A Protocol for Minimal Single Protein Labeling with CyDye Fluors for Live Cell Internalization Assays

Individual proteins chemically labeled with fluorescent dyes can be localized and tracked in real-time experiments in order to get insights about the site and molecular mechanism of action. Here, we have adapted a protocol that was originally developed for two-dimensional fluorescence difference gel electrophoresis (2D-DIGE) applications, to label proteins with CyDye fluors for single-molecule internalization assays in living cells. This “minimal labeling” method offers a number of advantages including specificity and known stoichiometry, simplicity, high reproducibility, and sensitivity and allows multiplexing while minimizing perturbations of the biological system. Moreover, since only a single lysine (Lys) residue per protein molecule is labeled, this method is also quantitative. To validate experimentally our protocol, we carried out the fluorescent labeling of IBB1, a major soybean protease isoinhibitor of the Bowman-Birk family that is currently being investigated as colorectal chemopreventive agent. Then, we analyzed the in vivo internalization dynamics of the labeled IBB1 protein in human colorectal adenocarcinoma HT29 cells

Combining Static and Dynamic Contract Checking for Curry

Static type systems are usually not sufficient to express all requirements on function calls. Hence, contracts with pre- and postconditions can be used to express more complex constraints on operations. Contracts can be checked at run time to ensure that operations are only invoked with reasonable arguments and return intended results. Although such dynamic contract checking provides more reliable program execution, it requires execution time and could lead to program crashes that might be detected with more advanced methods at compile time. To improve this situation for declarative languages, we present an approach to combine static and dynamic contract checking for the functional logic language Curry. Based on a formal model of contract checking for functional logic programming, we propose an automatic method to verify contracts at compile time. If a contract is successfully verified, dynamic checking of it can be omitted. This method decreases execution time without degrading reliable program execution. In the best case, when all contracts are statically verified, it provides trust in the software since crashes due to contract violations cannot occur during program execution.Comment: Pre-proceedings paper presented at the 27th International Symposium on Logic-Based Program Synthesis and Transformation (LOPSTR 2017), Namur, Belgium, 10-12 October 2017 (arXiv:1708.07854

Análisis del proteoma del exudado estigmático

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ReprOlive: a database with linked data for the olive tree (Olea europaea L.) reproductive transcriptome

Plant reproductive transcriptomes have been analyzed in different species due to the agronomical and biotechnological importance of plant reproduction. Here we presented an olive tree reproductive transcriptome database with samples from pollen and pistil at different developmental stages, and leaf and root as control vegetative tissues (http://reprolive.eez.csic.es). It was developed from 2,077,309 raw reads to 1,549 Sanger sequences. Using a pre-defined workflow based on open-source tools, sequences were pre-processed, assembled, mapped, and annotated with expression data, descriptions, GO terms, InterPro signatures, EC numbers, KEGG pathways, ORFs, and SSRs. Tentative transcripts (TTs) were also annotated with the corresponding orthologs in Arabidopsis thaliana from TAIR and RefSeq databases to enable Linked Data integration. It results in a reproductive transcriptome comprising 72,846 contigs with average length of 686 bp, of which 63,965 (87.8%) included at least one functional annotation, and 55,356 (75.9%) had an ortholog. A minimum of 23,568 different TTs was identified and 5,835 of them contain a complete ORF. The representative reproductive transcriptome can be reduced to 28,972 TTs for further gene expression studies. Partial transcriptomes from pollen, pistil, and vegetative tissues as control were also constructed. ReprOlive provides free access and download capability to these results. Retrieval mechanisms for sequences and transcript annotations are provided. Graphical localization of annotated enzymes into KEGG pathways is also possible. Finally, ReprOlive has included a semantic conceptualisation by means of a Resource Description Framework (RDF) allowing a Linked Data search for extracting the most updated information related to enzymes, interactions, allergens, structures, and reactive oxygen species.This work was supported by co-funding from the ERDF (European Regional Development Fund) and (i) Ministerio de Ciencia e Innovación [grant numbers BFU2011-22779 to JDA, and TIN2011-25840 and TIN2014-58304-R to JFAM], and (ii) Plan Andaluz de Investigación, Desarrollo e Innovación [grant numbers P10-CVI-6075 to MGC, P10-AGR-6274 and P11-CVI-7487 to JDA, and P11-TIC-7529 and P12-TIC-1519 to JFAM].Peer reviewe

Identification and localization of a caleosin in olive (Olea europaea L.) pollen during in vitro germination

In plant organs and tissues, the neutral storage lipids are confined to discrete spherical organelles called oil bodies. Oil bodies from plant seeds contain 0.6–3% proteins, including oleosins, steroleosins, and caleosins. In this study, a caleosin isoform of ∼30 kDa was identified in the olive pollen grain. The protein was mainly located at the boundaries of the oil bodies in the cytoplasm of the pollen grain and the pollen tube. In addition, caleosins were also visualized in the cytoplasm at the subapical zone, as well as in the tonoplast of vacuoles present in the pollen tube cytoplasm. The cellular behaviour of lipid bodies in the olive pollen was also monitored during in vitro germination. The number of oil bodies decreased 20-fold in the pollen grain during germination, whereas the opposite tendency occurred in the pollen tube, suggesting that oil bodies moved from one to the other. The data suggest that this pollen caleosin might have a role in the mobilization of oil bodies as well as in the reorganization of membrane compartments during pollen in vitro germination

Aspectos clínicos y epidemiológicos de los síndromes de Apert y Crouzon en España

Epidemiología y Teratología: Resultados sobre los datos del ECEMCApert and Crouzon syndromes are the most frequent ones among those syndromes with craniosynostosis. We have used data from the Spanish Collaborative Study of Congenital Malformations (ECEMC), to analyze some clinical and epidemiological characteristics of both syndromes. A total of 19 cases of Apert and 21 cases of Crouzon syndromes were identified among 1,914,726 liveborn infants. Their birth prevalence has diminished along the time although the decrease is only statistically significant for Crouzon syndrome. Mean parental ages, and mean differences between the parental ages, are significantly higher in Apert cases than in controls, what is indicating a relationship between paternal age and mutations for this syndrome. The differences between the parental ages of Crouzon cases and controls are not statistically significant. All the cases with Apert syndrome of our series were the first occurrence in the family, while 38.10% of the Crouzon cases were familial. We also observed a significant increasing linear trend in the frequency of infants with Apert syndrome, with the increasing paternal age, which is more noticeable since the age of 35 years. However, this is not observed for Crouzon cases whether sporadic or familial. Regarding the clinical aspects, Apert syndrome is more frequently associated to other defects than Crouzon syndrome. With respect to the affectation of hands in the Apert cases, the most frequent defect was the so-called "mitten hand", wich was present in 50% of our cases. In 28.57% the first finger was separated from the rest that are fused. Interestingly, in one case only the fifth finger was separated for the other four that remain fused.N

Long-term follow-up of certolizumab pegol in uveitis due to immune-mediated inflammatory diseases : multicentre study of 80 patients

Objectives To evaluate effectiveness and safety of certolizumab pegol (CZP) in uveitis due to immune-mediated inflammatory diseases (IMID). Methods Multicentre study of CZP-treated patients with IMID uveitis refractory to conventional immunosuppressant. Effectiveness was assessed through the following ocular parameters: best-corrected visual acuity, anterior chamber cells, vitritis, macular thickness and retinal vasculitis. These variables were compared between the baseline, and first week, first, third, sixth months, first and second year. Results We studied 80 (33 men/47 women) patients (111 affected eyes) with a mean age of 41.6±11.7 years. The IMID included were: spondyloarthritis (n=43), Behçet's disease (n=10), psoriatic arthritis (n=8), Crohn's disease (n=4), sarcoidosis (n=2), juvenile idiopathic arthritis (n=1), reactive arthritis (n=1), rheumatoid arthritis (n=1), relapsing polychondritis (n=1), Conclusions CZP seems to be effective and safe in uveitis related to different IMID, even in patients refractory to previous biological drugs

Fungal planet description sheets: 951–1041

Novel species of fungi described in this study include those from various countries as follows: Antarctica , Apenidiella antarctica from permafrost, Cladosporium fildesense fromanunidentifiedmarinesponge. Argentina , Geastrum wrightii onhumusinmixedforest. Australia , Golovinomyces glandulariae on Glandularia aristigera, Neoanungitea eucalyptorum on leaves of Eucalyptus grandis, Teratosphaeria corymbiicola on leaves of Corymbia ficifolia, Xylaria eucalypti on leaves of Eucalyptus radiata. Brazil, Bovista psammophila on soil, Fusarium awaxy on rotten stalks of Zea mays, Geastrum lanuginosum on leaf litter covered soil, Hermetothecium mikaniae-micranthae (incl. Hermetothecium gen. nov.)on Mikania micrantha, Penicillium reconvexovelosoi in soil, Stagonosporopsis vannaccii from pod of Glycine max. British Virgin Isles , Lactifluus guanensis onsoil. Canada , Sorocybe oblongispora on resin of Picea rubens. Chile, Colletotrichum roseum on leaves of Lapageria rosea. China, Setophoma caverna fromcarbonatiteinKarstcave. Colombia , Lareunionomyces eucalypticola on leaves of Eucalyptus grandis. Costa Rica, Psathyrella pivae onwood. Cyprus , Clavulina iris oncalcareoussubstrate. France , Chromosera ambigua and Clavulina iris var. occidentalis onsoil. French West Indies , Helminthosphaeria hispidissima ondeadwood. Guatemala , Talaromyces guatemalensis insoil. Malaysia , Neotracylla pini (incl. Tracyllales ord. nov. and Neotra- cylla gen. nov.)and Vermiculariopsiella pini on needles of Pinus tecunumanii. New Zealand, Neoconiothyrium viticola on stems of Vitis vinifera, Parafenestella pittospori on Pittosporum tenuifolium, Pilidium novae-zelandiae on Phoenix sp. Pakistan , Russula quercus-floribundae onforestfloor. Portugal , Trichoderma aestuarinum from salinewater. Russia , Pluteus liliputianus on fallen branch of deciduous tree, Pluteus spurius on decaying deciduouswoodorsoil. South Africa , Alloconiothyrium encephalarti, Phyllosticta encephalarticola and Neothyrostroma encephalarti (incl. Neothyrostroma gen. nov.)onleavesof Encephalartos sp., Chalara eucalypticola on leaf spots of Eucalyptus grandis × urophylla, Clypeosphaeria oleae on leaves of Olea capensis, Cylindrocladiella postalofficium on leaf litter of Sideroxylon inerme , Cylindromonium eugeniicola (incl. Cylindromonium gen. nov.)onleaflitterof Eugenia capensis , Cyphellophora goniomatis on leaves of Gonioma kamassi , Nothodactylaria nephrolepidis (incl. Nothodactylaria gen. nov. and Nothodactylariaceae fam. nov.)onleavesof Nephrolepis exaltata , Falcocladium eucalypti and Gyrothrix eucalypti on leaves of Eucalyptus sp., Gyrothrix oleae on leaves of Olea capensis subsp. macrocarpa , Harzia metro sideri on leaf litter of Metrosideros sp., Hippopotamyces phragmitis (incl. Hippopota- myces gen. nov.)onleavesof Phragmites australis , Lectera philenopterae on Philenoptera violacea , Leptosillia mayteni on leaves of Maytenus heterophylla , Lithohypha aloicola and Neoplatysporoides aloes on leaves of Aloe sp., Millesimomyces rhoicissi (incl. Millesimomyces gen. nov.) on leaves of Rhoicissus digitata , Neodevriesia strelitziicola on leaf litter of Strelitzia nicolai , Neokirramyces syzygii (incl. Neokirramyces gen. nov.)onleafspots o

Eventos adversos evitables en atención primaria. Estudio retrospectivo de cohortes para determinar su frecuencia y gravedad

Objetivo: Determinar la frecuencia de eventos adversos evitables (EAE) en atención primaria (AP). Diseño: Estudio retrospectivo de cohortes. Emplazamiento: consultas de medicina de familia y pediatría de Andalucía, Aragón, Castilla La Mancha, Cataluña, Madrid, Navarra y Comunidad Valenciana. Participantes: Se determinó revisar un mínimo de 2.397 historias clínicas (nivel de confianza del 95% y una precisión del 2%). La muestra se estratificó por grupos de edad de forma proporcional a su frecuentación y con revisión paritaria de historias de hombres y mujeres. Mediciones principales: Número y gravedad de los EAE identificados entre febrero de 2018 y septiembre de 2019. Resultados: Se revisaron un total de 2.557 historias clínicas (1.928, 75.4% de pacientes adultos y 629, 24.6% pediátricos). Se identificaron 182 EAE que afectaron a 168 pacientes (7,1%, IC 95% 6,1-8,1%); en adultos 7,6% (IC 95% 6,4-8,8%) y 5,7% (IC 95% 3,9-7,5%) en pacientes pediátricos. Las mujeres sufrieron más EAE que los hombres (p = 0,004). La incidencia de EAE en niños y niñas fue similar (p = 0,3). 6 (4.1%) de los EAE supusieron un daño permanente en pacientes adultos. Conclusiones: Buscar fórmulas para incrementar la seguridad en AP, particularmente en pacientes mujeres, debe seguir siendo un objetivo prioritario incluso en pediatría. Uno de cada 24 EAE supone un daño grave y permanente en el adulto