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1.  Analysis of mammalian gene function through broad based phenotypic screens across a consortium of mouse clinics 
de Angelis, Martin Hrabě | Nicholson, George | Selloum, Mohammed | White, Jacqui | Morgan, Hugh | Ramirez-Solis, Ramiro | Sorg, Tania | Wells, Sara | Fuchs, Helmut | Fray, Martin | Adams, David J | Adams, Niels C | Adler, Thure | Aguilar-Pimentel, Antonio | Ali-Hadji, Dalila | Amann, Gregory | André, Philippe | Atkins, Sarah | Auburtin, Aurelie | Ayadi, Abdel | Becker, Julien | Becker, Lore | Bedu, Elodie | Bekeredjian, Raffi | Birling, Marie-Christine | Blake, Andrew | Bottomley, Joanna | Bowl, Mike | Brault, Véronique | Busch, Dirk H | Bussell, James N | Calzada-Wack, Julia | Cater, Heather | Champy, Marie-France | Charles, Philippe | Chevalier, Claire | Chiani, Francesco | Codner, Gemma F | Combe, Roy | Cox, Roger | Dalloneau, Emilie | Dierich, André | Di Fenza, Armida | Doe, Brendan | Duchon, Arnaud | Eickelberg, Oliver | Esapa, Chris T | El Fertak, Lahcen | Feigel, Tanja | Emelyanova, Irina | Estabel, Jeanne | Favor, Jack | Flenniken, Ann | Gambadoro, Alessia | Garrett, Lilian | Gates, Hilary | Gerdin, Anna-Karin | Gkoutos, George | Greenaway, Simon | Glasl, Lisa | Goetz, Patrice | Da Cruz, Isabelle Goncalves | Götz, Alexander | Graw, Jochen | Guimond, Alain | Hans, Wolfgang | Hicks, Geoff | Hölter, Sabine M | Höfler, Heinz | Hancock, John M | Hoehndorf, Robert | Hough, Tertius | Houghton, Richard | Hurt, Anja | Ivandic, Boris | Jacobs, Hughes | Jacquot, Sylvie | Jones, Nora | Karp, Natasha A | Katus, Hugo A | Kitchen, Sharon | Klein-Rodewald, Tanja | Klingenspor, Martin | Klopstock, Thomas | Lalanne, Valerie | Leblanc, Sophie | Lengger, Christoph | le Marchand, Elise | Ludwig, Tonia | Lux, Aline | McKerlie, Colin | Maier, Holger | Mandel, Jean-Louis | Marschall, Susan | Mark, Manuel | Melvin, David G | Meziane, Hamid | Micklich, Kateryna | Mittelhauser, Christophe | Monassier, Laurent | Moulaert, David | Muller, Stéphanie | Naton, Beatrix | Neff, Frauke | Nolan, Patrick M | Nutter, Lauryl MJ | Ollert, Markus | Pavlovic, Guillaume | Pellegata, Natalia S | Peter, Emilie | Petit-Demoulière, Benoit | Pickard, Amanda | Podrini, Christine | Potter, Paul | Pouilly, Laurent | Puk, Oliver | Richardson, David | Rousseau, Stephane | Quintanilla-Fend, Leticia | Quwailid, Mohamed M | Racz, Ildiko | Rathkolb, Birgit | Riet, Fabrice | Rossant, Janet | Roux, Michel | Rozman, Jan | Ryder, Ed | Salisbury, Jennifer | Santos, Luis | Schäble, Karl-Heinz | Schiller, Evelyn | Schrewe, Anja | Schulz, Holger | Steinkamp, Ralf | Simon, Michelle | Stewart, Michelle | Stöger, Claudia | Stöger, Tobias | Sun, Minxuan | Sunter, David | Teboul, Lydia | Tilly, Isabelle | Tocchini-Valentini, Glauco P | Tost, Monica | Treise, Irina | Vasseur, Laurent | Velot, Emilie | Vogt-Weisenhorn, Daniela | Wagner, Christelle | Walling, Alison | Weber, Bruno | Wendling, Olivia | Westerberg, Henrik | Willershäuser, Monja | Wolf, Eckhard | Wolter, Anne | Wood, Joe | Wurst, Wolfgang | Yildirim, Ali Önder | Zeh, Ramona | Zimmer, Andreas | Zimprich, Annemarie | Holmes, Chris | Steel, Karen P | Herault, Yann | Gailus-Durner, Valérie | Mallon, Ann-Marie | Brown, Steve DM
Nature genetics  2015;47(9):969-978.
The function of the majority of genes in the mouse and human genomes remains unknown. The mouse ES cell knockout resource provides a basis for characterisation of relationships between gene and phenotype. The EUMODIC consortium developed and validated robust methodologies for broad-based phenotyping of knockouts through a pipeline comprising 20 disease-orientated platforms. We developed novel statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no prior functional annotation. We captured data from over 27,000 mice finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. Novel phenotypes were uncovered for many genes with unknown function providing a powerful basis for hypothesis generation and further investigation in diverse systems.
doi:10.1038/ng.3360
PMCID: PMC4564951  PMID: 26214591
2.  The Identification of Genes Important in Pseudomonas syringae pv. phaseolicola Plant Colonisation Using In Vitro Screening of Transposon Libraries 
PLoS ONE  2015;10(9):e0137355.
The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph) colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms) around plant cells. If the pathogen can suppress the plant’s natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn) mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP) genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction.
doi:10.1371/journal.pone.0137355
PMCID: PMC4556710  PMID: 26325299
3.  Specific cancer-associated mutations in the switch III region of Ras increase tumorigenicity by nanocluster augmentation 
eLife  null;4:e08905.
Hotspot mutations of Ras drive cell transformation and tumorigenesis. Less frequent mutations in Ras are poorly characterized for their oncogenic potential. Yet insight into their mechanism of action may point to novel opportunities to target Ras. Here, we show that several cancer-associated mutations in the switch III region moderately increase Ras activity in all isoforms. Mutants are biochemically inconspicuous, while their clustering into nanoscale signaling complexes on the plasma membrane, termed nanocluster, is augmented. Nanoclustering dictates downstream effector recruitment, MAPK-activity, and tumorigenic cell proliferation. Our results describe an unprecedented mechanism of signaling protein activation in cancer.
DOI: http://dx.doi.org/10.7554/eLife.08905.001
eLife digest
Cancer is a disease that develops when cells within the body acquire genetic mutations that allow them to grow and divide rapidly. Many human cancers have mutations in a gene that encodes a protein called Ras, which promotes cell growth and division by controlling the activities of other proteins.
Ras congregates at the membrane that surrounds the cell and can assemble into clusters (called nanoclusters) that each contain six to eight Ras proteins. The tight packing of the proteins in these nanoclusters increases the amount of Ras in the membrane locally, which allows Ras to interact with other proteins more efficiently to promote growth and cell division.
In normal cells, other proteins control when Ras is active. However, in many cancer cells, Ras is active all the time due to mutations that occur in three ‘hotspots’ within its gene. Other mutations in the gene that encodes Ras are also found in cancer cells, but these are less common and it is not clear how they alter the activity of the protein.
Here, Solman et al. used microscopy and biochemical techniques to study the effects of some of the less common mutations on Ras activity in human cells. The experiments show that several mutations that alter a region of Ras called the ‘switch III region’ moderately increase the activity of Ras. The mutations probably alter the way that Ras sits in the membrane, which in turn changes the way it interacts with other proteins and the membrane so that more Ras nanoclusters form.
Solman et al.'s findings reveal a new way that Ras can be activated in cancer cells. The next challenge is to develop drugs that block the formation of Ras nanoclusters and to find out if they have the potential to be used to treat cancer.
DOI: http://dx.doi.org/10.7554/eLife.08905.002
doi:10.7554/eLife.08905
PMCID: PMC4563131  PMID: 26274561
Ras; cancer; membrane; nanoclustering; signalling; human
4.  Histoplasmosis-Induced Hemophagocytic Syndrome: A Case Series and Review of the Literature 
Open Forum Infectious Diseases  2015;2(2):ofv055.
Background. Histoplasmosis-associated hemophagocytic lymphohistiocytosis (HLH) is a relatively rare disorder for which data are limited regarding optimal treatment and clinical outcomes in adults. We describe the clinical features, treatment, and outcomes of patients with histoplasmosis-associated HLH at our institution.
Methods. We performed a retrospective chart review of all inpatients at Parkland Hospital diagnosed with HLH associated with Histoplasma capsulatum from 2003 to 2013.
Results. Eleven cases of histoplasmosis-associated HLH over this time period were identified. Nine of eleven cases were males (82%). Nine of these patients had human immunodeficiency virus (HIV)/acquired immune deficiency syndrome (AIDS), 1 was a renal transplant patient on immunosuppressants, and the other had no documented immunocompromise. The most common HLH criteria were splenomegaly (n = 10), fever (n = 10), and ferritin >500 ng/dL (n = 9). Urine Histoplasma antigen was positive in every patient tested (n = 9 of 9), and most antibodies for Histoplasma were positive if checked (n = 4 of 5). A majority of patients received liposomal amphotericin B (n = 9) with an average treatment duration of 11 days, and 5 patients also received prednisone, intravenous immunoglobulin (IVIG), or both. Overall, 5 patients died within 30 days (45.5%), and 7 patients died within 90 days (63.6%). Of the 5 patients that received immunosuppression, 4 died (80%), whereas in the group not given additional immunosuppression (n = 5), 2 died (40%).
Conclusions. Histoplasmosis-associated HLH among adults is a lethal disease of highly immunocompromised patients, especially patients with HIV/AIDS. Clinical features such as splenomegaly, elevated ferritin, and cytopenias should prompt evaluation for HLH in this population. Further data are needed to define the role of immunosuppression, IVIG, and highly active antiretroviral therapy in treating this condition.
doi:10.1093/ofid/ofv055
PMCID: PMC4567100  PMID: 26380347
disseminated histoplasmosis; hemophagocytic syndrome; HIV
5.  Circles within circles: commentary on Ghosal et al. (2013) “Circ2Traits: a comprehensive database for circular RNA potentially associated with disease and traits” 
Frontiers in Genetics  2015;5:459.
doi:10.3389/fgene.2014.00459
PMCID: PMC4285795  PMID: 25610452
circular RNAs; MicroRNAs; Databases; Genetic; Parkinson disease; cancer
6.  Development of an estimation model for the evaluation of the energy requirement of dilute acid pretreatments of biomass☆ 
Biomass & Bioenergy  2015;72:28-38.
This study aims to develop a mathematical model to evaluate the energy required by pretreatment processes used in the production of second generation ethanol. A dilute acid pretreatment process reported by National Renewable Energy Laboratory (NREL) was selected as an example for the model's development. The energy demand of the pretreatment process was evaluated by considering the change of internal energy of the substances, the reaction energy, the heat lost and the work done to/by the system based on a number of simplifying assumptions. Sensitivity analyses were performed on the solid loading rate, temperature, acid concentration and water evaporation rate. The results from the sensitivity analyses established that the solids loading rate had the most significant impact on the energy demand. The model was then verified with data from the NREL benchmark process. Application of this model on other dilute acid pretreatment processes reported in the literature illustrated that although similar sugar yields were reported by several studies, the energy required by the different pretreatments varied significantly.
Highlights
•We developed a model for energy requirement evaluation for pretreatment processes.•NREL dilute acid pretreatment process was used as an example.•Around 1692 kJ kg−1 energy need to be supplied in order to carry out pretreatment.•The majority of the energy required was from the heating stage.•Solid loading rate was the key factor in influencing the energy requirements.
doi:10.1016/j.biombioe.2014.11.024
PMCID: PMC4461158  PMID: 26109752
Dilute acid pretreatment; Energy requirement; Lignocellulosic ethanol; Solids loading rate; Energy model
7.  Another Surprise from Metformin: Novel Mechanism of Action via K-Ras Influences Endometrial Cancer Response to Therapy 
Molecular cancer therapeutics  2013;12(12):10.1158/1535-7163.MCT-13-0439.
Metformin is an oral biguanide commonly used for the treatment of type II diabetes and has recently been demonstrated to possess anti-proliferative properties that can be exploited for the prevention and treatment of a variety of cancers. The mechanisms underlying this effect have not been fully elucidated. Using both in vitro and in vivo models, we examined the effects of metformin on endometrial tumors with defined aberrations in the PI3K/PTEN/mTOR and MAPK signaling pathways to understand metformin mechanism of action and identify clinically useful predictors of response to this agent. In vitro assays of proliferation, cytotoxicity, and apoptosis were used to quantify the effects of metformin on endometrial cancer cell lines with mutations in the PI3K/PTEN/mTOR and MAPK signaling pathways. The in vivo effects of oral metformin on tumor progression were further examined using xenograft mouse models of endometrial cancer. K-Ras localization was analyzed by confocal microscopy using GFP-labeled oncogenic K-Ras and by immunoblot following subcellular fractionation. Metformin inhibited cell proliferation, induced apoptosis, and decreased tumor growth in preclinical endometrial cancer models, with the greatest response observed in cells harboring activating mutations in K-Ras. Furthermore, metformin displaces constitutively active K-Ras from the cell membrane, causing uncoupling of the MAPK signaling pathway. These studies provide a rationale for clinical trials using metformin in combination with PI3K targeted agents for tumors harboring activating K-Ras mutations, and reveal a novel mechanism of action for metformin.
doi:10.1158/1535-7163.MCT-13-0439
PMCID: PMC3883498  PMID: 24077915
metformin; endometrial cancer; K-Ras; PTEN
8.  Caveolae regulate the nanoscale organization of the plasma membrane to remotely control Ras signaling 
The Journal of Cell Biology  2014;204(5):777-792.
Caveolae transduce mechanical stress into plasma membrane lipid alterations that disrupt Ras organization in an isoform-specific manner and modulate downstream signal transduction.
The molecular mechanisms whereby caveolae exert control over cellular signaling have to date remained elusive. We have therefore explored the role caveolae play in modulating Ras signaling. Lipidomic and gene array analyses revealed that caveolin-1 (CAV1) deficiency results in altered cellular lipid composition, and plasma membrane (PM) phosphatidylserine distribution. These changes correlated with increased K-Ras expression and extensive isoform-specific perturbation of Ras spatial organization: in CAV1-deficient cells K-RasG12V nanoclustering and MAPK activation were enhanced, whereas GTP-dependent lateral segregation of H-Ras was abolished resulting in compromised signal output from H-RasG12V nanoclusters. These changes in Ras nanoclustering were phenocopied by the down-regulation of Cavin1, another crucial caveolar structural component, and by acute loss of caveolae in response to increased osmotic pressure. Thus, we postulate that caveolae remotely regulate Ras nanoclustering and signal transduction by controlling PM organization. Similarly, caveolae transduce mechanical stress into PM lipid alterations that, in turn, modulate Ras PM organization.
doi:10.1083/jcb.201307055
PMCID: PMC3941050  PMID: 24567358
9.  Signal Integration by Lipid-Mediated Spatial Cross Talk between Ras Nanoclusters 
Molecular and Cellular Biology  2014;34(5):862-876.
Lipid-anchored Ras GTPases form transient, spatially segregated nanoclusters on the plasma membrane that are essential for high-fidelity signal transmission. The lipid composition of Ras nanoclusters, however, has not previously been investigated. High-resolution spatial mapping shows that different Ras nanoclusters have distinct lipid compositions, indicating that Ras proteins engage in isoform-selective lipid sorting and accounting for different signal outputs from different Ras isoforms. Phosphatidylserine is a common constituent of all Ras nanoclusters but is only an obligate structural component of K-Ras nanoclusters. Segregation of K-Ras and H-Ras into spatially and compositionally distinct lipid assemblies is exquisitely sensitive to plasma membrane phosphatidylserine levels. Phosphatidylserine spatial organization is also modified by Ras nanocluster formation. In consequence, Ras nanoclusters engage in remote lipid-mediated communication, whereby activated H-Ras disrupts the assembly and operation of spatially segregated K-Ras nanoclusters. Computational modeling and experimentation reveal that complex effects of caveolin and cortical actin on Ras nanoclustering are similarly mediated through regulation of phosphatidylserine spatiotemporal dynamics. We conclude that phosphatidylserine maintains the lateral segregation of diverse lipid-based assemblies on the plasma membrane and that lateral connectivity between spatially remote lipid assemblies offers important previously unexplored opportunities for signal integration and signal processing.
doi:10.1128/MCB.01227-13
PMCID: PMC4023829  PMID: 24366544
10.  Temporal Production of the Signaling Lipid Phosphatidic Acid by Phospholipase D2 Determines the Output of Extracellular Signal-Regulated Kinase Signaling in Cancer Cells 
Molecular and Cellular Biology  2014;34(1):84-95.
The Ras-extracellular signal-regulated kinase (ERK) cascade is an important signaling module in cells. One regulator of the Ras-ERK cascade is phosphatidic acid (PA) generated by phospholipase D (PLD) and diacylglycerol kinase (DGK). Using a newly developed PA biosensor, PASS (phosphatidic acid biosensor with superior sensitivity), we found that PA was generated sequentially by PLD and DGK in epidermal growth factor (EGF)-stimulated HCC1806 breast cancer cells. Inhibition of PLD2, one of the two PLD members, was sufficient to eliminate most of the PA production, whereas inhibition of DGK decreased PA production only at the later stages of EGF stimulation, suggesting that PLD2 precedes DGK activation. The temporal production of PA by PLD2 is important for the nuclear activation of ERK. While inhibition of both PLD and DGK had no effect on the overall ERK activity, inhibition of PLD2 but not PLD1 or DGK blocked the nuclear ERK activity in several cancer cell lines. The decrease of active ERK in the nucleus inhibited the activation of Elk1, c-fos, and Fra1, the ERK nuclear targets, leading to decreased proliferation of HCC1806 cells. Together, these findings reveal that PA production by PLD2 determines the output of ERK in cancer cell growth factor signaling.
doi:10.1128/MCB.00987-13
PMCID: PMC3911278  PMID: 24164897
11.  Commentary on Shimoyama et al. (2012): three ontologies to define phenotype measurement data 
doi:10.3389/fgene.2014.00093
PMCID: PMC4006037  PMID: 24795755
ontologies; phenotype; data representation; phenotype measurement; measurement ontologies
12.  Editorial: biological ontologies and semantic biology 
doi:10.3389/fgene.2014.00018
PMCID: PMC3912459  PMID: 24550936
semantic biology; biological ontologies; semantic web; data representation; data analysis
13.  Single-molecule analysis reveals self assembly and nanoscale segregation of two distinct cavin subcomplexes on caveolae 
eLife  2014;3:e01434.
In mammalian cells three closely related cavin proteins cooperate with the scaffolding protein caveolin to form membrane invaginations known as caveolae. Here we have developed a novel single-molecule fluorescence approach to directly observe interactions and stoichiometries in protein complexes from cell extracts and from in vitro synthesized components. We show that up to 50 cavins associate on a caveola. However, rather than forming a single coat complex containing the three cavin family members, single-molecule analysis reveals an exquisite specificity of interactions between cavin1, cavin2 and cavin3. Changes in membrane tension can flatten the caveolae, causing the release of the cavin coat and its disassembly into separate cavin1-cavin2 and cavin1-cavin3 subcomplexes. Each of these subcomplexes contain 9 ± 2 cavin molecules and appear to be the building blocks of the caveolar coat. High resolution immunoelectron microscopy suggests a remarkable nanoscale organization of these separate subcomplexes, forming individual striations on the surface of caveolae.
DOI: http://dx.doi.org/10.7554/eLife.01434.001
eLife digest
If you could look closely enough at the surface of some animal cells, especially fat or muscle cells, you would see that they are covered with pocket-like indents called ‘caveolae’. These structures are thought to help the cells communicate with the outside world, but they can also be used by viruses to gain entry into living cells.
Examining these caveolae even closer would reveal that these pockets contain proteins called caveolins that bind to each other—and also to cholesterol and fatty acids—to form a scaffold that help to maintain the shape of the caveolae from inside the cell. Each caveolae in a mammalian cell typically contains over 100 caveolin proteins. Caveolar coat proteins, or cavins for short, are also important building blocks for caveolae: however, we know relatively little about the interactions between caveolins and cavins.
Now, Gambin et al. have used powerful new single-molecule techniques to study these interactions. These experiments looked at the three main types of cavin proteins that associate with caveolae, and by tracking individual protein molecules they showed that cavin1 can interact with either cavin2 or cavin3, but that cavin2 and cavin3 do not interact with each other. Furthermore, cavin2 and cavin3 exist in separate stripes on a caveolae. Gambin et al. also stretched the cell membrane by forcing cells to take in extra water, and showed that this caused the cavin coat to peel away from the caveolae and break down into distinct cavin1-cavin2 and cavin1-cavin3 building blocks.
Faulty versions of caveolins and cavins have both been associated with several diseases in humans, including heart disease and muscle disorders. As such, an improved understanding of the formation and break down of caveolae may prove useful for developing treatments for these conditions.
DOI: http://dx.doi.org/10.7554/eLife.01434.002
doi:10.7554/eLife.01434
PMCID: PMC3903133  PMID: 24473072
caveolae; single-molecule; cell-free protein expression; human
14.  Ras nanoclusters 
Small GTPases  2013;4(1):57-60.
Ras proteins on the plasma membrane are laterally segregated into transient nanoclusters that are essential for high-fidelity signal transmission by the Ras/MAPK cascade. The dynamics of Ras nanocluster assembly and disassembly control MAPK signal output. BRaf inhibitors paradoxically activate CRaf and MAPK signaling in Ras-transformed cells. In our recent study, we showed that BRaf inhibition significantly enhances nanoclustering of oncogenic K- and N-Ras, but not H-Ras by increasing the frequency of Ras nanocluster formation. This disrupted spatiotemporal dynamics of Ras nanocluster fully accounts for the observed effects of Raf inhibitors on Ras signal transmission. Here together with other studies, we propose that the dynamics of Ras nanoclusters may represent a novel target for future therapeutic intervention.
doi:10.4161/sgtp.23145
PMCID: PMC3620104  PMID: 23419283
ras proteins; nanocluster; plasma membrane; BRaf inhibitors; pharmacological target
15.  A tale of two drug targets: the evolutionary history of BACE1 and BACE2 
Frontiers in Genetics  2013;4:293.
The beta amyloid (APP) cleaving enzyme (BACE1) has been a drug target for Alzheimer's Disease (AD) since 1999 with lead inhibitors now entering clinical trials. In 2011, the paralog, BACE2, became a new target for type II diabetes (T2DM) having been identified as a TMEM27 secretase regulating pancreatic β cell function. However, the normal roles of both enzymes are unclear. This study outlines their evolutionary history and new opportunities for functional genomics. We identified 30 homologs (UrBACEs) in basal phyla including Placozoans, Cnidarians, Choanoflagellates, Porifera, Echinoderms, Annelids, Mollusks and Ascidians (but not Ecdysozoans). UrBACEs are predominantly single copy, show 35–45% protein sequence identity with mammalian BACE1, are ~100 residues longer than cathepsin paralogs with an aspartyl protease domain flanked by a signal peptide and a C-terminal transmembrane domain. While multiple paralogs in Trichoplax and Monosiga pre-date the nervous system, duplication of the UrBACE in fish gave rise to BACE1 and BACE2 in the vertebrate lineage. The latter evolved more rapidly as the former maintained the emergent neuronal role. In mammals, Ka/Ks for BACE2 is higher than BACE1 but low ratios for both suggest purifying selection. The 5' exons show higher Ka/Ks than the catalytic section. Model organism genomes show the absence of certain BACE human substrates when the UrBACE is present. Experiments could thus reveal undiscovered substrates and roles. The human protease double-target status means that evolutionary trajectories and functional shifts associated with different substrates will have implications for the development of clinical candidates for both AD and T2DM. A rational basis for inhibition specificity ratios and assessing target-related side effects will be facilitated by a more complete picture of BACE1 and BACE2 functions informed by their evolutionary context.
doi:10.3389/fgene.2013.00293
PMCID: PMC3865767  PMID: 24381583
BACE1; BACE2; Alzheimer's Disease; type II diabetes; protein family evolution
16.  A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains 
Genome Biology  2013;14(7):R82.
Background
The mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.
Results
We undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems.
Conclusions
Comparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.
doi:10.1186/gb-2013-14-7-r82
PMCID: PMC4053787  PMID: 23902802
Mouse inbred lines; sequence variation; mouse phenotyping; gene knockout; C57BL/6
17.  Structure-based Reassessment of the Caveolin Signaling Model: Do Caveolae Regulate Signaling Through Caveolin-Protein Interactions? 
Developmental cell  2012;23(1):11-20.
Summary
Caveolin proteins drive formation of caveolae, specialized cell-surface microdomains that influence cell signaling. Signaling proteins are proposed to use conserved caveolin-binding motifs (CBMs) to associate with caveolae via the caveolin scaffolding domain (CSD). However, structural and bioinformatic analyses argue against such direct physical interactions: In the majority of signaling proteins, the CBM is buried and inaccessible. Putative CBMs do not form a common structure for caveolin recognition, are not enriched amongst caveolin-binding proteins, and are even more common in yeast, which lack caveolae. We propose that CBM/CSD-dependent interactions are unlikely to mediate caveolar signaling, and the basis for signaling effects should therefore be reassessed.
doi:10.1016/j.devcel.2012.06.012
PMCID: PMC3427029  PMID: 22814599
18.  Fendiline Inhibits K-Ras Plasma Membrane Localization and Blocks K-Ras Signal Transmission 
Molecular and Cellular Biology  2013;33(2):237-251.
Ras proteins regulate signaling pathways important for cell growth, differentiation, and survival. Oncogenic mutant Ras proteins are commonly expressed in human tumors, with mutations of the K-Ras isoform being most prevalent. To be active, K-Ras must undergo posttranslational processing and associate with the plasma membrane. We therefore devised a high-content screening assay to search for inhibitors of K-Ras plasma membrane association. Using this assay, we identified fendiline, an L-type calcium channel blocker, as a specific inhibitor of K-Ras plasma membrane targeting with no detectable effect on the localization of H- and N-Ras. Other classes of L-type calcium channel blockers did not mislocalize K-Ras, suggesting a mechanism that is unrelated to calcium channel blockade. Fendiline did not inhibit K-Ras posttranslational processing but significantly reduced nanoclustering of K-Ras and redistributed K-Ras from the plasma membrane to the endoplasmic reticulum (ER), Golgi apparatus, endosomes, and cytosol. Fendiline significantly inhibited signaling downstream of constitutively active K-Ras and endogenous K-Ras signaling in cells transformed by oncogenic H-Ras. Consistent with these effects, fendiline blocked the proliferation of pancreatic, colon, lung, and endometrial cancer cell lines expressing oncogenic mutant K-Ras. Taken together, these results suggest that inhibitors of K-Ras plasma membrane localization may have utility as novel K-Ras-specific anticancer therapeutics.
doi:10.1128/MCB.00884-12
PMCID: PMC3554123  PMID: 23129805
19.  Functional modelling of planar cell polarity: an approach for identifying molecular function 
Background
Cells in some tissues acquire a polarisation in the plane of the tissue in addition to apical-basal polarity. This polarisation is commonly known as planar cell polarity and has been found to be important in developmental processes, as planar polarity is required to define the in-plane tissue coordinate system at the cellular level.
Results
We have built an in-silico functional model of cellular polarisation that includes cellular asymmetry, cell-cell signalling and a response to a global cue. The model has been validated and parameterised against domineering non-autonomous wing hair phenotypes in Drosophila.
Conclusions
We have carried out a systematic comparison of in-silico polarity phenotypes with patterns observed in vivo under different genetic manipulations in the wing. This has allowed us to classify the specific functional roles of proteins involved in generating cell polarity, providing new hypotheses about their specific functions, in particular for Pk and Dsh. The predictions from the model allow direct assignment of functional roles of genes from genetic mosaic analysis of Drosophila wings.
doi:10.1186/1471-213X-13-20
PMCID: PMC3662592  PMID: 23672397
Planar polarity; PCP; Mathematical modelling; Self organisation; Drosophila; In-silico phenotyping
20.  Staurosporine 
The Ras GTPases comprising three main isoforms H-, N- and K-Ras operate at the plasma membrane as molecular switches in essential signaling pathways. Active concentration of the minor phospholipid phosphatidylserine in the inner leaflet of the plasma membrane contributes to the electrostatic potential that is required for K-Ras anchoring to the plasma membrane. We recently observed that staurosporine and related analogs: 7-oxostaurosporine, UCN-01 and UCN-02, long known as relatively non-specific protein kinase inhibitors, block endosomal sorting and recycling of phosphatidylserine, resulting in redistribution of phosphatidylserine to endosomes and endomembranes with concomitant mislocalization of K-Ras. Staurosporines are therefore a new tool to study phosphatidylserine trafficking. We discuss whether the mechanism of action of UCN-01, an FDA-approved staurosporine analog used as an anti-cancer therapeutic, is related to effects on phosphatidylserine subcellular distribution. Given the high prevalence of expression of constitutively active K-Ras in human cancers, we ask whether inhibitors of phosphatidylserine trafficking may have important therapeutic applications.
doi:10.4161/cib.24746
PMCID: PMC3737755  PMID: 23986809
Ras GTPase; phosphatidylserine; lipid recycling; staurosporine; UCN-01
21.  NucleoFinder: a statistical approach for the detection of nucleosome positions 
Bioinformatics  2013;29(6):711-716.
Motivation: The identification of nucleosomes along the chromatin is key to understanding their role in the regulation of gene expression and other DNA-related processes. However, current experimental methods (MNase-ChIP, MNase-Seq) sample nucleosome positions from a cell population and contain biases, making thus the precise identification of individual nucleosomes not straightforward. Recent works have only focused on the first point, where noise reduction approaches have been developed to identify nucleosome positions.
Results: In this article, we propose a new approach, termed NucleoFinder, that addresses both the positional heterogeneity across cells and experimental biases by seeking nucleosomes consistently positioned in a cell population and showing a significant enrichment relative to a control sample. Despite the absence of validated dataset, we show that our approach (i) detects fewer false positives than two other nucleosome calling methods and (ii) identifies two important features of the nucleosome organization (the nucleosome spacing downstream of active promoters and the enrichment/depletion of GC/AT dinucleotides at the centre of in vitro nucleosomes) with equal or greater ability than the other two methods.
Availability: The R code of NucleoFinder, an example datafile and instructions are available for download from https://sites.google.com/site/beckerjeremie/
Contact: cholmes@stats.ox.ac.uk
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/bts719
PMCID: PMC3597142  PMID: 23297036
22.  Nitric oxide signaling in plants 
doi:10.3389/fpls.2013.00553
PMCID: PMC3893618  PMID: 24474956
defense responses; hormones; nitric oxide; post-translational modifications; ROS; signaling
23.  Ras trafficking, localization and compartmentalized signalling 
Ras proteins are proto-oncogenes that are frequently mutated in human cancers. Three closely related isoforms, HRAS, KRAS and NRAS, are expressed in all cells and have overlapping but distinctive functions. Recent work has revealed how differences between the Ras isoforms in their trafficking, localization and protein-membrane orientation enable signalling specificity to be determined. We review the various strategies used to characterize compartmentalized Ras localization and signalling. Localization is an important contextual modifier of signalling networks and insights from the Ras system are of widespread relevance for researchers interested in signalling initiated from membranes.
doi:10.1016/j.semcdb.2011.09.002
PMCID: PMC3378476  PMID: 21924373
palmitoylation; GTPase; isoforms; organelle; plasma membrane; microdomains; nanoclusters
24.  Co-Regulation of Cell Polarization and Migration by Caveolar Proteins PTRF/Cavin-1 and Caveolin-1 
PLoS ONE  2012;7(8):e43041.
Caveolin-1 and caveolae are differentially polarized in migrating cells in various models, and caveolin-1 expression has been shown to quantitatively modulate cell migration. PTRF/cavin-1 is a cytoplasmic protein now established to be also necessary for caveola formation. Here we tested the effect of PTRF expression on cell migration. Using fluorescence imaging, quantitative proteomics, and cell migration assays we show that PTRF/cavin-1 modulates cellular polarization, and the subcellular localization of Rac1 and caveolin-1 in migrating cells as well as PKCα caveola recruitment. PTRF/cavin-1 quantitatively reduced cell migration, and induced mesenchymal epithelial reversion. Similar to caveolin-1, the polarization of PTRF/cavin-1 was dependent on the migration mode. By selectively manipulating PTRF/cavin-1 and caveolin-1 expression (and therefore caveola formation) in multiple cell systems, we unveil caveola-independent functions for both proteins in cell migration.
doi:10.1371/journal.pone.0043041
PMCID: PMC3418245  PMID: 22912783
25.  Entity/Quality-Based Logical Definitions for the Human Skeletal Phenome using PATO 
Conference Proceedings  2009;2009:7069-7072.
This paper describes an approach to providing computer-interpretable logical definitions for the terms of the Human Phenotype Ontology (HPO) using PATO, the ontology of phenotypic qualities, to link terms of the HPO to the anatomic and other entities that are affected by abnormal phenotypic qualities. This approach will allow improved computerized reasoning as well as a facility to compare phenotypes between different species. The PATO mapping will also provide direct links from phenotypic abnormalities and underlying anatomic structures encoded using the Foundational Model of Anatomy, which will be a valuable resource for computational investigations of the links between anatomical components and concepts representing diseases with abnormal phenotypes and associated genes.
doi:10.1109/IEMBS.2009.5333362
PMCID: PMC3398700  PMID: 19964203

Results 1-25 (89)