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1.  Design of Selective PAK1 Inhibitor G-5555: Improving Properties by Employing an Unorthodox Low-pKa Polar Moiety 
ACS Medicinal Chemistry Letters  2015;6(12):1241-1246.
Signaling pathways intersecting with the p21-activated kinases (PAKs) play important roles in tumorigenesis and cancer progression. By recognizing that the limitations of FRAX1036 (1) were chiefly associated with the highly basic amine it contained, we devised a mitigation strategy to address several issues such as hERG activity. The 5-amino-1,3-dioxanyl moiety was identified as an effective means of reducing pKa and logP simultaneously. When positioned properly within the scaffold, this group conferred several benefits including potency, pharmacokinetics, and selectivity. Mouse xenograft PK/PD studies were carried out using an advanced compound, G-5555 (12), derived from this approach. These studies concluded that dose-dependent pathway modulation was achievable and paves the way for further in vivo investigations of PAK1 function in cancer and other diseases.
doi:10.1021/acsmedchemlett.5b00398
PMCID: PMC4677365  PMID: 26713112
Kinase inhibitor; oncology; hERG; pKa
2.  Phosphoproteomic screening identifies Rab GTPases as novel downstream targets of PINK1 
The EMBO Journal  2015;34(22):2840-2861.
Abstract
Mutations in the PTEN‐induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser65) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1‐dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub‐family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser111) in response to PINK1 activation. Using phospho‐specific antibodies raised against Ser111 of each of the Rabs, we demonstrate that Rab Ser111 phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient‐derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser111 phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser111 phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser65. We further show mechanistically that phosphorylation at Ser111 significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser111 may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase‐mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.
doi:10.15252/embj.201591593
PMCID: PMC4654935  PMID: 26471730
Parkinson's disease; phosphoproteomics; PINK1; Rab GTPases; Membrane & Intracellular Transport; Methods & Resources; Post-translational Modifications, Proteolysis & Proteomics
3.  Tiered Human Integrated Sequence Search Databases for Shotgun Proteomics 
Journal of proteome research  2016;15(11):4091-4100.
The results of analysis of shotgun proteomics mass spectrometry data can be greatly affected by the selection of the reference protein sequence database against which the spectra are matched. For many species there are multiple sources from which somewhat different sequence sets can be obtained. This can lead to confusion about which database is best in which circumstances – a problem especially acute in human sample analysis. All sequence databases are genome-based, with sequences for the predicted gene and their protein translation products compiled. Our goal is to create a set of primary sequence databases that comprise the union of sequences from many of the different available sources and make the result easily available to the community. We have compiled a set of four sequence databases of varying sizes, from a small database consisting of only the ~20,000 primary isoforms plus contaminants to a very large database that includes almost all non-redundant protein sequences from several sources. This set of tiered, increasingly complete human protein sequence databases suitable for mass spectrometry proteomics sequence database searching is called the Tiered Human Integrated Search Proteome set. In order to evaluate the utility of these databases, we have analyzed two different data sets, one from the HeLa cell line and the other from normal human liver tissue, with each of the four tiers of database complexity. The result is that approximately 0.8%, 1.1%, and 1.5% additional peptides can be identified for Tiers 2, 3, and 4, respectively, as compared with the Tier 1 database, at substantially increasing computational cost. This increase in computational cost may be worth bearing if the identification of sequence variants or the discovery of sequences that are not present in the reviewed knowledge base entries is an important goal of the study. We find that it is useful to search a data set against a simpler database, and then check the uniqueness of the discovered peptides against a more complex database. We have set up an automated system that downloads all the source databases on the first of each month and automatically generates a new set of search databases and makes them available for download at http://www.peptideatlas.org/thisp/.
doi:10.1021/acs.jproteome.6b00445
PMCID: PMC5096980  PMID: 27577934
shotgun mass spectrometry; search databases; human
4.  Assessing outcomes of enhanced chronic disease care through patient education and a value-based formulary study (ACCESS)—study protocol for a 2×2 factorial randomized trial 
Background
Chronic diseases result in significant morbidity and costs. Although medications and lifestyle changes are effective for improving outcomes in chronic diseases, many patients do not receive these treatments, in part because of financial barriers, patient and provider-level knowledge gaps, and low patient motivation. The Assessing outcomes of enhanced chronic disease care through patient education and a value-based formulary study (ACCESS) will determine the impact of two interventions: (1) a value-based formulary which eliminates copayment for high-value preventive medications; and (2) a comprehensive self-management support program aimed at promoting health behavior change and medication adherence, combined with relay of information on medication use to healthcare providers, on cardiovascular events and/or mortality in low-income seniors with elevated cardiovascular risk.
Methods
The ACCESS study will use a parallel, open label, factorial randomized trial design, with blinded endpoint evaluation in 4714 participants who are over age >65 (and therefore have drug insurance provided by Alberta Blue Cross with 30 % co-payment); are at a high risk for cardiovascular events based on a history of any one of the following: coronary heart disease, prior stroke, chronic kidney disease, heart failure, or any two of the following: current cigarette smoking, diabetes mellitus, hypertension, or hypercholesterolemia; and have a household income
Discussion
Given identified gaps in care in chronic disease, and the frequency of financial and knowledge-related barriers in low-income Albertans, this study will test the impact of providing free high-value preventive medications (i.e., value-based insurance) and a tailored self-management education and facilitated relay strategy on outcomes and costs. By measuring the impact on both health outcomes and costs, as well as the impact on reducing health inequities in this vulnerable population, our study will facilitate informed policy decisions.
Trial registration
Clinicaltrials.gov: NCT02579655. Registered Oct 15, 2015.
Electronic supplementary material
The online version of this article (doi:10.1186/s13012-016-0491-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s13012-016-0491-6
PMCID: PMC5037634  PMID: 27671037
Randomized clinical trial; Chronic disease; Cost; Education; Drug insurance
The EMBO Journal  2015;34(17):2272-2290.
Lysosomes are essential organelles that function to degrade and recycle unwanted, damaged and toxic biological components. Lysosomes also act as signalling platforms in activating the nutrient-sensing kinase mTOR. mTOR regulates cellular growth, but it also helps to maintain lysosome identity by initiating lysosomal tubulation through a process termed autophagosome-lysosome reformation (ALR). Here we identify a lysosomal pool of phosphatidylinositol 3-phosphate that, when depleted by specific inhibition of the class III phosphoinositide 3-kinase VPS34, results in prolonged lysosomal tubulation. This tubulation requires mTOR activity, and we identified two direct mTOR phosphorylation sites on UVRAG (S550 and S571) that activate VPS34. Loss of these phosphorylation sites reduced VPS34 lipid kinase activity and resulted in an increase in number and length of lysosomal tubules. In cells in which phosphorylation at these UVRAG sites is disrupted, the result of impaired lysosomal tubulation alongside ALR activation is massive cell death. Our data imply that ALR is critical for cell survival under nutrient stress and that VPS34 is an essential regulatory element in this process.
doi:10.15252/embj.201590992
PMCID: PMC4585463  PMID: 26139536
lysosome; mTOR; tubule; UVRAG; VPS34
Objective
To determine the effect on inter-hospital patient sharing via transfers on the rate of Clostridium difficile infections (CDI) in a hospital.
Design
Retrospective cohort
Methods
Using data from the Healthcare Cost and Utilization Project California State Inpatient Database, 2005–2011, we identified 2,752,639 transfers. We then constructed a series of networks detailing the connections formed by hospitals. We computed two measures of connectivity, indegree and weighted indegree, measuring the number of hospitals from which transfers into a hospital arrive, and the total number of incoming transfers, respectively. We estimated a multivariate model of CDI cases using the log-transformed network measures as well as covariates for hospital fixed effects, log median length of stay, log fraction of patients aged 65 or older, quarter and year indicators as predictors.
Results
We found an increase of one in the log indegree was associated with a 4.8% increase in incidence of CDI (95% CI: 2.3–7.4) and an increase of one in log weighted indegree was associated with a 3.3% increase in CDI incidence (95% CI: 1.5–5.2). Moreover, including measures of connectivity in the models greatly improved their fit.
Conclusions
Our results suggest infection control is not under the exclusive control of a given hospital but is also influenced by the connections and number of connections that hospitals have with other hospitals.
doi:10.1017/ice.2015.130
PMCID: PMC4736722  PMID: 26072907
eLife  null;5:e12278.
The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1.
DOI: http://dx.doi.org/10.7554/eLife.12278.001
eLife digest
During digestion, proteins are broken down into their constituent parts called amino acids. Amino acids are transported in the bloodstream and are used to build up new cells and repair old ones. Optimal regulation of the cellular rates of amino acid uptake and protein synthesis is critical to the overall health of our bodies.
Inside each of our cells is a molecule called mammalian target of rapamycin (mTOR for short), which acts as a controller that receives information about amino acid availability. mTOR also senses how much of each amino acid the cell needs and calibrates the cell’s amino acid uptake and protein synthesis machineries accordingly.
When investigating an enzyme named ZNRF2, Hoxhaj et al. discovered that it interacts with mTOR on membranes inside cells. This raised questions about how ZNRF2 might work with mTOR to sense amino acid supplies and regulate cell growth.
Hoxhaj et al. found that when cells are provided with amino acids and growth-stimulating hormones, mTOR is activated and attaches a phosphate group to ZNRF2. This chemical modification promotes the release of ZNRF2 from membranes so that ZNRF2 separates from mTOR. In contrast, when cells are starved of amino acids, this phosphate group is removed from ZNRF2, which then returns to the membranes. On membranes, ZNRF2 also influences the activity of a pump called V-ATPase, which controls the internal acidity of the membrane-enclosed vesicles named lysosomes that help to recycle amino acids inside cells. The action of ZNRF2 on the pump may help to prime mTOR so that it is ready to sense amino acids.
These findings by Hoxhaj et al. suggest that ZNRF2 and mTOR may ‘tune’ each other, making constant to-and-fro adjustments to help ensure that levels of amino acid uptake and cell growth are set just right. However, many questions about ZNRF2 still remain to be addressed. For example, are genetic mutations in ZNRF2 involved in cancers, developmental disorders or growth syndromes? Is ZNRF2 most important in the brain, where it is particularly abundant? And how does ZNRF2 affect acidity within the lysosomes?
DOI: http://dx.doi.org/10.7554/eLife.12278.002
doi:10.7554/eLife.12278
PMCID: PMC4889327  PMID: 27244671
ZNRF2; mTORC1; amino acid sensing; V-ATPase; PP6; Ragulator; Human; Mouse
British Journal of Social Work  2015;46(5):1282-1300.
Decision makers in adult social care are increasingly interested in using evidence from research to support or shape their decisions. The scope and nature of the current landscape of adult social care research (ASCR) need to be better understood. This paper provides a bibliometric assessment of ASCR outputs from 1996 to 2011. ASCR papers were retrieved using three strategies: from key journals; using keywords and noun phrases; and from additional papers preferentially citing or being cited by other ASCR papers. Overall, 195,829 ASCR papers were identified in the bibliographic database Scopus, of which 16 per cent involved at least one author from the UK. The UK output increased 2.45-fold between 1996 and 2011. Among selected countries, those with greater research intensity in ASCR generally had higher citation impact, such as the USA, UK, Canada and the Netherlands. The top five UK institutions in terms of volume of papers in the UK accounted for 26 per cent of total output. We conclude by noting the limitations to bibliometric analysis of ASCR and examine how such analysis can support the strategic development of the field.
doi:10.1093/bjsw/bcv022
PMCID: PMC4985733  PMID: 27559228
Adult social care; bibliometrics; research policy; comparative analysis
Background
Guideline committees have identified the need for research to inform the provision of conservative care for older adults with stage 5 chronic kidney disease (CKD) who have a high burden of comorbidity or functional impairment. We will use both qualitative and quantitative methodologies to provide a comprehensive understanding of barriers and facilitators to care for these patients in primary care.
Objectives
Our objectives are to (1) interview primary care physicians to determine their perspectives of conservative care for older adults with stage 5 CKD and (2) survey primary care physicians to determine the prevalence of key barriers and facilitators to provision of conservative care for older adults with stage 5 CKD.
Design
A sequential exploratory mixed methods design was adopted for this study. The first phase of the study will involve fundamental qualitative description and the second phase will be a cross-sectional population-based survey.
Setting
The research is conducted in Alberta, Canada.
Participants
The participants are primary care physicians with experience in providing care for older adults with stage 5 CKD not planning on initiating dialysis.
Methods
The first objective will be achieved by undertaking interviews with primary care physicians from southern Alberta. Participants will be selected purposively to include physicians with a range of characteristics (e.g., age, gender, and location of clinical practice). Interviews will be recorded, transcribed verbatim, and analyzed using conventional content analysis to generate themes. The second objective will be achieved by undertaking a population-based survey of primary care physicians in Alberta. The questionnaire will be developed based on the findings from the qualitative interviews and pilot tested for face and content validity. Physicians will be provided multiple options to complete the questionnaire including mail, fax, and online methods. Descriptive statistics and associations between demographic factors and barriers and facilitators to care will be analyzed using regression models.
Limitations
A potential limitation of this mixed methods study is its cross-sectional nature.
Conclusions
This work will inform development of clinical resources and tools for care of older adults with stage 5 CKD, to address barriers and enable facilitators to community-based conservative care.
Electronic supplementary material
The online version of this article (doi:10.1186/s40697-016-0110-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s40697-016-0110-0
PMCID: PMC4819283  PMID: 27047667
Chronic kidney disease; Conservative care; Mixed methods; Non-dialysis care; Older adults; Primary care physicians
CMAJ Open  2016;4(2):E304-E308.
Background:
Patients with cardiovascular-related chronic diseases may face financial barriers to accessing health care, even in Canada, where universal health care insurance is in place. No current theory or framework is adequate for understanding the impact of financial barriers to care on these patients or how they experience financial barriers. The overall objective of this study is to develop a framework for understanding the role of financial barriers to care in the lives of patients with cardiovascular-related chronic diseases and the impact of such barriers on their health.
Methods:
We will perform an inductive qualitative grounded theory study to develop a framework to understand the effect of financial barriers to care on patients with cardiovascular-related chronic diseases. We will use semistructured interviews (face-to-face and telephone) with a purposive sample of adult patients from Alberta with at least 1 of hypertension, diabetes, heart disease or stroke. We will analyze interview transcripts in triplicate using grounded theory coding techniques, including open, focused and axial coding, following the principle of constant comparison. Interviews and analysis will be done iteratively to theoretical saturation. Member checking will be used to enhance rigour.
Interpretation:
A comprehensive framework for understanding financial barriers to accessing health care is instrumental for both researchers and clinicians who care for patients with chronic diseases. Such a framework would enable a better understanding of patient behaviour and nonadherence to recommended medical therapies and lifestyle modifications.
doi:10.9778/cmajo.20160030
PMCID: PMC4933648  PMID: 27398378
The World Health Organization reports noncommunicable disease as a global pandemic. While national and international health research/policy bodies, such as the World Health Organization and the Australian Institute of Health and Welfare, emphasize the importance of preventative health, there is a continuing distortion in the allocation of resources to curative health as a result of government failure. Government failure is, in part, the result of a political response to individual preference for certainty in receiving treatment for specific health conditions, rather than the uncertainty of population-based preventative intervention. This has led to a failure to engage with those primary causative factors affecting chronic disease, namely the psychosocial stressors, in which the socioeconomic determinants are an important component. Such causal factors are open to manipulation through government policies and joint government-government, government-private cooperation through application of nonmedical primary-preventative health policies. The health benefits of Aboriginal people in traditional land management, or caring-for-country, in remote to very remote Australia, is used to exemplify the social benefits of nonmedical primary-preventative health intervention. Such practices form part of the “healthy country, health people” concept that is traditionally relied upon by Indigenous peoples. Possible health and wider private good and public good social benefits are shown to occur across multiple disciplines and jurisdictions with the possibility of substantial economies. General principles in the application of nonmedical primary-preventative health activities are developed through consideration of the experience of Afboriginal people participation in traditional caring-for-country.
doi:10.3390/ijerph13040400
PMCID: PMC4847062  PMID: 27482574
chronic disease pandemic; Indigenous; social benefit; psychosocial stressors; environmental benefit; noncommunicable disease
We have engineered an integration-deficient lentiviral vector, LV305, to deliver the tumor antigen NY-ESO-1 to human dendritic cells in vivo through pseudotyping with a modified Sindbis virus envelop protein. Mice immunized once with LV305 developed strong, dose-dependent, multifunctional, and cytotoxic NY-ESO-1-specific cluster of differentiation 8 (CD8) T cells within 14 days post-immunization and could be boosted with LV305 at least twice to recall peak-level CD8 T-cell responses. Immunization with LV305 protected mice against tumor growth in an NY-ESO-1-expressing CT26 lung metastasis model, with the protective effect abrogated upon depletion of CD8 T cells. Adoptive transfer of CD8 T cells, alone or together with CD4 T cells or natural killer cells, from LV305-immunized donor mice to tumor-bearing recipient mice conferred significant protection against metastatic tumor growth. Biodistribution of injected LV305 in mice was limited to the site of injection and the draining lymph node, and injected LV305 exhibited minimal excretion. Mice injected with LV305 developed little to no adverse effects, as evaluated by toxicology studies adherent to good laboratory practices. Taken together, these data support the development of LV305 as a clinical candidate for treatment against tumors expressing NY-ESO-1.
doi:10.1038/mto.2016.10
PMCID: PMC5008268  PMID: 27626061
Journal of proteome research  2015;14(9):3461-3473.
The Human PeptideAtlas is a compendium of the highest quality peptide identifications from over 1000 shotgun mass spectrometry proteomics experiments collected from many different labs, all reanalyzed through a uniform processing pipeline. The latest 2015-03 build contains substantially more input data than past releases, is mapped to a recent version of our merged reference proteome, and uses improved informatics processing and the development of the AtlasProphet to provide the highest quality results. Within the set of ~20,000 neXtProt primary entries, 14,070 (70%) are confidently detected in the latest build, 5% are ambiguous, 9% are redundant, leaving the total percentage of proteins for which there are no mapping detections at just 16% (3166), all derived from over 133 million peptide-spectrum matches identifying more than 1 million distinct peptides using AtlasProphet to characterize and classify the protein matches. Improved handling for detection and presentation of single amino-acid variants (SAAVs) reveals the detection of 5,326 uniquely mapping SAAVs across 2,794 proteins. With such a large amount of data, the control of false positives is a challenge. We present the methodology and results for maintaining rigorous quality, along with a discussion of the implications of the remaining sources of errors in the build. We check our uncertainty estimates against a set of olfactory receptor proteins not expected to be present in the set. We show how the use of synthetic reference spectra can provide confirmatory evidence for claims of detection of proteins with weak evidence.
doi:10.1021/acs.jproteome.5b00500
PMCID: PMC4755269  PMID: 26139527
shotgun proteomics; tandem mass spectrometry; repositories; PeptideAtlas; Human Proteome Project; observed proteome
Background
Despite the growing evidence in the literature there is still a lack of consensus regarding the use of minimally invasive surgical technique (MIS) in total knee arthroplasty (TKA).
Methods
A prospective, randomized, international multicentre trial including 69 patients was performed to compare computer-assisted TKA (CAS-TKA) using either mini-midvastus (MIS group) or standard medial parapatellar approach (conventional group).
Patients from 3 centers (Maastricht, Zwickau, Adelaide) with end-stage osteoarthritis of the knee were randomized to either an MIS group with dedicated instrumentation or a conventional group to receive cruciate retaining CAS-TKA without patella resurfacing. The primary outcome was to compare post operative pain and range of motion (ROM). The secondary outcome was to measure the duration of surgery, blood loss, chair rise test, quadriceps strength, anterior knee pain, Knee Society Score (KSS),WOMAC scores, mechanical leg axis and component alignment.
Results
Patients in the MIS group (3.97 ± 2.16) had significant more pain at 2 weeks than patients in the conventional group (2.77 ± 1.43) p = 0.003. There was no significant difference in any of the other primary outcome parameters. Surgery time was significantly longer (p < 0.001) and there were significantly higher blood loss (p = 0.002) in the MIS group as compared to the conventional group. The difference of the mean mechanical leg alignment between the groups was not statistically significant (–0.43° (95 % CI –1.50 – 0.64); p = 0.43).
There was no significant difference of component alignment between the two surgical groups with respect to flexion/extension (p = 0.269), varus/valgus (p = 0.653) or rotational alignment (p = 0.485) of the femur component and varus valgus alignment (p = 0.778) or posterior slope (p = 0.164) of the tibial component.
Conclusion
There was no advantage of the MIS approach compared to a conventional approach CAS-TKA in any of the primary outcome measurements assessed, however the MIS approach was associated with longer surgical time and greater blood loss. MIS-TKA in combination with computer navigation is safe in terms of implant positioning.
Trial registration number
ClinicalTrials.gov NCT02625311 8 December 2015
doi:10.1186/s12891-016-0872-7
PMCID: PMC4711101  PMID: 26762175
Total knee arthroplasty; Navigation; Minimally invasive surgery; Blood loss; Accuracy
Scientific Reports  2015;5:17411.
Copepods are one of the most abundant metazoans in the marine ecosystem, constituting a critical link in aquatic food webs and contributing significantly to the global carbon budget, yet molecular mechanisms of their gene expression are not well understood. Here we report the detection of spliced leader (SL) trans-splicing in calanoid copepods. We have examined nine species of wild-caught copepods from Jiaozhou Bay, China that represent the major families of the calanoids. All these species contained a common 46-nt SL (CopepodSL). We further determined the size of CopepodSL precursor RNA (slRNA; 108-158 nt) through genomic analysis and 3′-RACE technique, which was confirmed by RNA blot analysis. Structure modeling showed that the copepod slRNA folded into typical slRNA secondary structures. Using a CopepodSL-based primer set, we selectively enriched and sequenced copepod full-length cDNAs, which led to the characterization of copepod transcripts and the cataloging of the complete set of 79 eukaryotic cytoplasmic ribosomal proteins (cRPs) for a single copepod species. We uncovered the SL trans-splicing in copepod natural populations, and demonstrated that CopepodSL was a sensitive and specific tool for copepod transcriptomic studies at both the individual and population levels and that it would be useful for metatranscriptomic analysis of copepods.
doi:10.1038/srep17411
PMCID: PMC4664967  PMID: 26621068
The EMBO Journal  2015;34(17):2272-2290.
Lysosomes are essential organelles that function to degrade and recycle unwanted, damaged and toxic biological components. Lysosomes also act as signalling platforms in activating the nutrient-sensing kinase mTOR. mTOR regulates cellular growth, but it also helps to maintain lysosome identity by initiating lysosomal tubulation through a process termed autophagosome-lysosome reformation (ALR). Here we identify a lysosomal pool of phosphatidylinositol 3-phosphate that, when depleted by specific inhibition of the class III phosphoinositide 3-kinase VPS34, results in prolonged lysosomal tubulation. This tubulation requires mTOR activity, and we identified two direct mTOR phosphorylation sites on UVRAG (S550 and S571) that activate VPS34. Loss of these phosphorylation sites reduced VPS34 lipid kinase activity and resulted in an increase in number and length of lysosomal tubules. In cells in which phosphorylation at these UVRAG sites is disrupted, the result of impaired lysosomal tubulation alongside ALR activation is massive cell death. Our data imply that ALR is critical for cell survival under nutrient stress and that VPS34 is an essential regulatory element in this process.
doi:10.15252/embj.201590992
PMCID: PMC4585463  PMID: 26139536
lysosome; mTOR; tubule; UVRAG; VPS34
The EMBO Journal  2015;34(22):2840-2861.
Mutations in the PTEN-induced kinase 1 (PINK1) are causative of autosomal recessive Parkinson's disease (PD). We have previously reported that PINK1 is activated by mitochondrial depolarisation and phosphorylates serine 65 (Ser65) of the ubiquitin ligase Parkin and ubiquitin to stimulate Parkin E3 ligase activity. Here, we have employed quantitative phosphoproteomics to search for novel PINK1-dependent phosphorylation targets in HEK (human embryonic kidney) 293 cells stimulated by mitochondrial depolarisation. This led to the identification of 14,213 phosphosites from 4,499 gene products. Whilst most phosphosites were unaffected, we strikingly observed three members of a sub-family of Rab GTPases namely Rab8A, 8B and 13 that are all phosphorylated at the highly conserved residue of serine 111 (Ser111) in response to PINK1 activation. Using phospho-specific antibodies raised against Ser111 of each of the Rabs, we demonstrate that Rab Ser111 phosphorylation occurs specifically in response to PINK1 activation and is abolished in HeLa PINK1 knockout cells and mutant PINK1 PD patient-derived fibroblasts stimulated by mitochondrial depolarisation. We provide evidence that Rab8A GTPase Ser111 phosphorylation is not directly regulated by PINK1 in vitro and demonstrate in cells the time course of Ser111 phosphorylation of Rab8A, 8B and 13 is markedly delayed compared to phosphorylation of Parkin at Ser65. We further show mechanistically that phosphorylation at Ser111 significantly impairs Rab8A activation by its cognate guanine nucleotide exchange factor (GEF), Rabin8 (by using the Ser111Glu phosphorylation mimic). These findings provide the first evidence that PINK1 is able to regulate the phosphorylation of Rab GTPases and indicate that monitoring phosphorylation of Rab8A/8B/13 at Ser111 may represent novel biomarkers of PINK1 activity in vivo. Our findings also suggest that disruption of Rab GTPase-mediated signalling may represent a major mechanism in the neurodegenerative cascade of Parkinson's disease.
doi:10.15252/embj.201591593
PMCID: PMC4654935  PMID: 26471730
Parkinson's disease; phosphoproteomics; PINK1; Rab GTPases
The Journal of Biological Chemistry  2015;290(50):30030-30041.
Diabetes is strongly associated with cognitive decline, but the molecular reasons are unknown. We found that fasting and peripheral insulin promote phosphorylation and dephosphorylation, respectively, of specific residues on brain proteins including cytoskeletal regulators such as slit-robo GTPase-activating protein 3 (srGAP3) and microtubule affinity-regulating protein kinases (MARKs), in which deficiency or dysregulation is linked to neurological disorders. Fasting activates protein kinase A (PKA) but not PKB/Akt signaling in the brain, and PKA can phosphorylate the purified srGAP3. The phosphorylation of srGAP3 and MARKs were increased when PKA signaling was activated in primary neurons. Knockdown of PKA decreased the phosphorylation of srGAP3. Furthermore, WAVE1, a protein kinase A-anchoring protein, formed a complex with srGAP3 and PKA in the brain of fasted mice to facilitate the phosphorylation of srGAP3 by PKA. Although brain cells have insulin receptors, our findings are inconsistent with the down-regulation of phosphorylation of target proteins being mediated by insulin signaling within the brain. Rather, our findings infer that systemic insulin, through a yet unknown mechanism, inhibits PKA or protein kinase(s) with similar specificity and/or activates an unknown phosphatase in the brain. Ser858 of srGAP3 was identified as a key regulatory residue in which phosphorylation by PKA enhanced the GAP activity of srGAP3 toward its substrate, Rac1, in cells, thereby inhibiting the action of this GTPase in cytoskeletal regulation. Our findings reveal novel mechanisms linking peripheral insulin sensitivity with cytoskeletal remodeling in neurons, which may help to explain the association of diabetes with neurological disorders such as Alzheimer disease.
doi:10.1074/jbc.M115.668103
PMCID: PMC4705965  PMID: 26499801
brain; cytoskeleton; insulin resistance; neurodegeneration; protein phosphorylation; MARKs; fasting; srGAP3
Science signaling  2015;8(372):ra35.
The deubiquitylating enzyme OTUB1 is present in all tissues and targets a multitude of substrates, both in the cytosol and nucleus. Here, we found that the phosphorylation of OTUB1 at Ser16 and its subsequent nuclear accumulation is mediated by casein kinase 2 (CK2). Whereas unphosphorylated OTUB1 was detected mainly in the cytosol, Ser16-phosphorylated OTUB1 was detected only in the nucleus. Pharmacological inhibition or genetic ablation of CK2 blocked the phosphorylation of OTUB1 at Ser16 and its nuclear localization in various cells. The phosphorylation of OTUB1 at Ser16 did not alter its catalytic activity in vitro, its ability to bind K63-linked ubiquitin chains in vitro and its ability to interact with the E2 enzyme UBE2N in vitro. The phosphorylation at Ser16 and subsequent nuclear localization of OTUB1 was essential for cells to repair ionizing radiation-induced DNA damage in osteosarcoma U2OS cells.
doi:10.1126/scisignal.aaa0441
PMCID: PMC4421874  PMID: 25872870
BMC Family Practice  2015;16:139.
Background
Despite Canada’s universal healthcare system, significant barriers impede individuals experiencing homelessness from accessing health services. Furthermore, there is a paucity in the qualitative literature describing how Canadians experiencing homelessness access health care services. Our objective was to qualitatively explore perceived healthcare needs and barriers among individuals experiencing homelessness in one large Canadian city – Calgary, Alberta.
Methods
We conducted a qualitative descriptive study that included open-ended interviews and focus groups with a variety of stakeholders who are involved in healthcare among Calgary’s homeless populations. These included individuals experiencing homelessness (n = 11) as well as employees from several healthcare service providers for those experiencing homelessness (n = 11). Transcripts from these interviews were thematically analyzed by two analysts.
Results
Stakeholder interviews yielded several pervasive themes surrounding the health care needs of the homeless and barriers to accessing care. Some of the primary health care needs which were identified included mental health, addictions, and allied health as well as care that addresses the social determinants of health. Notably, it was difficult for many stakeholders to pinpoint specific health care priorities, as they identified that the health care needs among Calgary’s homeless populations are diverse and complex, often even describing the needs as overwhelming. Types of barriers to primary care that were identified by stakeholders included: emotional, educational, geographical, financial and structural barriers, as well as discrimination.
Conclusions
Our findings highlight the diverse primary health care needs of Calgary’s homeless populations. Despite the fact that Canada has a universal publicly funded health care system, individuals experiencing homelessness face significant barriers in accessing primary care.
doi:10.1186/s12875-015-0361-3
PMCID: PMC4603688  PMID: 26463577
Homeless; Poverty; Qualitative research; Needs assessment
eLife  null;4:e07661.
We present a novel mass spectrometry-based high-throughput workflow and an open-source computational and data resource to reproducibly identify and quantify HLA-associated peptides. Collectively, the resources support the generation of HLA allele-specific peptide assay libraries consisting of consensus fragment ion spectra, and the analysis of quantitative digital maps of HLA peptidomes generated from a range of biological sources by SWATH mass spectrometry (MS). This study represents the first community-based effort to develop a robust platform for the reproducible and quantitative measurement of the entire repertoire of peptides presented by HLA molecules, an essential step towards the design of efficient immunotherapies.
DOI: http://dx.doi.org/10.7554/eLife.07661.001
eLife digest
The cells of the immune system protect us by recognizing telltale molecules produced by damaged and diseased cells, or by infection-causing microorganisms (which are also called pathogens). To help with this process, the cells in our bodies display small fragments of proteins (called peptides) on their surface that are then checked by the immune cells. Collectively, these peptides are referred to as the ‘immunopeptidome’, and deciphering the complexity of the human immunopeptidome is important for both basic research and medical science. Such an achievement would help to guide the development of next-generation vaccines and therapies against autoimmune disorders, infectious diseases and cancers.
In the past, immune peptides were mostly identified using a technique that is commonly called ‘shotgun’ mass spectrometry. However, this approach doesn't always provide reproducible results. In 2012, researchers reported the development of a new approach—which they called ‘SWATH’ mass spectrometry—that could yield more reproducible data.
Now, Caron et al.—including many of the researchers involved in the 2012 study—have developed a large collection of standardized tests that use SWATH mass spectrometry to analyze the human immunopeptidome. The workflow and the computational and data resources developed as part of this international effort are the first steps toward highly reproducible and measurable analyses of the immunopeptidome across many samples. Moreover, the large repository of assays generated by the project has been made public and will serve a large community of researchers, which should enable better collaborations.
In the future, SWATH mass spectrometry could be used as a robust technology for the reproducible detection and measurement of pathogen-specific or cancer-specific immune peptides. This could greatly help in the design of personalized immune-based therapies.
DOI: http://dx.doi.org/10.7554/eLife.07661.002
doi:10.7554/eLife.07661
PMCID: PMC4507788  PMID: 26154972
human leukocytes antigen; immunopeptidome; targeted mass spectrometry; SWATH-MS; DIA; human
PeptideAtlas, SRMAtlas and PASSEL are web-accessible resources to support discovery and targeted proteomics research. PeptideAtlas is a multi-species compendium of shotgun proteomic data provided by the scientific community, SRMAtlas is a resource of high-quality, complete proteome SRM assays generated in a consistent manner for the targeted identification and quantification of proteins, and PASSEL is a repository that compiles and represents selected reaction monitoring data, all in an easy to use interface. The databases are generated from native mass spectrometry data files that are analyzed in a standardized manner including statistical validation of the results. Each resource offers search functionalities and can be queried by user defined constraints; the query results are provided in tables or are graphically displayed. PeptideAtlas, SRMAtlas and PASSEL are publicly available freely via the website http://www.peptideatlas.org. In this protocol, we describe the use of these resources, we highlight how to submit, search, collate and download data.
doi:10.1002/0471250953.bi1325s46
PMCID: PMC4331073  PMID: 24939129
discovery proteomics; targeted proteomics; selected reaction monitoring (SRM); data repository; data resource; complete proteome library
It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL) in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02). VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector.
doi:10.1038/mtm.2015.17
PMCID: PMC4445008  PMID: 26029728
Evaporative and exhaust mobile source air toxic (MSAT) emissions of total volatile organic compounds, carbon monoxide, BTEX (benzene, toluene, ethylbenzene, and xylenes), formaldehyde, acetaldehyde, butadiene, methyl tertiary butyl ether, and ethanol were measured in vehicle-related high-end microenvironments (ME) under worst-case conditions plausibly simulating the >99th percentile of inhalation exposure concentrations in Atlanta (baseline gasoline), Chicago (ethanol-oxygenated gasoline), and Houston (methyl tertiary butyl either-oxygenated gasoline) during winter and summer seasons. High-end MSAT values as ratios of the corresponding measurements at nearby air monitoring stations exceeded the microenvironmental proximity factors used in regulatory exposure models, especially for refueling operations and MEs under reduced ventilation. MSAT concentrations were apportioned between exhaust and evaporative vehicle emissions in Houston where methyl tertiary butyl ether could be used as a vehicle emission tracer. With the exception of vehicle refueling operations, the results indicate that evaporative emissions are a minor component of high-end MSAT exposure concentrations.
doi:10.1007/s11869-015-0345-4
PMCID: PMC4837208  PMID: 27158280
Mobile source air toxics; Microenvironment; Exposure; VOC; BTEX; Evaporative emissions
Introduction
Breast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase (PAK)1. PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis.
Methods
PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n = 980 and 1,108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting.
Results
We demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P = 1.29 × 10−4 and P = 0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis.
Conclusions
Taken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-015-0564-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-015-0564-5
PMCID: PMC4445529  PMID: 25902869

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