID: 23966160
Cdk1; cell cycle; centrosome; nocodazole; paclitaxel; protein phosphatase 4; γ-tubulin
24.  The role of a student-run clinic in providing primary care for Calgary’s homeless populations: a qualitative study 
Despite the increasing popularity of Student-Run Clinics (SRCs) in Canada, there is little existing literature exploring their role within the Canadian healthcare system. Generalizing American literature to Canadian SRCs is inappropriate, given significant differences in healthcare delivery between the two countries. Medical students at the University of Calgary started a SRC serving Calgary’s homeless population at the Calgary Drop-In and Rehabilitation Centre (CDIRC). This study explored stakeholders’ desired role for a SRC within Calgary’s primary healthcare system and potential barriers it may face.
Individual and group semi-structured interviews were undertaken with key stakeholders in the SRC project: clients (potential patients), CDIRC staff, staff from other stakeholder organizations, medical students, and faculty members. Convenience sampling was used in the recruitment of client participants. Interview transcripts were analyzed using a coding template which was derived from the literature.
Participants identified factors related to the clinic and to medical students that suggest there is an important role for a SRC in Calgary. The clinic was cited as improving access to primary healthcare for individuals experiencing homelessness. It was suggested that students may be ideally suited to provide empathetic healthcare to this population. Barriers to success were identified, including continuity of care and the exclusion of some subsets of the homeless population due to location.
SRCs possess several unique features that may make them a potentially important primary healthcare resource for the homeless. Participants identified numerous benefits of the SRC to providing primary care for homeless individuals, as well as several important limitations that need to be accounted for when designing and implementing such a program.
PMCID: PMC3718696  PMID: 23866968
Primary care; Homeless persons; Medical student; Physician shortage areas
25.  Tsetse Fly Control in Kenya's Spatially and Temporally Dynamic Control Reservoirs: A Cost Analysis 
Human African trypanosomiasis (HAT) and animal African trypanosomiasis (AAT) are significant health concerns throughout much of sub-Saharan Africa. Funding for tsetse fly control operations has decreased since the 1970s, which has in turn limited the success of campaigns to control the disease vector. To maximize the effectiveness of the limited financial resources available for tsetse control, this study develops and analyzes spatially and temporally dynamic tsetse distribution maps of Glossina subgenus Morsitans populations in Kenya from January 2002 to December 2010, produced using the Tsetse Ecological Distribution Model. These species distribution maps reveal seasonal variations in fly distributions. Such variations allow for the identification of “control reservoirs” where fly distributions are spatially constrained by fluctuations in suitable habitat and tsetse population characteristics. Following identification of the control reservoirs, a tsetse management operation is simulated in the control reservoirs using capital and labor control inputs from previous studies. Finally, a cost analysis, following specific economic guidelines from existing tsetse control analyses, is conducted to calculate the total cost of a nationwide control campaign of the reservoirs compared to the cost of a nationwide campaign conducted at the maximum spatial extent of the fly distributions from January 2002 to December 2010. The total cost of tsetse management within the reservoirs sums to $14,212,647, while the nationwide campaign at the maximum spatial extent amounts to $33,721,516. This savings of $19,508,869 represents the importance of identifying seasonally dynamic control reservoirs when conducting a tsetse management campaign, and, in the process, offers an economical means of fly control and disease management for future program planning.
PMCID: PMC3347470  PMID: 22581989
Tsetse Fly; Kenya; Control Reservoirs; Control Simulation; Cost Analysis; African Trypanosomiasis

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