A rational quest for drug targets in the protein kinome of Trypanosoma brucei

Trypanosoma brucei is the protozoan parasite causing African trypanosomiasis, a neurological disease that affects humans and farm-stock in the impoverished sub-Saharan areas where tsetse fly transmission vector is endemic. Although it has great impact on public health and local economies, it has been neglected in drug discovery for almost a century. Current treatments are either toxic or of difficult administration, besides having serious risks of inducing resistance. Protein kinases are the primary set of signaling proteins in eukaryotes, including Trypanosoma brucei. Their druggability has been widely exploited in cancer research, and has been established in the parasite too. A recent kinome-wide RNAi screen with 176 individual cell lines of mammalian infective bloodstream forms of Trypanosoma brucei identified protein kinases required for proliferation in vitro. In order to investigate which protein kinases are also essential virulence factors in vivo, lines were pooled, inoculated into mice and screened for loss of fitness after 48 hours RNAi compared to uninduced controls. The presence of trypanosomes in the bloodstream was assessed using RNAi target sequencing (RITseq) and compared to an in vitro control. This revealed 49 protein kinases with a significant loss of fitness in vivo in two independent experiments, and a strong correlation between in vitro and in vivo loss of fitness for the majority. However, depletion of nine protein kinases affected more pronouncedly the growth in vivo than in vitro. Amongst these protein kinases were several with putative functions related with stress responses mediated through the PI3K/TOR or MAPK signaling cascades including CK2A2, a promiscuous protein kinase whose activity can be stress-induced; two MAP3Ks, involved in cell integrity upon osmotic shock; VPS15, component of the PI3K complex with roles in autophagosome formation and vesicular trafficking; BUD32, transducer of the PI3K/TOR pathway involved in translational regulation; and FAZ20, a parasite-specific pseudo-kinase localizing to the flagellum attachment zone. The other three have been implicated in repair of alkylation-induced cellular damage: SRPK1, a stress response RNA splicing regulator; AUK2, which acts during mitosis; and CAMKL, an AMPK with calcium-binding domains putatively involved in metabolic regulation. Identification of these virulence-associated protein kinases provides new insights in T. brucei-host interaction and reveals novel potential drug targets for protein kinase inhibitors. This RNAi screen revealed that the evolutionary divergent NEK kinase Repressor of Differentiation Kinase 2 (RDK2) has severe loss of fitness both in vitro and in vivo. Depletion of RDK2 had been shown previously to promote differentiation from bloodstream to procyclic-like forms causing the parasite’s death. Further investigation showed RDK2 to be an active protein kinase capable both of phosphorylating a substrate and to autophosphorylate. Protein kinase activity could be ablated by mutation of lysine 70 to methionine. Mutation of both serine residues (195 and 197), identified as sites of phosphorylation by phosphoproteomics, to alanine or glutamic acid, preventing and mimicking phosphorylation respectively, had no effect on protein kinase activity, suggesting they do not have a direct regulatory role on protein kinase activity. Introducing in the RNAi line a recoded RDK2 whose transcript eluded interference, permitted to some extent the rescue of the induced phenotype, while introducing a recoded inactive mutant did not. This may suggest that the lack of kinase activity was responsible for the RNAi phenotype and not depletion of the protein alone. RDK2 RNAi-differentiated cells could be maintained in conditioned procyclic form media for more than a week. However, they were unable to proliferate. Overexpression of RDK2 blocked the differentiation mediated by sequential treatment with 8-pCPT-cAMP and citrate/cis-aconitate (CCA). RNAi experiments in combination with known differentiation cues, suggested that when differentiation is triggered by the CCA signalling pathway, RDK2 inactivation happens downstream of the phosphatase TbPTP1. Differentiation caused by RDK2 inactivation could be traced in flow cytometry by the detection of EP procyclin expression. This was exploited in a cell-based mechanism-directed phenotypic screen for RDK2 inhibitors. A preliminary run with 518 drug-like molecules that had shown protein kinase inhibition, trypanocidal activity and/or activation of the EP procyclin promoter, unveiled 6 compounds triggering EP procyclin expression and parasite death in the low micromolar range. These compounds can be investigated further to assess whether RDK2 is their in vivo target

Profile of residents: attitude towards tourism in Benalmádena (Costa del Sol, Spain)

Tourism development and evolution along the time causes multiple impacts. Based on characteristic profiles of resident, these impacts may be assessed differently. The attitude of residents and the identification of profiles is a tool that allows to asses the status of destination and propose future strategies for improving the destination. This study aims to analyse the role of residents regarding the perception of tourism.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Así mismo, Esta investigación ha sido subvencionada por el programa Nacional de Investigación Básica 2012, CS2010-30840. "Geografías de la crisis: analisis urbano turístico de las Islas Baleares, Costa del Sol y principales destinos en el Caribe

Residents´ perceptions of tourism development in Benalmádena (Spain)

DOI: 10.1016/j.tourman.2015.11.007This study examines the residents´ perceptions of the impact of tourism in Benalmádena, and the profiles of the residents according to socio-demographic characteristics. A questionnaire assessed how these characteristics influence the residents' perceptions towards the environment, economy, and socio-cultural aspects. The survey was administered to a stratified sample of 770 residents in Benalmádena. Results show a significant effect of socio-demographic variables on perception of tourism impact. The educational background, place of birth and how long respondents had been living in the community explain a significant amount of the variance in overall attitudes. Interaction analyses revealed that place of birth moderated the relationship between the tourism dimensions and the years of residence. For instance, the respondents with less than five years of residence showed more positive attitude towards the impact of tourism. We offer a profile of these residents according to their perceptions of the impact of tourism in their community.This research has received funding from the Spanish Government, Fundamental Research Program (R+D) (CSO2012-30840), “Geographies of crisis: analysis of urban and tourist territories of the Balearic Islands, Costa del Sol and main tourist destinations of the Caribbean and Central America”. Andalucia Tech, Universidad de Málaga, Spain

A multi-parametric analysis of Trypanosoma cruzi infection: common pathophysiologic patterns beyond extreme heterogeneity of host responses

The extreme genetic diversity of the protozoan Trypanosoma cruzi has been proposed to be associated with the clinical outcomes of the disease it provokes: Chagas disease (CD). To address this question, we analysed the similarities and differences in the CD pathophysiogenesis caused by different parasite strains. Using syngeneic mice infected acutely or chronically with 6 distant parasite strains, we integrated simultaneously 66 parameters: parasite tropism (7 parameters), organ and immune responses (local and systemic; 57 parameters), and clinical presentations of CD (2 parameters). While the parasite genetic background consistently impacts most of these parameters, they remain highly variable, as observed in patients, impeding reliable one-dimensional association with phases, strains, and damage. However, multi-dimensional statistics overcame this extreme intra-group variability for each individual parameter and revealed some pathophysiological patterns that accurately allow defining (i) the infection phase, (ii) the infecting parasite strains, and (iii) organ damage type and intensity. Our results demonstrated a greater variability of clinical outcomes and host responses to T. cruzi infection than previously thought, while our multi-parametric analysis defined common pathophysiological patterns linked to clinical outcome of CD, conserved among the genetically diverse infecting strains

Genome-wide and protein kinase-focused RNAi screens reveal conserved and novel damage response pathways in Trypanosoma brucei

All cells are subject to structural damage that must be addressed for continued growth. A wide range of damage affects the genome, meaning multiple pathways have evolved to repair or bypass the resulting DNA lesions. Though many repair pathways are conserved, their presence or function can reflect the life style of individual organisms. To identify genome maintenance pathways in a divergent eukaryote and important parasite, Trypanosoma brucei, we performed RNAi screens to identify genes important for survival following exposure to the alkylating agent methyl methanesulphonate. Amongst a cohort of broadly conserved and, therefore, early evolved repair pathways, we reveal multiple activities not so far examined functionally in T. brucei, including DNA polymerases, DNA helicases and chromatin factors. In addition, the screens reveal Trypanosoma- or kinetoplastid-specific repair-associated activities. We also provide focused analyses of repair-associated protein kinases and show that loss of at least nine, and potentially as many as 30 protein kinases, including a nuclear aurora kinase, sensitises T. brucei to alkylation damage. Our results demonstrate the potential for synthetic lethal genome-wide screening of gene function in T. brucei and provide an evolutionary perspective on the repair pathways that underpin effective responses to damage, with particular relevance for related kinetoplastid pathogens. By revealing that a large number of diverse T. brucei protein kinases act in the response to damage, we expand the range of eukaryotic signalling factors implicated in genome maintenance activities

Gravity-driven Magnetic Field at ∼1000 au Scales in High-mass Star Formation

A full understanding of high-mass star formation requires the study of one of the most elusive components of the energy balance in the interstellar medium: magnetic fields. We report Atacama Large Millimeter/submillimeter Array (ALMA) 1.2 mm, high-resolution (700 au) dust polarization and molecular line observations of the rotating hot molecular core embedded in the high-mass star-forming region IRAS 18089-1732. The dust continuum emission and magnetic field morphology present spiral-like features resembling a whirlpool. The velocity field traced by the H13CO+ (J = 3-2) transition line reveals a complex structure with spiral filaments that are likely infalling and rotating, dragging the field with them. We have modeled the magnetic field and find that the best model corresponds to a weakly magnetized core with a mass-to-magnetic-flux ratio (λ) of 8.38. The modeled magnetic field is dominated by a poloidal component, but with an important contribution from the toroidal component that has a magnitude of 30% of the poloidal component. Using the Davis-Chandrasekhar-Fermi method, we estimate a magnetic field strength of 3.5 mG. At the spatial scales accessible to ALMA, an analysis of the energy balance of the system indicates that gravity overwhelms turbulence, rotation, and the magnetic field. We show that high-mass star formation can occur in weakly magnetized environments, with gravity taking the dominant role.Fil: Sanhueza, Patricio. National Astronomical Observatory Of Japan; JapónFil: Girart, Josep Miquel. Instituto de Estudios Espaciales de Cataluña; EspañaFil: Padovani, Marco. Osservatorio Astrofisico Di Arcetri; ItaliaFil: Galli, Daniele. Osservatorio Astrofisico Di Arcetri; ItaliaFil: Hull, Charles L. H.. Atacama Large Millimeter-submillimeter Array; ChileFil: Zhang, Qizhou. Harvard-Smithsonian Center for Astrophysics; Estados UnidosFil: Cortes, Paulo. National Radio Astronomy Observatory; Estados UnidosFil: Stephens, Ian. Worcester State University; Estados UnidosFil: Fernandez Lopez, Manuel. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas. Instituto Argentino de Radioastronomía. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto Argentino de Radioastronomía; ArgentinaFil: Jackson, James M.. NASA Ames Research Center; Estados UnidosFil: Frau, Pau. Csic. Instituto de Ciencias del Espacio; EspañaFil: Kock, Patrick M.. Academia Sinica; ChinaFil: Wu, Benjamin. National Astronomical Observatory Of Japan; JapónFil: Zapata, Luis A.. Instituto de Radioastronomía y Astrofísica; MéxicoFil: Olguin, Fernando. National Tsing Hua University; ChinaFil: Lu, Xing. National Astronomical Observatory Of Japan; JapónFil: Silva, Andrea. National Astronomical Observatory Of Japan; JapónFil: Tang, Ya Wen. Academia Sinica; ChinaFil: Sakai, Takeshi. The University Of Electro-communications; JapónFil: Guzmán, Andrés E.. National Astronomical Observatory Of Japan; JapónFil: Tatematsu, Ken'Ichi. National Astronomical Observatory Of Japan; JapónFil: Nakamura, Fumitaka. National Astronomical Observatory Of Japan; JapónFil: Chen, Huei Ru Vivien. National Tsing Hua University; Chin

Association of Candidate Gene Polymorphisms With Chronic Kidney Disease: Results of a Case-Control Analysis in the Nefrona Cohort

Chronic kidney disease (CKD) is a major risk factor for end-stage renal disease, cardiovascular disease and premature death. Despite classical clinical risk factors for CKD and some genetic risk factors have been identified, the residual risk observed in prediction models is still high. Therefore, new risk factors need to be identified in order to better predict the risk of CKD in the population. Here, we analyzed the genetic association of 79 SNPs of proteins associated with mineral metabolism disturbances with CKD in a cohort that includes 2, 445 CKD cases and 559 controls. Genotyping was performed with matrix assisted laser desorption ionizationtime of flight mass spectrometry. We used logistic regression models considering different genetic inheritance models to assess the association of the SNPs with the prevalence of CKD, adjusting for known risk factors. Eight SNPs (rs1126616, rs35068180, rs2238135, rs1800247, rs385564, rs4236, rs2248359, and rs1564858) were associated with CKD even after adjusting by sex, age and race. A model containing five of these SNPs (rs1126616, rs35068180, rs1800247, rs4236, and rs2248359), diabetes and hypertension showed better performance than models considering only clinical risk factors, significantly increasing the area under the curve of the model without polymorphisms. Furthermore, one of the SNPs (the rs2248359) showed an interaction with hypertension, being the risk genotype affecting only hypertensive patients. We conclude that 5 SNPs related to proteins implicated in mineral metabolism disturbances (Osteopontin, osteocalcin, matrix gla protein, matrix metalloprotease 3 and 24 hydroxylase) are associated to an increased risk of suffering CKD

Measurement of the production of a W boson in association with a charm quark in pp collisions at √s = 7 TeV with the ATLAS detector

The production of a W boson in association with a single charm quark is studied using 4.6 fb−1 of pp collision data at s√ = 7 TeV collected with the ATLAS detector at the Large Hadron Collider. In events in which a W boson decays to an electron or muon, the charm quark is tagged either by its semileptonic decay to a muon or by the presence of a charmed meson. The integrated and differential cross sections as a function of the pseudorapidity of the lepton from the W-boson decay are measured. Results are compared to the predictions of next-to-leading-order QCD calculations obtained from various parton distribution function parameterisations. The ratio of the strange-to-down sea-quark distributions is determined to be 0.96+0.26−0.30 at Q 2 = 1.9 GeV2, which supports the hypothesis of an SU(3)-symmetric composition of the light-quark sea. Additionally, the cross-section ratio σ(W + +c¯¯)/σ(W − + c) is compared to the predictions obtained using parton distribution function parameterisations with different assumptions about the s−s¯¯¯ quark asymmetry

Search for a CP-odd Higgs boson decaying to Zh in pp collisions at √s=8TeV with the ATLAS detector

A search for a heavy, CP-odd Higgs boson, A, decaying into a Zboson and a 125GeV Higgs boson, h, with the ATLAS detector at the LHC is presented. The search uses proton–proton collision data at a centre-of-mass energy of 8TeV corresponding to an integrated luminosity of 20.3fb−1. Decays of CP-even hbosons to ττor bbpairs with the Zboson decaying to electron or muon pairs are considered, as well as h →bbdecays with the Zboson decaying to neutrinos. No evidence for the production of an Aboson in these channels is found and the 95% confidence level upper limits derived for σ(gg→A) ×BR(A →Zh) ×BR(h →f¯f)are 0.098–0.013pb for f=τand 0.57–0.014pb for f=bin a range of mA=220–1000GeV. The results are combined and interpreted in the context of two-Higgs-doublet models. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons