Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process

<p>Abstract</p> <p>Background</p> <p>The elucidation of gene expression patterns leads to a better understanding of biological processes. Real-time quantitative RT-PCR has become the standard method for in-depth studies of gene expression. A biologically meaningful reporting of target mRNA quantities requires accurate and reliable normalization in order to identify real gene-specific variation. The purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from RNA to cDNA, or differences between tissues or cells in overall transcriptional activity. The validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. In the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time RT-PCR.</p> <p>Results</p> <p>In this study, we assess the expression stability of 7 traditional and 4 novel housekeeping genes in a set of 27 samples representing different tissues and organs of tomato plants at different developmental stages. First, we designed, tested and optimized amplification primers for real-time RT-PCR. Then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. Our analysis suggests that SGN-U314153 (<it>CAC</it>), SGN-U321250 (<it>TIP41</it>), SGN-U346908 ("<it>Expressed</it>") and SGN-U316474 (<it>SAND</it>) genes provide superior transcript normalization in tomato development studies. We recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process.</p> <p>Conclusion</p> <p>This work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time RT-PCR studies of gene expression during tomato development process. From our study a tool-kit of control genes emerges that outperform the traditional genes in terms of expression stability.</p

New records of moths (Insecta, Lepidoptera) from urban gardens on Terceira Island with new data on recently introduced species to the Azores

The urban Duque da Terceira Garden, in the main town of Angra do Heroísmo (Terceira, Azores), was investigated to describe the species composition of moths (Insecta, Lepidoptera). Moths were sampled with two methods: SLAM traps (between April and September 2017) and light trapping (two sessions in the summer of 2017). A total of 42 taxa were sampled with the addition of 19 new records for Duque da Terceira Garden, five of which were also new records for Terceira Island. The five species recorded as new for Terceira were subjected to an exhaustive taxonomic analysis. In addition, we revised the colonization status of some species. Our results show that urban gardens may help the establishment of exotic species and that it is necessary to monitor and control how these species established in urban environments.info:eu-repo/semantics/publishedVersio

Molecular analysis of menadione-induced resistance against biotic stress in Arabidopsis

19 páginas, 6 figuras, 2 tablas.Menadione sodium bisulphite (MSB) is a water-soluble derivative of vitamin K3, or menadione, and has been previously demonstrated to function as a plant defence activator against several pathogens in several plant species. However, there are no reports of the role of this vitamin in the induction of resistance in the plant model Arabidopsis thaliana. In the current study, we demonstrate that MSB induces resistance by priming in Arabidopsis against the virulent strain Pseudomonas syringae pv. tomato DC3000 (Pto) without inducing necrosis or visible damage. Changes in gene expression in response to 0.2 mm MSB were analysed in Arabidopsis at 3, 6 and 24 h post-treatment using microarray technology. In general, the treatment with MSB does not correlate with other publicly available data, thus MSB produces a unique molecular footprint. We observed 158 differentially regulated genes among all the possible trends. More up-regulated genes are included in categories such as 'response to stress' than the background, and the behaviour of these genes in different treatments confirms their role in response to biotic and abiotic stress. In addition, there is an over-representation of the G-box in their promoters. Some interesting functions are represented among the individual up-regulated genes, such as glutathione S-transferases, transcription factors (including putative regulators of the G-box) and cytochrome P450s. This work provides a wide insight into the molecular cues underlying the effect of MSB as a plant resistance inducer.This work was partially funded by an INVESCAN, S.L. grant (No.OTT2001438) to the CSIC and by a BIO2006-02168 grant of MICINN to PT. The microarrays were funded in part by the “Genome España” Foundation. MER was supported by a research contract (ID-TF-06/002) from the Consejería de Industria, Comercio y Nuevas Tecnologías (Gobierno de Canarias). The authors thank CajaCanarias for their research support. We also thank Lorena Perales for her help in performing the bacterial growth curves, Dr. Héctor Cabrera for his useful advice on writing the manuscript, the English translation service of the Universidad Politécnica de Valencia and Mrs. Pauline Agnew whose endeavoured to edit the English translation of this paper.Peer reviewe

Melanoma, ancestralidad y variantes MC1R en la población mestizada uruguaya

Malignant melanoma (MM) is the most dangerous type of skin cancer and the main cause of death produced by skin diseases. In Uruguay, the incidence rate is 3.8/100,000, one of the highest in Latin America. We analyzed the contribution of ancestry and MC1R as a candidate gene for sporadic melanoma in Uruguay. Our objective wasto investigate the possible associations between ancestry and the MC1R gene with sporadic melanoma in the Uruguayan population. To that end, one hundred patients with sporadic MM and 107 controls were recruited. Phenotypic factors and lifestyle were evaluated as risk factors. At the same time, we analyzed fiveancestry informative markers, the MC1R variants (R151, R160 and D294H) and five tag-SNPs. Phototype, atypical nevi, sunburns and recreational exposure were the main risk factors for MM in the Uruguayan population. We confirmed 16q as a candidate region for MM. R151C, and R160W showed an important association with risk of melanoma (OR= 3.85, P= 1 x 10-2; OR= 10.15, P= 7 x 10-3, respectively). Furthermore, three novel MC1R haplotypes from the promoter region were detected,and the two most common haplotypes for the coding region were different to the ones found in Europeans through HapMap. However, MC1R coding region haplotypes revealed a highly similar frequency to that of the Spanish population. Our results showed that the chromosomal 16q region confers susceptibility to MM risk in the Uruguayan population. In addition, the admixed genome structure of the MC1R region could be part of the explanation of melanoma etiology.El melanoma maligno (MM) es uno de los más peligrosos, y la principal causa de muerte producida por tumores de piel. En el Uruguay, la tasa de incidencia es de 3,8/100.000, una de las más altas de América Latina. En este trabajo analizamos la ancestría y el gen candidato MC1R entre los pacientes con MM del Uruguay. Nuestro objetivo fue investigar la posible asociación entre ancestría y el gen MC1R en pacientes con melanoma esporádico en la población uruguaya. Con tal finalidad, se reclutaron 100 pacientes con MM esporádico y 107 controles. Se evaluó el riesgo de factores fenotípicos y de estilo de vida. Además se analizaron cinco marcadores informativos de ancesdencia, variantes del gen MC1R (R151, R160 y D294H) y cinco tagSNPs. El fototipo, los nevos atípicos, quemaduras solares y la exposición recreativa fueron los principales factores de riesgo para MM en la población uruguaya. La región cromosómica 16q es candidata para MM, mientras que R151C y R160W mostraron una importante asociación con el riesgo para MM (OR= 3,85, P= 1 x 10-2; OR= 10,15, P= 7 x 10-3, respectivamente). Por otra parte, se detectaron tres nuevos haplotipos en la región promotora y los dos haplotipos más frecuentes en la región codificante son diferentes a los encontrados en la población europea. Sin embargo, los haplotipos de la región codificante presentan una frecuencia muy similar a las encontradas en la población española. Los resultados muestran que la región cromosómica 16q confiere susceptibilidad al riesgo de MM en la población uruguaya. Por otra parte, la estructura genómica mestizada de la región del MC1R podría explicar la etiología del melanoma

Durabilidad del Concreto en Ambiente Urbanos y Urnbano/Marinos de México y España

Trabajo presentado en el III Congreso Nacional ALCONPAT (Asociación Latinoamericana de Control de Calidad, Patología y Recuperación de la Construcción), celebrado en Caracas (Venezuela), en noviembre de 200

Brown dog tick, Rhipicephalus sanguineus sensu lato, infestation of susceptible dog hosts is reduced by slow release of semiochemicals from a less susceptible host

Domestic dog breeds are hosts for the brown dog tick, Rhipicephalus sanguineus sensu lato, but infestation levels vary among breeds. Beagles are less susceptible to tick infestations than English cocker spaniels due to enhanced production of 2-hexanone and benzaldehyde that act as volatile tick repellents. We report the use of prototype slow-release formulations of these compounds to reduce the burden of R. sanguineus s. l. on English cocker spaniel dogs. Twelve dogs were randomly assigned to two groups with six dogs each. The treated group received collars with slow-release formulations of the compounds attached, while the control group received collars with clean formulations attached. Five environmental infestations were performed, with the number of ticks (at all stages) on the dogs being counted twice a day for 45 days. The counts on the number of tick stages found per dog were individually fitted to linear mixed effects models with repeated measures and normal distribution for errors. The mean tick infestation in the treated group was significantly lower than in the control group. For larvae and nymphs, a decrease in tick infestation was observed at the fifth count, and for adults, lower average counts were observed in all counts. The compounds did not interfere with the distribution of the ticks on the body of the dogs, as a similar percentage of ticks was found on the anterior half of the dogs (54.5% for the control group and 56.2% for the treated group). The biological and reproductive parameters of the ticks were not affected by the repellents. This study highlights for the first time the potential use of a novel allomone (repellent)-based formulation for reduction of tick infestation on susceptible dogs

Bioinformatics Discovery of Vertebrate Cathelicidins from the Mining of Available Genomes

Due to the worrying increase in antimicrobial resistance to conventional antibiotics, the search for alternatives is becoming increasingly important. Antimicrobial peptides (AMPs), originating from natural resources, have been recognised as a novel class of antibiotics. An advantage of peptides over antibiotics is that the resistance is more difficult to attain than for conventional antibiotics. With the increasing number of genomes sequenced and available in the public domain, one alternative methodology to obtain novel AMPs is to analyse genes and proteins from genomic databases to predict and identify amino acid sequences that share similarities and molecular features with natural bioactive antimicrobial peptides. In this chapter, we summarise some of our recent results on the production of antimicrobial peptides, particularly, how we managed to identify a family of antimicrobial peptides: cathelicidins, through bioinformatics tools, from the genomes of two lower vertebrates (a reptile and a bird) available in public databases. We hope that our preliminary investigation with these novel peptides could be useful for the design of future strategies that pursue the production of antimicrobial peptides through biotechnology

Multiclasificadores basados en aprendizaje automático como herramienta para la evaluación del perfil neurotóxico de líquidos iónicos

Los líquidos iónicos poseen un perfil fisicoquímico único, el cual los provee de un amplio rango de aplicaciones. Su variabilidad estructural casi ilimitada permite su diseño para tareas específicas. Sin embargo, su sustentabilidad, específicamente su seguridad desde el punto de vista toxicológico, ha sido frecuentemente cuestionada. Este último aspecto limita significativamente el cumplimiento de las regulaciones establecidas por la Unión Europea para el registro, evaluación, autorización y restricción de compuestosquímicos (REACH), así como su aplicación final. Debido a que la mayoría de los líquidos iónicos no han sido sintetizados, se hace evidente la importancia del desarrollo de herramientas quimioinformáticas que, de forma eficiente, permitan evaluar el potencial toxicológico de estos compuestos. En este sentido, el uso combinado de múltiples clasificadores ha demostrado superar las limitaciones de desempeño asociadas al uso de clasificadores individuales. En el presente trabajo fueron evaluadas varias estrategias alternativas de multiclasificadores basados en técnicas de aprendizaje automático supervisado, como herramientas para la evaluación del perfil neurotóxico de líquidos iónicos basado en la inhibición de la enzima acetilcolinesterasa, como indicador de neurotoxicidad. Se obtuvieron dos multiclasificadores con una alta capacidad predictiva sobre un conjunto de validación externa (no utilizado en el proceso de aprendizaje de los modelos). De acuerdo a los resultados obtenidos el 96% de un conjunto de nuevos líquidos iónicos podrá ser correctamente clasificado con la utilizaciónde estos multiclasificadores, los cuales constituyen herramientas de toma de decisión útiles en el campo del diseño y desarrollo de nuevos líquidos iónicos sustentables

Dogs are resistant to prion infection, due to the presence of aspartic or glutamic acid at position 163 of their prion protein

Unlike other species, prion disease has never been described in dogs even though they were similarly exposed to the bovine spongiform encephalopathy (BSE) agent. This resistance prompted a thorough analysis of the canine PRNP gene and the presence of a negatively charged amino acid residue in position 163 was readily identified as potentially fundamental as it differed from all known susceptible species. In the present study, the first transgenic mouse model expressing dog prion protein (PrP) was generated and challenged intracerebrally with a panel of prion isolates, none of which could infect them. The brains of these mice were subjected to in vitro prion amplification and failed to find even minimal amounts of misfolded prions providing definitive experimental evidence that dogs are resistant to prion disease. Subsequently, a second transgenic model was generated in which aspartic acid in position 163 was substituted for asparagine (the most common in prion susceptible species) resulting in susceptibility to BSE‐derived isolates. These findings strongly support the hypothesis that the amino acid residue at position 163 of canine cellular prion protein (PrPC) is a major determinant of the exceptional resistance of the canidae family to prion infection and establish this as a promising therapeutic target for prion diseases.MINECO/FEDER. Grant Numbers: AGL2015‐65046‐C2‐1‐R, AGL2008‐05296‐C02 Interreg. Grant Number: POCTEFA EFA148/1

Radon and material radiopurity assessment for the NEXT double beta decay experiment

The Neutrino Experiment with a Xenon TPC (NEXT), intended to investigate the neutrinoless double beta decay using a high-pressure xenon gas TPC filled with Xe enriched in 136Xe at the Canfranc Underground Laboratory in Spain, requires ultra-low background conditions demanding an exhaustive control of material radiopurity and environmental radon levels. An extensive material screening process is underway for several years based mainly on gamma-ray spectroscopy using ultra-low background germanium detectors in Canfranc but also on mass spectrometry techniques like GDMS and ICPMS. Components from shielding, pressure vessel, electroluminescence and high voltage elements and energy and tracking readout planes have been analyzed, helping in the final design of the experiment and in the construction of the background model. The latest measurements carried out will be presented and the implication on NEXT of their results will be discussed. The commissioning of the NEW detector, as a first step towards NEXT, has started in Canfranc; in-situ measurements of airborne radon levels were taken there to optimize the system for radon mitigation and will be shown too.Comment: Proceedings of the Low Radioactivity Techniques 2015 workshop (LRT2015), Seattle, March 201