Cistatinas de cebada: proteínas de defensa contra artrópodos

Las cistatinas son un grupo de proteínas identificadas en animales, plantas y algunos microorganismos que inhiben cisteín proteasas C1A, de la familia de la papaína. Las cistatinas de plantas o fitocistatinas tienen un papel endógeno relacionado con el intercambio proteico y la muerte celular programada y se ha demostrado que actúan como proteínas de defensa frente a plagas y patógenos. En cebada, Hordeum vulgare L., se ha identificado la familia completa de cistatinas compuesta por trece miembros, HvCPI-1 a 13. Siete de ellos habían sido previamente caracterizados. En este trabajo se ha completado la caracterización de todos los miembros de esta familia de cistatinas y se ha evaluado su actividad frente a artrópodos plaga. Todas las cistatinas de cebada mantienen conservados el motivo LARFAV, exclusivo de vegetales, y los tres motivos implicados en la unión enzima-inhibidor, aunque en algunos casos con pequeñas variaciones. Además, se ha determinado que la cistatina HvCPI-4 posee una extensión carboxilo terminal con un motivo conservado implicado en la inhibición de cisteín proteasas C13 de la familia de legumaínas. Las estructuras tridimensionales de las cistatinas, se han modelizado a partir de la estructura cristalizada de la orizacistatina I de arroz. Los patrones de expresión obtenidos mediante RT-qPCR han indicado que las cistatinas se expresan principalmente en semilla aunque sus mensajeros también se acumulan en hojas y raíces. Además, se ha determinado la localización subcelular de las trece cistatinas, básicamente detectada en el retículo endoplásmico y el aparato de Golgi, aunque la cistatina HvCPI-4 aparece también en el núcleo y la HvCPI-1 en el núcleo y el citoplasma. Ensayos de inhibición in vitro frente a cisteín proteasas comerciales tipo catepsina B, H, L y legumaína han mostrado una capacidad inhibitoria diferencial y específica dentro de la familia de cistatinas de cebada. En relación con su función de defensa, se han estudiado los efectos de las 13 cistatinas sobre diferentes especies de artrópodos cuyos enzimas digestivos más abundantes son de tipo cisteín proteasa, dianas potenciales de las cistatinas. Concretamente, se han seleccionado: el escarabajo de la patata, Leptinotarsa decemlineata (Say) y su depredador natural, Podisus maculiventris (Say), la crisomela del maíz, Diabrotica virgifera (LeConte), el ácaro, Tetranychus urticae Koch y dos pulgones, Myzus persicae (Sulzer) y Acyrthosiphon pisum (Harris). Las proteasas digestivas del ácaro y de los pulgones no se conocían de forma detallada, por lo que se ha realizado una caracterización enzimática de estas especies. En todas ellas se ha detectado la presencia de catepsinas L y B y leucin aminopeptidasa pero además, en el caso del ácaro y del pulgón M. persicae se ha detectado también actividad catepsina D y en el caso del pulgón A. pisum se ha detectado actividad carboxipeptidasa. En ninguno de los casos se ha detectado actividad serín proteasa. Se ha determinado la capacidad inhibitoria in vitro de las 13 cistatinas frente a la actividad catepsina L y catepsina B presente en extractos proteicos de estos seis artrópodos, utilizando substratos específicos. Las cistatinas han mostrado diferente especificidad por la actividad de los artrópodos aunque, en general, todas son mejores inhibidores de su actividad tipo catepsina L. La cistatina HvCPI-6 ha resultado ser el inhibidor más potente y ha sido seleccionado para realizar ensayos in vivo con 3 de los artrópodos anteriormente mencionados: T. urticae, M. persicae y A. pisum. Para estudiar la toxicidad en pulgones, se han preparado dietas artificiales suplementadas con cistatina HvCPI-6 y se han analizado la mortalidad y las alteraciones en sus proteasas digestivas. En el caso de A. pisum la ingestión de la cistatina en altas concentraciones provoca la muerte del insecto, muerte que esta asociada a la inhibición de cisteín proteasas digestivas. Por el contrario, en M. persicae no se observa mortalidad a ninguna de las dosis ensayadas, lo que parece estar relacionada con la sobreproducción de proteasas digestivas. Además, se han generado plantas transgénicas de Arabidopsis thaliana (L.) Heyn, que expresan la cistatina HvCPI-6, para estudiar el efecto que produce sobre M. persicae. Pulgones alimentados con las plantas transgénicas han mostrado un retraso en el desarrollo aunque no se han observado alteraciones en la descendencia. Dado que A. pisum no se alimenta de plantas de arabidopsis, se han realizado bioensayos combinados dieta/plantas de haba para estudiar los efectos de la cistatina sobre el desarrollo y la descendencia de este pulgón. Igual que en el caso de M. persicae se ha observado un retraso en el desarrollo, pero no hay modificaciones en la descendencia. El objetivo final de la utilización de la cistatina como transgen es la obtención de variedades agrícolas más protegidas frente al ataque de plagas y enfermedades. Con este propósito se han generado plantas transgénicas de maíz, Zea mays L., que expresan la cistatina HvCPI-6. Mediante bioensayos se ha podido determinar que la expresión de la cistatina en plantas provoca un retraso en el desarrollo de los ácaros que se han alimentado de las plantas transgénicas, que se correlaciona con una disminución de la actividad tipo catepsina B. Los resultados anteriores, indican que las cistatinas de cebada y en particular la cistatina HvCPI-6, suponen una vía alternativa para el control de plagas y que un estudio exhaustivo de las mismas permitiría considerar la expresión piramidal (“gene pyramiding”) de los genes que codifican estos inhibidores junto con otros genes con carácter insecticida, para aumentar la resistencia de las plantas al ataque de artrópodos y evitar la adaptación de los mismos. SUMMARY Cystatins are a group of proteins identified in animals, plants and microorganisms that inhibit cysteine proteases C1A from the papain family. Plant cystatins or phytocystatins have an endogenous function related with the protein turnover and the programmed cell death and it has been demonstrated that they have a defence role against pests ad pathogens. In barley, Hordeum vulgare L., it has been identified the whole family of cystatins which includes 13 members, HvCPI-1 to 13. Seven of these members had been previously characterized. In this work, the characterization of all the members of this family has been completed and it has been also evaluated their activity against arthropods. All barley cystatins have the motif LARFAV, specific from plants, and three conserved motifs involved in the enzyme-inhibitor interaction, although in some cases show little variations. It has been also determined that the cystatin HvCPI-4 has a carboxy terminal extensión, with a conserved motif involved in the inhibition of the cysteine proteases C13 of the legumain family. The tridimensional structures of the thirteen cystatins have been modelized from the crystalized structure of the oryzacystatin I from rice. Expression patterns obtained from RT-qPCR have indicated that barley cystatins are expressed mainly on seeds, although their messengers are also acumulated in leaves and roots. Moreover, the subcellular localization of the thirteen cystatins has been analysed. Basically, they have been detected in the endoplasmatic reticulum and Golgi apparatus, with the exception of the cystatin HvCPI-4 which appears also in the nucleus and the HvCPI-1 cystatin which has been localized in the nucleus and the cytoplasm. In vitro inhibition assays against commercial cysteine proteases: cathepsin B-, L-, H-like and legumain have demonstrated a differencial and specific inhibitory capacity within the family of barley cystatins. The defence function has been studied analysing the effects of the 13 cystatins against different species of arthropods which their digestive enzymes are mainly cysteine proteases. It has been selected: the Colorado potato beetle Leptinotarsa decemlineata (Say) and its natural predator Podisus maculiventris (Say), the western corn rootworm Diabrotica virgifera (LeConte), the two-spotted spider mite Tetranychus urticae Koch and two aphids, Myzus persicae (Sulzer) and Acyrthosiphon pisum (Harris). It has been determined in detail the enzymatic characterization of the mite and the two aphids unknown up to know. In all of them, the presence of cathepsin L- and Blike and leucine aminopeptidase activities has been detected. Moreover, cathepsin D activity was also found in the mite and in the aphid M. persicae and carboxipeptidase activity in the aphid A. pisum. Serine proteases have not been detected in any of them. The in vitro inhibitory capacity of the 13 cystatins has been determined against protein extracts from the six arthropods. Cystatins have resulted better inhibitors of cathepsin L-like activity than B-like activity. The cystatin HvCPI-6 has been the strongest inhibitor of protein extracts derived from all the arthropods tested, and it has been selected to do in vivo assays with T. urticae, M. persicae y A. pisum. To study the effects on both aphids, artificial diets supplemented with the HvCPI-6 protein have been prepared. In the case of A. pisum, the ingestion of the cystatin at high concentration has provoked the death of the insect. This death can be associated to the inhibition of its cysteine proteases. On the contrary, it has not been observed any mortality of M. persicae fed with the HvCPI-6 diet. Transgenic plants of Arabidopsis thaliana (L.) Heyn expressing the HvCPI-6 cystatin have been generated to study their effects on M. persicae. Aphids fed on transgenic plants suffered a delay on their development but no alterations in their fecundity were observed. Since A. pisum does not fed on arabidopsis, combined assays using artificial diet and bean plants have been done to study the effects of the cystatin HvCPI-6 on this aphid. A delay in its development was observed but no alterations in its fecundity were detected. The final aim of the use of the cystatin as a transgen is to obtain crop varieties more protected against pests and pathogens. With this purpose transgenic plants of maize, Zea mays L., expressing the HvCPI-6 cystatin were developed to perform bioassays with mites. Again, a delay on the development of mites fed on the transgenic plants was detected. This delay has been related with a reduction in the cathepsin B-like activity. All these results indicate that barley cystatins, and particularly the HvCPI-6 cystatin, suppose and alternative for pest control. A gene pyramiding approach using cystatins together with other insecticidal genes could be considered to overcome arthropod adaptation and to get pest resistant plants

Differential in vitro and in vivo effect of barley cysteine and serine protease inhibitors on phytopathogenic microorganisms

Protease inhibitors from plants have been involved in defence mechanisms against pests and pathogens. Phytocystatins and trypsin/α-amylase inhibitors are two of the best characterized protease inhibitor families in plants. In barley, thirteen cystatins (HvCPI-1 to 13) and the BTI-CMe trypsin inhibitor have been previously studied. Their capacity to inhibit pest digestive proteases, and the negative in vivo effect caused by plants expressing these inhibitors on pests support the defence function of these proteins. Barley cystatins are also able to inhibit in vitro fungal growth. However, the antifungal effect of these inhibitors in vivo had not been previously tested. Moreover, their in vitro and in vivo effect on plant pathogenous bacteria is still unknown. In order to obtain new insights on this feature, in vitro assays were made against different bacterial and fungal pathogens of plants using the trypsin inhibitor BTI-CMe and the thirteen barley cystatins. Most barley cystatins and the BTI-CMe inhibitor were able to inhibit mycelial growth but no bacterial growth. Transgenic Arabidopsis plants independently expressing the BTI-CMe inhibitor and the cystatin HvCPI-6 were tested against the same bacterial and fungal pathogens. Neither the HvCPI-6 expressing transgenic plants nor the BTI-CMe ones were more resistant to plant pathogen fungi and bacteria than control Arabidopsis plants. The differences observed between the in vitro and in planta assays against phytopathogenic fungi are discusse

Service-learning educational approach for undergraduate students: development of an outreach workshop for high school students

In the last decade, research institutes and universities have strengthened the development of outreach activities in the biomedical field, involving researchers and professors as well as graduate students, but with little or no implication of undergraduate students. However, the development of this type of activities, using the Service-Learning educational approach, could be a valuable tool that would manage the acquisition of learning competencies by undergraduate students of Health Science Degrees and would put science at the service of society. In this project, we present the development of the workshop entitled “Exploring the human body”, in which 205 students in their first and second year of a Degree in Nursing or Medicine (University of Málaga, Spain) acted as mentors of 753 high school students (15 to 16 years old) in several school years (since 2016-2017, excluding 2019-2020 and 2020-2021 due to the COVID-19 pandemic). The workshop consisted of five work stations. Each station featured a set of different experiments and activities that were designed to teach the multiple levels by which the human body, and particularly the nervous system, can be studied: biomolecules, cells, tissues, organs and systems. Both high school and undergraduate students gave an evaluation of the workshop via questionnaires (Likert scale-based and short-answer questions) and a debriefing with the university professors. Data showed an overall score of 4.6 out of 5 points for the workshop by both high school and undergraduate students. In addition, undergraduate students pointed out that their participation had a positive impact on their academic background (4.8 out of 5 points), mainly due to the improvement of their oral communication skills (78 students) and self-confidence (58 students).Universidad de Málaga. Servicio de Publicaciones y Divulgación Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

YUCCA8 and YUCCA9 overexpression reveals a link between auxin signaling and lignification through the induction of ethylene biosynthesis.

Auxin is associated with the regulation of virtually every aspect of plant growth and development. Many previous genetic and biochemical studies revealed that, among the proposed routes for the production of auxin, the so-called indole-3-pyruvic acid (IPA) pathway is the main source for indole-3-acetic acid (IAA) in plants. The IPA pathway involves the action of 2 classes of enzymes, tryptophan-pyruvate aminotransferases (TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS 1(TAA1)/TRYPTOPHAN AMINOTRANSFERASE RELATED (TAR)) and flavin monooxygenases (YUCCA). Both enzyme classes appear to be encoded by small gene families in Arabidopsis consisting of 5 and 11 members, respectively. We recently showed that it is possible to induce transcript accumulation of 2 YUCCA genes, YUC8 and YUC9, by methyl jasmonate treatment. Both gene products were demonstrated to contribute to auxin biosynthesis in planta.1 Here we report that the overexpression of YUC8 as well as YUC9 led to strong lignification of plant aerial tissues. Furthermore, new evidence indicates that this abnormally strong secondary growth is linked to increased levels of ethylene production

Identification of Solanum pimpinellifolium genome regions for increased resilience to nitrogen deficiency in cultivated tomato

14 Pág.High-quality crop production with minimal fertilizer inputs is a key goal for the agriculture of the future. Globally, tomato is one of the most important vegetable crops and its intensive production and breeding has been based on the application of large quantities of nitrogen (N) fertilizers. Therefore, the development of N use efficient (NUE) cultivars with low N inputs needs to be addressed. Some variability in plant growth, fruit quality and NUE traits among tomato (Solanum lycopersicum L.) varieties under low N supply has been reported, however, the relevance of wild relatives of tomato has not yet been assessed. In this study, we found that S. pimpinellifolium accession To-937 (SP) may be a suitable resource to increase NUE in tomato. We studied a set of 29 introgression lines (IL) from SP into the Moneymaker cultivar (MM) in different seasons to investigate the potential of SP introgressions to maintain the tomato plant performance during the growth cycle under low N input in greenhouse conditions. We identified specific regions in the SP genome, on chromosomes 1, 3 and 10, involved in the responses to N inputs of fruit production and fruit quality. Notably, the line SP_10-4 maintained vegetative biomass and fruit yield production under limiting N supply. The introgressed region contained putative candidate genes as sucrose phosphate phosphatase (SPP), invertases (INV) and glutamine synthase 1 (GS1) genes, implicated in C and N metabolism. Genomic and expression analyses revealed differences in coding and non-coding sequences as well as in mRNA levels in SP_10-4, suggesting that these genes might well contribute to the reported biomass responses to N. Additionally, line SP_1-4 showed stable fruit amino acid contents under both sufficient and limiting N supplies, indicating that assimilated N partitioning to the fruit is maintained in response to N. Altogether, our results confirmed the suitability of SP as a source of NUE related traits and the interest in the studied ILs for developing new tomato cultivars with improved NUE under sustainable fertilization conditions.This work was supported by the Ministerio de Ciencia e Innovación (MCIN) and the Agencia Estatal de Investigación (grants RTA2015-00014-c02-00 to SGN and JM, PID2020-114165RR-C21 to JM, and PID2022-136541OB-I00 to SGN), the Agroalnext program (MCIN with funding of NextGenEU-PRTR-C17.I1 Generalitat Valenciana AGROALNEXT/2022/056 to SGN), Vicerrectorado de Investigación de la Universitat Politècnica de València (PAID-11-21 to SGN; PAID-10-20 and PAID-PD-22 to RM), Ministerio de Ciencia e Innovación (TED2021-129296B-I00 to SGN) and European Commission H2020 research and innovation program through HARNESSTOM grant agreement no. 101000716 (to AG) and RoxyCOST CA18210 for networking activities (to AG). We also want to acknowledge the "Severo Ochoa Program for Centers of Excellence in R&D" (CEX2020-000999-S) supported by MCIN/AEI/10.13039/501100011033. We thank Mike Bennett for revising the English language and Javier Forment from the IBMCP Bioinformatics service for support on genomic analyses.With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2020‐000999‐S)Peer reviewe

Universidad y sociedad: comunicación e integración en empresas e instituciones públicas y organizaciones no lucrativas. Nuevos avances

Depto. de Teorías y Análisis de la ComunicaciónFac. de Ciencias de la InformaciónFALSEsubmitte

Adverse maternal, fetal, and newborn outcomes among pregnant women with SARS-CoV-2 infection: an individual participant data meta-analysis

Introduction Despite a growing body of research on the risks of SARS-CoV-2 infection during pregnancy, there is continued controversy given heterogeneity in the quality and design of published studies. Methods We screened ongoing studies in our sequential, prospective meta-analysis. We pooled individual participant data to estimate the absolute and relative risk (RR) of adverse outcomes among pregnant women with SARS-CoV-2 infection, compared with confirmed negative pregnancies. We evaluated the risk of bias using a modified Newcastle-Ottawa Scale. Results We screened 137 studies and included 12 studies in 12 countries involving 13 136 pregnant women. Pregnant women with SARS-CoV-2 infection—as compared with uninfected pregnant women—were at significantly increased risk of maternal mortality (10 studies; n=1490; RR 7.68, 95% CI 1.70 to 34.61); admission to intensive care unit (8 studies; n=6660; RR 3.81, 95% CI 2.03 to 7.17); receiving mechanical ventilation (7 studies; n=4887; RR 15.23, 95% CI 4.32 to 53.71); receiving any critical care (7 studies; n=4735; RR 5.48, 95% CI 2.57 to 11.72); and being diagnosed with pneumonia (6 studies; n=4573; RR 23.46, 95% CI 3.03 to 181.39) and thromboembolic disease (8 studies; n=5146; RR 5.50, 95% CI 1.12 to 27.12). Neonates born to women with SARS-CoV-2 infection were more likely to be admitted to a neonatal care unit after birth (7 studies; n=7637; RR 1.86, 95% CI 1.12 to 3.08); be born preterm (7 studies; n=6233; RR 1.71, 95% CI 1.28 to 2.29) or moderately preterm (7 studies; n=6071; RR 2.92, 95% CI 1.88 to 4.54); and to be born low birth weight (12 studies; n=11 930; RR 1.19, 95% CI 1.02 to 1.40). Infection was not linked to stillbirth. Studies were generally at low or moderate risk of bias. Conclusions This analysis indicates that SARS-CoV-2 infection at any time during pregnancy increases the risk of maternal death, severe maternal morbidities and neonatal morbidity, but not stillbirth or intrauterine growth restriction. As more data become available, we will update these findings per the published protocol

Adverse maternal, fetal, and newborn outcomes among pregnant women with SARS-CoV-2 infection: an individual participant data meta-analysis.

INTRODUCTION Despite a growing body of research on the risks of SARS-CoV-2 infection during pregnancy, there is continued controversy given heterogeneity in the quality and design of published studies. METHODS We screened ongoing studies in our sequential, prospective meta-analysis. We pooled individual participant data to estimate the absolute and relative risk (RR) of adverse outcomes among pregnant women with SARS-CoV-2 infection, compared with confirmed negative pregnancies. We evaluated the risk of bias using a modified Newcastle-Ottawa Scale. RESULTS We screened 137 studies and included 12 studies in 12 countries involving 13 136 pregnant women.Pregnant women with SARS-CoV-2 infection-as compared with uninfected pregnant women-were at significantly increased risk of maternal mortality (10 studies; n=1490; RR 7.68, 95% CI 1.70 to 34.61); admission to intensive care unit (8 studies; n=6660; RR 3.81, 95% CI 2.03 to 7.17); receiving mechanical ventilation (7 studies; n=4887; RR 15.23, 95% CI 4.32 to 53.71); receiving any critical care (7 studies; n=4735; RR 5.48, 95% CI 2.57 to 11.72); and being diagnosed with pneumonia (6 studies; n=4573; RR 23.46, 95% CI 3.03 to 181.39) and thromboembolic disease (8 studies; n=5146; RR 5.50, 95% CI 1.12 to 27.12).Neonates born to women with SARS-CoV-2 infection were more likely to be admitted to a neonatal care unit after birth (7 studies; n=7637; RR 1.86, 95% CI 1.12 to 3.08); be born preterm (7 studies; n=6233; RR 1.71, 95% CI 1.28 to 2.29) or moderately preterm (7 studies; n=6071; RR 2.92, 95% CI 1.88 to 4.54); and to be born low birth weight (12 studies; n=11 930; RR 1.19, 95% CI 1.02 to 1.40). Infection was not linked to stillbirth. Studies were generally at low or moderate risk of bias. CONCLUSIONS This analysis indicates that SARS-CoV-2 infection at any time during pregnancy increases the risk of maternal death, severe maternal morbidities and neonatal morbidity, but not stillbirth or intrauterine growth restriction. As more data become available, we will update these findings per the published protocol

The Caldera. No. 25 Special Edition 2022. 70 years

Especial: Instituto Caldas, 70 años de historia En las siguientes páginas, los estudiantes rinden homenaje a nuestra querida Institución, a través, especialmente, de la Palabra. Mi eterna gratitud a cada uno de ellos, jóvenes lectores, escritores, dibujantes pero, sobre todo, Caldistas de CORAZÓN: Laura Camila Herrera, Victoria Chacón Gil, María Paula Méndez, Steven Gómez Guerrero, Constanza Narváez Solano, Yatana Zea Arenas, Carolina Valencia Neira, Valery Samara Meza, Steffy Picón Carrillo y Gabriela Portilla Castellanos.Especial 70 años. Instituto Caldas 6 Reloj Solar 2021 43 IX Concurso de Lectura en Voz Alta 46 La Cuna de Excélsior 48 Equipo de Maestros 51 Perfiles Caldistas 54 Expresiones Caldistas 58 Día Caldista 98 Galería de Imágenes 103Special: Instituto Caldas, 70 years of history In the following pages, the students pay homage to our beloved Institution, especially through the Word. My eternal gratitude to each of them, young readers, writers, cartoonists but, above all, Caldistas of HEART: Laura Camila Herrera, Victoria Chacón Gil, María Paula Méndez, Steven Gómez Guerrero, Constanza Narváez Solano, Yatana Zea Arenas, Carolina Valencia Neira, Valery Samara Meza, Steffy Picón Carrillo and Gabriela Portilla Castellanos