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26.  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
27.  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
28.  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
29.  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
30.  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.
Background
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.
Methods
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.
Results
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.
Interpretation
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.
doi:10.9778/cmajo.20130045
PMCID: PMC3985957  PMID: 25077122
31.  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
32.  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
33.  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.
doi:10.3390/ijerph10115971
PMCID: PMC3863881  PMID: 24217181
smoking; alcohol abuse; health; behavioral choice; psychosocial determinants; human capital
34.  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
35.  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
36.  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
37.  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
38.  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
39.  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
40.  Development of a Community-Sensitive Strategy to Increase Availability of Fresh Fruits and Vegetables in Nashville’s Urban Food Deserts, 2010–2012 
Background
Food deserts, areas that lack full-service grocery stores, may contribute to rising rates of obesity and chronic diseases among low-income and racial/ethnic minority residents. Our corner store project, part of the Centers for Disease Control and Prevention’s Communities Putting Prevention to Work initiative, aimed to increase availability of healthful foods in food deserts in Nashville, Tennessee.
Community Context
We identified 4 food deserts in which most residents are low-income and racially and ethnically diverse. Our objectives were to develop an approach to increase availability of fresh fruits and vegetables, low-fat or nonfat milk, and 100% whole-wheat bread in Nashville’s food deserts and to engage community members to inform our strategy.
Methods
Five corner stores located in food deserts met inclusion criteria for our intervention. We then conducted community listening sessions, proprietor surveys, store audits, and customer-intercept surveys to identify needs, challenges to retailing the products, and potential intervention strategies.
Outcome
Few stores offered fresh fruits, fresh vegetables, low-fat or nonfat milk, or 100% whole-wheat bread, and none stocked items from all 4 categories. Major barriers to retailing healthful options identified by community members are mistrust of store owners, history of poor-quality produce, and limited familiarity with healthful options. Store owners identified neighborhood crime as the major barrier. We used community input to develop strategies.
Interpretation
Engaging community residents and understanding neighborhood context is critical to developing strategies that increase access to healthful foods in corner stores.
doi:10.5888/pcd10.130008
PMCID: PMC3725846  PMID: 23886044
41.  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
42.  Atg13 and FIP200 act independently of Ulk1 and Ulk2 in autophagy induction 
Autophagy  2011;7(12):1424-1433.
Under normal growth conditions the mammalian target of rapamycin complex 1 (mTORC1) negatively regulates the central autophagy regulator complex consisting of Unc-51-like kinases 1/2 (Ulk1/2), focal adhesion kinase family-interacting protein of 200 kDa (FIP200) and Atg13. Upon starvation, mTORC1-mediated repression of this complex is released, which then leads to Ulk1/2 activation. In this scenario, Atg13 has been proposed as an adaptor mediating the interaction between Ulk1/2 and FIP200 and enhancing Ulk1/2 kinase activity. Using Atg13-deficient cells, we demonstrate that Atg13 is indispensable for autophagy induction. We further show that Atg13 function strictly depends on FIP200 binding. In contrast, the simultaneous knockout of Ulk1 and Ulk2 did not have a similar effect on autophagy induction. Accordingly, the Ulk1-dependent phosphorylation sites we identified in Atg13 are expendable for this process. This suggests that Atg13 has an additional function independent of Ulk1/2 and that Atg13 and FIP200 act in concert during autophagy induction.
doi:10.4161/auto.7.12.18027
PMCID: PMC3327613
Atg13; autophagy; FIP200; Ulk1; Ulk2
43.  Change in Hematologic Indices over Time in Pediatric Inflammatory Bowel Disease Treated with Azathioprine 
Drugs in R&d  2012;10(4):213-217.
Azathioprine leads to changes in mean corpuscular volume (MCV) and white blood cell (WBC) indices reflecting efficacy or toxicity. Understanding the interactions between bone marrow stem cells and azathioprine could highlight abnormal response patterns as forerunners for hematologic malignancies. This study gives a statistical description of factors influencing the relationship between MCV and WBC in children with inflammatory bowel disease treated with azathioprine. We found that leukopenia preceded macrocytosis. Macrocytosis is therefore not a good predictor of leukopenia. Further studies will be necessary to determine the subgroup of patients at increased risk of malignancies based on bone marrow response.
doi:10.2165/11539930-000000000-00000
PMCID: PMC3586161  PMID: 21171667
44.  Change in Hematologic Indices over Time in Pediatric Inflammatory Bowel Disease Treated with Azathioprine 
Drugs in R&D  2012;10(4):213-217.
Azathioprine leads to changes in mean corpuscular volume (MCV) and white blood cell (WBC) indices reflecting efficacy or toxicity. Understanding the interactions between bone marrow stem cells and azathioprine could highlight abnormal response patterns as forerunners for hematologic malignancies. This study gives a statistical description of factors influencing the relationship between MCV and WBC in children with inflammatory bowel disease treated with azathioprine. We found that leukopenia preceded macrocytosis. Macrocytosis is therefore not a good predictor of leukopenia. Further studies will be necessary to determine the subgroup of patients at increased risk of malignancies based on bone marrow response.
doi:10.2165/11539930-000000000-00000
PMCID: PMC3586161  PMID: 21171667
45.  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
46.  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
47.  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
48.  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
49.  Early migration characteristics of a hydroxyapatite-coated femoral stem: an RSA study 
International Orthopaedics  2009;35(4):483-488.
Measurement of early stem subsidence can be used to predict the likelihood of long-term femoral component loosening and clinical failure. Data that examines the early migration pattern of clinically proven stems will provide clinicians with useful baseline data with which to compare new stem designs. This study was performed to evaluate the early migration pattern of a hydroxyapatite-coated press-fit femoral component that has been in use for over ten years. We enrolled 30 patients who underwent THA for osteoarthritis. The median age was 70 years (range, 55–80 years). Patients were clinically assessed using the Harris hip score. Radiostereometric analysis was used to evaluate stem migration at three to four days, six months, one year and two years. We observed a mean subsidence of 0.73 mm at six months, 0.62 mm at one year and 0.58 mm at two years and a mean retroversion of 1.82° at six months, 1.90° at one year and 1.59° at two years. This data suggests that subsidence is confined to the first six months after which there was no further subsidence. The results from this study can be compared with those from novel cementless stem designs to help predict the long-term outcome one may expect from new cementless stem designs.
doi:10.1007/s00264-009-0913-z
PMCID: PMC3066322  PMID: 20012862
50.  Heparin-Induced Thrombocytopenia Associated with Massive Intracardiac Thrombosis: A Case Report 
Case Reports in Hematology  2012;2012:257023.
A 60-years old patient was admitted to a community hospital with septic arthritis. He was treated with antibiotics and subcutaneous unfractionated heparin (UH) was used for venous thromboprophylaxis. After three days, he developed leg deep venous thrombosis and was treated with IV heparin. One day later, the patient developed pulmonary emboli, which was found using ventilation/perfusion scan. He was transferred to the University Hospital for further management. Upon arrival, antibiotic and intravenous UH were continued. Trans-Esophageal Echocardiogram showed a thrombus in the right atrium, a small portion of which extended to the left atrium through a patent foramen ovale. Another large thrombus was noted in the right ventricle, which extended to the pulmonary artery. Review of the patient's medical records revealed a halving of his platelet count three days following the heparin administration. Therefore, HIT seemed very likely. Intravenous UH was stopped and an emergency thrombectomy was performed. ELISA testing of HIT antibodies came negative. This made HIT diagnosis unlikely and the patient received dalteparin. A week later, as the platelet count declined again, HIT antibodies' testing using ELISA and C-14 serotonin release was repeated, and both assays were positive. Argatroban was restarted and the platelet count normalized.
doi:10.1155/2012/257023
PMCID: PMC3420555  PMID: 22937322

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