Caracterización molecular del mutante riso 1508 de cebada : modificaciones transcripcionales y posttranscripcionales en el desarrollo de la semilla

La semilla es el principal órgano reproductivo de las plantas espermatofitas, permitiendo la dispersión de las poblaciones y asegurando su supervivencia gracias a su tolerancia a la desecación y a su capacidad para germinar bajo condiciones ambientales óptimas. El rendimiento y valor económico de los cereales, que constituyen la primera cosecha mundial, depende, en buena medida, de la eficacia con que se acumulan en la semilla sustancias de reserva: proteínas, carbohidratos y lípidos. El principal carbohidrato acumulado en la semilla de cebada es el almidón y la fracción mayoritaria de proteínas es la de las prolaminas (solubles en etanol al 70%); estas proteínas tienen muy bajo contenido en lisina, un aminoácido esencial en la dieta de animales monogástricos. Con el fin de mejorar el valor nutricional de la semilla de cebada, se han obtenido diferentes mutantes con un mayor contenido en este aminoácido. Riso 1508 es un mutante de cebada rico en lisina cuya mutación lys3a, de efectos pleiotrópicos, segrega como un único gen mendeliano. Entre otros, presenta una reducción drástica de la expresión de algunos genes que codifican proteínas de reserva de tipo prolamina, en concreto, presenta reducida la expresión de los genes que codifican B-, C- y ϒ-Hordeínas y del inhibidor de tripsina CMe, pero no tiene alterada la expresión del gen que codifica las D-Hordeínas. Este último gen carece en su promotor del motivo GLM (5’‐(G/A)TGA(G/C)TCA(T/C)‐3’), que es reconocido por factores transcripcionales bZIP. En este trabajo, el mutante de cebada Riso 1508 se ha utilizado como herramienta para profundizar en el conocimiento de la regulación génica en semillas durante las fases de la maduración y la germinación. Para ello, en una primera aproximación, se llevó a cabo un análisis transcriptómico comparando el genotipo mutante con el silvestre durante la maduración de la semilla. Además de confirmar variaciones en los genes que codifican proteínas de reserva, este análisis indicó que también estaban afectados los genes relacionados con metabolismo de carbohidratos. Por ello se decidió caracterizar la familia multigénica de sacarosas sintasa (SUSy) en cebada. Se anotaron dos nuevos genes, HvSs3 y HvSs4, cuya expresión se comparó con la de los genes HvSs1 y HvSs2, previamente descritos en el laboratorio. La expresión de los cuatro genes en tejidos diferentes y su respuesta a estreses abióticos se analizó mediante RT-qPCR. HvSs1 y HvSs2 se expresaron preferencialmente durante el desarrollo del endospermo, y HvSs1 también fue un tránscrito abundante durante la germinación. HvSs1 se indujo en hojas en condiciones de anoxia y HvSs3 por estrés hídrico, y ambos genes se indujeron por tratamientos de frío. La localización subcelular de las cuatro isoformas no fue sólo citoplásmica, sino que también se localizaron en zonas próximas a retículo endoplásmico y en la cara interna de la membrana plasmática; además, se observó una co-localización de HvSS1 con el marcador de mitocondrias. Estos datos sugieren un papel distinto aunque parcialmente solapante de las cuatro Sacarosa Sintasas de cebada, descritas hasta la fecha. Las cinéticas de expresión de los genes que codifican los TFs más importantes implicados en la regulación génica durante el desarrollo del endospermo de cebada, se analizaron por RT-qPCR en ambos genotipos, demostrando que los TFs de la clase DOF aparecieron desregulados durante todo el proceso en Riso 1508 comparado con el cv. Bomi, aunque también se observaron diferencias significativas en algunos de los que codifican bZIPs. Estudios previos indicaban que el ortólogo de BLZ2 en maíz, O2, se regula post-traduccionalmente mediante un mecanismo de fosforilación/defosforilación reversible, y que la forma defosforilada es la fisiológicamente activa. En este trabajo se demostró que BLZ2 está sujeto a este tipo de regulación y que la proteín-fosfatasa HvPP2C2 está implicada en el proceso. La interacción de HvPP2C2 y BLZ2 tiene lugar en el núcleo celular únicamente en presencia de 100 μM ABA. En el mutante Riso 1508, BLZ2 se encuentra en un estado hiperfosforilado tanto durante la maduración como durante la germinación de la semilla, lo que dificultaría la unión de BLZ2 a las secuencias GLM en los promotores de los genes que codifican B-, C-,y ϒ- Hordeínas y CMe. Summary The seed is the main reproductive organ of spermatophyte plants allowing the spread of populations and ensuring their survival through its desiccation tolerance and because of their ability to germinate under optimum environmental conditions. Yield and economic value of cereal crops, that constitute the first world crop, depend largely on the efficiency with which they accumulate in the seed reserve substances: proteins, carbohydrates and lipids. The main carbohydrate accumulated in the barley seed is starch and the major protein fraction is that of prolamins (soluble in 70% ethanol); these proteins have a very low lysine content, an essential amino-acid for the diet of monogastric animals. In order to improve the nutritional value of the barley seed, different mutants have been obtained with a higher content of this amino-acid. Riso 1508 is one lysine-rich mutant whose mutation (lys3a) segregates as a single Mendelian gene with pleiotropic effects, such as a drastic reduction of genes encoding the trypsin inhibitor CMe and the B-, C-and ϒ-hordeins, but has not altered the expression of the gene encoding the D-hordeins. This latter gene lacks in its promotor the GLM motif (5’‐(G/A)TGA(G/C)TCA(T/C)‐3’), that is recognised by bZIP transcription factors In this work we have used the barley mutant Riso 1508 as a tool for better understanding gene regulation in seeds during the maturation and germination phases. To this aim, a transcriptomic analysis was performed comparing wild and mutant genotypes during seed maturation. Besides confirming variations in the expression of genes encoding reserve proteins, this analysis indicated that some genes related with carbohydrate metabolism were also affected. It was therefore decided to characterize the multigene family of sucrose synthases (SUSy) in barley. Two new genes were annotated, HvSs3 and HvSs4, and its expression was compared with that of genes HvSs1 and HvSs2, previously described in our laboratory. The expression of the four genes in different tissues and in response to abiotic stresses was analyzed by RTqPCR. HvSs1 and HvSs2 were preferentially expressed during the development of the endosperm, and the HvSs1 transcript was also abundant upon germination. HvSs1 was induced in leaves by anoxic conditions, HvSs3 by water stress, and both genes were induced by cold treatments. The subcellular localization of all four isoforms was not only cytoplasmic, but they could be found along the endoplasmic reticulum and at the inner side of the cell membrane; HvSS1, was also associated with the mitochondrial marker. These data suggest a distinct but partially overlapping roles for the barley sucrose synthases, described so far. The expression kinetics of the genes encoding the most important TFs involved in gene regulation during barley endosperm development was analyzed by RT-qPCR in both genotypes. These data show that the genes encoding DOF TFs were mis-regulated throughout the process in Riso 1508, although significant differences were also found among some of those encoding bZIPs. Previous studies indicated that the BLZ2 orthologue in maize, O2, was post-translationally regulated by reversible phosphorylation/dephosphorylation and that the dephosphorylated protein is the physiologically active form. In this work we demostrate that BLZ2 is under a similar regulation and that the proteinphosphatase HvPP2C2 is implicated in the process. The interaction between HvPP2C2 and BLZ2 takes place in the cell nucleus only in the presence of 100 μM ABA. In the Riso 1508 mutant, BLZ2 is found in a hyperphosphorylated state in the maturation phase and upon seed germination; because of this, the BLZ2 binding to the GLM promoter sequences of genes encoding B-, C- y ϒ- Hordeins and CMe would be decreased in the mutant

Actuaciones administrativas en el marco del derecho administrativo global. Estudio de casos.

El Derecho administrativo global ha surgido en nuestro medio como una posición jurídica que permite entender las distintas formas de administración global y los impactos generados por estas. Desde la óptica de construcción del mismo, planteamos una lectura paralela de la teoría clásica del surgimiento del Derecho administrativo y la olvidada teoría de la Administración Pública, como punto de partida, ya que no es posible asumir el estudio de esta amplia y difusa disciplina jurídica, desconociendo la esencia real de su existencia, ahora ampliada al espacio supraestatal. Es en este momento cuando debemos preferir la integralidad del mismo, incluyendo una novedosa y necesaria lectura, producto de las dinámicas que plantean actores externos a los propios estados, lo que permite la inserción de una visión más allá de lo nacional recogida en el Global Administrative Law –GAL-, el que está llamado a responder a varias de las preguntas actuales sobre la legitimidad o legalidad de un sin número de actuaciones o acciones que se presentan por parte de las instituciones, organizaciones o entidades de múltiple naturaleza jurídica, dentro de un esquema conceptual fundado en la gobernanza global, cuyo campo de acción lo encuentra en un único espacio administrativo-jurídico global. Así las cosas, tenemos que situar la aparición del GAL, analizar sus fundamentos, conceptos y principios con las teorías norteamericanas e italianas, sin dejar de lado las apreciaciones que emiten algunos pensadores y doctrinantes al respecto. Por último, escogimos dos casos de transcendencia local y global, sobre los cuales vertimos las teorías estudiadas para, en uno de ellos arribar a una conclusión diferente a la conocida en nuestro medio, y con el otro, elaborar un esquema novedoso de análisis desde la interpretación global de actuación. Finalmente comprendemos que esta postura, aún en construcción, debe nutrirse a partir de la interdisciplinariedad académica para ser llevada a la práctica del ejercicio del DerechoThe Global Administrative Law has emerged in our environment as a legal position that allows us to understand the different forms of global administration and the impacts generated by them. From the construction perspective of the same, we propose a parallel reading of the classical theory of the emergence of administrative law and the forgotten theory of public administration, as a starting point, which is impossible to examine this information and diffuse, ignoring the essence real of its existence, now extended to the supra-state space. It is at this moment when we should prefer the integrality of the same, including a novel and reading test, the product of the dynamics posed by actors external to the states themselves, which allows the insertion of a vision beyond the national recovery in the administrative world Law -GAL-, which is called to answer several questions about the legitimacy or legality of a number of actions or actions that are presented by institutions, organizations or entities of multiple legal nature, within a well-founded conceptual scheme in global governance, whose field of action is found in a single global administrative-legal space. So, we have to situate the appearance of the LAG, analyze its foundations, concepts and principles with the American and Italian theories, without neglecting the assessments made by some thinkers and doctrinants about it. Finally we chose two cases of local and global transcendence, on which the vertices studied the theories, in one of them to arrive at a conclusion different from the one known in our environment, and with the other, to elaborate a novel scheme of analysis of the global issue of action. Finally, we understand that this position, even in construction, must be nourished by academic interdisciplinarity in order to be taken to the practice of law practic

Multidrug Efflux Pumps at the Crossroad between Antibiotic Resistance and Bacterial Virulence

Multidrug efflux pumps can be involved in bacterial resistance to antibiotics at different levels. Some efflux pumps are constitutively expressed at low levels and contribute to intrinsic resistance. In addition, their overexpression may allow higher levels of resistance. This overexpression can be transient, in the presence of an effector (phenotypic resistance), or constitutive when mutants in the regulatory elements of the expression of efflux pumps are selected (acquired resistance). Efflux pumps are present in all cells, from human to bacteria and are highly conserved, which indicates that they are ancient elements in the evolution of different organisms. Consequently, it has been suggested that, besides antibiotic resistance, bacterial multidrug efflux pumps would likely contribute to other relevant processes of the microbial physiology. In the current article, we discuss some specific examples of the role that efflux pumps may have in the bacterial virulence of animals’ and plants’ pathogens, including the processes of intercellular communication. Based in these evidences, we propose that efflux pumps are at the crossroad between resistance and virulence of bacterial pathogens. Consequently, the comprehensive study of multidrug efflux pumps requires addressing these functions, which are of relevance for the bacterial–host interactions during infection.Work in our laboratory is supported by grants from the Spanish Ministry of Economy and Competitiveness (BIO2014-54507-R and JPI Water StARE JPIW2013-089-C02-01); from Madrid Autonomous Community [S2010/BMD2414 (PROMPT)]; and from the Instituto de Salud Carlos III [Spanish Network for Research on Infectious Diseases (REIPI RD12/0015)]. MA-R and PB are recipients of FPI fellowships from MINECO.Peer reviewedPeer Reviewe

The family of DOF transcription factors in Brachypodium distachyon: phylogenetic comparison with rice and barley DOFs and expression profiling

ABSTRACT: Transcription factors (TFs) are proteins that have played a central role both in evolution and in domestication, and are major regulators of development in living organisms. Plant genome sequences reveal that approximately 7% of all genes encode putative TFs. The DOF (DNA binding with One Finger) TF family has been associated with vital processes exclusive to higher plants and to their close ancestors (algae, mosses and ferns). These are seed maturation and germination, light-mediated regulation, phytohormone and plant responses to biotic and abiotic stresses, etc. In Hordeum vulgare and Oryza sativa, 26 and 30 different Dof genes, respectively, have been annotated. Brachypodium distachyon has been the first Pooideae grass to be sequenced and, due to its genomic, morphological and physiological characteristics, has emerged as the model system for temperate cereals, such as wheat and barley. RESULTS: Through searches in the B. distachyon genome, 27 Dof genes have been identified and a phylogenetic comparison with the Oryza sativa and the Hordeum vulgare DOFs has been performed. To explore the evolutionary relationship among these DOF proteins, a combined phylogenetic tree has been constructed with the Brachypodium DOFs and those from rice and barley. This phylogenetic analysis has classified the DOF proteins into four Major Cluster of Orthologous Groups (MCOGs). Using RT-qPCR analysis the expression profiles of the annotated BdDof genes across four organs (leaves, roots, spikes and seeds) has been investigated. These results have led to a classification of the BdDof genes into two groups, according to their expression levels. The genes highly or preferentially expressed in seeds have been subjected to a more detailed expression analysis (maturation, dry stage and germination). CONCLUSIONS: Comparison of the expression profiles of the Brachypodium Dof genes with the published functions of closely related DOF sequences from the cereal species considered here, deduced from the phylogenetic analysis, indicates that although the expression profile has been conserved in many of the putative orthologs, in some cases duplication followed by subsequent divergence may have occurred (neo-functionalization)

Low Ciprofloxacin concentrations select Multidrug-Resistant Mutants overproducing efflux pumps in clinical isolates of Pseudomonas Aeruginosa

Low antibiotic concentrations present in natural environments are a severe and often neglected threat to public health. Even if they are present below their MICs, they may select for antibiotic-resistant pathogens. Notably, the minimal subinhibitory concentrations that select resistant bacteria, and define the respective sub-MIC selective windows, differ between antibiotics. The establishment of these selective concentrations is needed for risk-assessment studies regarding the presence of antibiotics in different habitats. Using short-term evolution experiments in a set of 12 Pseudomonas aeruginosa clinical isolates (including high-risk clones with ubiquitous distribution), we have determined that ciprofloxacin sub-MIC selective windows are strain specific and resistome dependent. Nonetheless, in all cases, clinically relevant multidrug-resistant (MDR) mutants emerged upon exposure to low ciprofloxacin concentrations, with these concentrations being below the levels reported in ciprofloxacin-polluted natural habitats where P. aeruginosa can be present. This feature expands the conditions and habitats where clinically relevant quinolone-resistant mutants can emerge. In addition, we established the lowest concentration threshold beyond which P. aeruginosa, regardless of the strain, becomes resistant to ciprofloxacin. Three days of exposure under this sub-MIC "risk concentration" led to the selection of MDR mutants that displayed resistance mechanisms usually ascribed to high selective pressures, i.e., the overproduction of the efflux pumps MexCD-OprJ and MexEF-OprN. From a One-Health viewpoint, these data stress the transcendent role of low drug concentrations, which can be encountered in natural ecosystems, in aggravating the antibiotic resistance problem, especially when it comes to pathogens of environmental origin. IMPORTANCE It has been established that antibiotic concentrations below MICs can select antibiotic-resistant pathogens, a feature of relevance for analyzing the role of nonclinical ecosystems in antibiotic resistance evolution. The range of concentrations where this selection occurs defines the sub-MIC selective window, whose width depends on the antibiotic. Herein, we have determined the ciprofloxacin sub-MIC selective windows of a set of Pseudomonas aeruginosa clinical isolates (including high-risk clones with worldwide distribution) and established the lowest concentration threshold, notably an amount reported to be present in natural ecosystems, beyond which this pathogen acquires resistance. Importantly, our results show that this ciprofloxacin sub-MIC selects for multidrug-resistant mutants overproducing clinically relevant efflux pumps. From a One-Health angle, this information supports that low antimicrobial concentrations, present in natural environments, may have a relevant role in worsening the antibiotic resistance crisis, particularly regarding pathogens with environmental niches, such as P. aeruginosa. It has been established that antibiotic concentrations below MICs can select antibiotic-resistant pathogens, a feature of relevance for analyzing the role of nonclinical ecosystems in antibiotic resistance evolution. The range of concentrations where this selection occurs defines the sub-MIC selective window, whose width depends on the antibiotic

Regulación transcripcional en semillas de cebada cv. bomi y su mutante alto en lisina RISO-1508

En este trabajo se presenta un estudio de expresión global de los genes diferencialmente expresados (GDE) en Bomi y Riso 1508 en semillas en desarrollo a 15-18 dap (días después de la polinización), mediante hibridaciones de micromatrices de DNA (GeneChip Barley Genome Array, Affymetrix), donde están representados 25500 genes de cebada

Mutational Evolution of Pseudomonas aeruginosa Resistance to Ribosome-Targeting Antibiotics

The present work examines the evolutionary trajectories of replicate Pseudomonas aeruginosa cultures in presence of the ribosome-targeting antibiotics tobramycin and tigecycline. It is known that large number of mutations across different genes – and therefore a large number of potential pathways – may be involved in resistance to any single antibiotic. Thus, evolution toward resistance might, to a large degree, rely on stochasticity, which might preclude the use of predictive strategies for fighting antibiotic resistance. However, the present results show that P. aeruginosa populations evolving in parallel in the presence of antibiotics (either tobramycin or tigecycline) follow a set of trajectories that present common elements. In addition, the pattern of resistance mutations involved include common elements for these two ribosome-targeting antimicrobials. This indicates that mutational evolution toward resistance (and perhaps other properties) is to a certain degree deterministic and, consequently, predictable. These findings are of interest, not just for P. aeruginosa, but in understanding the general rules involved in the evolution of antibiotic resistance also. In addition, the results indicate that bacteria can evolve toward higher levels of resistance to antibiotics against which they are considered to be intrinsically resistant, as tigecycline in the case of P. aeruginosa and that this may confer cross-resistance to other antibiotics of therapeutic value. Our results are particularly relevant in the case of patients under empiric treatment with tigecycline, which frequently suffer P. aeruginosa superinfections

Wildlife and Antibiotic Resistance

Antibiotic resistance is a major human health problem. While health care facilities are main contributors to the emergence, evolution and spread of antibiotic resistance, other ecosystems are involved in such dissemination. Wastewater, farm animals and pets have been considered important contributors to the development of antibiotic resistance. Herein, we review the impact of wildlife in such problem. Current evidence supports that the presence of antibiotic resistance genes and/or antibiotic resistant bacteria in wild animals is a sign of anthropic pollution more than of selection of resistance. However, once antibiotic resistance is present in the wild, wildlife can contribute to its transmission across different ecosystems. Further, the finding that antibiotic resistance genes, currently causing problems at hospitals, might spread through horizontal gene transfer among the bacteria present in the microbiomes of ubiquitous animals as cockroaches, fleas or rats, supports the possibility that these organisms might be bioreactors for the horizontal transfer of antibiotic resistance genes among human pathogens. The contribution of wildlife in the spread of antibiotic resistance among different hosts and ecosystems occurs at two levels. Firstly, in the case of non-migrating animals, the transfer will take place locally; a One Health problem. Paradigmatic examples are the above mentioned animals that cohabit with humans and can be reservoirs and vehicles for antibiotic resistance dissemination. Secondly, migrating animals, such as gulls, fishes or turtles may participate in the dissemination of antibiotic resistance across different geographic areas, even between different continents, which constitutes a Global Health issue. Copyright © 2022 Laborda, Sanz-García, Ochoa-Sánchez, Gil-Gil, Hernando-Amado and Martínez

Circunferencia escrotal y parámetros morfométricos en machos Bubalus bubalis de la raza Murrah

In the present study the scrotal circumference of male buffaloes (Bubalus bubalis, Murrah race) was characterized and its relationship with other morphometric parameters was defined. The study was realized at “El Mosquito” farm, in San Marcos municipality in Sucre, Colombia. Fourteen (14) male buffaloes were taken from their weaning to twenty months old. These animals were subjected to testis measures (SC, testis longitude), body weight, and croup height, every twenty-eight days. To establish SC of the group under study, descriptive statistics was used, mean and standard deviation. In the same way, simple correlation analysis between SC with age, weight, croup height and testicular length (right and left). SC increased during experimental monitoring from 12, 4 ± 1, 0 cm to 20, 2 ± 1,4 cm. The weight ranged from 162, 1 ± 24, 9 kg to 323, 8 ± 39,8 kg. LTD fluctuated from 4,0 ± 0,7 cm to 7,4±0,7 cm. LTI began from 4,2 ± 0,5 cm and finished in 6,9 ± 0,6 cm and height rump at 10 months, was 104 ± 4,4 ± 3,4 cm to 118 cm at 20 months old male buffaloes studied. In conclusion, correlations between CE and age, weight, right and left testis length height and height haunch were: r = 0, 84; 0, 90; 0, 93; 0.91, and 0, 71 respectively. It´s concluded growth to SC was not uniform, also a high correlation was found between SC and morphometric parameters studied.Objetivo: Caracterizar la circunferencia escrotal (CE) y su relación con otros parámetros morfometricos en machos bufalinos (Bubalus bubalis) de la raza Murrah en la Hacienda Mosquito, Municipio de San Marcos – Sucre – Colombia. Metodología: Se tomaron 14 machos a partir del destete hasta los 20 meses de edad, se les realizaron medidas testiculares (CE, longitud testicular), peso corporal y alzada al anca, cada 28 días. Para establecer la CE del grupo en estudio se utilizó estadística descriptiva, media y desviación estándar. Igualmente análisis de correlaciones simples entre CE con edad, peso, alzada al anca y longitud testicular (derecha e izquierda). Resultados: La CE aumentó durante el seguimiento experimental de 12,4 ± 1,0 cm a 20,2 ± 1,4 cm. El peso osciló de 162,1 ± 24,9 kg a 323,8 ± 39,8 kg. La LTD fluctúo de 4,0 ± 0,7 cm a 7,4 ± 0,7 cm. La LTI inició con 4,2 ± 0,5 cm y culminó con 6,9 ± 0,6 cm y la alzada al anca a los 10 meses fue de 104 ± 4,4 cm a 118 ± 3,4 cm a los 20 meses de edad de los animales estudiados. Las correlaciones entre CE con edad, peso, longitud testicular derecha e izquierda y alzada al anca fueron: r= 0,4, 0,90; 0,93; 0,91, y 0,71 respectivamente. Se concluye, que el crecimiento de la CE no fue uniforme, además se encontró una alta correlación entre la CE y los parámetros morfometricos estudiados

The family of DOF transcription factors in Brachypodium distachyon: BdDOF24 in germinating seeds

The DOF (DNA binding with One Finger) transcription factor (TF) family is characterized by a binding domain of 52 amino acid residues that is structured as a Cys2/Cys2 Zn2+ finger that recognizes the common core 5?-T/AAAAG-3? in the promoter regions of their target genes. DOF TFs have been associated with biological processes exclusive to higher plants and their close ancestors (algae, mosses and ferns)