Degradation of pheromone and plant volatile components by a same Odorant-Degrading Enzyme in the cotton leafworm, Spodoptera littoralis

Background: Odorant-Degrading Enzymes (ODEs) are supposed to be involved in the signal inactivation step within the olfactory sensilla of insects by quickly removing odorant molecules from the vicinity of the olfactory receptors. Only three ODEs have been both identified at the molecular level and functionally characterized: two were specialized in the degradation of pheromone compounds and the last one was shown to degrade a plant odorant. Methodology: Previous work has shown that the antennae of the cotton leafworm Spodoptera littoralis , a worldwide pest of agricultural crops, express numerous candidate ODEs. We focused on an esterase overexpressed in males antennae, namely SlCXE7. We studied its expression patterns and tested its catalytic properties towards three odorants, i.e. the two female sex pheromone components and a green leaf volatile emitted by host plants. Conclusion: SlCXE7 expression was concomitant during development with male responsiveness to odorants and during adult scotophase with the period of male most active sexual behaviour. Furthermore, SlCXE7 transcription could be induced by male exposure to the main pheromone component, suggesting a role of Pheromone-Degrading Enzyme. Interestingly, recombinant SlCXE7 was able to efficiently hydrolyze the pheromone compounds but also the plant volatile, with a higher affinity for the pheromone than for the plant compound. In male antennae, SlCXE7 expression was associated with both long and short sensilla, tuned to sex pheromones or plant odours, respectively. Our results thus suggested that a same ODE could have a dual function depending of it sensillar localisation. Within the pheromone-sensitive sensilla, SlCXE7 may play a role in pheromone signal termination and in reduction of odorant background noise, whereas it could be involved in plant odorant inactivation within the short sensilla.

Enhanced degradation of metalaxyl in agricultural soils of São Paulo State, Brazil

This work investigated the effect of repeated applications on enhanced degradation of metalaxyl in two different agricultural soils used for cultivation of orange and lemon from Casa Branca and Itapetininga districts of São Paulo State, Brazil. Soil samples were collected from areas repeatedly treated with commercial ridomil 50GR for six successive years, and from other areas never exposed to this fungicide. At the laboratory, soil samples received a 14C-metalaxyl solution and its degradation was studied through radiometric techniques to measure biomineralization and recovery of extractable- and soil-bound products. Enhanced degradation was verified only in one soil, although partial degradation and mineralization of the fungicide were detected in both soils. The different rates and patterns of metalaxyl degradation in the soils were probably due to their different physical, chemical, and biological characteristics.

Photoelectron spectroscopy for surface analysis: X-ray and UV excitation

This article summarizes the basic principles of photoelectron spectroscopy for surface analysis, with examples of applications in material science that illustrate the capabilities of the related techniques.

RFID Tags for Detecting Concrete Degradation in Bridge Decks, RB08-011, 2013

Steel reinforcing bar (rebar) corrosion due to chlorine ingress is the primary degradation mechanism for bridge decks. In areas where rock salt is used as a de-icing agent, salt water seeps into the concrete through cracks, causing corrosion of the rebar and potentially leading to catastrophic failure if not repaired. This project explores the use of radio frequency identification (RFID) tags as low-cost corrosion sensors. RFID tags, when embedded in concrete, will fail due to corrosion in the same manner as rebar after prolonged exposure to salt water. In addition, the presence of salt water interferes with the ability to detect the tags, providing a secondary mechanism by which this method can work. During this project, a fieldable RFID equipment setup was constructed and tested. In addition to a number of laboratory experiments to validate the underlying principles, RFID tags were embedded and tested in several actual bridge decks. Two major challenges were addressed in this project: issues associated with tags not functioning due to being in close proximity to rebar and issues associated with portland concrete coming in direct contact with the tags causing a detuning effect and preventing the tags from operating properly. Both issues were investigated thoroughly. The first issue was determined to be a problem only if the tags are placed in close proximity to rebar. The second issue was resolved by encapsulating the tag. Two materials, polyurethane spray foam and extruded polystyrene, were identified as providing good performance after testing, both in the lab and in the field.

Beta-oxidation in fatty acid degradation and beyond.

The degradation of fatty acids in plants occurs primarily in the peroxisomes through the beta-oxidation cycle. Enzymes that are involved in various aspects of beta-oxidation have been identified recently and shown to act biochemically on a diversity of fatty acids and derivatives. Analysis of several mutants has revealed essential roles for beta-oxidation in the breakdown of reserve triacylglycerols, seed development, seed germination and post-germinative growth before the establishment of photosynthesis. Beta-oxidation has also a considerable importance during the vegetative and reproductive growth phases, and plays a role in plant responses to stress, particularly in the synthesis of jasmonic acid.

Analysis of the contribution of the beta-oxidation auxiliary enzymes in the degradation of the dietary conjugated linoleic acid 9-cis-11-trans-octadecadienoic acid in the peroxisomes of Saccharomyces cerevisiae.

Beta-oxidation of the conjugated linoleic acid 9-cis,11-trans-octadecadienoic acid (rumenic acid) was analyzed in vivo in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanoate is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxyacyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The amount of polyhydroxyalkanaote synthesized from the degradation of rumenic acid was found to be similar to the amount synthesized from the degradation of 10-trans,12-cis-octadecadienoic acid, oleic acid or 10-cis-heptadecenoic acid. Furthermore, the degradation of 10-cis-heptadecenoic acid was found to be unaffected by the presence of rumenic acid in the media. Efficient degradation of rumenic acid was found to be independent of the Delta(3,5),Delta(2,4)-dienoyl-CoA isomerase but instead relied on the presence of Delta(3),Delta(2)-enoyl-CoA isomerase activity. The presence of the unsaturated monomer 3-hydroxydodecenoic acid in polyhydroxyalkanoate derived from rumenic acid degradation was found to be dependent on the presence of a Delta(3),Delta(2)-enoyl-CoA isomerase activity. Together, these data indicate that rumenic acid is mainly degraded in vivo in S. cerevisiae through a pathway requiring only the participation of the auxiliary enzymes Delta(3),Delta(2)-enoyl-CoA isomerase, along with the enzyme of the core beta-oxidation cycle.

Selective ablation of photovoltaic materials with UV laser sources for monolithic interconnection of devices based on a-Si:H

Lasers are essential tools for cell isolation and monolithic interconnection in thin-film-silicon photovoltaic technologies. Laser ablation of transparent conductive oxides (TCOs), amorphous silicon structures and back contact removal are standard processes in industry for monolithic device interconnection. However, material ablation with minimum debris and small heat affected zone is one of the main difficulty is to achieve, to reduce costs and to improve device efficiency. In this paper we present recent results in laser ablation of photovoltaic materials using excimer and UV wavelengths of diode-pumped solid-state (DPSS) laser sources. We discuss results concerning UV ablation of different TCO and thin-film silicon (a-Si:H and nc-Si:H), focussing our study on ablation threshold measurements and process-quality assessment using advanced optical microscopy techniques. In that way we show the advantages of using UV wavelengths for minimizing the characteristic material thermal affection of laser irradiation in the ns regime at higher wavelengths. Additionally we include preliminary results of selective ablation of film on film structures irradiating from the film side (direct writing configuration) including the problem of selective ablation of ZnO films on a-Si:H layers. In that way we demonstrate the potential use of UV wavelengths of fully commercial laser sources as an alternative to standard backscribing process in device fabrication.

Glucocorticoid-induced tumor necrosis factor receptor is a p21Cip1/WAF1 transcriptional target conferring resistance of keratinocytes to UV light-induced apoptosis.

Glucocorticoid-induced tumor necrosis factor receptor (GITR) is a member of the tumor necrosis factor receptor superfamily, is expressed in T lymphocytes, and exerts an anti-apoptotic function in these cells. We reported that GITR is also highly expressed in the skin, specifically in keratinocytes, and that it is under negative transcriptional control of p21(Cip1/WAF1), independently from the cell cycle. Although GITR expression is higher in p21-deficient keratinocytes and skin, it is down-modulated with differentiation and in response to UVB. The combined analysis of keratinocytes with increased GITR expression versus normal keratinocytes and skin of mice with a disruption of the GITR gene indicates that this protein protects keratinocytes from UVB-induced apoptosis both in vitro and in vivo.

UV irradiation of human keratinocytes activates the inflammasome

Human keratinocytes represent a potent source of the pro-inflammatory cytokines pro-interleukin(IL)-1α and -β. ProIL-1β requires processing by caspase-1 (IL-1β-converting enzyme, ICE) for activation and receptor binding. ProIL-1α and -β lack a signal peptide and leave the cell via the alternative secretion pathway, which is independent of the classical ER/Golgi pathway. Both cytokines are stored in the cytoplasm and can be activated and released upon UV irradiation. In macrophages maturation of proIL-1β requires the activation of inflammasomes, innate multiprotein immune complexes, which are essential for the activation of caspase-1 and thereby for processing of proIL-1β. However, the intracellular pathways, which are responsible for activation of proIL-1β and secretion of IL-1β in keratinocytes, are unknown. We show that human keratinocytes express inflammasome proteins in vitro and in vivo. UVB irradiation of keratinocytes results in an increase of cytoplasmic Ca2+ from intracellular stores. This shift is required for inflammasome-dependent activation of caspase-1 and subsequent processing of proIL-1β and secretion of IL-1β. In contrast to macrophages, caspase-1 cannot activate proIL-18 in keratinocytes, although secretion of this cytokine is also induced by UVB irradiation. In vivo, caspase-1 is also essential for UVB-induced inflammation in the skin, since caspase-1 knockout mice showed a strongly reduced inflammatory response after UVB irradiation. Our results suggest that keratinocytes are important immuno-competent cells under physiological and pathological conditions.

Aspergillus protein degradation pathways with different secreted protease sets at neutral and acidic pH.

Aspergillus fumigatus grows well at neutral and acidic pH in a medium containing protein as the sole nitrogen source by secreting two different sets of proteases. Neutral pH favors the secretion of neutral and alkaline endoproteases, leucine aminopeptidases (Laps) which are nonspecific monoaminopeptidases, and an X-prolyl dipeptidase (DppIV). Acidic pH environment promotes the secretion of an aspartic endoprotease of pepsin family (Pep1) and tripeptidyl-peptidases of the sedolisin family (SedB and SedD). A novel prolyl peptidase, AfuS28, was found to be secreted in both alkaline and acidic conditions. In previous studies, Laps were shown to degrade peptides from their N-terminus until an X-Pro sequence acts as a stop signal. X-Pro sequences can be then removed by DppIV, which allows Laps access to the following residues. We have shown that at acidic pH Seds degrade large peptides from their N-terminus into tripeptides until Pro in P1 or P'1 position acts as a stop for these exopeptidases. However, X-X-Pro and X-X-X-Pro sequences can be removed by AfuS28 thus allowing Seds further sequential proteolysis. In conclusion, both alkaline and acidic sets of proteases contain exoprotease activity capable of cleaving after proline residues that cannot be removed during sequential digestion by nonspecific exopeptidases.

Água aquecida e radiação UV-C no controle pós-colheita de Cryptosporiopsis perennans em maçãs

O objetivo deste trabalho foi avaliar a colonização de Cryptosporiopsis perennans na epiderme de maçãs e a eficiência da aplicação de água aquecida e radiação UV-C no controle desse patógeno. Em maçãs submetidas à inoculação de C. perennans, a colonização de lenticelas e das áreas adjacentes pelo patógeno foi avaliada por microscopia eletrônica de varredura. A sensibilidade dos conídios de C. perennans aos tratamentos foi avaliada em suspensão aquosa, às temperaturas de 28, 45, 50 e 55ºC, por 15 e 30 s, e às doses de radiação UV-C de 0,018, 0,037, 0,075, 0,150, 0,375, 0,750, 1,500 e 3,000 kJ m-2. Em maçãs submetidas à inoculação de C. perennans, foram avaliados os efeitos de 0,375, 0,750 e 1,500 kJ m-2 de radiação UV-C e da aspersão de água aquecida à 50ºC, por 15 e 30 s no controle do patógeno. O fungo produziu abundante micélio e conídios nas lenticelas e nas áreas adjacentes, na epiderme das maçãs. A água aquecida a 50ºC por 15 s e à dose de radiação de UV-C de 0,750 kJ m-2 reduzem em mais de 99% a sobrevivência de conídios. A aspersão de água aquecida a 50ºC por 15 s e à dose de radiação de UV-C de 0,375 kJ m-2, controlam C. perennans em maçãs.

Functional diversity of bacterial genes associated with aromatic hydrocarbon degradation in anthropogenic dark earth of Amazonia

The objective of this work was to evaluate the catabolic gene diversity for the bacterial degradation of aromatic hydrocarbons in anthropogenic dark earth of Amazonia (ADE) and their biochar (BC). Functional diversity analyses in ADE soils can provide information on how adaptive microorganisms may influence the fertility of soils and what is their involvement in biogeochemical cycles. For this, clone libraries containing the gene encoding for the alpha subunit of aromatic ring-hydroxylating dioxygenases (α-ARHD bacterial gene) were constructed, totaling 800 clones. These libraries were prepared from samples of an ADE soil under two different land uses, located at the Caldeirão Experimental Station - secondary forest (SF) and agriculture (AG) -, and the biochar (SF_BC and AG_BC, respectively). Heterogeneity estimates indicated greater diversity in BC libraries; and Venn diagrams showed more unique operational protein clusters (OPC) in the SF_BC library than the ADE soil, which indicates that specific metabolic processes may occur in biochar. Phylogenetic analysis showed unidentified dioxygenases in ADE soils. Libraries containing functional gene encoding for the alpha subunit of the aromatic ring-hydroxylating dioxygenases (ARHD) gene from biochar show higher diversity indices than those of ADE under secondary forest and agriculture.

The DNA damage response to adeno-associated virus : centrosome overduplication and induction of cell death independently of the spindle checkpoint

Summary: Adeno-associated virus type 2 (AAV2) is a small virus containing single-stranded DNA of approximately 4.7kb in size. Both ends of the viral genome are flanked with inverted terminal repeat sequences (ITRs), which serve as primers for viral replication. Previous work in our laboratory has shown that AAV2 DNA with ultraviolet radiation-generated crosslinks (UV-AAV2) provokes a DNA damage response in the host cell by mimicking a stalled replication fork. Infection of cells with UV-AAV2 leads to a p53-and Chk1-mediated cell cycle arrest at the G2/M border of the cell cycle. However, tumour cells lacking the tumour suppressor protein p53 cannot sustain this arrest and enter a prolonged impaired mitosis, the outcome of which is cell death. The aim of my thesis was to investigate how UV-inactivated AAV2 kilts p53-deficient cancer cells. I found that the UV-AAV2-induced DNA damage signalling induces centriole overduplication in infected cells. The virus is able to uncouple the centriole duplication cycle from the cell cycle, leading to amplified centrosome numbers. Chk1 colocalises with centrosomes in the infected cells and the centrosome overduplication is dependent on the presence of Chk1, as well as on the activities of ATR and Cdk kinases and on the G2 arrest. The UV-AAV2-induced DNA damage signalling inhibits the degradation of cyclin B 1 and securin by the anaphase promoting complex, suggesting that the spindle checkpoint is activated in these mitotic cells. Interference with the spindle checkpoint components Mad2 and BubR1 revealed that the UV-AAV2-provoked mitotic catastrophe occurs independently of spindle checkpoint function, This work shows that, in the p53 deficient cells, UV-AAV2 triggers mitotic catastrophe associated with a dramatic Chk1-dependent overduplication of centrioles and the consequent formation of multiple spindle poles in mitosis. Résumé Le virus associé à l'adénovirus type 2 (AAV2) est un petit virus contenant un simple brin d'ADN d'environ 4.7kb. Des expériences antérieures dans notre laboratoire ont montré que les liens intramoléculaires sur l'ADN de AAV2 provoqués paz l'irradiation aux ultraviolets (UV) ressemblent à une fourche de réplication bloquée, ce qui provoque une réponse aux dommages à l'ADN dans la cellule hôte. L'infection des cellules avec UV-AAV2 résulte en un arrêt du cycle cellulaire à la transition G2/M entraîné par les protéines ATR et Chk1. Cependant, les cellules tumorales auxquelles il manque le suppresseur de tumeur p53 ne peuvent pas tenir cet arrêt et entrent dans une mitose anormale et prolongée qui se terminera par la mort cellulaire. Le but de ma thèse était d'étudier comment l'AAV2 inactivé par l'irradiation UV tue les cellules cancéreuses n'ayant pas p53. Je montre ici que le signal de dommages à l'ADN induit par UV-AAV2 génère une surduplication des centrioles dans les cellules infectées. Le virus est capable de dissocier le cycle de duplication du centriole du cycle cellulaire ce qui crée un nombre amplifié de centrosomes. Chk1 est co-localisé avec le centrosome dans les cellules infectées et la swduplication du centrosome est dépendante de la présence de Chk1, de l'activité des kinases ATR et Cdk et de l'arrêt en G2 de la cellule. Le signal d'ADN endommagé induit par UV-AAV2 réprime la dégradation des protéines cycline B1 et securine par le complexe promoteur de l'anaphase (APC), ce qui suggère que le point de contrôle du fuseau mitotique est activé dans ces cellules en mitose. L'étude d'interférence avec des éléments du point de contrôle du fuseau mitotique, Mad2 et BubR1, a révélé que la catastrophe mitotique provoquée paz UV-AAV2 survient indépendamment du point de contrôle du fuseau mitotique. Ce travail montre que dans les cellules déficientes en p53, UV-AAV2 induit une catastrophe mitotique associée à une surduplication des centrioles dépendant de Chk1 et ayant pour conséquence dramatique la formation de multiples fuseaux mitotiques dans la cellule en mitose.