Light Regulation of Metabolic Networks in Microbes

[EN] Light Regulation of Metabolic Networks inMicrobes Day and night dominate our life and profoundly influence society. We feel the importance of the differences between day and night if we need sleep or if we have a jetlag. Both phenomena are triggered by our circadian clock, which can be influenced by light. Deprivation of light as well as perturbation of the circadian clock leads to severe health problem

Selecció clonal i sanitària de la varietat trepat a la denominació d'origen Conca de Barberà

El trepat, varietat de raïm cultivada principalment a la Denominació d'Origen (DO) Conca de Barberà, és considerat una varietat autòctona catalana. La selecció clonal i sanitària s'ha realitzat per a permetre la certificació i multiplicació dels millors clons, amb una elevada adaptació a les condicions ambientals i amb una producció per a l'obtenció de vins de qualitat. Aquest programa de selecció també permet la conservació de la diversitat intravarietal procurant mantenir el nivell de variabilitat que presenta encara la població actual.El trepat, variedad de uva cultivada principalmente en la Denominación de Origen (D. O.) Conca de Barberà, se considera una variedad autóctona catalana. La selección clonal y sanitaria se ha realizado para permitir la certificación y multiplicación de los mejores clones, con una elevada adaptación a las condiciones ambientales y con una producción para la obtención de vinos de calidad. Este programa de selección también permite la conservación de la diversidad intravarietal procurando mantener el nivel de variabilidad que aun presenta la población actual

Functional analysis of the Phycomyces carRA gene encoding the enzymes phytoene synthase and lycopene cyclase.

[EN] Phycomyces carRA gene encodes a protein with two domains. Domain R is characterized by red carR mutants that accumulate lycopene. Domain A is characterized by white carA mutants that do not accumulate significant amounts of carotenoids. The carRA-encoded protein was identified as the lycopene cyclase and phytoene synthase enzyme by sequence homology with other proteins. However, no direct data showing the function of this protein have been reported so far. Different Mucor circinelloides mutants altered at the phytoene synthase, the lycopene cyclase or both activities were transformed with the Phycomyces carRA gene. Fully transcribed carRA mRNA molecules were detected by Northern assays in the transformants and the correct processing of the carRA messenger was verified by RT-PCR. These results showed that Phycomyces carRA gene was correctly expressed in Mucor. Carotenoids analysis in these transformants showed the presence of ß-carotene, absent in the untransformed strains, providing functional evidence that the Phycomyces carRA gene complements the M. circinelloides mutations. Co-transformation of the carRA cDNA in E. coli with different combinations of the carotenoid structural genes from Erwinia uredovora was also performed. Newly formed carotenoids were accumulated showing that the Phycomyces CarRA protein does contain lycopene cyclase and phytoene synthase activities. The heterologous expression of the carRA gene and the functional complementation of the mentioned activities are not very efficient in E. coli. However, the simultaneous presence of both carRA and carB gene products from Phycomyces increases the efficiency of these enzymes, presumably due to an interaction mechanism

Protein-DNA interactions in the promoter region of the Phycomyces carB and carRA genes correlate with the kinetics of their mRNA accumulation in response to light.

[EN] Carotene biosynthesis in Phycomyces is photoinducible and carried out by phytoene dehydrogenase (encoded by carB) and a bifunctional enzyme possessing lycopene cyclase and phytoene synthase activities (carRA). A light pulse followed by periods of darkness produced similar biphasic responses in the expression of the carB and carRA genes, indicating their coordinated regulation. Specific binding complexes were formed between the carB-carRA intergenic region and protein extracts from wild type mycelia grown in the dark or 8min after irradiation. These two conditions correspond to the points at which the expression of both genes is minimal, suggesting that these binding complexes are involved in the down-regulation of photocarotenogenesis in Phycomyces. Protein extracts from carotene mutants failed to form the dark retardation complex, suggesting a role of these genes in the regulation of photocarotenogenesis. In contrast, protein extracts from phototropic mutants formed dark retardation complexes identical to that of the wild type.Junta de Castilla y León; Spanish Ministerio de Educación y Ciencia/FEDE

RNY3 modulates cell proliferation and IL13 mRNA levels in a T lymphocyte model: a possible new epigenetic mechanism of IL-13 regulation

[EN] llergic asthma is the most common type of asthma. It is characterized by TH2 cell–driven infammation in which interleukin-13 (IL-13) plays a pivotal role. Cytoplasmic RNAs (Y-RNAs), a variety of non-coding RNAs that are dysregulated in many cancer types, are also diferentially expressed in patients with allergic asthma. Their function in the development of the disease is still unknown. We investigated the potential role of RNY3 RNA (hY3) in the TH2 cell infammatory response using the Jurkat cell line as a model. hY3 expression levels were modulated to mimic the upregulation efect in allergic disease. We evaluated the efect of hY3 over cell stimulation and the expression of the TH2 cytokine IL13. Total RNA was isolated and retrotranscribed, and RNA levels were assessed by qPCR. In Jurkat cells, hY3 levels increased upon stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. When transfecting with high levels of hY3 mimic molecules, cell proliferation rate decreased while IL13 mRNA levels increased upon stimulation compared to stimulated control cells. Our results show the efect of increased hY3 levels on cell proliferation and the levels of IL13 mRNA in Jurkat cells. Also, we showed that hY3 could act over other cells via exosomes. This study opens up new ways to study the potential regulatory function of hY3 over IL-13 production and its implications for asthma development.Publicación en abierto financiada por el Consorcio de Bibliotecas Universitarias de Castilla y León (BUCLE), con cargo al Programa Operativo 2014ES16RFOP009 FEDER 2014-2020 DE CASTILLA Y LEÓN, Actuación:20007-CL - Apoyo Consorcio BUCLE

PTGDR gene expression and response to dexamethasone treatment in an in vitro model

[EN]Asthma is a multifactorial pathology influenced by environmental and genetic factors. Glucocorticoid treatment decreases symptoms by regulating genes involved in the inflammatory process through binding to specific DNA sequences. Polymorphisms located in the promoter region of the Prostaglandin D Receptor (PTGDR) gene have been related to asthma. We aimed to analyze the effect of PTGDR promoter haplotypes on gene expression and response to corticosteroid therapy. A549 lung epithelial cells were transfected with vectors carrying four different PTGDR haplotypes (CTCT, CCCC, CCCT and TCCT), and treated with dexamethasone. Different approaches to study the promoter activity (Dual Luciferase Reporter System), gene expression levels (qPCR) and cytokine secretion (Multiplexed Bead-based Flow Cytometric) were used. In addition, in silico analysis was also performed. Cells carrying the TCCT haplotype showed the lowest promoter activity (p-value<0.05) and mRNA expression levels in basal conditions. After dexamethasone treatment, cells carrying the wild-type variant CTCT showed the highest response, and those carrying the TCCT variant the lowest (p-value<0.05) in luciferase assays. Different transcription factor binding patterns were identified in silico. Moreover, differences in cytokine secretion were also found among different promoter haplotypes. Polymorphisms of PTGDR gene influence basal promoter activity and gene expression, as well as the cytokine secretory pattern. Furthermore, an association between these positions and response to corticoid treatment was observed

Phycomyces MADB interacts with MADA to form the primary photoreceptor complex for fungal phototropism

The fungus Phycomyces blakesleeanus reacts to environmental signals, including light, gravity, touch, and the presence of nearby objects, by changing the speed and direction of growth of its fruiting body (sporangiophore). Phototropism, growth toward light, shares many features in fungi and plants but the molecular mechanisms remain to be fully elucidated. Phycomyces mutants with altered phototropism were isolated ≈40 years ago and found to have mutations in the mad genes. All of the responses to light in Phycomyces require the products of the madA and madB genes. We showed that madA encodes a protein similar to the Neurospora blue-light photoreceptor, zinc-finger protein WC-1. We show here that madB encodes a protein similar to the Neurospora zinc-finger protein WC-2. MADA and MADB interact to form a complex in yeast 2-hybrid assays and when coexpressed in E. coli, providing evidence that phototropism and other responses to light are mediated by a photoresponsive transcription factor complex. The Phycomyces genome contains 3 genes similar to wc-1, and 4 genes similar to wc-2, many of which are regulated by light in a madA or madB dependent manner. We did not detect any interactions between additional WC proteins in yeast 2-hybrid assays, which suggest that MADA and MADB form the major photoreceptor complex in Phycomyces. However, the presence of multiple wc genes in Phycomyces may enable perception across a broad range of light intensities, and may provide specialized photoreceptors for distinct photoresponses

Actuación para el seguimiento de la adquisición de conocimientos por parte de los alumnos de la asignatura de Genética en el Grado de Biología

Memoria ID-032. Ayudas de la Universidad de Salamanca para la innovación docente, curso 2020-2021

TWEAK/Fn14 and non-canonical NF-kappaB signaling in kidney disease

The incidence of acute kidney injury (AKI) and chronic kidney disease (CKD) is increasing. However, there is no effective therapy for AKI and current approaches only slow down, but do not prevent progression of CKD. TWEAK is a TNF superfamily cytokine. A solid base of preclinical data suggests a role of therapies targeting the TWEAK or its receptor Fn14 in AKI and CKD. In particular TWEAK/Fn14 targeting may preserve renal function and decrease cell death, inflammation, proteinuria, and fibrosis in mouse animal models. Furthermore there is clinical evidence for a role of TWEAK in human kidney injury including increased tissue and/or urinary levels of TWEAK and parenchymal renal cell expression of the receptor Fn14. In this regard, clinical trials of TWEAK targeting are ongoing in lupus nephritis. Nuclear factor-kappa B (NF-κB) activation plays a key role in TWEAK-elicited inflammatory responses. Activation of the non-canonical NF-κB pathway is a critical difference between TWEAK and TNF. TWEAK activation of the non-canonical NF-κB pathways promotes inflammatory responses in tubular cells. However, there is an incomplete understanding of the role of non-canonical NF-κB activation in kidney disease and on its contribution to TWEAK actions in vivo.Grant support: ISCII and FEDER funds FIS PS09/00447, ISCIII-RETIC REDinREN/RD06/0016, RD12/0021, Comunidad de Madrid/CIFRA/S2010/BMD-2378. Salary support: FIS to MDSN, Programa Intensificación Actividad Investigadora (ISCIII/Agencia Laín-Entralgo/CM) to AO, FPU (Ministerio de Educación, Cultura y Deporte) to JP, Fundacion Conchita Rabago to LCT

Modulation of the Serum Cytokine Expression Pattern in Hymenoptera Allergic Patients Treated with Specific Venom Immunotherapy

[EN] Venom immunotherapy (VIT) is an adequate model to explore the immune mechanisms underlying this type of treatment. We have investigated the use of protein arrays to detect variations in the levels of cytokines in patients receiving VIT. In the present study we selected 11 non-atopic patients with systemic reactions after Hymenoptera sting that received VIT during at least three years. In order to evaluate the success of VIT all of them should have tolerated a sting field after VIT. Serum samples were obtained before initiating VIT and after at least three years of successful VIT. We analyzed 42 serum proteins corresponding to a Th1/Th2 panel using protein array methodology. We observed a significant increase of Interleukin 10, Myeloid Macrophage Colony Stimulation Factor, Macrophage Derived Chemokine, Interleukin 1-α, Vascular Endothelial Growing Factor and Stem Cell Factor serum levels after successful VIT. We discuss the usefulness and normalization of this array method to analyze cytokines and other serum proteins. Monitoring these serum cytokines could help to predict the response and to elucidate the mechanisms underlying immunotherapy.Fundación para la Investigación de la Sociedad Española de Alergología e Inmunología Clínic