Identificação e caracterização de sequências codificadoras de peptídeos antimicrobianos (PAM) nas células da hemolinfa de espécies nativas de peneídeos marinhos

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em BiotecnologiaProteínas ou peptídeos antimicrobianos (PAM) são componentes essenciais do sistema imune inato e encontram-se amplamente distribuídos entre vertebrados, invertebrados e plantas. São moléculas relativamente pequenas, de caráter anfipático e catiônico, podendo apresentar uma atividade microbicida rápida e potente contra um amplo espectro de microrganismos. Em camarões peneídeos, três classes de PAM foram isoladas e caracterizadas molecular e funcionalmente, a partir de suas células sangüíneas ou hemócitos: peneidinas (PEN), crustinas (CRUS) e os fatores anti-lipopolissacarídeos (ALF). O objetivo desse estudo foi o de detectar, clonar e caracterizar molecularmente seqüências semelhantes a PEN, CRUS e ALF nos hemócitos de três espécies nativas de camarões peneídeos Farfantepenaeus paulensis, F. subtilis e Litopenaeus schmitti, além de elaborar filogramas a partir das seqüências obtidas. Através da utilização de iniciadores desenhados a partir de regiões de consenso de seqüências disponíveis em bancos gênicos públicos, foi possível amplificar fragmentos de cDNA correspondentes às três classes de PAM acima mencionadas nos diferentes peneídeos. Em F. subtilis foi clonada uma seqüência semelhante à PEN do subgrupo 2 denominada de Farsub PEN2-1 (GenBank: EF450742), codificando para um peptídeo maduro de 54 aminoácidos, altamente catiônico e com alta identidade aminoacídica com as PEN2 de F. paulensis (91-93%). Em L. schmitti, mas não nos outros peneídeos, foi possível obter um produto de amplificação correspondendo a uma seqüência de ALF, cujo peptídeo maduro consiste de 98 aminoácidos, com similaridades aminoacídicas de 93 e 95% com F. chinensis e L. vannamei, respectivamente. Esta seqüência, denominada Litsch ALF (GenBank: DQ991357) codifica para uma molécula altamente catiônica e contém, assim como outras seqüências de ALF, uma região anfipática entre dois resíduos conservados de cisteína. Já, seqüências similares a CRUS foram obtidas nas três espécies de peneídeos analisados, sendo denominadas de Farsub CRUS (127 aminoácidos, GenBank: EF450744), Farpau CRUS (150 aminoácidos, GenBank: EF182747) e Litsch CRUS (148 aminoácidos, GenBank: EF182748). As três seqüências codificam para peptídeos contendo uma região N-terminal hidrofóbica rica em resíduos de glicina e uma região C-terminal com doze resíduos conservados de cisteína, onde se encontra um domínio WAP. Todas as seqüências de CRUS obtidas apresentaram alta similaridade aminoacídica com as diferentes isoformas de CRUS do camarão L. vannamei (em torno de 85%). Este foi o primeiro relato da presença de ALF e CRUS em camarões peneídeos nativos brasileiros. A PEN já havia sido detectada em F. paulensis e L. schmitti. Atualmente, alguns dos PAM identificados estão em fase de expressão em sistema recombinante, para posteriormente determinar seu espectro de ação frente a diferentes grupos de microrganismos e verificar seu potencial como agente terapêutico em saúde humana, veterinária e aqüicultura. Antimicrobial proteins or peptides (AMP) are major components of the immune system, which are widely distributed among vertebrates, invertebrates and plants. They are small cationic and amphipatic molecules that exhibit a rapid and potent activity against a broad range of microorganisms. In penaeid shrimp, three different classes of AMP have been isolated and molecular and functionally characterized from the blood cells or hemocytes: penaeidins (PEN), crustins (CRUS) and anti-lipopolysaccharide factors (ALF). The main purpose of this study was to detect, clone and molecularly characterize, gene sequences similar to PEN, CRUS and ALF from the hemocytes of the indigenous penaeid shrimp Farfantepenaeus paulensis, F. subtilis and Litopenaeus schmitti and also construct philograms from the obtained sequences It was possible to amplify cDNA sequences of the three AMP classes mentioned above, using primers based on consensus regions of corresponding sequences available at the GenBank. In F. subtilis, a PEN-like sequence, belonging to the subgroup 2, was cloned and named Farsub PEN2-1 (GenBank: EF450742). It coded for a mature peptide composed of 54 amino acids, highly cationic, and exhibited a high amino acidic identity with PEN2 isoforms of F. paulensis (91-93%). In L. schmitti, but not in other penaeids, one amplification product corresponding to an ALF-like sequence was obtained and called Litsch ALF (GenBank: DQ991357). It coded for a mature peptide of 98 amino acids exhibiting 93 and 95% of amino acid similarity with the ALF of F. chinensis and L. vannamei respectively. On the other hand, CRUS-like sequences were obtained in all three penaeid species, and were named Farsub CRUS (127 amino acids, GenBank: EF450744), Farpau CRUS (150 amino acids, GenBank: EF182747) and Litsch CRUS (148 amino acids, GenBank: EF182748). The three CRUS sequences coded for peptides containing a hydrophobic N-terminal sequence rich in glycine residues and a C-terminal sequence with twelve conserved cysteine residues and a WAP domain. All obtained CRUS sequences had amino acid similarity with different isoforms of L. vannamei CRUS (about 85%). This is the first report of ALF and CRUS sequences in indigenous Brazilian penaeid shrimp. PEN sequences were already described in F. paulensis e L. schmitti. Some of the cloned AMP are presently under expression through recombinant system, for further evaluate their antimicrobial activity spectra and their potential use as therapeutic agents for human and veterinary health and also aquaculture

Host defense effectors expressed by hemocytes shape the bacterial microbiota from the Scallop Hemolymph

The interaction between host immune response and the associated microbiota has recently become a fundamental aspect of vertebrate and invertebrate animal health. This interaction allows the specific association of microbial communities, which participate in a variety of processes in the host including protection against pathogens. Marine aquatic invertebrates such as scallops are also colonized by diverse microbial communities. Scallops remain healthy most of the time, and in general, only a few species are fatally affected on adult stage by viral and bacterial pathogens. Still, high mortalities at larval stages are widely reported and they are associated with pathogenic Vibrio. Thus, to give new insights into the interaction between scallop immune response and its associated microbiota, we assessed the involvement of two host antimicrobial effectors in shaping the abundances of bacterial communities present in the scallop Argopecten purpuratus hemolymph. To do this, we first characterized the microbiota composition in the hemolymph from non-stimulated scallops, finding both common and distinct bacterial communities dominated by the Proteobacteria, Spirochaetes and Bacteroidetes phyla. Next, we identified dynamic shifts of certain bacterial communities in the scallop hemolymph along immune response progression, where host antimicrobial effectors were expressed at basal level and early induced after a bacterial challenge. Finally, the transcript silencing of the antimicrobial peptide big defensin ApBD1 and the bactericidal/permeability-increasing protein ApLBP/BPI1 by RNA interference led to an imbalance of target bacterial groups from scallop hemolymph. Specifically, a significant increase in the class Gammaproteobacteria and the proliferation of Vibrio spp. was observed in scallops silenced for each antimicrobial. Overall, our results strongly suggest that scallop antimicrobial peptides and proteins are implicated in the maintenance of microbial homeostasis and are key molecules in orchestrating host-microbiota interactions. This new evidence depicts the delicate balance that exists between the immune response of A. purpuratus and the hemolymph microbiota.info:eu-repo/semantics/publishedVersio

Simbología gráfica de la nacionalidad Shuar, aplicación en un sistema señalético turístico del cantón Morona - Macas

Se elaboró un diccionario de los signos y símbolos más representativos de la nacionalidad Shuar, seguidamente se aplicó en un sistema señalético turístico del cantón Morona -Macas de la provincia de Morona Santiago. Careciendo un sistema señalético turístico, mediante método analítico se elaboró un diccionario de símbolos y signos Shuar que constituyó la base para desarrollar su aplicación. Se produjeron nuevas propuestas gráficas empleando un computador Macbook Pro, programas Adobe Ilustrator y adobe Phtoshop en su versión Cs5, tableta gráfica Wacom Bamboo en impresora Samsung 6555; posteriormente se implementaron montajes digitales para simular su implementación. La población de Macas que según el INEC es de 47.137 habitantes entre hombres y mujeres, se ha considerado a la población mayor a 20 años que representa el 49,44% es decir 23.305 habitantes. Se realizaron 96 encuestas. Determinando que el nivel de funcionalidad gráfico tiene un 94% de legibilidad, 91% de ubicación adecuada, 95% de material adecuado, 86% de altura adecuada, 91% de apreciación a luz del día, 89% de apreciación en la noche, el sistema es funcional. La significación reflejo que el 91% de la población se siente identificado, el sistema de iconos y color que constituyen el sistema señalético es significativo. Se concluye que el diccionario de signos y símbolos Shuar constituyó la base para la elaboración del sistema señalético turístico del cantan Morona - Macas de la provincia de Morona Santiago. Recomiendo al ministerio de turismo del cantón Morona- Macas de la Provincia de Morona Santiago, pueda basarse en esta tesis para la elaboración de sus sistemas señaléticos turísticos y su posible aplicación en diferentes formatos el mismo que facilitará los recorridos turísticos

Gas exchange acclimation to elevated CO2 in upper-sunlit and lower-shaded canopy leaves in relation to nitrogen acquisition and partitioning in wheat grown in field chambers.

Growth at elevated CO2 often decreases photosynthetic capacity (acclimation) and leaf N concentrations. Lower-shaded canopy leaves may undergo both CO2 and shade acclimation. The relationship of acclimatory responses of flag and lower-shaded canopy leaves of wheat (Triticum aestivum L.) to the N content, and possible factors affecting N gain and distribution within the plant were investigated in a wheat crop growing in field chambers set at ambient (360 μmol mol-1) and elevated (700 μmol mol-1) CO2, and with two amounts of N fertilizer (none and 70 kg ha-1 applied on 30 April). Photosynthesis, stomatal conductance and transpiration at a common measurement CO2, chlorophyll and Rubisco levels of upper-sunlit (flag) and lower-shaded canopy leaves were significantly lower in elevated relative to ambient CO2-grown plants. Both whole shoot N and leaf N per unit area decreased at elevated CO2, and leaf N declined with canopy position. Acclimatory responses to elevated CO2 were enhanced in N-deficient plants. With N supply, the acclimatory responses were less pronounced in lower canopy leaves relative to the flag leaf. Additional N did not increase the fraction of shoot N allocated to the flag and penultimate leaves. The decrease in photosynthetic capacity in both upper-sunlit and lower-shaded leaves in elevated CO2 was associated with a decrease in N contents in above-ground organs and with lower N partitioning to leaves. A single relationship of N per unit leaf area to the transpiration rate accounted for a significant fraction of the variation among sun-lit and shaded leaves, growth CO2 level and N supply. We conclude that reduced stomatal conductance and transpiration can decrease plant N, leading to acclimation to CO2 enrichment.This work was funded by the Spanish ‘Plan Nacional de Investigación y Desarrollo’ (grant N° BFI2000-0871). A. Del Pozo was the recipient of a fellowship from the Spanish Ministry of Education for a sabbatical leave. R. Morcuende had a Ramón y Cajal research contract from the Spanish Ministry of Education. The technical cooperation of A.L. Verdejo in gas exchange measurements, chlorophyll and Rubisco activity determination is acknowledged. We thank the staff of the experimental farm of IRNASA for assistance in crop husbandry.Peer reviewe

Gas exchange acclimation to elevated CO2 in upper-sunlit and lower-shaded canopy leaves in relation to nitrogen acquisition and partitioning in wheat grown in field chambers.

Growth at elevated CO2 often decreases photosynthetic capacity (acclimation) and leaf N concentrations. Lower-shaded canopy leaves may undergo both CO2 and shade acclimation. The relationship of acclimatory responses of flag and lower-shaded canopy leaves of wheat (Triticum aestivum L.) to the N content, and possible factors affecting N gain and distribution within the plant were investigated in a wheat crop growing in field chambers set at ambient (360 μmol mol-1) and elevated (700 μmol mol-1) CO2, and with two amounts of N fertilizer (none and 70 kg ha-1 applied on 30 April). Photosynthesis, stomatal conductance and transpiration at a common measurement CO2, chlorophyll and Rubisco levels of upper-sunlit (flag) and lower-shaded canopy leaves were significantly lower in elevated relative to ambient CO2-grown plants. Both whole shoot N and leaf N per unit area decreased at elevated CO2, and leaf N declined with canopy position. Acclimatory responses to elevated CO2 were enhanced in N-deficient plants. With N supply, the acclimatory responses were less pronounced in lower canopy leaves relative to the flag leaf. Additional N did not increase the fraction of shoot N allocated to the flag and penultimate leaves. The decrease in photosynthetic capacity in both upper-sunlit and lower-shaded leaves in elevated CO2 was associated with a decrease in N contents in above-ground organs and with lower N partitioning to leaves. A single relationship of N per unit leaf area to the transpiration rate accounted for a significant fraction of the variation among sun-lit and shaded leaves, growth CO2 level and N supply. We conclude that reduced stomatal conductance and transpiration can decrease plant N, leading to acclimation to CO2 enrichment.This work was funded by the Spanish ‘Plan Nacional de Investigación y Desarrollo’ (grant N° BFI2000-0871). A. Del Pozo was the recipient of a fellowship from the Spanish Ministry of Education for a sabbatical leave. R. Morcuende had a Ramón y Cajal research contract from the Spanish Ministry of Education. The technical cooperation of A.L. Verdejo in gas exchange measurements, chlorophyll and Rubisco activity determination is acknowledged. We thank the staff of the experimental farm of IRNASA for assistance in crop husbandry.Peer reviewe

Changes in leaf morphology and composition with future increases in CO2 and temperature revisited. Wheat in field chambers

Whether leaf morphology is altered by future increases in atmospheric CO2 and temperature has been re-examined during three years in wheat grown in field chambers at two levels of nitrogen supply. Flag leaf fresh and dry mass, area, volume, and ratios of these parameters, as well as the contents of water, chlorophyll, non-structural carbohydrates and nitrogen compounds have been determined at anthesis and 14 days later. High CO2 decreased, rather than increased as reported in the literature, leaf mass per area and leaf density, and increased water content per area and volume and water percentage. Warmer temperatures also decreased leaf mass per area, but did not affect density or water per area or volume, while they increased water percentage. Nitrogen supply did not change CO2 and temperature effects on leaf morphology. Non-structural carbohydrates increased and nitrogen compounds decreased in elevated CO2, and the sum of these compounds decreased with warmer temperatures. These changes in composition did not account for modifications of leaf morphology. We conclude that increases in atmospheric CO2 and temperature after leaf initiation can decrease leaf mass per area, and elevated CO2 can also decrease leaf density, due to decreases in leaf structural compounds. The functional significance of these changes is probably a decrease in photosynthetic capacity per unit leaf area.E.G. and D.G. were the recipients of I3P-European Social Fund and Junta de Castilla y León fellowships, respectively. We thank the staff of this Institute’s experimental farm for technical assistance in crop husbandry. This work has been funded by the Spanish National Research and Development Programme- European Regional Development Fund, ERDF (Project BFI2003-01277).Peer reviewe

Indications and contraindications of dental implants in medically compromised patients: update

The aim of this study was to review the current scientific literature in order to analyse the indications and contrain - dications of dental implants in medically compromised patients. A reference research was carried out on PubMed using the key words "implant" AND (oral OR dental) AND (systemic disease OR medically compromised), in articles published between 1993 and 2013. The inclusion criteria were the following: clinical studies in which, at least, 10 patients were treated, consensus articles, reviewed articles and meta-analysis performed in humans treated with dental implants, and which included the disease diagnosis. A total of 64 articles were found, from which 16 met the inclusion criteria. Cardiac systemic diseases, diabetic endocrine pathologies or controlled metabolic disorders do not seem to be a total or partial contraindication to the placement of dental implants. Tobacco addiction, and head and neck radiotherapy are correlated to a higher loss of dental implants. Patients suffering from osteoporosis undergoing biphosphonates therapy show an increased risk of developing bone necrosis after an oral surgery, especially if the drugs are administered intravenously or they are associated to certain concomitant medication

Copulatory behaviour increases sperm viability in female spiders

One remarkable reproductive feature in animals with internal fertilization is a reduction in sperm viability over time in females. Whether this reduction is driven by male-male competition and/or cryptic female choice is unclear. From the perspective of cryptic female choice, we postulated that sperm viability is affected by a particular male copulatory behaviour. In this study, we investigated the following aspects: (1) sperm viability in mated females vs. males; (2) whether sperm viability varies temporally after mating; and (3) whether male copulatory behaviour covaries positively with sperm viability within females. We used the spider Holocnemus pluchei, whose males use several copulatory behaviours to court females. We found that females that stored sperm for 4 or 15 days showed no difference in sperm viability but had lower sperm viability compared with males, and males that performed a longer post-insemination behaviour had higher sperm viability inside the female. It is unclear how sperm viability is reduced and how male post-insemination behaviour affects this. It is possible that extending copulation allows males to induce females to keep sperm alive for longer. This result is predicted by theory whereby males induce females to facilitate sperm to reach and fertilize eggs based on male postcopulatory behaviour.Fil: Cargnelutti, Franco Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; ArgentinaFil: Calbacho Rosa, Lucía Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; ArgentinaFil: Uñates, Diego Rafael. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; ArgentinaFil: Costa Schmidt, Luiz Ernesto. Universidade Federal de Pelotas; Brasil. Universidade do Vale do Rio dos Sinos, Rio Grande do Sul; BrasilFil: Córdoba-Aguilar, Alex. Universidad Nacional Autónoma de México. Instituto de Ecología; MéxicoFil: Peretti, Alfredo Vicente. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Diversidad y Ecología Animal. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas Físicas y Naturales. Instituto de Diversidad y Ecología Animal; Argentin

Modelling soil organic carbon stocks in global change scenarios: a CarboSOIL application

Global climate change, as a consequence of the increasing levels of atmospheric CO2 concentration, may significantly affect both soil organic C storage and soil capacity for C sequestration. CarboSOIL is an empirical model based on regression techniques and developed as a geographical information system tool to predict soil organic carbon (SOC) contents at different depths. This model is a new component of the agro-ecological decision support system for land evaluation MicroLEIS, which assists decision-makers in facing specific agro-ecological problems, particularly in Mediterranean regions. In this study, the CarboSOIL model was used to study the effects of climate change on SOC dynamics in a Mediterranean region (Andalusia, S Spain). Different downscaled climate models were applied based on BCCR-BCM2, CNRMCM3, and ECHAM5 and driven by SRES scenarios (A1B, A2 and B2). Output data were linked to spatial data sets (soil and land use) to quantify SOC stocks. The CarboSOIL model has proved its ability to predict the short-medium- and long-term trends (2040s, 2070s and 2100s) of SOC dynamics and sequestration under projected future scenarios of climate change. Results have shown an overall trend towards decreasing of SOC stocks in the upper soil sections (0–25 cm and 25–50 cm) for most soil types and land uses, but predicted SOC stocks tend to increase in the deeper soil section (0–75 cm). Soil types as Arenosols, Planosols and Solonchaks and land uses as “permanent crops” and “open spaces with little or no vegetation” would be severely affected by climate change with large decreases of SOC stocks, in particular under the medium–high emission scenario A2 by 2100. The information developed in this study might support decision-making in land management and climate adaptation strategies in Mediterranean regions, and the methodology could be applied to other Mediterranean areas with available soil, land use and climate data.Ministerio de Economía y CompetitividadJunta de Andalucí

A new antibiotic-loaded sol-gel can prevent bacterial intravenous catheter-related infections

The aim of this study was to evaluate the effectiveness of a moxifloxacin-loaded organic-inorganic sol-gel (A50) by locally preventing the catheter-related bloodstream infection (CRBSI) provoked by Staphylococcus epidermidis (S. epidermidis) and the effect resulting from its hydrolytic degradation on coagulation by using a rabbit in-vivo model. A50 coating can completely inhibit growth and would locally prevent CRBSI provoked by S. epidermidis. None of the coagulation blood parameters showed a significant difference constant over time between the control catheter group and the A50-coated catheter group, despite the visible silica release resulting from physiological A50 sol-gel degradation detected in serum at least during the first week. At pathological level, foreign body reaction was present in both of types of catheter, and it was characterized by the presence of macrophages and foreign body giant cell. However, this reaction was different in each group: the A50-coated catheter group showed a higher inflammation with histiocytes, which were forming granuloma-like aggregates with an amorphous crystalline material inside, accompanied by other inflammatory cells such as plasma cells, lymphocytes and mast cells. In conclusion, A50 coating a venous catheter showed excellent bactericidal anti-biofilm response since it completely inhibited S. epidermidis biofilm development and, far from showing procoagulant effects, showed slightly anticoagulant effects.This research received financial support from the Mutua Madrileña Foundation (04078/001). J.J.A.-C. was funded by an FPI grant from the Spanish Ministry of Economics and Competitiveness (BES-2014-069007)