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1.  Noninvasive Image Texture Analysis Differentiates K-ras Mutation from Pan-Wildtype NSCLC and Is Prognostic 
PLoS ONE  2014;9(7):e100244.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1371/journal.pone.0100244
PMCID: PMC4079229  PMID: 24987838
2.  Wear of a 5 Megarad Cross-linked Polyethylene Liner: A 6-year RSA Study 
Background
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.
Questions/purposes
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1007/s11999-013-2789-x
PMCID: PMC3676600  PMID: 23334705
3.  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.
doi:10.4161/cbt.24342
PMCID: PMC3813564  PMID: 23760490
AKT; EGFR; MK-2206; cetuximab; acquired cetuximab-resistance; non-small cell lung cancer; MAPK
4.  The Mtb Proteome Library: A Resource of Assays to Quantify the Complete Proteome of Mycobacterium tuberculosis 
Cell host & microbe  2013;13(5):602-612.
SUMMARY
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.
doi:10.1016/j.chom.2013.04.008
PMCID: PMC3766585  PMID: 23684311
5.  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.
doi:10.1042/BJ20140334
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
6.  The Association of Income with Health Behavior Change and Disease Monitoring among Patients with Chronic Disease 
PLoS ONE  2014;9(4):e94007.
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1371/journal.pone.0094007
PMCID: PMC3983092  PMID: 24722618
7.  Association between Drug Insurance Cost Sharing Strategies and Outcomes in Patients with Chronic Diseases: A Systematic Review 
PLoS ONE  2014;9(3):e89168.
Background
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.
Objective
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).
Methods
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.
Findings
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.
Conclusion
Lowering cost sharing in patients with chronic diseases may improve adherence, but the impact on clinical and economic outcomes is uncertain.
doi:10.1371/journal.pone.0089168
PMCID: PMC3965394  PMID: 24667163
8.  Machine-learning prediction of cancer survival: a retrospective study using electronic administrative records and a cancer registry 
BMJ Open  2014;4(3):e004007.
Objectives
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.
Setting
A regional cancer centre in Australia.
Participants
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).
Results
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.
Conclusions
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.
doi:10.1136/bmjopen-2013-004007
PMCID: PMC3963101  PMID: 24643167
Cancer; Survival; Prediction; Machine Learning; Electronic Medical Record
9.  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.
doi:10.1038/nature11835
PMCID: PMC3951219  PMID: 23334424
S. cerevisiae; selected reaction monitoring; SRM; MRM; spectral library; peptide library; mass spectrometric map; protein QTL
10.  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.
doi:10.1042/BJ20131478
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
11.  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.
doi:10.1371/journal.pgen.1004007
PMCID: PMC3916237  PMID: 24516393
12.  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.
doi:10.1098/rsob.130210
PMCID: PMC3938053  PMID: 24554596
bone morphogenetic protein; SMAD1; FAM83G; PAWS1; ALK3; BMPR1
13.  A Rev-Independent gag/pol Eliminates Detectable psi-gag Recombination in Lentiviral Vectors 
BioResearch Open Access  2013;2(6):421-430.
Abstract
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.
doi:10.1089/biores.2013.0037
PMCID: PMC3869434  PMID: 24380052
gene therapy; immunotherapy; retrovirus; viral vectors
14.  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.
doi:10.1002/pmic.201100515
PMCID: PMC3832291  PMID: 22318887
data repository; MRM; software; SRM; targeted proteomics
15.  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.
Background
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.
Methods
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.
Results
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).
Conclusions
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.
doi:10.1186/1471-2334-13-422
PMCID: PMC3847688  PMID: 24010891
Helicobacter pylori; Socio-economic status; Measles; Chicken pox; Mumps
16.  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.
doi:10.1038/onc.2012.90
PMCID: PMC3381861  PMID: 22430206
nuclear EGFR; SFK; Yes; Lyn
17.  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.
doi:10.4161/cc.25919
PMCID: PMC3899200  PMID: 23966160
Cdk1; cell cycle; centrosome; nocodazole; paclitaxel; protein phosphatase 4; γ-tubulin
18.  The role of a student-run clinic in providing primary care for Calgary’s homeless populations: a qualitative study 
Background
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.
Methods
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.
Results
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.
Conclusions
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.
doi:10.1186/1472-6963-13-277
PMCID: PMC3718696  PMID: 23866968
Primary care; Homeless persons; Medical student; Physician shortage areas
19.  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.
doi:10.1016/j.apgeog.2011.11.005
PMCID: PMC3347470  PMID: 22581989
Tsetse Fly; Kenya; Control Reservoirs; Control Simulation; Cost Analysis; African Trypanosomiasis
20.  The anti-inflammatory drug BAY 11-7082 suppresses the MyD88-dependent signalling network by targeting the ubiquitin system 
Biochemical Journal  2013;451(Pt 3):427-437.
The compound BAY 11-7082 inhibits IκBα [inhibitor of NF-κB (nuclear factor κB)α] phosphorylation in cells and has been used to implicate the canonical IKKs (IκB kinases) and NF-κB in >350 publications. In the present study we report that BAY 11-7082 does not inhibit the IKKs, but suppresses their activation in LPS (lipopolysaccharide)-stimulated RAW macrophages and IL (interleukin)-1-stimulated IL-1R (IL-1 receptor) HEK (human embryonic kidney)-293 cells. BAY 11-7082 exerts these effects by inactivating the E2-conjugating enzymes Ubc (ubiquitin conjugating) 13 and UbcH7 and the E3 ligase LUBAC (linear ubiquitin assembly complex), thereby preventing the formation of Lys63-linked and linear polyubiquitin chains. BAY 11-7082 prevents ubiquitin conjugation to Ubc13 and UbcH7 by forming a covalent adduct with their reactive cysteine residues via Michael addition at the C3 atom of BAY 11-7082, followed by the release of 4-methylbenzene-sulfinic acid. BAY 11-7082 stimulated Lys48-linked polyubiquitin chain formation in cells and protected HIF1α (hypoxia-inducible factor 1α) from proteasomal degradation, suggesting that it inhibits the proteasome. The results of the present study indicate that the anti-inflammatory effects of BAY 11-7082, its ability to induce B-cell lymphoma and leukaemic T-cell death and to prevent the recruitment of proteins to sites of DNA damage are exerted via inhibition of components of the ubiquitin system and not by inhibiting NF-κB.
doi:10.1042/BJ20121651
PMCID: PMC3685219  PMID: 23441730
lymphoma; linear ubiquitin assembly complex (LUBAC); myeloid differentiation factor 88 (MyD88); nuclear factor κB (NF-κB); proteasome; ubiquitin conjugating 13 (Ubc13); DAPI, 4′,6-diamidino-2-phenylindole; DLBCL, diffuse large B-cell lymphoma; DMEM, Dulbecco’s modified Eagle’s medium; ERK, extracellular-signal-regulated kinase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; HEK, human embryonic kidney; HIF1α, hypoxia-inducible factor 1α; HOIP, haem-oxidized IRP2 ligase-1-interacting protein; HRMS, high-resolution mass spectra; HTLV-1, human T-cell lymphotropic virus 1; IL, interleukin; IL-1R, IL-1 receptor; IRAK, IL-receptor-associated kinase; IκB, inhibitor of NF-κB; IKK, IκB kinase; JNK, c-Jun N-terminal kinase; K48-pUb, Lys48-linked polyubiquitin; K63-pUb, Lys63-linked polyubiquitin; LPS, lipopolysaccharide; LUBAC, linear ubiquitin assembly complex; MALDI–TOF, matrix-assisted laser-desorption ionization–time-of-flight; MAPK, mitogen-activated protein kinase; MS/MS, tandem MS; MyD88, myeloid differentiation factor 88; NEDD8, neural-precursor-cell-expressed developmentally down-regulated 8; NEMO, NF-κB essential modifier; NF-κB, nuclear factor κB; PAMP, pathogen-associated molecular pattern; pUb, polyubiquitin; RBR, RING-between-RING, TAB, TAK1-binding protein; TAK1, transforming growth factor β-activated kinase 1; TBK1, tumour-necrosis-factor-receptor-associated factor-associated NF-κB activator-binding kinase 1; TRAF, tumour-necrosis-factor-receptor-associated factor; Ubc, ubiquitin conjugating; UBE, ubiquitin-activating enzyme
21.  Association of enrolment in primary care networks with diabetes care and outcomes among First Nations and low-income Albertans 
Open Medicine  2012;6(4):e155-e165.
Background
The prevalence of diabetes mellitus and its complications is higher among First Nations people and people with low socio-economic status (SES). Previous studies in Alberta have shown that provision of care through Primary Care Networks (PCNs) is associated with better quality of care and better outcomes for people with diabetes, possibly because of greater utilization of chronic disease management programs. However, it is unknown whether First Nations individuals and those in lower SES groups experience these benefits.
Methods
We used administrative and laboratory data for a population-based cohort analysis of Alberta residents under 65 years of age with diabetes. The primary outcome, assessed over a 1-year period, was admission to hospital or emergency department visit for a diabetes-specific ambulatory care sensitive condition (ACSC). Secondary outcomes were 2 quality-of-care indicators (likelihood of measurement of glycated hemoglobin [HbA1c] and or retinal screening) and 2 measures of health care utilization (visits to specialist and primary care physicians). We used negative binomial regression to determine the association between care within a PCN and hospital admission or emergency department visit for diabetes-specific ACSCs. We also assessed outcomes in 3 populations of interest (individuals receiving a health care subsidy [household income less than $39 250 and not eligible for Income Support], those receiving Income Support, and First Nations individuals) relative to the remainder of the population, controlling for whether care was provided in a PCN and adjusting for several baseline characteristics.
Results
We identified a total of 106 653 patients with diabetes eligible for our study, of whom 43 327 (41%) received care in a PCN. Receiving care through a PCN was associated with lower rates of ACSC-related hospital admission or emergency department visits for all groups of interest, which suggests that PCNs had similar effects across each group. However, regardless of where care was provided, First Nations and low-SES patients had more than twice the adjusted rates of hospital admission or emergency department visits for diabetes-specific ACSCs than the general population and were less likely to receive guideline-recommended care, including measurement of HbA1c and retinal screening.
Interpretation
Care in a PCN was associated with lower risks of hospital admission or emergency department visits for diabetes-specific ACSCs, even within vulnerable groups such as First Nations people and those of low SES. However, differences in outcomes and quality-of-care indicators persisted for First Nations individuals and those of low SES, relative to the general population, irrespective of where care was provided.
PMCID: PMC3654512  PMID: 23687531
22.  Identification of the Amino Acids 300–600 of IRS-2 as 14-3-3 Binding Region with the Importance of IGF-1/Insulin-Regulated Phosphorylation of Ser-573 
PLoS ONE  2012;7(8):e43296.
Phosphorylation of insulin receptor substrate (IRS)-2 on tyrosine residues is a key event in IGF-1/insulin signaling and leads to activation of the PI 3-kinase and the Ras/MAPK pathway. Furthermore, phosphorylated serine/threonine residues on IRS-2 can induce 14-3-3 binding. In this study we searched IRS-2 for novel phosphorylation sites and investigated the interaction between IRS-2 and 14-3-3. Mass spectrometry identified a total of 24 serine/threonine residues on IRS-2 with 12 sites unique for IRS-2 while the other residues are conserved in IRS-1 and IRS-2. IGF-1 stimulation led to increased binding of 14-3-3 to IRS-2 in transfected HEK293 cells and this binding was prevented by inhibition of the PI 3-kinase pathway and an Akt/PKB inhibitor. Insulin-stimulated interaction between endogenous IRS-2 and 14-3-3 was observed in rat hepatoma cells and in mice liver after an acute insulin stimulus and refeeding. Using different IRS-2 fragments enabled localization of the IGF-1-dependent 14-3-3 binding region spanning amino acids 300–600. The 24 identified residues on IRS-2 included several 14-3-3 binding candidates in the region 300–600. Single alanine mutants of these candidates led to the identification of serine 573 as 14-3-3 binding site. A phospho-site specific antibody was generated to further characterize serine 573. IGF-1-dependent phosphorylation of serine 573 was reduced by inhibition of PI 3-kinase and Akt/PKB. A negative role of this phosphorylation site was implicated by the alanine mutant of serine 573 which led to enhanced phosphorylation of Akt/PKB in an IGF-1 time course experiment. To conclude, our data suggest a physiologically relevant role for IGF-1/insulin-dependent 14-3-3 binding to IRS-2 involving serine 573.
doi:10.1371/journal.pone.0043296
PMCID: PMC3422239  PMID: 22912850
23.  Distribution, genetic analysis and conservation priorities for rare Texas freshwater molluscs in the genera Fusconaia and Pleurobema (Bivalvia: Unionidae) 
Aquatic Biosystems  2012;8:12.
Background
Freshwater bivalves in the order Unionoida are considered to be one of the most endangered groups of animals in North America. In Texas, where over 60% of unionids are rare or very rare, 15 species have been recently added to the state’s list of threatened species, and 11 are under consideration for federal listing. Due to insufficient survey efforts in the past decades, however, primary data on current distribution and habitat requirement for most of these rare species are lacking, thus challenging their protection and management. Taxonomic identification of endemic species based on shell morphology is challenging and complicates conservation efforts. In this paper we present historic and current distributional data for three rare Texas species, Fusconaia askewi, F. lananensis, and Pleurobema riddellii, collected during our 2003–2011 state-wide surveys and suggest appropriate conservation measures. In addition, we tested the genetic affinities of Fusconaia and similar species collected from eastern Texas and western Louisiana using cox1 and nad1 sequences.
Results
We found that F. askewi still inhabits four river basins in eastern and northeastern Texas and can be locally abundant, while P. riddellii was found only in one river basin. Pleurobema riddellii was well-separated from F. askewi and grouped with the P. sintoxia clade. The sequences for F. lananensis were very similar to those for F. askewi, with a maximum difference of just over 1% for nad1 and only 0.7% for cox1, similar to the variation between F. askewi alleles. Except for one low difference (1.55%) with the partial cox1 sequence for F. burkei, all other Fusconaia populations, including those from the Calcasieu drainage, differed by over 2.3% for both genes.
Conclusions
Our study suggested that F. lananensis is not a valid species, and it is likely that only one Fusconaia species (F. askewi or its probable senior synonym F. chunii) is currently present in East Texas, thus simplifying conservation efforts. Distribution range of both these regional endemics (F. askewi and P. riddellii) has been reduced in the last 80 years.
doi:10.1186/2046-9063-8-12
PMCID: PMC3422191  PMID: 22731520
Freshwater molluscs; Fusconaia askewi; Fusconaia lananensis; Pleurobema riddellii; Molecular identification; Taxonomy; Distribution; Habitat requirements; Conservation priorities
24.  Morbidity of the Arterial Switch Operation 
The Annals of Thoracic Surgery  2012;93(6):1977-1983.
Background
The arterial switch operation (ASO) has become a safe, reproducible surgical procedure with low mortality in experienced centers. We examined morbidity, which remains significant, particularly for complex ASO.
Methods
From 2003 to 2011, 101 consecutive patients underwent ASO, arbitrarily classified as “simple” (n = 52) or “complex” (n = 49). Morbidity was measured in selected complications and postoperative hospitalization. Three outcomes were analyzed: ventilation time, postextubation hospital length of stay, and a composite morbidity index, defined as ventilation time + postextubation hospital length of stay + occurrence of selected major complications. Complexity was measured with the comprehensive Aristotle score.
Results
The operative mortality was zero. Twenty-five major complications occurred in 23 patients: 6 of 25 (12%) in simple ASO and 19 of 49 (39%) in complex ASO (p = 0.002). The most frequent complication was unplanned reoperation (15 vs 6, p = 0.03). No patients required permanent pacing. The complex group had a significantly higher morbidity index and longer ventilation time and postextubation hospital length of stay. In multivariate analysis, factors independently predicting higher morbidity were the comprehensive Aristotle score, arch repair, bypass time, and malaligned commissures. Myocardial infarction caused one sudden late death at 3 months. Late coronary failure was 2%. Overall survival was 99% at a mean follow-up of 49 ± 27 months.
Conclusions
In this consecutive series without operative mortality, morbidity was significantly higher in complex ASO. The only anatomic incremental risk factors for morbidity were aortic arch repair and malaligned commissures, but not primary diagnosis, weight less than 2.5 kg, or coronary patterns.
doi:10.1016/j.athoracsur.2011.11.061
PMCID: PMC3381339  PMID: 22365263
25.  PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65 
Open Biology  2012;2(5):120080.
Summary
Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser65. We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser65. We further show that phosphorylation of Parkin at Ser65 leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser65 or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr257, which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser65 and/or PINK1 at Thr257 represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.
doi:10.1098/rsob.120080
PMCID: PMC3376738  PMID: 22724072
PINK1; Parkin; Parkinson's disease

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