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It is well known that plants respond to various stresses by releasing volatile organic compounds (VOCs). Green leaf volatiles (GLVs), which are commonly produced across different plant species after tissue damage, comprise an important group within VOCs. Among them, (Z)-3-hexenyl butyrate (HB) was described as a natural stomatal closure inducer, playing an important role in stomatal immunity, although its mechanism of action is still unknown. Here, through different genetic, pharmacological, and biochemical approaches we uncover that HB perception initiates different defense signaling events such as activation of Ca2+ permeable channels, mitogen-activated protein kinases (MPKs) and production of NADPH oxidase-mediated reactive oxygen species (ROS). Furthermore, HB-mediated stomata closure resulted to be independent of abscisic acid (ABA) biosynthesis and signaling. Additionally, exogenous treatments with this volatile alleviate water stress and improve fruit productivity in tomato plants. HB efficacy was also tested under open field conditions, leading to enhanced resistance against Phytophthora spp. and Pseudomonas syringae infection in potato and tomato plants, respectively. Collectively, our results provide insights into HB signaling transduction pathway, confirming its role in stomatal closure and plant immune system activation, and proposing HB as a new phytoprotector for the sustainable control of biotic and abiotic stresses in agriculture.

Objective: COVID-19 pandemic required optimization of hospital institutional flow, especially regarding the use of intensive care unit (ICU) beds. The aim of this study was to assess whether the individualization of the indication for postoperative recovery from pulmonary surgery in ICU beds was associated with more perioperative complications. Method: retrospective analysis of medical records of patients undergoing anatomic lung resections for cancer in a tertiary hospital. The sample was divided into: Group-I, composed of surgeries performed between March/2019 and February/2020, pre-pandemic, and Group-II, composed of surgeries performed between March/2020 and February/2021, pandemic period in Brazil. We analyzed demographic data, surgical risks, surgeries performed, postoperative complications, length of stay in the ICU and hospital stay. Preventive measures of COVID-19 were adopted in group-II. Results: 43 patients were included, 20 in group-I and 23 in group-II. The groups did not show statistical differences regarding baseline demographic variables. In group-I, 80% of the patients underwent a postoperative period in the ICU, compared to 21% in group-II. There was a significant difference when comparing the average length of stay in an ICU bed (46 hours in group-I versus 14 hours in group-II - p

[EN] Viroids are naked RNAs that do not code for any known protein and yet are able to infect plants causing severe diseases. Because of their RNA nature, many studies have focused on the involvement of viroids in RNA-mediated gene silencing as being their pathogenesis mechanism. Here, the alterations caused by the Citrus exocortis viroid (CEVd) on the tomato translation machinery were studied as a new aspect of viroid pathogenesis. The presence of viroids in the ribosomal fractions of infected tomato plants was detected. More precisely, CEVd and its derived viroid small RNAs were found to co-sediment with tomato ribosomes in vivo, and to provoke changes in the global polysome profiles, particularly in the 40S ribosomal subunit accumulation. Additionally, the viroid caused alterations in ribosome biogenesis in the infected tomato plants, affecting the 18S rRNA maturation process. A higher expression level of the ribosomal stress mediator NAC082 was also detected in the CEVd-infected tomato leaves. Both the alterations in the rRNA processing and the induction of NAC082 correlate with the degree of viroid symptomatology. Taken together, these results suggest that CEVd is responsible for defective ribosome biogenesis in tomato, thereby interfering with the translation machinery and, therefore, causing ribosomal stress., Spanish Ministry of Science, Innovation and Universities [BIO2009-11818, BIO2015-70483-R to A.F.]; Spanish Ministry of Science, Innovation and Universities [BFU2009-11958]; Generalitat Valenciana (Valencia, Spain) [AICO/2017/048]; Natural Sciences and Engineering Research Council of Canada [155219-17 to J.-P.P.]; The RNA group is supported by a grant from the Universite de Sherbrooke; J.-P.P. holds the Research Chair of the Universite de Sherbrooke in RNA Structure and Genomics, and is a member of the Centre de Recherche du CHUS; B.B.-P. was a recipient of a VALi+d postdoctoral contract of the Generalitat Valenciana [APOSTD/2017/039]; Schleiff group is funded through the Deutsche Forschungsgemeinschaft [SFB 902]. Funding for open access charge: Spanish Ministry of Science, Innovation and Universities.

Objective: COVID-19 pandemic required optimization of hospital institutional flow, especially regarding the use of intensive care unit (ICU) beds. The aim of this study was to assess whether the individualization of the indication for postoperative recovery from pulmonary surgery in ICU beds was associated with more perioperative complications. Method: retrospective analysis of medical records of patients undergoing anatomic lung resections for cancer in a tertiary hospital. The sample was divided into: Group-I, composed of surgeries performed between March/2019 and February/2020, pre-pandemic, and Group-II, composed of surgeries performed between March/2020 and February/2021, pandemic period in Brazil. We analyzed demographic data, surgical risks, surgeries performed, postoperative complications, length of stay in the ICU and hospital stay. Preventive measures of COVID-19 were adopted in group-II. Results: 43 patients were included, 20 in group-I and 23 in group-II. The groups did not show statistical differences regarding baseline demographic variables. In group-I, 80% of the patients underwent a postoperative period in the ICU, compared to 21% in group-II. There was a significant difference when comparing the average length of stay in an ICU bed (46 hours in group-I versus 14 hours in group-II - p

New strategies of control need to be developed with the aim of economic and environmental sustainability in plant and crop protection. Metabolomics is an excellent platform for both understanding the complex plant–pathogen interactions and unraveling new chemical control strategies. GC-MS-based metabolomics, along with a phytohormone analysis of a compatible and incompatible interaction between tomato plants and Fusarium oxysporum f. sp. lycopersici, revealed the specific volatile chemical composition and the plant signals associated with them. The susceptible tomato plants were characterized by the over-emission of methyl- and ethyl-salicylate as well as some fatty acid derivatives, along with an activation of salicylic acid and abscisic acid signaling. In contrast, terpenoids, benzenoids, and 2-ethylhexanoic acid were differentially emitted by plants undergoing an incompatible interaction, together with the activation of the jasmonic acid (JA) pathway. In accordance with this response, a higher expression of several genes participating in the biosynthesis of these volatiles, such as MTS1, TomloxC,TomloxD, and AOS, as well as JAZ7, a JA marker gene, was found to be induced by the fungus in these resistant plants. The characterized metabolome of the immune tomato plants could lead to the development of new resistance inducers against Fusarium wilt treatment.

This article belongs to the Section Molecular Plant Sciences., Infectious viroid clones consist of dimeric cDNAs used to generate transcripts which mimic the longer-than-unit replication intermediates. These transcripts can be either generated in vitro or produced in vivo by agro-inoculation. We have designed a new plasmid, which allows both inoculation methods, and we have compared them by infecting Solanum lycopersicum and Solanum melongena with clones of Citrus exocortis virod (CEVd), Tomato chlorotic dwarf viroid (TCDVd), and Potato spindle tuber viroid (PSTVd). Our results showed more uniform and severe symptoms in agro-inoculated plants. Viroid accumulation and the proportion of circular and linear forms were different depending on the host and the inoculation method and did not correlate with the symptoms, which correlated with an increase in PR1 induction, accumulation of the defensive signal molecules salicylic (SA) and gentisic (GA) acids, and ribosomal stress in tomato plants. The alteration in ribosome biogenesis was evidenced by both the upregulation of the tomato ribosomal stress marker SlNAC082 and the impairment in 18S rRNA processing, pointing out ribosomal stress as a novel signature of the pathogenesis of nuclear-replicating viroids. In conclusion, this updated binary vector has turned out to be an efficient and reproducible method that will facilitate the studies of viroid–host interactions., This work was supported by the Spanish Ministry of Economy and Competitiveness (co-supported by FEDER) Grants BIO2017-88321-R (V.P.) and PID2019-104126RB-I00 (G.G.).

[ES] Los consumidores son conscientes del peligro derivado del uso de antioxidantes y antimicrobianos sintéticos en la industria agroalimentaria, demandando alternativas más seguras y ecológicas. En este estudio, se ha determinado la actividad antioxidante de aceites esenciales comerciales mediante el método DPPH y su efecto antimicrobiano frente a la bacteria Pseudomonas syringae y el hongo fitopatógeno Fusarium oxysporum a través del empleo del método estandarizado de disco. Los aceites esenciales de clavo, ajedrea, canela y orégano, así como carvacrol, mostraron la máxima actividad antioxidante, comparable a antioxidantes establecidos. El aceite esencial de gaulteria fue el más potente inhibidor del crecimiento de P. syringae en las dosis más altas (20 y 10 µL) ensayadas. El aceite esencial de orégano, así como su componente principal carvacrol, detuvieron el crecimiento de la bacteria incluso a la dosis más baja ensayada (1 µL). Los aceites esenciales de canela, orégano y menta inhibieron el desarrollo de F. oxysporum en todas las dosis (20, 10 y 5 µL) aplicadas. En general, la mayoría de aceites esenciales mostraron más actividad antifúngica que antibacteriana y antioxidante., [EN] Consumers are aware of the dangers arising from the use of synthetic antioxidants and antimicrobials in the agrifood industry, demanding safer and "greener" alternatives. In this study, the antioxidant activity of commercial essential oils through DPPH method, their antimicrobial effects against the bacterium Pseudomonas syringae and the phytopathogenic fungus Fusarium oxysporum by means of the standardized disk method were determined. Clove along with winter savory, cinnamon and oregano essential oils as well as carvacrol showed the highest antioxidant activity comparable to reference standards. Wintergreen essential oil was the most potent inhibitor against P. syringae growth at the highest doses (20 and 10 µL). Oregano essential oil and its main component carvacrol were able to stop the bacterium growth even at the lowest treatment (1 µL). Cinnamon, oregano and peppermint essential oils inhibited F. oxysporum development at all doses (20, 10 and 5 µL) assayed. In general, most of the essential oils displayed more antifungal than antibacterial and antioxidant activities.

© 2020 by the authors., Citrus exocortis viroid (CEVd) is known to cause different symptoms in citrus trees, and its mechanism of infection has been studied in tomato as an experimental host, producing ribosomal stress on these plants. Some of the symptoms caused by CEVd in tomato plants resemble those produced by the phytohormone ethylene. The present study is focused on elucidating the relationship between CEVd infection and ethylene on disease development. To this purpose, the ethylene insensitive Never ripe (Nr) tomato mutants were infected with CEVd, and several aspects such as susceptibility to infection, defensive response, ethylene biosynthesis and ribosomal stress were studied. Phenotypic characterization revealed higher susceptibility to CEVd in these mutants, which correlated with higher expression levels of both defense and ethylene biosynthesis genes, as well as the ribosomal stress marker SlNAC082. In addition, Northern blotting revealed compromised ribosome biogenesis in all CEVd infected plants, particularly in Nr mutants. Our results indicate a higher ethylene biosynthesis in Nr mutants and suggest an important role of this phytohormone in disease development and ribosomal stress caused by viroid infection.

[EN] Background: Secondary metabolites play an important role in the plant defensive response. They are produced as a defence mechanism against biotic stress by providing plants with antimicrobial and antioxidant weapons. In higher plants, the majority of secondary metabolites accumulate as glycoconjugates. Glycosylation is one of the commonest modifications of secondary metabolites, and is carried out by enzymes called glycosyltransferases. Results: Here we provide evidence that the previously described tomato wound and pathogen-induced glycosyltransferase Twi1 displays in vitro activity toward the coumarins scopoletin, umbelliferone and esculetin, and the flavonoids quercetin and kaempferol, by uncovering a new role of this gene in plant glycosylation. To test its activity in vivo, Twi1-silenced transgenic tomato plants were generated and infected with Tomato spotted wilt virus. The Twi1- silenced plants showed a differential accumulation of Twi1 substrates and enhanced susceptibility to the virus. Conclusions: Biochemical in vitro assays and transgenic plants generation proved to be useful strategies to assign a role of tomato Twi1 in the plant defence response. Twi1 glycosyltransferase showed to regulate quercetin and kaempferol levels in tomato plants, affecting plant resistance to viral infection., This work was supported by grant BIO2012-33419 from the Direccion General de Programas y Transferencia de Conocimiento, Spanish Ministry of Science and Innovation, and grant AICO/2017/048 from the Valencian Local Government (Generalitat Valenciana, Spain). LC was supported by a predoctoral fellowship (ACIF/2010/231) from the Valencian Local Government (Generalitat Valenciana, Spain). None of the funding bodies was involved in the design of the study, the collection, analysis, and interpretation of the data, nor in the writing of the manuscript, which was made entirely by the authors.

[EN] Volatile organic compounds (VOCs) emitted by plants are secondary metabolites that mediate the plant interaction with pathogens and herbivores. These compounds may perform direct defensive functions, i. e., acting as antioxidant, antibacterial, or antifungal agents, or indirectly by signaling the activation of the plant's defensive responses. Using a non-targeted GC-MS metabolomics approach, we identified the profile of the VOCs associated with the differential immune response of the Rio Grande tomato leaves infected with either virulent or avirulent strains of Pseudomonas syringae DC3000 pv. tomato. The VOC profile of the tomato leaves infected with avirulent bacteria is characterized by esters of (Z)-3-hexenol with acetic, propionic, isobutyric or butyric acids, and several hydroxylated monoterpenes, e. g., linalool, a -terpineol, and 4-terpineol, which defines the profile of an immunized plant response. In contrast, the same tomato cultivar infected with the virulent bacteria strain produced a VOC profile characterized by monoterpenes and SA derivatives. Interestingly, the differential VOCs emission correlated statistically with the induction of the genes involved in their biosynthetic pathway. Our results extend plant defense system knowledge and suggest the possibility for generating plants engineered to over-produce these VOCs as a complementary strategy for resistance., This work was funded by Grant BIO2012-33419 from the Spanish Ministry of Economy and Competitiveness.

A new method is proposed for solving the glottal inverse filtering (GIF) problem. The goal of GIF is to separate an acoustical speech signal into two parts: the glottal airflow excitation and the vocal tract filter. To recover such information one has to deal with a blind deconvolution problem. This ill-posed inverse problem is solved under a deterministic setting, considering unknowns on both sides of the underlying operator equation. A stable reconstruction is obtained using a double regularization strategy, alternating between fixing either the glottal source signal or the vocal tract filter. This enables not only splitting the nonlinear and nonconvex problem into two linear and convex problems, but also allows the use of the best parameters and constraints to recover each variable at a time. This new technique, called alternating minimization glottal inverse filtering (AM-GIF), is compared with two other approaches: Markov chain Monte Carlo glottal inverse filtering (MCMC-GIF), and iterative adaptive inverse filtering (IAIF), using synthetic speech signals. The recent MCMC-GIF has good reconstruction quality but high computational cost. The state-of-the-art IAIF method is computationally fast but its accuracy deteriorates, particularly for speech signals of high fundamental frequency (F0). The results show the competitive performance of the new method: With high F0, the reconstruction quality is better than that of IAIF and close to MCMC-GIF while reducing the computational complexity by two orders of magnitude.

Quality of life is related to one of the basic human desires, which is to live well and feel good. The scope of this study was to evaluate the quality of life of psychoactive substance users and relatives, compared to non-users, analyzed by socioeconomic strata. A cross-sectional study with users of psychoactive substances, relatives, and other individuals who called the Information and Orientation Service regarding drug abuse. Data collection took place between November 2009 and December 2010. Data was collected from users, relatives, and non-users, including socioeconomic characteristics and data regarding substance consumption when appropriate. In addition to this the abbreviated version of the World Health Organization Quality of Life (WHOQOL-BREF) questionnaire was given to 347 individuals. Among the 138 users (70%) used alcohol, 76 (39%), marijuana, 111 (57%) tobacco, 78 (40%) cocaine and 70 (36%) crack. Control subjects had higher, scores than the relatives of users and users in all areas of the questionnaire (p < 0.05). Psychoactive substance users scored lower in almost all domains and overall score in the WHOQOL-BREF questionnaire in comparison with the sample of non-drug users. These findings reflect poor quality of life of patients and their relatives.

[EN] The importance of salicylic acid (SA) in the signal transduction pathway of plant disease resistance has been well documented in many incompatible plant-pathogen interactions, but less is known about signalling in compatible interactions. In this type of interaction, tomato plants have been found to accumulate high levels of 2,5-dihydroxybenzoic acid (gentisic acid, GA), a metabolic derivative of SA. Exogenous GA treatments induce in tomato plants a set of PR proteins that differ from those induced by salicylic acid. While SA accumulates in tomato plants mainly as 2-O-beta-D-glucoside, GA has only been found as 5-O-beta-D-xyloside. To characterize this step of the GA signalling pathway further, the present work focuses on the study of the GA-conjugating activity in tomato plants. A gentisate glycosyltransferase (GAGT) cDNA has been isolated and overexpressed in Pichia pastoris, and GA-conjugating activity was confirmed by detecting the xylosylated GA. The purified plant protein is highly specific for GA, showing no activity toward many other phenolic compounds, including SA. In addition, it shows an outstanding selectivity for UDP-xylose as the sugar donor, which differentiates this enzyme from most glycosyltransferases. Both the GA-conjugating activity and the corresponding mRNA show a strong, rapid, and transient induction upon treatment of tomato plants with GA or SA. Furthermore, its expression is rapidly induced by compatible infections. However, neither the gene nor the activity seems to respond to incompatible infections or wounding. The unique properties of this new glycosyltransferase suggest a specific role in regulating the free GA levels in compatible plant-pathogen interactions., This work has been supported by the Spanish Ministry of Science and Innovation (grant numbers BMC2003-07837 and BFU2006-11546).

[EN] In the present work, we have looked for the nature of the phenylpropanoids biosynthesized during the plant-pathogen reaction of two systems, Cucumis sativus and Cucumis melo infected with either prunus necrotic ringspot virus (PNRSV) or melon necrotic spot virus (MNSV), respectively. An accumulation of p-coumaric, caffeic and/or ferulic acids was observed in infected plant extracts hydrolysed with P-glucosidase or esterase. Analysis of undigested samples by HPLC/ESI revealed that these compounds are mainly forming esters with glucose: 1-O-coumaroyl-beta-glucose, 1-O-caffeoyl-beta-glucose, and 1-O-fer-uloyl-beta-glucose. Cinnamic acid 4-hydroxylase (C4H, EC 1.14.13.11), the second enzyme of the plant phenylpropanoid pathway, plays a pivotal role in the synthesis of these hydroxycinnamic acids. Thus, we have isolated and characterised a cDNA clone encoding this enzyme from PNRSV-infected cucumber, and a partial cDNA from MNSV-infected melon leaves. The deduced amino acid sequence revealed a notable degree of identity with homologous C4H enzymes from other plant species. In agreement with the induction of the phenylpropanoids presently described, it is reported that in cucumber and melon leaves, both viral infections studied induced C4H mRNA expression. A similar induction was observed for the first pherylpropanoid biosynthetic enzyme, phyenylalanine ammonia-lyase (PAL, EC 4.3.1.5), and for chitinase and peroxidase defence-related genes., We gratefully acknowledge Dr. Lynne Yenush for critical reading of the manuscript and helpful discussions, and L. Latorre, and L. Coracha´n for the technical support in the greenhouse. The authors want to acknowledge the detailed suggestions of the referees. This work was supported by Grant BMC2000-1136 from Comisio´n Interministerial de Ciencia y Tecnologı´a, Spanish Ministry of Science and Technology

[EN] We describe the efficacy of Bacillus subtilis strain IAB/BS03 in reducing disease incidence of B. subtilis IAB/BS03 as a foliar treatment against Botrytis cinerea and Pseudomonas syringae on greenhouse-grown tomato (Solanum lycopersicon) plants. We also tested the effect of foliar treatments on lettuce (Lactuca sativa) against lettuce downy mildew caused by Bremia lactucae in multiple trials under different field conditions. All the assays indicated that B. subtilis IAB/BS03 reduced disease. To ascertain the mechanism of action, the induction of pathogenesis-related (PR) proteins, the accumulation of salicylic acid and the activation of peroxidase caused by foliar or root treatments with B. subtilis IAB/BS03 were studied in tomato. A salicylic acid-independent induction of the antifungal protein PR1 was observed after treatment with B. subtilis IAB/BS03, with the strongest induction due to root treatment compared with foliar application. A metabolic analysis of B. subtilis IAB/BS03 culture broth using Ultra Performance Liquid Chromatography coupled with ultraviolet and mass spectrometric detection determined surfactin and iturin A isomers. These compounds have been described as antifungal and antibiotic lipopeptides. The results indicated that B. subtilis IAB/BS03 could be effectively used as a biocontrol agent., This work was funded by IAB S. L. (Investigaciones y Aplicaciones Biotecnologicas, S. L.), and by grant BIO2012-33419 from the Spanish Ministry of Economy and Competitiveness. Mayte Castellano was the recipient of a research grant also funded by IAB S. L. The authors would like to thank Cristina Torres (IBMCP, UPV-CSIC) for her excellent technical assistance.

[EN] Moringa oleifera seeds contain a cationic coagulant protein that can be used either for drinking water clarification or wastewater treatment. The seeds contain 30-40% (w/w) oil, which represents a valuable resource with industrial, food and renewable energy applications. However, the oil also increases the organic matter added to water and avoids storage of treated water and its consumption within 24h. So oil extraction is recommended as a way to purify the extract. This work proposes solvent extraction using three solvents (hexane, acetone, ethanol) and two procedures (batch and Soxhlet) and analyses for the first time the influence that these factors have on the coagulant efficiency of the extract prepared using defatted seeds. Results show that solvent-defatted seed extracts have a coagulant efficiency comparable to that observed for polyaluminium chloride coagulant (up to 88%), while non-defatted seed extract efficiency is 30% lower. Moreover, ethanol and Soxhlet method for oil extraction allows obtaining a defatted extract that requires doses of coagulant protein 5-33 times lower than hexane or acetone defatted extract to clarify turbid water, reducing in consequence the organic load added. SDS-PAGE analysis showed no influence of the solvent on the nature of the coagulant protein present in the extracts. The results of the study provide an oil extraction procedure that recovers M. oleifera oil for industrial purposes and enhances the defatted seeds obtaining an improved coagulant for drinking water treatment., The authors thank V. Conejero and A. Saurí from Instituto Mixto Universitario de Biología Molecular y Celular de Plantas (IBMCP UPV-CSIC), for their excellent technical assistance, and M.D. Climent, X. Climent and A. Sepulcre for their collaboration on this research. The authors wish to thank CCD-UPV for the financial support given to this research, through ADSI-DEO 2012 call and to Support Programme for Research and Development (PAID-06-12) from Universitat Politècnica de València

The total least squares (TLS) method is a successful approach for linear problems if both the right-hand side and the operator are contaminated by some noise. For ill-posed problems, a regularisation strategy has to be considered to stabilise the computed solution. Recently a double regularised TLS method was proposed within an infinite dimensional setup and it reconstructs both function and operator, reflected on the bilinear forms Our main focuses are on the design and the implementation of an algorithm with particular emphasis on alternating minimisation strategy, for solving not only the double regularised TLS problem, but a vast class of optimisation problems: on the minimisation of a bilinear functional of two variables.

Proteinaceous aspartic proteinase inhibitors are rare in nature and are described in only a few plant species. One of them corresponds to a family of cathepsin D inhibitors (CDIs) described in potato (Solanum tuberosum), involving up to 15 isoforms with a high sequence similarity. In this work, we describe a tomato (Solanum lycopersicum) wound-inducible protein called jasmonic-induced protein 21 (JIP21). Sequence analysis of its cDNA predicted a putative function as a CDI. The JIP21 gene, whose protein has been demonstrated to be glycosylated, is constitutively expressed in flowers, stem, and fruit, and is inducible to high levels by wounding and methyl jasmonate in leaves of tomato plants. The genomic sequence of JIP21 shows that the gene is intronless and reveals the presence of both a methyl jasmonate box (TGACT) and a G-box (CACGT) in the promoter. In contrast to the presumed role of JIP21 based on sequence analysis, a detailed biochemical characterization of the purified protein uncovers a different function as a strong chymotrypsin inhibitor, which questions the previously predicted inhibitory activity against aspartic proteinases. Moreover, Egyptian cotton worm (Spodoptera littoralis) larvae fed on transgenic tomato plants overexpressing JIP21 present an increase in mortality and a delay in growth when compared with larvae fed on wild-type plants. These larvae belong to the Lepidoptera family whose main digestive enzymes have been described as being Ser proteases. All these results support the notion that tomato JIP21 should be considered as a chymotrypsin inhibitor belonging to the Ser proteinase inhibitors rather than a CDI. Therefore, we propose to name this protein tomato chymotrypsin inhibitor 21 (TCI21).

In a previous report [Mol. Gen. Genet. 228 (1991) 281], carboxypeptidase inhibitor protein (CPI) mRNA was found to accumulate in leaves of wounded tomato plants, but CPI protein could not be detected. In contrast, we found that CPI protein does accumulate in tomato leaves in response to wounding, and also in response to treatment with either systemin, methyl jasmonate (MeJ), oligogalacturonic acid, or chitosan. Identification of CPI protein was confirmed by its inhibition of metallo-carboxypeptidase A (CPAase), which was used as an assay during purification of the inhibitor from leaves of MeJ-treated tomato plants. Amino acid sequence analysis and mass spectroscopic analyses of the pure protein confirmed its identity as CPI. The pure protein inhibited CPAase in a 1:1 stoichimetric interaction. Time course analyses of the induction of CPI mRNA in tomato leaves in response to wounding indicated that the gene is a member of the group of "late genes" that code for defensive proteins synthesized in leaves in response to herbivore attack.

[EN] Hydroxycinnamic acid amides (HCAA) are secondary metabolites involved in plant development and defense that have been widely reported throughout the plant kingdom. These phenolics show antioxidant, antiviral, antibacterial, and antifungal activities. Hydroxycinnamoyl-CoA: tyramine N-hydroxycinnamoyl transferase (THT) is the key enzyme in HCAA synthesis and is induced in response to pathogen infection, wounding, or elicitor treatments, preceding HCAA accumulation. We have engineered transgenic tomato plants overexpressing tomato THT. These plants displayed an enhanced THT gene expression in leaves as compared with wild type (WT) plants. Consequently, leaves of THT-over-expressing plants showed a higher constitutive accumulation of the amide coumaroyltyramine (CT). Similar results were found in flowers and fruits. Moreover, feruloyltyramine (FT) also accumulated in these tissues, being present at higher levels in transgenic plants. Accumulation of CT, FT and octopamine, and noradrenaline HCAA in response to Pseudomonas syringae pv. tomato infection was higher in transgenic plants than in the WT plants. Transgenic plants showed an enhanced resistance to the bacterial infection. In addition, this HCAA accumulation was accompanied by an increase in salicylic acid levels and pathogenesis-related gene induction. Taken together, these results suggest that HCAA may play an important role in the defense of tomato plants against P. syringae infection., The authors are grateful to C. Torres, A. Sauri, and T. Caballero for technical support. We also thank C. Gomez-Mena for her assistance in tomato flower sampling. This work was supported by Grant BIO2012-33419 from the Direccion General de Programas y Transferencia de Conocimiento, from the Spanish Ministry of Science and Innovation. L. Campos was the recipient of a predoctoral fellowship ACIF/2010/231 from Generalitat Valenciana (Spain). The work presented here was carried out in collaboration between all authors. J. M. Belles defined the research theme. L. Campos, L. Zacares and M. P. Lopez-Gresa carried out the laboratory experiments. P. Lison and I. Rodrigo contributed to the experimental design and the interpretation of the data. L. Campos drafted the article. M. P. Lopez-Gresa, P. Lison and I. Rodrigo participated in revising it critically for important intellectual content. J. M. Belles and V. Conejero gave the final approval of the version to be published. Each author has participated sufficiently in the work to take public responsibility for the content.

[EN] We have observed that treatments with salicylic acid (SA) or gentisic acid (GA) induced resistance to RNA pathogens such as ToMV and CEVd in tomato and Gynura auriantiaca, respectively. Accumulation of SA and GA has been found to occur in plants infected by these pathogens, thus pointing out a possible defence role of both molecules. To study the molecular basis of the observed induced resistance to RNA pathogens the induction of silencing-related genes by SA and GA was considered. For that purpose, we searched for tomato genes which were orthologous to those described in Arabidopsis thaliana, such as AtDCL1, AtDCL2, AtDCL4, AtRDR1, AtRDR2 and AtRDR6, and we tracked their induction in tomato along virus and viroid infections. We observed that CEVd significantly induced all these genes in tomato, with the exception of ToRDR6, being the induction of ToDCL4 the most outstanding. Regarding the ToMV asymptomatic infection, with the exception of ToRDR2, we observed a significant induction of all the indicated silencing-related genes, being ToDCL2 the most induced gene. Subsequently, we analyzed their transcriptional activation by SA and at the time when ToMV was inoculated on plants. ToDCL2, ToRDR1 and ToRDR2 were significantly induced by both SA and GA, whereas ToDCL1 was only induced by SA. Such an induction resulted more effective by SA treatment, which is in agreement with the stronger SAinduced resistance observed. Our results suggest that the observed delay in the RNA pathogen accumulation could be due to the pre-induction of RNA silencing-related genes by SA or GA. 2014 Elsevier Masson SAS. All rights reserved, The authors are grateful to Cristina Torres and Asuncion Sauri for technical support. This work was supported by Grant BFU2009-11958 from Direccion General de Programas y Transferencia de Conocimiento, from Spanish Ministry of Science and Innovation, and Grants PAID-06-08-3295 and SP20120576 from Universitat Politecnica de Valencia (UPV). Laura Campos was the recipient of a predoctoral fellowship ACIF/2010/231 from Generalitat Valenciana (Spain). Ma Pilar Lopez Gresa held a postdoctoral fellowship JAE-Doc_08_00402 from the Consejo Superior de Investigaciones Cientificas (Spain).

Phytophthora citrophthora is the most widely spread oomycete plant pathogen over all the citrus growing areas and represents one of the major causes of crop losses. Constitutive over-expression of genes encoding proteins involved in plant defence mechanisms to disease is one of the strategies proposed to increase plant tolerance to oomycete and fungal pathogens. P23 (PR-5), a 23-kDa pathogenesis-related protein similar to osmotins, is induced in tomato (Lycopersicon esculentum Mill. cv. Rutgers) plants when they are infected with citrus exocortis viroid, and its antifungal activity has been demonstrated in in vitro assays. We have successfully produced transgenic orange (Citrus sinensis L. Obs. cv. Pineapple) plants bearing a chimeric gene construct consisting of the cauliflower mosaic virus 35S promoter and the coding region of the tomato pathogenesis-related PR-5. Nine regenerated transgenic lines constitutively expressed the PR protein. They were challenged with Phytophthora citrophthora using a detached bark assay. A significant reduction in lesion development was consistently observed in one transgenic line in comparison to the control plants. This same line achieved plant survival rates higher than control plants when transgenic trees were inoculated with oomycete cultures. These results provide evidence for the in vivo activity of the tomato PR-5 protein against Phytophthora citrophthora, and suggest that this may be employed as a strategy aimed at engineering Phytophthora disease resistance in citrus.

Quality of life is related to one of the basic human desires, which is to live well and feel good. The scope of this study was to evaluate the quality of life of psychoactive substance users and relatives, compared to non-users, analyzed by socioeconomic strata. A cross-sectional study with users of psychoactive substances, relatives, and other individuals who called the Information and Orientation Service regarding drug abuse. Data collection took place between November 2009 and December 2010. Data was collected from users, relatives, and non-users, including socioeconomic characteristics and data regarding substance consumption when appropriate. In addition to this the abbreviated version of the World Health Organization Quality of Life (WHOQOL-BREF) questionnaire was given to 347 individuals. Among the 138 users (70%) used alcohol, 76 (39%), marijuana, 111 (57%) tobacco, 78 (40%) cocaine and 70 (36%) crack. Control subjects had higher, scores than the relatives of users and users in all areas of the questionnaire (p < 0.05). Psychoactive substance users scored lower in almost all domains and overall score in the WHOQOL-BREF questionnaire in comparison with the sample of non-drug users. These findings reflect poor quality of life of patients and their relatives. Qualidade de vida está relacionada a um dos desejos humanos básicos, que é viver bem e se sentir bem. O objetivo deste estudo foi avaliar a qualidade de vida dos usuários de substâncias psicoativas e familiares, em comparação aos não-usuários, analisados por estratos socioeconômicos. Foi realizado um estudo transversal com usuários de substâncias psicoativas, parentes e outras pessoas que chamaram o Serviço de Informação e Orientação sobre o abuso de drogas (VIVAVOZ). A coleta de dados ocorreu entre novembro de 2009 e dezembro de 2010. Foram coletados dados de usuários, parentes e não-usuários, incluindo as características socioeconômicas e dados sobre consumo de substâncias, quando apropriado, além disso, foi administrado o questionário WHOQOL-BREF. Responderam ao WHOQOL-Bref 347 pessoas, 138 (70%) usuários de álcool , 76 (39%) maconha, 111 (57%) tabaco, 78 (40%) de cocaína e 70 (36%) crack. Os controles tiveram escores maiores de qualidade de vida, que os familiares de usuários e usuários em todas as áreas do questionário (p < 0,05). Usuários de substâncias psicoativas tiveram escores mais baixos em quase todos os domínios e escore geral do WHOQOL-Bref em comparação com a amostra de não usuários de drogas. Estes resultados refletem baixa qualidade de vida dos pacientes e seus familiares.

Viroids are single-stranded, circular, noncoding RNAs that infect plants, causing devastating diseases. In this work, we employed 2D DIGE, followed by MS identification, to analyze the response of tomato plants infected by Citrus exocortis viroid (CEVd). Among the differentially expressed proteins detected, 45 were successfully identified and classified into different functional categories. Validation results by RT-PCR allowed us to classify the proteins into two expression groups. First group included genes with changes at the transcriptional level upon CEVd infection, such as an endochitinase, a β-glucanase, and pathogenesis-related proteins, PR10 and P69G. All these defense proteins were also induced by gentisic acid, a pathogen-induced signal in compatible interactions. The second group of proteins showed no changes at the transcriptional level and included several ribosomal proteins and translation factors, such as the elongation factors 1 and 2 and the translation initiation factor 5-alpha. These results were validated by 2D Western blot, and possible PTMs caused by CEVd infection were detected. Moreover, an interaction between eukaryotic elongation factor 1 and CEVd was observed by 2D Northwestern. The present study provides new protein-related information on the mechanisms of plant resistance to pathogens., We would like to thank the Proteomic Service of the IBMCP (Instituto de Biologia Molecular y Celular de Plantas, Valencia, Spain) for the technical assistance. We also thank Dr. Alejandro Ferrando (Instituto de Biologia Molecular y Celular de Plantas UPV-CSIC) for critical reading of the manuscript, discussions, and for kindly providing us with the eIF5A antisera. We are also grateful to Dr. Brenda Hunter (University of Arizona) for both eEF1A and eEF2 antiserum. This work was supported by Grant BFU2009-11958 from Direccion General de Programas y Transferencia de Conocimiento, from Spanish Ministry of Science and Innovation. Laura Campos was the recipient of a predoctoral fellowship ACIF/2010/231 from Generalitat Valenciana (Spain). M. Pilar Lopez-Gresa held a postdoctoral fellowship JAEDoc_08_ 00402 from the Consejo Superior de Investigaciones Cientificas (Spain).

[EN] 1H nuclear magnetic resonance (NMR)-based metabolomics has been applied to study the compatible interaction between tomato plants and Tomato Mosaic Virus (ToMV). A detailed time course of metabolic fingerprinting of ToMV-inoculated and non-inoculated systemically infected tomato leaves has provided a fundamental understanding of the metabolic state of the plant not only in response to ToMV infection, but also under various physiological conditions. By this analytical platform a total of 32 metabolites including amino/organic acids, sugars, phenylpropanoids, flavonoids and other miscellaneous compounds were detected. Using multivariate data analysis, we have identified a subset of metabolites induced during the plant defence response and metabolites whose accumulation was dependent on the developmental stage, the position of the leaf on the stem, and the harvesting time. Specifically, a general time-dependent decrease in organic acids, amino acids (excluding asparagine), phenylpropanoids and rutin was observed in individual leaves. In addition, metabolite alterations were also found to correlate with the developmental stage of the leaf: high levels of organic acids, some amino acids, phenylpropanoids, and flavonoids were found in lower leaves while elevated amounts of sugars were present in the upper ones. Moreover, a marked variation in the content of some metabolites was also observed to be associated to the asymptomatic ToMV infection both in inoculated and systemically infected leaves. While flavonoids accumulated in virus-inoculated leaves, increased levels of phenylpropanoids were observed in non-inoculated leaves where ToMV actively replicates. Finally, diurnal changes in the metabolite content were also observed: an increase of amino acids and organic acids (except glutamic acid) were observed in the samples collected in the morning, whereas sugars and secondary metabolite levels increased in the tomato leaves harvested in the evening, The authors are grateful to Cristina Torres Vidal for technical support and Dr. Lynne Yenush for critical reading of the manuscript and exciting intellectual discussions. This work was supported by Grant BFU 2009-11958 from Secretaria de Estado de Investigacion from Spanish Ministry of Science and Innovation. M.P.L.G held a postdoctoral fellowship JAEDoc 08 00402 from the Consejo Superior de Investigaciones Cientificas (Spain) and was recipient of a postdoctoral fellowship (BEST/2010/111) from Generalitat Valencia (Spain).

[EN] In the present work, we aimed to define the chemical nature of the phenylpropanoids induced during the interaction of the tomato plant. cultivar Rutgers, with the bacterial pathogen Pseudomonas syringae pv. tomato. Using LC-MS and NMR spectroscopy, we determined the chemical structures of this class of metabolites, which accumulated in response to exposure to the pathogen. A rapid accumulation of phytoalexin hydroxycinnamic acid amides (HCAA) of noradrenaline (cis/trans N-p-coumaroylnoradrenaline and cis/trans N-feruloylnoradrenline) and octopamine (cis/trans N-p-coumaroyloctopamine and cis/trans N-feruloyloctopamine), chlorogenic acid (CGA), and the flavonoid rutin was detected after bacterial infiltration. Among them, cis N-p-coumaroylnoradrenaline, cis N-p-coumaroyloctopamine, and isomers cis/trans of N-feruloylnoradrenaline have never been described in the literature. Moreover, cis/trans isomers of N-feruloyloctopamine are identified for the first time as being associated with the tomato/P. syringae interaction. Inhibition of the P. syringae-induced ethylene by 2-aminoethoxyvinylglycine, markedly impaired the accumulation of HCAA in inoculated tomato leaves, but it had no effect on CGA or rutin synthesis. On the other hand, the lack of accumulation of SA in NahG transgenic tomato plants, overexpressing a bacterial salicylic hydroxylase, did not prevent the enhancing of HCAA produced by P. syringae infection. Taken together, our results indicate that ethylene, but not SA, is essential for the synthesis of HCAA in response to bacterial infection of tomato leaves. Antibiotic activities of the induced metabolites were also studied. CGA induced a notable expression of the defence-related genes PR1 and P23. Moreover, trans-HCAA of noradrenaline and octopamine showed a potent free radical scavenging competence. In particular, trans N-feruloylnoradrenaline presented a very outstanding antioxidant activity, thus indicating that these compounds may play a role in the defence response of tomato plants against bacterial infection. (C) 2011 Elsevier B.V. All rights reserved., The authors want to thank Dr. Lynne Yenush for critical reading of the manuscript and exciting intellectual discussions. Dominique Melck, Estrella Mateos (ICB and ITQ NMR Service) for recording NMR spectra and Letizia Ciavatta for interpretation of the 600-MHz NMR spectra. This work was supported by Grant BFU2006-11546 from Direccion General de Programas y Transferencia de Conocimiento, from Spanish Ministry of Science and Innovation. M.P.L.G. was the recipient of a postdoctoral fellowship APOSTD/2007/128 from Generalitat Valencia (Spain).

Beltran Herrera Javier Dario, Pregrado, Biólogo(a)

No presente trabalho são apresentadas soluções analíticas das equações de difusão de nêutrons bidimensionais com dois grupos de energia, a saber, nêutrons rápidos e térmicos em uma placa com propriedades homogêneas. Alem disso, são resolvidos detalhadamente os problemas onde a placa homogênea é substituída por duas e quatro regiões, tornando-os não-homogêneos. A partir da aplicação da transformada de Laplace e da Técnica da Transformada Integral Generalizada (GITT), respectivamente, é resolvida em uma forma analítica o problema de autovalor resultante para o fluxo de nêutrons. No problema heterogêneo são usados filtros para homogenizar as condições de contorno não-homogêneas. Esta é a condição para a aplicação da GITT. Os três problemas mencionados acima são resolvidos aplicando primeiramente a GITT, o qual reduz a dimensão da equação de difusão, seguida da aplicação da transformada de Laplace, o qual reduz a ordem da equação. Deste procedimento, resulta um sistema de equações algébricas dependente das constantes de integração. 0 sistema é resolvido usando a técnica da eliminação de Gauss. Os fluxos transformados pela GITT são recuperados invertendo-se analiticamente a transformada de Laplace usando a expansão de Heaviside, os quais ainda dependem das constantes de integração. A partir da aplicação das condições de contorno e de interface (para os problemas não-homogêneos) obtém-se um sistema de equações algébricas homogêneas, de onde é determinado o fator de multiplicação efetivo Keff pelo método da bissecção. As constantes de integração são determinadas fazendo use da potencia prescrita da placa. Assim, os fluxos de nêutrons transformados pela GITT ficam determinados e os fluxos de nêutrons rápidos e térmicos são recuperados através da formula da inversa da GITT, usando a expansão do potencial. Resultados são comparados com a solução do método de diferenças finitas. In the present work we present analytical solutions of the bi-dimensional neutron diffusion equation with two energy groups, i.e. fast and thermal neutrons in a sheet with homogeneous properties. Further we solve the detailed problem where the homogeneous sheet is substituted by two and four regions, rendering the problem a non-homogeneous one. Upon application of the Laplace transform and Generalized Integral Transform Tecnique (GITT), respectively, we solve in an analytical fashion the resulting eigenvalue problem for the neutron flux. In the heterogeneous problem, we use filter functions in order to homogenize the non-homogeneous boundary conditions. This is a condition for the application of GITT. We solve the three problems mentioned above applying first GITT, which reduces the dimension of the diffusion equation followed by the Laplace transform, which reduces the order of the equation. This procedure yields a non-homogeneous algebraic system depending on integration constants. The system is solved using the elimination technique by Gauss. The transformed fluxes by GITT are recovered upon inverting analytically the Laplace transform using Heaviside's expansion which depend still on the integration constants. Upon application of the boundary and interface conditions (for the non-homogeneous problem) one obtains a system of homogeneous algebraic equations, where we determine the effective multiplication factor keff by the bisection method. The integration constants are determined making use of the predefined power of the sheet. Thus the neutron fluxes transformed by GITT are determined and the fast and thermal neutron flux are recovered by the inverse formula of GITT, using the potential expansion. Results are compared to the solution by the finite difference method.

P23, a 23 kDa pathogenesis-related (PR) protein, was purified from citrus exocortis viroid (CEVd)-infected tomato leaves. Partial amino acid sequencing of this protein including the N-terminal and nine additional tryptic fragments covering about 50% of its primary structure revealed extensive homologies to the members of the family of plant thaumatin-like proteins. Sequence alignment revealed that tomato P23 is the previously described NP24 protein found to be associated to osmotic stress in tomato. In view of this fact the possible role of pathogenesis-related P23 protein as a component of a general mechanism of response of the plant is discussed.

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Físicas e Matemáticas. Programa de Pós-graduação de Matemática e Computação Científica Uma técnica de regularização que vem ganhando destaque na comunidade de problemas inversos é a regularização de Tikhonov com termo de penalização dado pela seminorma de variação limitada. Esse método de regularização busca aproximar a solução "exata"' do problema por funções em BV, um espaço de Banach. O método de Tikhonov é largamente utilizado para problemas inversos formulados em espaços de Hilbert, situação para qual vários resultados teóricos são conhecidos. Esse método de regularização tem como característica fornecer soluções suaves, o que se torna uma desvantagem em certas aplicações em processamento de imagens, quando a imagem a ser reconstruída apresenta grandes gradientes ou quando é descontínua. Neste trabalho apresentamos um método tipo Tikhonov que visa obter soluções de problemas inversos mal-postos num contexto mais geral. Com essa generalização procuramos resultados teóricos para o tratamento de uma equação mal-posta em que o operador envolvido é definido entre espaços de Banach, além de utilizar uma penalização não diferenciável. Dessa maneira, o método investigado corresponde a uma generalização da teoria clássica de Tikhonov, a qual pode ser utilizada no espaço de funções de variação limitada. Após introduzirmos as condições necessárias para garantir a existência de uma solução para o problema regularizado, damos início ao estudo da qualidade das soluções obtidas por esse método. Exibimos resultados de estabilidade e taxas de convergência entre uma solução regularizada e uma solução "exata" do problema inverso. Tal análise de convergência é obtida com base na distância de Bregman. Operadores lineares e não lineares são considerados. Para problemas não lineares, investigamos também um método de Tikhonov iterado.

In the present work we have studied the accumulation of gentisic acid (2,5-dihydroxybenzoic acid, a metabolic derivative of salicylic acid, SA) in the plant-pathogen systems, Cucumis sativus and Gynura aurantiaca, infected with either prunus necrotic ringspot virus (PNRSV) or the exocortis viroid (CEVd), respectively. Both pathogens produced systemic infections and accumulated large amounts of the intermediary signal molecule gentisic acid as ascertained by electrospray ionization mass spectrometry (ESI-MS) coupled on line with high performance liquid chromatography (HPLC). The compound was found mostly in a conjugated (beta-glucoside) form. Gentisic acid has also been found to accumulate (although at lower levels) in cucumber inoculated with low doses of Pseudomonas syringae pv. tomato, producing a nonnecrotic reaction. In contrast, when cucumber was inoculated with high doses of this pathogen, a hypersensitive reaction occurred, but no gentisic-acid signal was induced. This is consistent with our results supporting the idea that gentisic-acid signaling may be restricted to nonnecrotizing reactions of the host plant (Belles et al. in Mol Plant-Microbe Interact 12:227-235, 1999). In cucumber and Gynura plants, the activity of gentisic acid as inducing signal was different to that of SA, thus confirming the data found for tomato. Exogenously supplied gentisic acid was able to induce peroxidase activity in both Gynura and cucumber plants in a similar way as SA or pathogens. However, gentisic-acid treatments strongly induced polyphenol oxidase activity in cucumber, whereas pathogen infection or SA treatment resulted in a lower induction of this enzyme. Nevertheless, gentisic acid did not induce other defensive proteins which are induced by SA in these plants. This indicates that gentisic acid could act as an additional signal to SA for the activation of plant defenses in cucumber and Gynura plants.