Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria Revista de los másteres de Biotecnología de la UPO es-ES Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2254-3821 Increase of blastocyst rate when environment conditions are improved during handling of human oocytes and pre-embryos in an IVF treatment <p><strong>Motivation:</strong> There is not yet a standardized system of human pre-embryo culture during in vitro fertilization (IVF) treatments and several technical improvements are continually emerging. We have recently performed a study showing better success rates when pre-embryos are culture in group at low oxygen tension (5% O2) in a benchtop incubator instead of individually at atmospheric oxygen tension (20% O2) in a conventional incubator. We have continued this line of research using these updated culture conditions in order to evaluate the efficacy of a new ART station with a closed environment in comparison of an open flow cabinet. This new system keeps a controlled environment maintaining a carbon dioxide (CO2) concentration of 6% and a temperature of 37ºC. The present study aims to assess whether oocyte and embryo handling within a closed station improves the embryo quality (measured as the probability of transferring or freezing an embryo) compared to an open flow cabinet.</p> <p><strong>Methods:</strong> Prospective randomized study performed from 2016 to the present. 57 patients have been included to date, who signed the corresponding consent for the study and met the inclusion and exclusion criteria. During their IVF treatments, pre-embryos were cultured in group at low oxygen in the K-MINC incubator. The population of patients was assigned randomly to the following groups:</p> <p>-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Control group: oocyte and pre-embryo handling was performed inside a conventional open flow cabinet without a controlled environment.</p> <p>-&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Study group: Oocyte and pre-embryo handling was performed inside a closed station with a controlled environment: 6% CO2 and 37ºC.</p> <p><strong>Results:</strong> To date, 30 patients have been included in the control group with 301 pre-embryos in total while 27 patients have been included in the study group with 224 pre-embryos in total. Blastocyst rate (67.4% vs. 61.8%, p = 0.18), good quality blastocyst rate (31.3% vs. 25.9%, p = 0.18) and viable blastocyst rate (54.9% vs. 48.2%, p = 0.13) have been higher in the study group compared with the control group. No significant differences were found in the fertilization rate, in the implantation rate or in the clinical pregnancy rate.</p> <p><strong>Conclusions:</strong> Blastocyst rate, good quality blastocyst rate and viable blastocyst rate were increased in more than a 5% when oocytes and pre-embryos were handled in a closed station. However, this improvement is not yet significant and the recruitment of patients must continue.</p> Esther Santamaría-López Myriam Ruíz-Pérez Víctor Blasco-Rodríguez Manuel Fernández-Sánchez Nicolás Prados-Dodd Derechos de autor 0 2020-03-19 2020-03-19 9 Conserved gene modules regulate light signals in algae and plants <p><strong>In the Plant Development Unit (PDU) we aim to discover mechanisms that allowed photosynthetic organisms to reach the level of developmental complexity shown today. Plants are particularly good models as they have been evolving as light autotrophs for millions of years, ever since the first bacteria developed oxygenic photosynthesis and killed 99% of existing species in the process. But light is not only the main source of energy for plants, it is also one of the main regulators of their development, as endo-symbiotic cyanobacteria (chloroplasts) perfected their physiological synchronization with the emerging eukaryotes (1). Another important aspect of plant evolution was the transit to the aerial world and the acquisition of characteristics that allowed them to successfully colonise this new habitat (2). In the PDU we have followed the evolution of the day length response (photoperiod) that coordinates the daily physiological activities of plants and can be also used to regulate seasonal behaviours such as winter recesses or flowering time (3). When gene expression networks from photoperiod experiments from microalgae, bryophytes and higher plants are compared, a common nodular structure is discovered (4). Following these discoveries, we have isolated ancestor algal genes that show the same function as higher plants in the response to photoperiod such as the CONSTANS-DOF module (5). We are currently investigating common regulatory mechanisms in photoperiod sensing such as the effect on the circadian clock, senescence, retrograde signalling (6) and protein stability (7)</strong></p> Pedro de los Reyes M Teresa Ruiz Gloria Serrano-Bueno Francisco J Romero-Campero José M Romero Federico Valverde Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-01 2020-04-01 9 Development of the technique of isoelectricfocusing and immunodetection of immunoglobulin A in the healthcare context as support for the diagnosis of Multiple Sclerosis <p><strong>Motivation:</strong> Multiple sclerosis (MS) is characterized by demyelination and subsequent degeneration of myelin sheaths that line the axons of neurons, producing multifocal and temporarily dispersed damage to the central nervous system (CNS) [1] that leads to neuronal damage and axonal loss [2]. MS is a complex disease in which the exact etiology remains unknown, although our current understanding of the natural history of MS and its immunopathogenesis points to an immune deregulation resulting from an interaction between genetic predispositions and environmental factors [2]. Given this uncertainty, there is a need for the identification of biomarkers in both cerebrospinal fluid and peripheral blood that help us to have a more complete view of the disease, and at the same time could contribute to a faster and more accurate diagnosis. This project arises from the importance and need to study immunoglobulin A as a biomarker for MS.</p> <p><strong>Methods:</strong> Isoelectric focusing is one of the most powerful techniques used to solve complex mixtures of proteins [2].</p> <p>The main objective of this project is the development of the isoelectroenfocusing and immunodetection technique for oligoclonal bands of immunoglobulin A and the IgA reduction protocol [3][4] to establish a subsequent relationship between patients diagnosed with MS and intrathecal secretion of immunoglobulin A.</p> <p><strong>Results:</strong> For the implementation of the technique, a set of cerebrospinal fluid (CSF) samples previously extracted by lumbar puncture were selected, presenting the patient with a diagnostic suspicion of demyelinating disease, and presenting a high quantification of intrathecal secretion of IgA, routinely carried out in the Clinical Biochemistry Unit, immunology laboratory. A total of 23 samples were selected based on IgA concentrations in both CSF and serum.</p> <p><strong>Conclusions:</strong> It should be noted that today the CSF remains the most interesting fluid for the study of degenerative neurological diseases. The CSF is the best representative of the clinical manifestations that are confined to the central nervous system, hence its characterization is, increasingly, of high interest for a more accurate diagnosis. With this set-up it is intended to give new information to the clinician to help diagnose the disease. Its value as a marker of the disease will be derived from subsequent observations and studies that clinicians perform in their cohort.</p> Elena Gálvez-Salinas Marta Isabel García-S´´anchez Derechos de autor 2020 2020-03-19 2020-03-19 9 Molecular basis of liver fibrosis <p><strong>Motivation:</strong> Liver fibrosis is the result of an exacerbated scarring response after continuing damage of the tissue. (1). Hepatic stellate cells (HSCs) have been described as a key factor for this process. In the normal liver, HSCs remain quiescent and are only activated by an injury trigger (2). Previously, our group reported that GATA4 is a crucial factor for the maintenance of HSCs quiescence and the inhibition of fibrosis.&nbsp; Therefore, unraveling the molecular pathways mediated by GATA4 could be useful for promoting fibrosis regression (3). In this project, we perform a lineage tracing assay in order to explore GATA4 expression during regeneration after inducing fibrosis with tetrachloride (CCl4) in mice. In addition, we study the potential transcriptional repression of Hif2α by GATA4, a mechanism that could explain, at least in part, the role of GATA4 in the inhibition of liver fibrosis.</p> <p><strong>Methods:</strong> CCl4 injections were used to induce liver fibrosis in a mouse model with constitutive GFP expression in HSCs. Livers were extracted and analyzed by immunofluorescence right at the end of the treatment and after 4 weeks of recovery. A conserved Hif2α intronic region, which contains two putative GATA sites were cloned into the pGL3 luciferase reporter plasmid. Transient transfections experiments were performed using pGl3-Hif2α plasmid and a plasmid for Gata4 constitutive expression to study the potential transcriptional repression of Hif2 by GATA4.&nbsp; Dual-Luciferase ® Reporter Assay System (Promega) was used for luciferase measurement.</p> <p><strong>Results:</strong> Preliminary results showed that reversion of activated HSCs to a quiescent state is accompanied by reactivation of GATA4 expression in mice recovering from CCl4 treatment. These results pointed to a crucial role of GATA4 for reversion of HSCs phenotype. On the other hand, 293T cells transfected with GATA4 expression plasmids showed reduced luciferase activity, which likely indicates a repression of Hif2α by GATA4 via the conserved GATA binding sites in the HIF2α enhancer region.</p> <p><strong>Conclusions:</strong> There is growing evidence that GATA4 not only orchestrates the preservation of normal tissue organization, but also actively contributes to fibrosis regression. Moreover, our work proposes genetical regulation of Hif2α by GATA4 could be decisive for this process.</p> Pilar Rodríguez-Martín Noelia Arroyo-De Alba Anabel Rojas Derechos de autor 0 2020-03-19 2020-03-19 9 Generation of induced pluripotent stem cell (iPSC) lines from urine samples. <p><strong>Motivation:</strong></p> <p>Induced pluripotent stem cell (iPSC) lines arise from the need of seeking alternative sources of stem cells for cellular and genetic therapies to avoid ethical aspects related to the use of embryonic stem cells. Urine represents a rich source of epitelial cell that can undergo a dedifferentiation process to generate iPSCs (Zhou et al, 2011). Interestingly, the method to obtain iPSC from urine derived cells (UDC) requiers a non-invasive technique that avoids biopsies and other surgical techniques.</p> <p>In others reports, iPSC derived from UDC are been used to produce cardiomyocites (Feng et al , 2018), retinal cells (Clegga et Foltza, 2019) and beta cells (Maehr, 2011) , for the treatment of such as myocardial infarctions, degenerative &nbsp;retinal diseases and type 1 diabetes, respectively, among other diseases.</p> <p>&nbsp;</p> <p><strong>Methods:</strong></p> <p>For isolation and cultivation of UDC, we used the protocol described by Zhou et al. Specific culture media were used for the selection of UDC colonies suitable for transfection and for iPSC culture maintenance. The most remarkable tecnique used was a commercial ,non-integrative, viral system, named Sendai, for the transfection of UDC with transcription factors (hKlf4, hOct3/4, hSox2, cMyc).</p> <p>Once iPSC line was stabilized, it was characterized by techniques such as alkaline phosphatase assay, PCR, immunofluorescence, flow cytometry for nuclear (OCT-4, SOX-2, NANOG, TELO) and surface (TRA-1-60, TRA-1-81 and SSEA4) pluripotency markers. Differentiation potential of iPSCs into the three germ layers was performed using STEMdiff Trilineage Differentiation Kit protocol (STEMCELL Technologies)</p> <p>&nbsp;</p> <p><strong>Results: </strong></p> <p>Cultured urine epithelial cells were successfully transfected and formed colonies iPSC-like. Characterization of these colonies confirmed expression of pluripotency markers and alkaline phosphatase. The absence of exogenous pluripotency vector expression was verified by PCR.</p> <p>&nbsp;</p> <p><strong>Conclusions:</strong></p> <p>So far, we can accept that it has been possible to isolate, cultivate and transform UDCs into iPSCs. Now, we need to further characterize our iPSC line, by studying its karyotype and testing its ability to form teratomas in vivo. To apply this procedure in cell therapy, we should repeat all culture processes under Good Manufacturing Practices‎ (GMP). The generation of iPSC lines represents a great advance for cell therapy and tissues bioengineering.</p> María Romero-Muñoz Vivian Capilla-González Nuria Mellado-Damas Yolanda Aguilera Derechos de autor 0 2020-03-19 2020-03-19 9 Effect of cell dedifferentiation and genetic p53 profile in the expression of Bcl-2 family members in tyrosine kinase inhibitor-treated liver cancer cells <p><strong>Motivation:</strong> Hepatocellular carcinoma (HCC) is the fifth most common type of liver cancer and the second most frequent cause of cancer‐related death worldwide [1]. The recommended first‐line treatment for patients with locally advanced disease and well-preserved liver function is Sorafenib with a mean overall survival of 11 months [2]. Other drugs have been developed to increase the therapeutic arsenal of treatments such as Lenvatinib [3], Regorafenib [4]&nbsp; and Cabozantinib [5]. The Bcl-2 protein family plays a central part in the control of apoptosis [6]. Bcl-2, Bcl-XL and Mcl-1 are antiapoptotic members Bax, Bak, Bid, and Bim are proapoptotic members. The cytoprotective function of Bcl-2 proteins stems from their ability to antagonize Bax and Bak, and thus prevent apoptosis. [7]. The aim of the present study was the comparative analysis of the tyrosine kinase inhibitors in cell death and proliferation, and the expression of Bcl-2 family members according to the differentiation degrees and p53 genetic profile in liver cancer cells.</p> <p><strong>Methods:</strong> Sorafenib, Regorafenib, Cabozantinib, and Lenvatinib were obtained commercially from Carbosynth Limited. Parameters were assessed in differentiated cells: HepG2 (ATCC/LGC Standards, SLU, Barcelona, Spain) and Huh7 (Apath LLC, Brooklyn, USA), and dedifferentiated cells: JHH2 and JHH4 cell lines obtained from the Japanese Collection of Research Bioresources Cell Bank (Tokyo, Japan). Cells were negative for mycoplasma contamination. HCC cell lines were maintained in supplemented Minimum Essential Medium with Earle's Balanced Salts at 37°C in a humidified incubator with 5 % CO2. Cells were seeded at a density of 10^5 cells/cm2 in 2D culture. Different parameters related to cell death and proliferation were associated with the expression of Bcl-2 family members assessed by Western-blot analysis.</p> <p><strong>Results:</strong> The administration of tyrosine kinase inhibitors induced cell death and reduced cell proliferation. This effect was associated with an upregulation of tBid and Bim expression in differentiated liver cancer cells (HepG2 and Huh7) compared to dedifferentiated cells (JHH2 and JHH4, respectively). In addition, the lack of p53 in liver cancer cells (Huh7 and JHH4) had a lower degree in the expression compared to their p53 wild type counterpart (HepG2 and JHH).</p> <p><strong>Conclusions:</strong> The dedifferentiation of cancer cells and mutated p53 reduce the upregulaupregulation of Bim and Bid induced by Sorafenib and Regorafenib in liver cancer cells.</p> Raúl Fernández-Rodríguez Matilde Revuelta-González Jordi Muntané Derechos de autor 0 2020-03-19 2020-03-19 9 Combined application of chemical and biological compounds for the reduction of phosphorus in wastewater and biological fanges <p><strong>Motivation:</strong> Nowaday wastewater treatment &nbsp;is a really important topic &nbsp;that is receiving a lot of attention due to the need of continuously finding and improving mechanisms and new techniques that allow us to eliminate as much organic matter, metals, and other toxic compounds as possible of the drinking waters and that are discharged to the riverbeds. Phosphorus is one of the elements that usually cause more problems when treating these waters. The joint use of chemical and organic compounds that have coagulant and flocculant capacity can help to reduce the use of these chemical compounds, which can leave certain contaminating residues, such as the cost in these processes. This could be a viable and cheap alternative in the field of wastewater treatments to reduce the presence of these organic compounds.</p> <p><strong>Methods:</strong> The Jar-test technique was used to carry out the wastewater &nbsp;tests, whereby increasing concentrations of different chemical and organic compounds were added to a fixed amount of decanted water to verify the change in phosphorus concentration that was given in each of the mixtures and check if there is a reduction in the concentration of this element and if this happens to obtain the optimal dose, with the aim of optimizing the process. Complementarily, the pH, turbidity and conductivity were measured to see how these parameters were affected.</p> <p><strong>Results:</strong> The results obtained show that various organic compounds, like cellulose and starch, that are very cheap and easy to produce (some are even considered as wastes for some industries), can be used to decrease the concentration of phosphorus in sewage from wastewater treatment plants when used together with chemical compounds. However, more studies are required due to the characteristics of each water and the different organic and chemical compounds.</p> <p><strong>Conclusions:</strong> The use of biological coagulants in combination with chemical ones could mean a new study field that has an enormous economic and environmental potential. However, the few studies carried out require further investigation of combinations with greater potential as well as in which type of water each one works better in order to optimize the process in each treatment plant according to their needs.</p> Juan Manuel Sánchez Santos Eva Rodriguez González Ana Moral Rama Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-02 2020-04-02 9 Effect of confinement on the solubility of salt in water: A simulation study on zeolites <p><strong>Motivation:</strong> Salinity is an important concept to understand the environmental conditions and organisms that can be found in water as well and depends on the solubility of salts in water. The effect of temperature and pressure on the solubility of salts (as sodium chloride) in water and other solvents has been widely studied. However, there is scarce studies on the solubility of salts in confined systems, and how the confinement affects to the solvation of salts. Zeolites are a group of crystalline porous solids based on silica and they exhibit high surface area, high thermal stability, and high exchange capacity. Pure silica zeolites are hydrophobic, but the presence of cations can change this nature making them suitable for a variety of applications such as drying of refrigerants, removal of atmospheric pollutants, separation of air components, recovering radioactive ions from waste solutions, catalysis of hydrocarbon reactions, and many others. In this work, we study the influence of the confinement of water in two types of zeolite on the solubility of sodium chloride.<br><strong>Methods:</strong> To study the influence that confinement of water in zeolites has on the dissociation of NaCl clusters, we carry out molecular simulation using two techniques, Molecular Dynamics (MD) and Monte Carlo methods (MC). All simulations are performed using the RASPA simulation code and the force fields and models used for water and the ions are taken from the literature and previously validated. As initial step, we use MD to analyse the effect of temperature and system size on the dissociation of salt. Then, the effect of confinement is analysed using MC simulations. For this purpose we calculated the adsorption of water in two commercial zeolites (MFI and FAU) and after obtaining the adsorption capacity of water for these structures, we study the dissociation of salt in the adsorbed water. We focus on the effect exerted by the number of cations on the zeolites, the topology and the initial concentration of salt.<br><strong>Results:</strong> Our results show that the size of the simulating box has an impact on the dissociation of ions, obtaining that the larger the box, the better the dissociation. In the confined systems, we found that in the pure silica MFI zeolite (longitudinal channels connected to zig-zag channels) dissociation is enhanced and the same occurs in the pure FAU zeolite (cages connected throw windows). Nevertheless, for the FAU zeolite with aluminium atoms, the dissociation of ions is hampered when increasing the number of Al substitutions. On the contrary, the presence of aluminium in the MFI does not alter the dissociation of ions.</p> Teresa Ramos Carreño Juan José Gutierrez Sevillano Sofía Calero Díaz Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-05 2020-04-05 9 Influence of agitation rate on a semi-industrial photoreactor for olive mill wastewater treatment by photo-Fenton reaction <p><strong>Motivation:</strong> Olive mill wastewaters (OMWs) generated in the Mediterranean countries present an <br>environmental problem due to its high organic load and the presence of toxic and inhibition growth <br>compounds such as residual oil matter and phenolic compounds. The various attempts to treat OMWs by <br>conventional methods have not yielded satisfactory results due to the recalcitrant nature of the <br>organic material contained in OMWs (Thanekar, Panda and Gogate, 2018).</p> <p style="text-align: justify;">Advanced oxidation processes (AOPs) have the capacity to degrade biodegradable and <br>non-biodegradable compounds, in addition to a wide number of persistent organic compounds (Oller, <br>Malato and Sánchez-Pérez, 2011). Photo-degradation with artificial UV-irradiation lamps can <br>accelerate the degradation of pollutants more than dark degradation (Fenton Reaction) (García and <br>Hodaifa, 2017).</p> <p style="text-align: justify;"><strong>Methods:</strong> In this work, experiments were performed in a semi-industrial batch stirred photo-reactor. <br>The agitation rates were varied in each experiment (60, 150, 300, 400 and 500 rpm). The common <br>operating conditions were initial pH = 3, hydrogen peroxide amount equal to that determined by the <br>reaction stoichiometry, oxidant/catalyst ratio = 0.03, temperature = 20 ºC and artificial <br>ultraviolet irradiation.</p> <p style="text-align: justify;"><strong>Results:</strong> The highest photo-degradation percentage (90%) was registered in the experiment with 500 <br>rpm. In all cases, the TOC removal percentages were increased throughout the reaction. The <br>global removal percentages were decreased when the agitation rate exceeded 300 rpm.</p> <p style="text-align: justify;"><strong>Conclusions:</strong> Finally, it can be concluded that at this scale the best agitation rate used is 300 <br>rpm and the photo-degradation time needed is higher than 60 min. In these conditions, the TOC <br>removal percentages expected are in the range from 76% to<br>90%.</p> <p><br><br></p> Nathalia Basegio-Castellani Gassan Hodaifa Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-05 2020-04-05 9 Optimization of super-resolution microscopy to the visualization of biotechnological samples <p><strong>Motivation: </strong>Super-resolution microscopy (SRM) allows the resolution of cellular structures in the range of tens of nanometers being able to useful for biotechnological researchs, like the study of the chromosomal structure of the fission yeast Schizosaccharomyces pombe. Nevertheless these techniques need an optimization and the preservation of the structures of study.</p> <p>In this investigation we want optimize the SRM protocol to be useful to the visualization of structures in the mencionated scale.</p> <p>Specifically, we want define more precisely the position of telomeres and centromeres that make the Rabl configuration of chromosomes. Commonly is used the fluorescence microscopy (FM) for the cell research, but it shows less foci than the real number of this structures. However, using cells with the inactivated Linker of nucleoskeleton and cytoskeleton complex (LINC) the Rabl configuration can change, realeasing the centromere-LINC contacts. This separation could allow discern more centromeres with FM than in wild type cells, but could affect the telomeres.</p> <p>In this study we will compare the chromosomal distribution between wild type cells and inactivated LINC cells using FM and SRM.</p> <p><strong>Methods:</strong> Different strains are generated with centromeres, telomeres and nuclear membrane tagged with fluorescence proteins to study through FM the possible alterations in the Rabl configuration in inactivated LINC cells.</p> <p>This strains together with inmunofluorescence techniques allow an approach to the optimization of a SRM protocol. Then to study the structures of interest with SRM, strains with HA-tag are used in combination with the inmunofluorescence protocol. In addition, protein extraction and western blots was performed to test the union of the antibody to the target protein and check the efficient of the union of primary and secondary antibody.</p> <p><strong>Results:</strong> Optimization of the immunofluorescence protocol has been achieved with the tag GFP, necessary for the refinement of the SRM. The best results in FM have been reached with cells fixed with 4% paraformadehyde and tagged with nanobodies, being unnecessary the cellular wall digestion to permeabilize it.</p> <p>Using MF to analyze the Rabl configuration, wild type and inactivated LINC cells shown a similar distribution of telomeres, being the most common the cells with 2 telomeres, followed by 1 and 3 telomeres. Moreover the 27% of LINC mutants show 2 centromeres versus the wild type that only show 1 centromere.</p> David Delgado Gestoso Alejandro Campoy López Ana María Brokate Alfonso Fernandez Alvarez Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-06 2020-04-06 9 In vivo study of the effect of Ulipristal Acetate (UPA) on endometrial receptivit <p><strong>Motivation:</strong> Ulipristal Acetate (UPA) is a drug used as an emergency contraceptive (EA). Its mechanism of action is associated with suppression of ovulation, although its impact on endometrial receptivity in embryonic implantation cannot be ruled out. In this study, the effect of UPA is evaluated by gene expression using a panel of 192 genes related to the endometrial receptivity. The comparison between the gene expression profile in 4 patients who received 30 mg of UPA on LH+2 day with their baseline state and one month post treatment could shed light on the receptivity and the implantation process</p> <p>&nbsp;</p> <p><strong>Methods:</strong> Three consecutive cycles of 4 healthy patients surgically sterilized were analyzed. The first cycle without the intake of UPA (B), the second cycle with intake of 30 mg of UPA on the day LH+2 (T), and the third cycle one month after treatment and without the intake of UPA (PT). In all cycles the endometrial biopsy was taken on day LH+6 to 8. Gene expression of each sample was determined by quantitative PCR using a panel of 192 genes related to endometrial receptivity. Fold-Change (FC) was calculated to determine the differential expression of genes in different conditions regarding to the control cycle. Statistical analyzes were performed using SPSS and functional enrichment analysis GO (Gene Ontology).</p> <p>&nbsp;</p> <p><strong>Results:</strong> The FC data indicate a repression of the genes in the T regarding to B cycle. Statistical analysis showed that there are 11 genes that explain the variance between B, T and PT and also, the three conditions are well differentiated from each other. As for the FC, those genes with FC&gt; 0.2 in absolute value were considered. GO enrichment showed that the genes that are deregulated in the T cycle with respect to B are associated with the response to ions such as zinc, copper, metals and aging. The PT-B comparison shows deregulation of transport genes and ion homeostasis and tissue remodeling. PT-T showed deregulation of sexual reproduction genes and positive growth regulation</p> <p>&nbsp;</p> <p><strong>Conclusions:</strong> There is repression of the genes after treatment with UPA, the transcriptomic profiles of the three populations B, T and PT are different from each other, with the profile of T being the furthest, and B and PT the closest to each other. This would suggest that the administration of UPA in LH+2 would have a repressive effect on genes associated with uterine receptivity (mostly involved in ionic transport). This effect seems to persist until the following month.</p> Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-03-19 2020-03-19 9 Resveratrol derivates for age-related macular degeneration treatment <p><strong>Motivation:</strong> Age-related macular degeneration (AMD) is a condition characterized by the acumulation of deposits called drusen between the retinal pigment epitheliun (RPE) and Bruch's membrane. As the disease progresses, occurs the degeneration of RPE and photoreceptors cells (dry AMD) or the infiltration of blood vessels from the choroidal vasculature into the retina (wet AMD). To date AMD has no effective treatment. This condition is the leading cause of blindness among elderly individuals worldwide and the total number of patients is expected to increase to 288 million affected individuals in 2040.</p> <p>Resveratrol has shown to have protective effects in others ophthalmologic disorders as diabetic retinopathy, likely through upregulation of Sirt1. The aim of this work is to test some small molecules derivated from resveratrol to see if there is a protective effect against the progression of AMD.</p> <p><strong>Methods:</strong> The compounds were tested in vitro using the RPE cell line ARPE-19. Posible toxic effects were studied using RealTime-Glo™ MT Cell Viability Assay and RealTime-Glo™ Annexin V Apoptosis and Necrosis Assay. The efects in the cell cycle were annalised by flow cytometry. Changes in Sirt1 enzymatic activity were messured using SIRT-Glo™ Assay System and Sirt1 expression has been annalised by inmunofluorescence (IF) and western blot (WB)</p> <p><strong>Results:</strong> The compounds did not show significant toxic effects or effects for the cell cycle in the concentration range annalised except resveratrol, that seems to cause cell cycle arrest at S and G2/M phase and cell apoptosis. Sirt1 enzymatic activity and Sirt1 expression in the cells do not seem to be increased in response to resveratrol derivates treatment at high doses compared to vehicle control.</p> <p><strong>Conclusions:</strong> Resveratrol derivates seem to be safer and less toxic than resveratrol, at least for in vitro use. Further studies are needed to determinate their possible therapeutic applications.</p> Ana Cristina Almansa-García Helena Lucena-Pardós Adoración Montero-Sánchez Francisco Javier Díaz Corrales Derechos de autor 0 2020-03-19 2020-03-19 9 Development of a reporter system for screening anti-biofilm activities <p class="AbstractText"><span style="font-size: 12.0pt; font-family: 'Calibri',sans-serif; color: black;">Biofilm formation is responsible for increasing antibiotic tolerance in pathogenic bacteria. It is estimated that approximately 80% of chronic infections are associated with this phenomenon. Therefore, the search for therapeutic agents with specific biofilm targets has become of vital importance. One of the main strategies is the search for enzymes that degrade the different components of the extracellular matrix. However, since the composition of the matrix varies among the different microorganisms, an alternative would be to interfere with the signaling cascades that lead to the formation of the biofilm or stimulate its dispersion.</span></p> Ana Díaz-Navarro Aroa López-Sánchez Fernando Govantes-Romero Derechos de autor 0 2020-03-19 2020-03-19 9 Characterization of retinal cells derived from iPSCs of a patient with PRPF31 associated retinitis pigmentosa <p><strong>Motivation: </strong>Retinitis pigmentosa (RP) is a group of hereditary retinal dystrophies caused by mutations in different genes with a prevalence of 1 in 4000. It is an untreatable disease with a variable clinical evolution in which patients develop severe visual impairment or total blindness. Mutations in pre-mRNA splicing PRPF31 gene have been described as the second most common cause of autosomal dominant RP. Previous studies relate mutations in PRPF31 with dysfunction and degeneration of the retinal pigment epithelium (RPE). Thanks to the ability to obtain and differentiate induced pluripotent stem cells (iPSC), retinal models can be generated to study the disease mechanism and to evaluate new therapies. This work is based on a personalized cellular model obtained by differentiating RPE from iPSCs of a patient with PRPF31 c.165G mutation, which will be used to study the mechanism of the disease.</p> <p><strong>Methods:</strong> iPSCs and previously differentiated RPE cells have been cultured and imaged. The characterization of the RNA level expression of specific genes of both cell types has been performed by RT-PCR. Expression at the protein level has been analyzed by Western blot. At the physiological level, the ability of the cellular model to establish an epithelial barrier has been evaluated by transepithelial electrical resistance (TER).</p> <p><strong>Results:</strong> Phase contrast images showed a characteristic and distintive morphology of iPSC and RPE cells. RT-PCR showed the silencing of pluripotency genes such as NANOG in RPE cells, as well as the exclusive expression of specific genes such as CRALBP and RPE65 in RPE. In the comparative study of the cellular models of patient and healthy control, it was observed a variation in the expression levels of the PRPF31 and RPE65 genes. In Western blot, the PRPF31 protein detected in the patient's RPE showed a different band pattern compared with healthy control and iPSCs. Finally, TER showed a similar maturation of the two cell models compared, indicating that PRPF31 c.165G mutation does not affect the cells adhesions.</p> <p><strong>Conclusions:</strong> The cellular model of RPE with PRPF31 c.165G mutation has been correctly differentiated, allowing the study of the consequences at the cellular level of this genetic defect. The decrease found in RPE65 gene expression suggests that this could be the mechanism by wich PRPF31 c.165G mutation causes RP, because RPE65 insufficiency is a known cause of blindness.</p> Carla Victoria Jiménez-Medina Alberto Cañibano-Hernández Berta De la Cerda-Haynes Derechos de autor 0 2020-03-19 2020-03-19 9 In vitro effect of ceftazidime-avibactam pressure on ceftazidime-avibactam resistance in KPC-producing Klebsiella pneumoniae clinical isolates. <p><strong>Motivation:</strong> Infections caused by KPC-producing Klebsiella pneumoniae represent a challenge due to the limited available treatement choices. In this context, ceftazidime-avibactam (CAZ-AVI) is postulated as an alternative treatment effective against class A beta-lactamases such as KPC [1]. But, recent data reported the failure of CAZ-AVI treatment of infections by KPC-producing K. pneumoniae due to the development of CAZ-AVI resistance [2]. However, little is known concernig the CAZ-AVI resistance developement by CAZ-AVI selective pressure. Here, we aimed to determinate in vitro whether the exposure of KPC-producing K. pneumoniae clinical isolates to CAZ-AVI subinhibitory concentrations could lead the selection of CAZ-AVI resistant isolates.</p> <p><strong>Methods:</strong> Seventeen KPC-producing K. pneumoniae clinical isolates (7 KPC-2, 9 KPC-3 and 1 KPC-11) were analyzed. Minimum inhibitory concentrations (MICs) of CAZ-AVI were determined by broth microdilution using a fixed AVI cocentration of 4 mg/L [3]. Moreover, these isolates were further exposed to increasing concentrations of CAZ and fixed 4 mg/L of AVI, from a sub-MIC up to 256/4 mg/L of CAZ-AVI (or the concentration able to kill the bacterial isolate) at 37ºC with shaking during 24h. New MICs to CAZ-AVI were determined in each condition and after 15 days without CAZ-AVI pressure. Therefore, in order to demonstrated that blaKPC gene is responsible for acquisition of CAZ-AVI resistance in KPC-producing K. pneumoniae, blaKPC-2 and blaKPC-3 were cloned into a reference K. pneumoniae CECT 997 strain. Resistance or susceptibility were determined according to EUCAST criteria [3].</p> <p><strong>Results:</strong> All (17/17, 100%) KPC-producing K. pneumoniae isolates were able to grow at high concentrations of CAZ-AVI (≥64/4 mg/L), increasing their resistance to CAZ-AVI ≥8-fold. Likewise, fifteen of the 17 (88.2%) resistant isolates maintained the acquired CAZ-AVI resistance 15 days after without CAZ-AVI pressure. In addition, the CECT 997 mutants with blaKPC-2 or blaKPC-3 were able to grow up to 256/4 mg/L of CAZ-AVI, displaying and maintaining CAZ-AVI MIC shift from &lt;0.01/4 mg/L (susceptible) to 512/4 mg/L (resistant).</p> <p><strong>Conclusions:</strong> These data suggest that exposure of KPC-producing K. pneumoniae to subinhibitory CAZ-AVI concentrations could lead to the selection of CAZ-AVI resistance and this resistance is stable over the time.</p> Daniel Díaz-Roblizo Ángel Rodríguez-Villodres José Antonio Lepe Jerónimo Pachón Younes Smani Derechos de autor 0 2020-03-19 2020-03-19 9 PLA2G6-associated neurodegeneration (PLAN): Characterization of patients and drug screening <p>Neurodegeneration with brain iron accumulation (NBIA) envolve a group of rare neurodegenerative disorders characterized by brain iron accumulation, progressive extrapyramidal dysfunction (dystonia, stiffness, choreoathetosis), and presence of axonal spheroids, usually limited by the central nervous system.</p> <p>&nbsp;</p> <p>Within the differents subtypes of NBIA, in this study we focussed in PLA2G6-associated neurodegeneration (PLAN), diseases caused by a mutation in the phospholipase A2 group VI (PLA2G6). PLA2G6 encodes the enzyme iPLA2b, a calcium-independent phospholipase A2 which is involved in lipid metabolism. The loss of iPLA2b’s function result in mitochondrial abnormalities and synaptic transmission impairment in neurons among other alterations.</p> <p>&nbsp;</p> <p>In the current work we studied the pathophysiology of three confirmed cases of PLAN using fibroblasts derived from the patients: PLAN 10 (heterozygous mutation), PLAN 11(heterozygous mutation) and PLAN 8 (homozygous mutation). The aim of this study is to characterize, in patient-derived fibroblasts, the pathological alterations produced by PLA2G6 mutations. Main methods used were Western blot and Prussian Blue Staining. Our results confirm iron accumulation in patients-derived fibroblasts, impaired autophagy and ferritinophagy, especially in the patient that suffers the homozygous mutation (Plan 8). The purpose is to carry out a drug screening able to reverse the pathophysiology observed.</p> Diana Reche-López Irene Villalón-García José Antonio Sánchez-Alcázar Derechos de autor 0 2020-03-19 2020-03-19 9 Identification of bioactive compounds in diabetes and infertility models of C. elegans <p>Polycystic ovary syndrome (POS) is a complex and heterogeneous endocrine disease characterized by hyperandrogenism, oligo-anovulation and metabolic disorders, including insulin resistance, obesity and type II diabetes. Additionally, POS causes infertility in childbearing age women, making of great interest the search of new treatments and compounds for the improvement of the symptoms of this chronic disease. Despite the definitive pathological mechanism of POS is still unknown, as mentioned before there is a strong relation between the syndrome and different alterations in the insulin signaling pathway. This pathway is evolutionary conserved and it has a homologue pathway in the organism Caenorhabditis elegans. Mutations in the insulin receptor homologue daf-2 gene or in the phosphatidylinositol 3-kinase (PI3K) homologue age-1, result in reduction of fertility, being the decrease in progeny sharper in the allele age-1(mg305) mutant, in addition to a less pleiotropic effect. We consider this mutant of C. elegans a good model to study the insulin pathway, and specifically POS, due to the high degree of conservation of the pathway and the similarities between the phenotype caused by the mutation and the human symptoms.</p> E Olaizola-Bárcena M J Muñoz-Ruíz J M Monje-Moreno A M Brokate Llanos Derechos de autor 0 2020-03-19 2020-03-19 9 Ensemble and Greedy Approach for the Reconstruction of Large Gene Co-Expression Networks <p>In the recent years, the vast amount of genetic information generated by high-throughput approaches, have led to the need of new methods for data handling. The integrative analysis of diverse-nature gene information could provide a much-sought overview to study complex biological systems and processes. In this sense, Co-expression Gene Networks (CGN) have become a powerful tool in the comprehensive analysis of gene expression. Such networks represent relationships between genes (or gene products) by means of a graph composed of nodes and edges, where nodes represent genes and edges the relationships among them. Amongst the main features of CGN, sparseness and scale-free topology may notably affect the latter network analysis. Within this framework, structure optimization techniques are also important in the reduction of the size of the networks, not only improving their topology but also keeping a positive prediction ratio. On the other hand, ensemble strategies have significantly improved the precision of results by combining different measures or methods.</p> <p>In this work, we present Ensemble and Greedy networks (EnGNet), a novel two-step method for CGN inference. First, EnGNet uses an ensemble strategy for co-expression networks generation. Final score is estimated by major voting among three different methdos, i.e. Spearman and Kendall coefficients and Normalized Mutual Information. Second, a greedy algorithm optimizes both the size and the topological features of the network. Not only do achieved results show that this method is able to obtain reliable networks, but also that it significantly improves the topology of the networks.</p> <p>Moreover, the usefulness of the method is proven by an application to a human dataset on post-traumatic stress disorder (PTSD), revealing an innate immunity-mediated response to this pathology in accordance with previous studies. These results are indicative of the potential of CGN, and EnGNet in particular, in the unveiling of the genetic causes for complex diseases. Finally, the implications of CGN in biomarkers discovery, could lead research towards earlier detection and effective treatment of these diseases.</p> Francisco Gómez-Vela Fernando M Delgado-Chaves Domingo S Rodríguez-Baena Miguel García-Torres Davinia Federico Derechos de autor 0 2020-03-19 2020-03-19 9 Study of phytopathogenesis regulation through chromatine modification <p>Chromatin is a structure formed by DNA and protein found in eukaryotic cells. This structure serves to package and condense the DNA. Histones are the main proteins forming part of this structure. The histones have aminoacidic tails which modifications are highly involved in transcriptional control of genetic programs that lead essentials processes like mitosis or more complex processes as can be cell differentiation. We have done a phylogenetic study and have found 3 genes which can encode the enzymes in charge of histone H3 methylation at its lysine resides 9 (KMT1) and 36 (KMT3 and KMT2H). The lysine 9 methylation in H3 is the main hallmark of constitutive heterochromatin (1,2) and the H3 lysine 36 methylation is generally associated with active transcription (3). We have deleted these putative methyltransferases in Ustilago maydis. While KMT1 deletion has not showed a significant virulence phenotype, both H3K36 methyltransferases mutants present infection defects. Interestingly, meanwhile KMT3 deletion shows a reduction in virulence, KMT2H deletion improve pathogenesis. In addition, by western blot analysis we have observed a reduction of H3K36 trimethylation in both mutants respect the wild type strain, indicating there are two H3K36 methyltransferases with opposite roles in infection. These observations are in agreement with a recent publication where the fungus Fusarium fujikuroi is methylating H3K36 in heterochromatin and euchromatin area through two different methyltransferases (4). We are currently studying the differences between these methyltrasnferases at the molecular and cellular level during the pathogenesis process.</p> Ismael Fernández-Portillo Ramón Ramos-Barrales Derechos de autor 0 2020-03-19 2020-03-19 9 Crispr-cpf1 edition in the fission yeast to characterize the regulatory function of 3´UTR RNA loops in transcription termination <p>The wos2 gene encodes for the protein p23, which acts as a co-chaperone in the presence of the protein Hsp90. Their function is to fold proteins during a heat shock stress. This process is very similar both in human and the fission yeast<em> S.pombe</em>, our model organism in this project a. It was previously discovered that there exist three mRNA sizes depending on the 3’UTR length. Interestingly, the abundance of each depends on growth temperature. We then hypothesized that the secondary structure of the 3´UTR RNA could dictate the termination site depending on temperature. We are trying to demonstrate this assumption as well as to develop a temperature sensitive switch to control gene expression by inserting the wos2 3’UTR in the 5’UTR of rpl42S59Q gene marker. This allele is resistant to cycloheximide whilst the deletion is not. This way we may easily asses gene expression. To this end, we are using the CRISPR/Cpf1 technology, to engineer the genome region of interest. We are in the process of checking engineered candidates.</p> Jesús María Catalán-Casas Víctor Álvarez Tallada Derechos de autor 0 2020-03-19 2020-03-19 9 The study of the effect of mannose in GALE Caenorhabditis elegans mutants using as food source Escherichia Coli mutants defective in mannose metabolism pathway <p>Type III galactosemia is a rare disease characterized by mutations in the GALE gene that encodes the enzyme UDP-galactose 4-epimerase. The deficit of this enzyme gives rise to various physical and mental problems in humans (Walter et al. 1999). Several studies in gale-1 mutants of C. elegans worms have shown a&nbsp; positive effect of mannose (Brokate-Llanos et al. 2014), on the longevity and development of these mutants.</p> <p>These worms have as their main diet E. coli (OP50), so that the positive effects of mannose can be influenced by the transformation these bacteria can produce through metabolic transformation of mannose. Trying to eliminate this possible influence of OP50 mannose metabolism on these experiments, UV radiation inactivated&nbsp; E.coli&nbsp; has been used as worm food in the presence of different concentrations of mannose. This approach presents, among others, the problem that by inactivating bacteria by UV radiation we are eliminating their replication, but not their complete metabolism,demanding other experimental approaches as using E. coli strains unable to metabolize mannose to feed the worms.</p> <p>Therefore, the objective of this work is to study the effects of mannose in C. elegans, fed with OP50 mutants defective in different steps of mannose metabolism to assess the direct effects of mannose on C. elegans GALE mutants.</p> <p>- Construction of OP50 isogenic strains defective in different steps of mannose metabolism by transformation with mutant alleles obtained from E. coli (K12) mutant strains. OP50 was transformed by homologous recombination with fragments containing deletions in ΔmanA (gene for the enzyme of the sugars metabolism mannose derived pathway), ΔmanB (gene for the enzyme of the lipopolysaccharide synthesis mannose derived pathway) and ΔmanX (gene for one of the mannose transporters within the cell). These deleted genes were replaced by a kanamycin resistance gene.</p> <p>- Preparation of eggs of C. elegans and incubation in plates with different concentrations of mannose (0%, 1%, 2%, 3%).The E. coli used are the different mutant strains obtained and a control (wild type OP50).</p> <p>OP50 mutants for the genes indicated above were correctly obtained. They were tested with the GALE worms, and we observed that ΔmanA and ΔmanX show a great improvement in GALE mutants growth and development with respect to the wt OP50 control. Nevertheless, we did not observe these changes with ΔmanB. It seems that LPS pathway is probably important for the mannose asimilation.</p> José Ramón Sánchez-Santiago Patricia Lucas-Rodríguez Manuel Muñoz-Ruíz Ana María Brokate-Llanos Andrés Garzón-Villar Derechos de autor 0 2020-03-19 2020-03-19 9 Impact of Myo-inositol supplementation in the prevention of neural tube defects <p><strong>Motivation:</strong> Neural tube defects (NTDs) occur during early development by failure of neural tube closure and cause severe birth problems like spina bifida. It has been demonstrated that the mechanisms required during neural tube closure are regulated by genetic and environmental factors, such as maternal diet. Folic acid supplementation during pregnancy prevents the appearance of NTDs in 70% of the cases and the remaining 30% are considered folate resistant. For that reason, there is a need to find new supplements that could help preventing NTDs. One compound that is being tested is inositol, a simple carbon six sugar alcohol that participates in a diverse range of cellular functions. In clinical trials, inositol was used to prevent NTDs and the offspring from mothers that included inositol and folic acid in their diet during pregnancy did not develop NTDs. In our laboratory we are using Loop-tail, mutant of the member of the Wnt-PCP Vangl2, that in heterozygosity presents an incidence of 6% spina bifida. Previous studies using Loop-tail, revealed that a cellular aggregate originates in neural tube dorsal zone of heterozygous embryos. This aggregate, Sox10 positive, is formed by cells from the neural crest which did not migrate correctly. Besides, this cellular aggregate shares similarities with lipomyelomeningocele, the most common type of spina bifida occulta. In order to prevent the appearance of this cellular aggregate our laboratory previously used D-Chiro-inositol in Loop-tail mice as a supplement. Although the aggregate size and prevalence was reduced, crown-rump length of heterozygous embryos was significantly shorter than control embryos. Therefore, we are currently testing Myo-inositol, the isomeric form of inositol used in the human trials designed to prevent NTDs.</p> <p><strong>Methods:</strong> Myo-inositol was administered in the drinking water to pregnant females from day E1.5 of gestation until E11,5 and embryos were collected at stage E12,5. From each litter, data referring to number of implants, resorptions and genotype of the embryos was registered for possible effects on embryotoxicity. In situ hybridization using a Sox10 probe, neural crest marker, was later performed in order to study the presence and intensity of cellular aggregates.</p> José Moral-Rodríguez Beatriz María Fernández-Santos Patricia Ybot-González Derechos de autor 0 2020-03-19 2020-03-19 9 Membrane protein Oca3 is essential to keep structural integrity of mitochondria and endoplasmic reticulum <p>Mitochondrial function is tightly conserved through evolution since it becomes essential for the fitness of any eukaryotic cell. Defective function of this organelle represents the cellular basis of some severe diseases in humans. Thus, the characterization of genes involved in the correct mitochondrial structure and function is critical to understand and treating these diseases. In our laboratory, using the fission yeast as a model, we are characterising the function of <em>oca3</em> gene, the ortholog of EMC2 gene in human. This gene is predicted to be a member of the ER membrane protein complex involved in the mitochondrion-endoplasmic reticulum membrane tethering<sup>1</sup>. We find the protein in the non-aqueous phase in cell extracts and Oca3-mCherry tagging actually decorates most cell membranes. Oca3 over-expression cause lethality<sup>2</sup> and the gene deletion becomes cold-sensitive. In both situations aberrant mitochondria aggregations are observed and endoplasmic reticulum seems disorganised. Interestingly, addition of Tween20 restores the viability of <em>oca3</em> deletion at low temperature. This result suggests that Oca3 may have a role in membrane fluidity homeostasis. In addition to this, we will analyse different gene interaction between some of the EMC complex members to clarify both, the importance of Oca3 for the complex and the importance of the complex itself for the cell.</p> M Berraquero V A Tallada J Jiménez Derechos de autor 0 2020-03-19 2020-03-19 9 Relevance of p53 in the regulation of pro and antiapoptotic factors from the Bcl-2 family during the treatment with tirosine kinase inhibitors <p><strong>Motivation:</strong></p> <p>Liver cancer is the sixth most common cancer and the fourth most frequent cause of cancer-related death worldwide [1]. Nowadays, only one third of patients diagnosed with HCC are in the earliest BCLC 0-A stages, with a high proportion being in more advanced stages of the disease (BCLC B-C). Patients with the presence of poor prognostic factors such as vascular invasion, extrahepatic metastases and/or impaired hepatic function are considered in the advanced stage of the disease (BCLC C). Sorafenib is the standard of care for advanced HCC stage demonstrated in two large-scale trials [2]. Other drugs have been developed to increase the therapeutic arsenal of treatments. A phase III clinical trial comparing Lenvatinib and Sorafenib demonstrated that Lenvatinib was statistically non-inferior to Sorafenib in overall survival as a first-line treatment in patients with advanced HCC. Regorafenib and Cabozantinib have been shown to be effective as second-line therapy. [2]</p> <p><strong>&nbsp;</strong></p> <p><strong>Methods:</strong></p> <p>Sorafenib, Regorafenib, Cabozantinib and Lenvatinib were obtained commercially from Carbosynth Limited (Berkshire, UK). HepG2, Hep3B and Huh7 cell lines were obtained from American Type Culture Collection (ATCC; LGC Standards, S.L.U., Barcelona, Spain). HCC cell lines were maintained in supplemented Minimum Essential Medium with Earle's Balanced Salts (MEM/EBSS) at 37°C in a humidified incubator with 5 % CO2. Cells were seeded at a density of 100,000 cells/cm2 in 2D culture. Different parameters related to cell death and proliferation were associated with the expression of proapoptotic (Bak, Bax, tBid and Bim) antiapoptotic (Bcl-2, Mcl-1 and Bcl-xL) and regulatory (Beclin-1) Bcl-2 family members assessed by Western-blot analysis.</p> <p>&nbsp;</p> <p><strong>Results:</strong></p> <p>The administration of tyrosine kinase inhibitors induced cell death and reduced cell proliferation. This effect was associated with an upregulation of tBid and Bim expression in liver cancer cells. This effect was not observed in Hep3B and Huh7 which were less responsiveness to the proapoptotic and antiproliferative properties of tyrosine kinase inhibitors.</p> <p>&nbsp;</p> <p><strong>Conclusions:</strong></p> <p>The induction of cell death and antiproliferative properties of tyrosine kinase inhibitors were associated with the increase of the expression of different proapoptotic Bcl-2 family members. This expression appeared to be regulated by p53.</p> M I Caballos S Salas-Pino J Muntané Derechos de autor 0 2020-03-19 2020-03-19 9 Identification of compounds and genes that affect neurodegenerative diseases in Caenorhabditis elegans. <p><strong>Motivation:</strong> Neurodegenerative diseases have become a global health problem that need more research to find a solution. Our group has described in the organism model Caenorhabditis elegans that the loss of function of the sul-2 gene, which encodes a steroid hormone sulfatase homologous STS human, improvement the symptoms of neurodegenerative diseases. Likewise, the compound STX64, an inhibitor of steroid hormone sulphatases activity, mimics the lack of sul-2 function. It has been described that ssu-1, the only C. elegans gene orthologous to human steroid hormone sulfotransferase, is expressed in ASJ amphids neurons. The initial hypothesis is to see if the improvement of the symptoms of neurodegenerative diseases in the sul-2 mutant and with the STX treatment is due to the accumulation of sulfated hormone, or on the contrary, to the decrease of the non-sulfated hormone.</p> N Bellido-Alocolea M M Pérez-Jim´énez M J Muñoz Derechos de autor 0 2020-03-19 2020-03-19 9 Validation of STAT1 variants found in patients with primary immunodeficiencies and evaluation of the effect of JAK inhibition using an in vitro model <p>Regulation of cellular responses to interferons, cytokines, growth factors and hormones is mediated by signal transducer and activator of transcription (STAT) proteins. In the immune system binding of a cytokine (e.g. IFN) to the corresponding surface receptor, Janus kinase (JAK) molecules are phosphorylated, resulting in the docking and phosphorylation of the associated STAT proteins. The STATs will form homo or heterodimers and translocate to regulate transcription of pro-inflammatory target genes. Mutations in STAT1 are known to result in immunodeficiency and/or immune dysregulation syndromes.</p> <p>In this project, the functional impact of variants in the STAT1 gain-of-function gene (STAT1 GOF) will be analyzed on a protein level. The effect of a directed treatment approach targeting the JAK-STAT pathway (JAK inhibitors) will be evaluated in an in vitro model. Freshly isolated PBMCs or whole blood samples from patients and healthy controls were obtained. The cells were stimulated with IFNg and the treated with the JAK inhibitor Ruxolitinib. Extra and intracellular staining with anti-human fluorochrome conjugated antibodies was performed in order to determine the expression of STAT1 and pSTAT1 on monocytes by means of flow cytometry.</p> <p>Two pediatric patients and one related adult patient were studied and the pathogenicity of the variants was confirmed as STAT1 and pSTAT1 levels in the patients at baseline as well as after IFNg stimulation were markedly increased, when compared with healthy controls. The in vitro administration of different concentrations of the JAK inhibitor Ruxolitinib resulted in the normalization of pSTAT1 levels in the cells obtained from patients with STAT1 GOF mutations. Patients with STAT1 GOF mutations show a severe clinical phenotype with recurrent bacterial and fungal infections. Currently no specific treatment options are available. However recent case reports suggested the benefit of JAK inhibition. Therefore we studied the effect of this drug using primary cells in an in vitro model on a molecular level. Importantly two patients have been started on this medication and have achieved a significant clinical response. We are currently evaluating the capacity of our protocol to identify patients with alterations of the JAK-STAT pathway eligible for this targeted treatment approach.</p> Paloma Guisado-Hernández Pilar Blanco-Lobo Beatriz De Felipe Peter Olbrich Olaf Neth Derechos de autor 0 2020-03-19 2020-03-19 9 Scaling and variability of embryoid symmetry breaking <p>&nbsp;</p> <p><strong>Motivation:</strong> The formation of the main embryonic axis in mouse is thought to be controlled by extra-embryonic signals. Recent studies however, have shown that three-dimensional aggregates of mouse embryonic stem cells – embryoids – can spontaneously break their initial symmetry to establish an axis in the absence of extra-embryonic signals. This process is characterized by a moving front of mesendodermal fates marked by a region of high Nodal and Wnt signaling. Theoretical analysis suggests that this propagating front is established by a self-organizing bistable reaction-diffusion system. However, it is still unclear how a self-organizing system can robustly generate an embryonic axis independently of size and temporal variability.</p> <p><strong>Methods:</strong> To address this question, we are using a multi-disciplinary approach that complements experiments with modeling to understand and predict the dynamics of axis self-organization. On the theoretical side, we use mathematical analysis and simulations to identify parameters that control axis-formation scalability. In addition, we are exploring how external signals can control axis self-organization and reduce its variability. On the experimental side, we perform high-throughput live imaging of embryoids to monitor axis formation in normal situations and upon perturbations.</p> <p><strong>Results:</strong> Our model predicts that activating external signals can promote the emergence of a propagating mesendodermal front with an intrinsic temporal variability. In normal conditions, the front expands across the whole embryoid. In contrast, when inhibitory signals are added, the propagation of the mesendodermal front can be stopped. Crucially, the model predicts that the time between activation and inhibition determines the final extension of the axis. To test this prediction, we are imaging embryoids generated with a reporter cell line of Brachyury, one of the earliest markers of axis formation. In agreement with the model, our data shows that the axis emerges with an intrinsic variability of ten hours and propagates over the whole embryoid. Upon application of a small-molecule inhibitor of Nodal, the propagation of the axis is stopped at different positions depending on the time of axis formation.</p> <p><strong>Conclusions:</strong> To reduce this variability, we are devising an automated image analysis approach to apply the pharmacological inhibitor of Nodal depending on the time of axis formation. This project will demonstrate how external signals and self-organization are coupled together to achieve robust axis-formation during embryonic development.</p> Rosana Cáceres-Carrillo Jelena Raspopvic Luciano Marcon Derechos de autor 0 2020-03-19 2020-03-19 9 Proteaosome dynamics during quiescence in Schizosaccharomyces pombe <p>The Proteasome is one of the largest protein complexes present in eukaryotic cells and it is responsible for 90% of total protein degradation. Thus, proteasome availability and correct function are key elements in eukaryotic cells form yeast to human to deal with unfolded or unwanted proteins.</p> <p>When the concentration of cells in culture increases, cells initiate a metabolic reprogramming in order to become quiescent. During this developmental state of no proliferation, the proteasome is sequestered in cytoplasmic granules, so they are readily available when those cells resume growth. However, the mechanisms involved in proteasome storage and recycling are poorly understood.</p> <p>Here, we use the fission yeast as eukaryotic model to study proteasome dynamics. Previous work done in our laboratory have shown that the existence of two separate proteasome pools: one cytoplasmic and one nuclear.</p> <p>The objective of this work is to study the formation, composition and dissolution of the proteasome storage granules (PSGs), and to analyze consequences for cell survival in conditions in which PSGs are not formed during quiescence.</p> <p>To accomplish this, we are using a collection of deletion of kinases to try to interfere with proteasome storage signalling, and a mutant required for the synthesis of ubiquitin, the molecule that target proteins for degradation via proteasome. All these mutants were expressing the proteasome subunits tagged with either GFP or tomato as proteasome marker. We used confocal imaging, fluorescence recovery after photobleaching (FRAP) experiments and cell survival assays in cells exposed to low glucose concentration to promote quiescence to induce PSGs and study their localization and dynamics.</p> <p>Our result show that whereas none of the kinases assayed so far present significant defects in PSG formation, a mutant defective in the polyubiquitin gene (ubi4), shows a severe reduction in the number of cells capable of generating these PSGs in low glucose. This result suggests an important role of the ubiquitin in the formation and/or composition of PSGs. We are currently checking deletion of additional kinases, analyzing the rate of PSGs dissolution upon refeeding with rich media, as well as setting up assays to determine the rate of cell survival in conditions of defective PSG formation.</p> Yosu Odriozola-Gil Gabriel Romero-Ruíz Rafael Rodríguez-Daga Derechos de autor 0 2020-03-19 2020-03-19 9 Membrane protein Oca3 is essential to keep structural integrity of mitochondria and endoplasmic reticulum <p>Mitochondrial function is tightly conserved through evolution since it becomes essential for the fitness of any eukaryotic cell. Defective function of this organelle represents the cellular basis of some severe diseases in humans. Thus, the characterization of genes involved in the correct mitochondrial structure and function is critical to understand and treating these diseases. In our laboratory, using the fission yeast as a model, we are characterising the function of <em>oca3</em> gene, the ortholog of EMC2 gene in human. This gene is predicted to be a member of the ER membrane protein complex involved in the mitochondrion-endoplasmic reticulum membrane tethering<sup>1</sup>. We find the protein in the non-aqueous phase in cell extracts and Oca3-mCherry tagging actually decorates most cell membranes. Oca3 over-expression cause lethality<sup>2</sup> and the gene deletion becomes cold-sensitive. In both situations aberrant mitochondria aggregations are observed and endoplasmic reticulum seems disorganised. Interestingly, addition of Tween20 restores the viability of <em>oca3</em> deletion at low temperature. This result suggests that Oca3 may have a role in membrane fluidity homeostasis. In addition to this, we will analyse different gene interaction between some of the EMC complex members to clarify both, the importance of Oca3 for the complex and the importance of the complex itself for the cell.</p> M Berraquero V. A. Tallada J Jiménez Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-03-19 2020-03-19 9 Analytical method validation: determination of power of hydrogen (pH) and electrolytic conductivity in drinking water and surface water <p>On many occasions, when it is necessary to measure the constituents or physicochemical properties of samples from multiple sources, quality control companies require a measurement methodology. Its development needs a considerable investment of time to ensure the reliability of the analytical assay. Quality control companies through pilot studies supported by the scientific method achieve this reliability. This process is known as analytical method validation. Validation will demonstrate that the analytical method complies with the criteria applicable for the relevant performance characteristics.<br>Nowadays, quality control companies analyze water sample from many sources. The present situation highlights the need to standardize methods capable of assessing or quantifying the parameters that measure the level of water quality. Thus, the legislation in force has included all necessary criteria, measurements and parameters to guarantee its quality, safety and cleanliness to protect people and environment from the adverse effects that water pollution may cause for all living beings.<br>pH measures the activity of hydrogen ions in solution. Electrolytic conductivity is defined as the flow of electricity through a solution. pH measurement is important in drinking water treatment because it affects process controls such as chlorination, coagulation and corrosion. Water conductivity is often related to the concentration of dissolved mineral salts or filterable residue. Deviations from normal conductivity may be a sign of changes in the mineral composition of the source water, periodic variations, chemical fluctuations or the forced entry of industrial waste. Measuring electrolytic conductivity can help determine the amount of ionic reagent needed to affect precipitation or neutralization reactions in water treatment</p> Álvaro Aranda Torrecillas Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-05 2020-04-05 9 uning and validation of an analytical method for the determination of petroleum hydrocarbons in water using gas chromatography and FID detector. <p>Total oil hydrocarbons (TPHs) are released into the environment mainly by polluting the water on the surface, especially in areas close to production and storage places, but also during the handling, transport and processing of those products. The assessment of this contamination is carried out by measuring the concentrations of petroleum products in water [1]. The most commonly used methods for determining TPHs are infrared absorption (IR) and gas chromatography (GC). However, the cancellation of the IR absorption method in Europe due to the ban on the use of freons (necessary for the extraction of hydrocarbons in the sample) resulted in the GC being the most commonly used, in addition to its high sensitivity, selectivity and wide range of hydrocarbons capable of detecting [2].</p> <p>This study aims to fine-tune and validate a method capable of identifying and quantifying chain hydrocarbons between C7-C40 using gaseous chromatography with a flame ionization detector (GC-FID) in all types of water, in the one that everybody consumes and in non-drinking water. After an exhaustive analysis of the available bibliography, different methods that are currently being used for the quantification of TPHs have been compared and analyzed, finally selecting a method described by UNE-EN ISO 9377-22:2001 [3].</p> <p>This regulation includes the extraction and chromatographic method. Regarding the first, the extraction method is carried out thanks to a purification column with Florisil and using dichloromethane as a solvent, as well as evaporation techniques for its concentration. For the chromatographic method, a chromatograph adjustment and a calibration line shall be made with pattern solutions formed by a mixture of n-alkane mineral oils. This is ultimately intended to validate the method so that the analytical laboratory can offer its customers reliable and reproducible results.</p> Brinidilda Dolores Cofrade Romero María de la Menta Ballesteros Martin María del Mar González Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-05 2020-04-05 9 Microalgal biomass production in bubble column using urban wastewater from secondary treatment <p><strong>Motivation:</strong> Currently, urban wastewater treatment plants generate treated effluent, which contained pollutants that are discharged into aquatic resources. Microalgae for its growth could use these contaminants/nutrients. This use open the possibility to achieve three goals: i) Complete wastewater treatment, ii) The generation of algal biomass rich in energetic compounds, and iii) the incorporation of atmosphere or industrial carbon dioxide to the algal culture by its injection into the culture media with the aim to reduce the greenhouse effect (Malvis et al., 2019).</p> <p><strong>&nbsp;</strong><strong>Methods:</strong> For the complete urban wastewater treatment, microalgal cultures in bubble columns were proposed. In this sense, all the experiments were performed under natural environmental conditions. The microalga Chlorella vulgaris was used under different aeration rates (varied from 1 L/min to 5 L/min). The common operating conditions were pH of the culture media 9.0, ambient temperature and solar light.</p> <p><strong>&nbsp;</strong><strong>Results:</strong> The algal culture media in urban wastewater from secondary treatment registered variation in the values of maximum specific growth rate (0.00390-0.00942 1/h) and volumetric biomass productivity (0.0261-0.439 mg/(L h)) under different aeration rates. Both parameters were registered its highest value at aeration rate equal to 3 L/min. TOC removal was decreased with the aeration rate increase and varied in the range 14 % to 62.3 %.</p> <p><strong>&nbsp;</strong></p> <p><strong>Conclusions:</strong> The use of Chlorella vulgaris for its growth in urban wastewater from secondary treatment was verified. The highest values for maximum specific growth rate (0.00943 1/h) and biomass productivity (0.439 mg/(L h)) were determined in culture worked with aeration rate equal to 3 L/min</p> Gassan Hodaifa Mayra A. Vargas-Porras Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-05 2020-04-05 9 CRISPR/Cas9 system optimization in Pseudomona syringae using a heterologous repair system <p><strong>Motivation:</strong> The methods of genetic manipulation in most Pseudomonas species, such as Pseudomonas syringae, remain a laborious endeavor. However, the application of the CRISPR-Cas9 system in this bacterium would allow a means of fast and directed genetic edition. The CRISPR-Cas9 system produces a doubles-stranded DNA break by the Cas9 protein. This break can be repaired by the NHEJ machinery in a very low frecuence either by the homologous repair system, which is widely spread amoung prokaryotes. The bacteria Pseudomona syringae pv. phaseolicola and pv. tomato are phytopathogenic causing diseases that affect crops of high economic value. Therefore, the optimization of a genetic modification system for these bacteria is very important.</p> <p><strong>Methods:</strong> In this study we report the development of the CRISPR Cas9 technology in Pseudomonas syringae pv tomato and pv. phaseolicola by using through the transformation of this bacteria with standardized pSEVA plasmids. After incorporating, the plasmids expressing the Cas9 nuclease and the guide RNA (gRNA), the Cas9 protein is complexed with the gRNA designed to direct a double-strand break to the pyrF gene. This lesion might be repaired by the bacterium intrinsic repair systems, but with a very low frequency. In order to solve this problem, a plasmid family has been used for expression in Pseudomonas syringae of three bacterial NHEJ systems from Sphingopyxis granuli, Mycobacterium tuberculosis and Mycobacterium smegmatis.</p> <p><strong>Results:</strong> The results shown in this study provide a step ahead towards the genetic modification in Gram negative bacteria by using the CRISPR Cas9 system and a non homologos end joining system together. Nevertheless it should be emphasised that the Cas9 protein can be toxic in this bacteria producing non-targeted mutations, because of that is very important keep the nuclease cas9 as little time as possible in the bacterium</p> Estela Sanz Martí Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-05 2020-04-05 9 Wastewater and biological fanges treatment: reduction of phosphorus with chemical and biological products <p><strong>Motivation:</strong><br>Wastewater treatment is both an important and difficult topic nowadays due to the negative effects it can cause on the environment and people’s health. One of these problems is he presenc of organic nutrients such as nitrates, nitrites and phosphates.<br>In our project we have studied the possibility of combining some of the already used chemical coagulants with natural and biodegradable coagulants to remove phosphorus in wastewater by alternative cogualation-flocculation processes.<br><strong>Methods:</strong><br>Within the methodology used, we have used the Jar- test together with different chemical and biological reagents to perform the coagulation-flocculation process. In this process, the solids and nutrients of the water sample decant obtaining the clarified to be analyzed. Once obtained, filtration tests are performed and the amount of soluble phosphorus is analyzed by spectophotometry as well as the pH, turbidity and conductivity to determine the effectiveness of the coagulants used.<br><strong>Results:</strong><br>Among the results, we can add roughly that the chemical caogulants already used work better in conjunction with biologic coagulants not used until now, such as agar, different types of cellulose and starches. The use of these components are also beneficial for industrial companies which work with these kind of wastes because they are cheaper and easier to obtain.<br><strong>Conclusions:</strong><br>Biological coagulants are more effective for reducing the percentage of phosphorus in water, so there are many that have not yet been studied and that could be more beneficial for the environment and for the performance of companies, so it would be interesting that research lines continue to open in order to deepen this issue.</p> Macarena Gata Montero Eva Rodríguez González Ana Moral Rama Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-05 2020-04-05 9 Smart bactericides, design, synthesis and characterization. <p>Preventing microbial resistance to antibiotics is one of the most important challenges of our times, because multiresistant microorganisms are increasingly being reported. An approach based on silver nanoparticles is promising, given that this type of particles has been proven to exhibit antimicrobial activity. In addition, green strategies would be desirable, in which harmful chemicals are replaced by natural products to generate nanoparticles. Specifically, tannic acid (a phenolic metabolite present in many plants) has been used in addition to silver, and Coppo E et al. (2014) report their antimicrobial effects against various types of bacteria, including Escherichia coli (bacteria used in the model). Several synthesis methods have been previously described in combination with characterization by Raman spectroscopy (Dadosh 2009; Cao et al. 2014). However, we have found out that it is essential that the tannic acid solution used in the synthesis is neutralized before adding it to the silver solution to obtain the desired nanoparticles in a green synthesis. We have characterized our nanoparticles by UV-Vis spectrophotometry, and measured their hydrodynamic size and electrostatic stability by dynamic light scattering, which revealed an average size of 10-12 nm and a Zeta potential below -30mV. We have measured the antimicrobial activity using the minimum inhibitory concentration method, which, according to our preliminary results, indicate that said nanoparticles have a high antibacterial power against E.coli Dh5-α at low concentrations of the order of (15-20) μg of nanoparticles/ml. Our goals are to further adjust the range of concentrations and determine what would be the optimum concentration to ensure permanent antibacterial activity. With all the aforementioned, it can be stated that this type of nanoparticles is a very interesting proposal for the challenge of microbial resistance to antibiotics.</p> María Dolores Cortés Sánchez Patrick Jacques Merkling Ana Paula Zaderenko Partida Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-05 2020-04-05 9 Increasing oil accumulation in olive mesocarp: Cloning and characterization of phospholipid: diacylglycerol acyltransferase (PDAT) genes from olive fruit <p><strong>Motivation:</strong> To identify, characterize and study the regulation of genes and enzymes involved in the biosynthesis of triacylglycerols, in order to increase oil accumulation in olive mesocarp. In particular, to investigate the contribution of phospholipid:diacylglycerol acyltransferase (PDAT) genes (Dahlqvist et al., 2000). &nbsp;</p> <p><strong>Methods:</strong> Isolation of full-length cDNA clones by PCR, sequencing of amplified fragments, and sequence analysis using bioinformatic tools. Gene expression analysis using quantitative real-time PCR (qRT-PCR). Functional expression in a mutant of Saccharomyces cerevisiae (Sandager et al., 2002), using a yeast vector containing an inducible GAL1 promoter.</p> <p><strong>Results:</strong> A cDNA sequence, designated OepPDAT2, encoding phospholipid:diacylglycerol acyltransferase (PDAT) has been isolated from olive. Gene expression analysis has been performed in Picual and Arbequina cultivars using different olive tissues and in olive mesocarp under different abiotic stresses such as draught, low and high temperature, darkness and wounding. Overexpression in yeast is being carried out in a quadruple mutant of S. cerevisiae that cannot synthesize triacylglycerols to confirm gene identity.</p> <p><strong>Conclusions:</strong> Sequence analysis suggests that the OepPDAT2 gene encodes a PDAT enzyme. Gene expression analysis in olive seed and mesocarp tissues indicates that OePDAT2 participates in triacylglycerol synthesis during development and ripening of olive fruit, and it is transcriptionally regulated by different environmental factors</p> Úrsula García-Conde M. Dolores Sicardo José M. Martínez-Rivas Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-06 2020-04-06 9 Development of vectors and specialized Streptomyces strains for functional metagenomics <p><strong>Motivation:</strong> Antimicrobial resistance (AMR) are one of the most important existing threats to public health aggravated by the<br>overuse of antimicrobials. It is estimated that the mortality from drug-resistance infections would increase up to 10 millions by<br>2050. One of the major hurdles in microbial ecology is the inability to culture most of the microbial diversity present in ecosystems<br>under laboratory conditions. The molecular analysis strategies used to examine the microbial community DNA, also known as<br>the metagenome, have been denoted as metagenomics techniques. With the use of functional metagenomics we are focus our<br>efforts on searching new antimicrobials. For the expression of genes from metagenomics DNA, Escherichia coli is the host most<br>used for function-based screening. However, the potential of Streptomyces as a surrogate host for the production of heterologous<br>proteins has shown to be interesting for expression of proteins that are difficult to express in other bacterial host system.<br><strong>Methods:</strong> Basing on previous work, we are using a construction of specialized vectors with modified heterologous expression<br>system that incorporate viral components. The construction consist in the T7 RNA-polymerase gene (gene1), synthesized with<br>a codon optimization for Streptomyces, that encodes the phage RNA-polimerase which is insensitive to many of the bacterial<br>termination signals. The gene1 expression is regulated by a inducible expression system based on PnitA-NitR regulatory system.<br>To test that our system work properly, we have introduced in the cosmid for the metagenomic library construction, which is<br>replicative in Streptomyces, a reporter gene (xylE) whose gene product is a catechol dioxygenase which converts the colourless<br>substrate catechol to an intensely yellow oxidation product.<br><strong>Results:</strong> This part of the project aims to evaluate the efficience of heterologous expression measuring the catechol dioxygenase<br>activity in solid and liquid media. In solid media we have seen that the Streptomyces colonies turned yellow when catechol was<br>applied indicating that the expression system is working efficiently. The main objective of this project is to develop a metagenomic<br>library, with an improved heterologous expression, from DNA originating in several strains of Streptomyces in order to search<br>for new antimicrobial resistance.</p> Álvaro Cebrián García Eva María Camacho Fernández Amando Flores Díaz Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-06 2020-04-06 9 Role of phenylacetaldehyde reductase (OePAR) in olive fruit phenolic metabolism <p><strong>Motivation:</strong> Virgin olive oil (VOO) is a key component of the Mediterranean diet very rich in phenolic compounds related to important health benefits [1]. These phenolic compounds have antioxidant biological activity and they also provide organoleptic properties to VOO. Oleuropein, with a complex secoiridoid structure containing an hydroxytyrosol residue, is the most abundant phenolic compound in the olive fruit mesocarp. Moreover, oleuropein is the precursor of the most important phenols found in VOO [2].<br>In this study, the molecular and biochemical characterization of two enzymes phenylacetaldehyde reductases (OePAR1.1 and 1.2) related to hydroxytyrosol and tyrosol biosynthesis in olive fruit, has been carried out. These enzymes use as substrates, 3,4-dihydroxyphenylacetaldehyde (3,4-HPAA) and 4-hydroxyphenylacetaldehyde (4-HPAA) to form hydroxytyrosol and tyrosol, respectively [2,3].<br><strong>Methods:</strong> Two genes encoding for enzymes with putative phenylacetaldehyde reductase activity have been identified from an olive transcriptome which was obtained from seven olive varieties with different phenolic profiles grown under different biotic and abiotic stresses [2]. They have been synthesized, cloned and expressed in Escherichia coli. The extraction and purification of their encoded proteins has been optimized. Purity of the recombinant proteins was evaluated by electrophoresis (SDS-PAGE) and densitometry analysis. Concentration of the purified recombinant proteins was determined by Bradford assay. The functional identity of these proteins have been determined by in vitro activity assays further analyzed by HPLC to quantity substrates and products in the reaction media. The developed methodology is being used to complete the kinetic characterization of both proteins. In addition, complementary studies of gene expression will be performed by RT-qPCR.<br><strong>Results:</strong> Both recombinant OePARs display reductase activity forming hydroxytyrosol after incubation with its precursor 3,4-HPAA. Furthermore, OePAR1.2 has shown significantly higher specific activity than OePAR1.1. Results from functional genomic, expression analysis and metabolomic studies carried out will be analyzed together to determine the role of these enzymes in the olive fruit phenolic metabolism and to establish the catalytic differences between them.</p> Cristina Bahamonde Pereira Mª Begoña Herrera Rodríguez Mª Rosario Sánchez Rodríguez Ana Gracia Pérez Rubio Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-06 2020-04-06 9 Analysis of biological aging yeast in Andalusian wines with competitivity molecular markers. <p>Biologically aged sherry and sherry-like wines (namely fino and manzanilla wines) age by an aerobic process which depends on the oxidative activity of flor strains of Saccharomyces cerevisiae. Upon depletion of fermentable carbon or nitrogen sources these yeasts aggregate, creating a floating biofilm on the wine surface which is known as flor or veil. Wineries that produce this type of wine are very interested in the preservation of a distinctive aroma and flavor of their own wines. Choosing the right flor yeast to colonize their casks could result in a better control of wine properties. Moreover, the veil needs to be formed as fast as possible each time wine is removed or added to the cask, to avoid wine oxidation. Because of that, the implantation of a desired yeast strain in the shortest period of time would be very convenient.</p> <p>In our study we are analyzing the microbial population of flor yeasts present in 5 different wines from a winery at PDO Jerez-Xeres-Sherry. Our main goal will be to identify yeasts providing specific characteristics to the aging wine.</p> <p>In addition, we want to study the best way of preadapting or conditioning these strains, so they can form the biofilm over the wine faster than using conventional methods, avoiding oxidation and creating a controlled microbial environment to get a wine with consistent, desired properties.</p> <p>On this poster we present our results on the diversity found in wine flor biofilm from this winery using nuclear microsatellite markers and mitochondrial RFLP. We also present results on the influence of the preadapting culture pH and of inoculum size in the rate of biofilm formation.</p> <p>Further experiments will be made to determine the best way to incorporate the yeast into the wine (such as lyophilized, dehydrated or compressed) and to make them more competitive</p> Alejandro Cubilla Parejo Juan Quintero Blanco Andrés Garzón Villar Juan Jiménez Martinez Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-06 2020-04-06 9 Characterization of plant growth promoting bacteria isolated from red fruits. Studies on growth promotion and fruit quality in strawberries plants <p>Microoganisms associated to the rizosphere of cultivated plants used for human consumption are scarcely analyzed. However, nowadays, organic farming, where the use of microbial inoculants is essential, has arisen as an emergent alternative with great commercial interest.</p> <p>In this study, a collection of bacterial strains isolated from strawberry and blueberry rhizosphere (healthy and infected with Macrophomina phaseolina) as well as from the inside of stolons of strawberries plants (endophytic bacteria) has been constructed and characterized by their PGP and biocontrol properties.</p> <p>Three PGP properties have been determinate: auxin and siderophores production and phosphate solubilization. Regarding biocontrol activities, the presence of five enzymatic activities have been determined: protease, chitinase, cellulose, amylase and β-Glucosidase. On the other hand, the ability of the isolated strains to inhibit under in vitro conditions the growth of two pathogenic fungi of rump fruits, TOR 102 and TOR 872 (both belonging to the specie M. phaseolina) was tested. Strains reaching the better results were sequenced and identified as: Cupriavidius metalliduras, Bacillus proteolyticus, Arthrobacter pascens, Bacillus amyloliquefaciens, Raoultella planticola, Enterobacter roggernkampii, Bacillus megaterium, Pseudomonas multiresinivorans, Bacillus invictae, Pseudomonas aeruginosa, Chryseobacterium cucumelis, Klebsiella pneumonia, Achromobacter denitrificans, Bacillus velezenvelezensis, Burkholdelia contaminans, Bacillus niacin, Pantoea annatis, and Bacillus frigoritolerans.</p> <p>After that, a strawberry growth promotion assay was performed under controlled conditions. Strawberries plants were inoculated with three bacterial strains previously characterized by its high level of auxin production, namely Enterobacter rooggenkampii (AC8), Chryseobacterium cucumelis (ACH2) and Klebsiella pneumoniae (ACH7t). A greenhouse assay was carried out, with 6 replicates per treatment, including three strains as well as an uninoculated control. Biometric parameters (flowering precocity, number and weight or fruits, root and shoot dry weight), as well as quality ones (fruit size and Brix degrees) were determined at the end of the assay. Results showed that strains ACH7t was significantly superior in flowering precocity and number of fruits, while strains AC8 and ACH7t showed Brix values significantly different than the other treatments.</p> Inés Canosa Pérez-Fragero Maria Camacho Soledad Cárdenas Rodríguez Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-06 2020-04-06 9 A universal new generation vaccine against type I Porcine Respiratory and Reproductive Syndrome Virus <p>Porcine respiratory-reproductive syndrome (PRRS) is a devastating disease for the global porcine industry. PRRS leads to economic losses above 664 million dollars per year only in the USA and showing an increasing trend. The disease is affecting with a similar magnitude the porcine industry of the European Union. PRRS is caused by an enveloped, single stranded RNA virus (PRRSV) from the Arteviridae family. PRRSV interacts with porcine monocytes, weakening the immune system of the host and leading to pathologies affecting the respiratory and reproductive systems. Because of their immaturity, piglets are very vulnerable to PRRSV infection. Treatment and quarantine are not viable choices for the porcine industry. Therefore, prophylactic vaccines are the best alternative to prevent PRRS. However, the currently marketed vaccines are failing in protecting piglets in all regions of the world. The causes of failure are basically two. On one hand, the use of live attenuated vaccines needed for a complete antigenic presentation is preserving the immune evasion mechanisms of the wild-type virus. On the other hand, high divergence between viral serotypes leads to almost no cross-reactivity of the immune response between viral strains.<br>This project aims to develop a new PRRS vaccine capable of creating a complete immune response in piglets that neutralizes early-stage infection by any of the viral serotypes running in the USA and the EU. These premises could be possible thanks to the flexibility typical of a recombinant subunit vaccine, which has been designed to improve the usual low antigenic presentation of this type of vaccines. We have successfully produced a recombinant baculovirus for accumulation of the vaccine’s active ingredient in insect cells, in the form of protein bodies formed by using a tag derived from vegetal zein proteins (Zera® technology). Expression tests in insect cells confirmed the viability of this production system, obtaining 13 mg/L of purified antigen. The preliminary immunogenicity tests in murine model showed that the new vaccine is immunogenic in vivo and that it could be manufactured formulating the final product in a lyophilized format. We have made experimental batches of the vaccine and designed an experimental trial with challenge in piglets, in order to test the efficacy of our vaccine in comparison with commercial gold-standards. The trial will begin in March 2020 and the results will be part of this work.</p> Sergio Andrade Raquel Cid Juan José Infante Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-06 2020-04-06 9 Role of polyphenol oxidase in olive metabolism <p>Virgin olive oil (VOO) is the most important fat in the Mediterranean diet, which has many nutritional benefits and excellent organoleptic properties. These characteristics are mainly linked to the phenolic compounds present in VOO. The content of phenolic compounds in VOO is related to the initial content of phenolic glycosides in the olive fruit, which are later transformed by hydrolytic and oxidative enzymes to form the main phenolic components of VOO [1].<br>The main enzyme involved in the oxidative degradation of phenolic compounds during the oil extraction process is the polyphenol oxidase (PPO). This enzyme may catalyse two different reactions, hydroxylation of monophenols to form orthodiphenols (monophenolase activity), or oxidation of these orthodiphenols to quinones (diphenolase activity). Thus, PPO could display an important role both in the degradation and biosynthesis pathways, which means that these pathways could be connected [1]. In consequence, the functional characterization of olive PPOs is needed to understand the metabolism of phenolic compounds.<br>Two polyphenol oxidase genes (OePPO1 and OePPO2) have been identified from a transcriptome obtained from diverse olive varieties, with different phenolic contents, and submited to various biotic and abiotic stresses [3]. Both genes were synthesized, cloned and expressed in E. coli (BL21). Extraction and purification protocols of the recombinant proteins have been optimized using affinity chromatography [2]. Similarly, specific activity assays and HPLC analysis methods have been designed for the biochemical characterization of the recombinant proteins.<br>The functional identity of both proteins has been verified, since both of them have shown to be active against orthodiphenols (hydroxytyrosol and oleuropein), and their catalytic characterization is being completed using natural phenolic substrates from olive fruit (oleuropein and verbascoside) and VOO (hydroxytyrosol, tyrosol and acetate of hydroxytyrosol).<br>Later, expression studies by qRT-PCR will be carried out and, finally, the expression, functional genomics and metabolomics data will be analyzed together to determine the specific role of these enzymes in the metabolism of phenolic compounds in olive trees.</p> Laura Arroyo Morales María Begoña Herrera Rodríguez Rosario Sánchez Rodríguez Ana Gracia Pérez Rubio Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-06 2020-04-06 9 Comparative study of different methods for determining moisture in differents food matrices <p>The determination of moisture in food is a very important process because the moisture content affects the processing capacity, the shelf life, and the quality of the product. Therefore, moisture content determination plays a key role in ensuring quality in many industries, such as food, pharmaceutical and chemical.<br>The objective of this study was to evaluate the differences between the internal method used in Laboratorios Vital and different external methods of the Official Association of Analytical Chemists (AOAC) or the International Organization for Standardization (ISO). External methods evaluated were: ISO 662:2016, ISO 3727-1:2001, ISO 665:2001, AOAC 984.25, AOAC 931.04, AOAC 926.08, AOAC 968.11, AOAC 934.01, AOAC 941.08, AOAC 925,45C, ISO 5537:2004, ISO 579:2013 and AOAC 934.06. By means of this study, what is intended is to demonstrate the validity of the internal method being that each external method of the AOAC or ISO has associated the use of a specific temperature and time which would require more time to perform the moisture determination.<br>To carry out this study, 12 different food matrices were introduced in a Dinko D60D stove: butter, oil seeds, frozen chips, chocolate, cheese, roasted coffee, tea, ice cream, honey, milk powder, coke and dried fruit.<br>Moisture analysis were performed for 5 non-consecutive days and every day 2 replicates of each food matrix were analyzed.<br>After carrying out the different tests, the results were compared and it was observed that there were no significant differences in the determination of the percentage of moisture. This allows to confirm that the internal method used by the company is reliable for the determination of this parameter, which has clear economic advantages for the analytical laboratory that reduces the time of delivery of data to customers by almost a week.</p> María de la Menta Ballesteros Martín Fernando Andrada Franco María De La Rosa Herrera Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-06 2020-04-06 9 Validation of the standard method EN ISO 19343:2017 for detection and quantification of histamine in different matrices of fish and fishery products using high performance liquid chromatography <p><strong>Motivation:</strong> Histamine, a biogenic amine produced by bacterial decarboxylation of histidine, is the main cause of scombroid fish poisoning and is strictly regulated by the European Union in fish and fishery products [1]. Therefore, an adequate histamine quantification method is necessary in order to assess current regulations and prevent health problems. In this sense, High Performance Liquid Chromatography (HPLC) have been widely used for the detection of histamine as it provides good specificity and sensitivity when coupled with a highly sensitive detector. The standard protocol EN ISO 19343 is the current in use and it was developed to meet with 2073/2005 European Regulation about microbiological criteria for food products. However, there is still room for improvement in terms of resolution, sensitivity and discrimination and the validation of this protocol is a necessary step in laboratories to prove the reliability of results and a previous step to get ISO certification [2].<br><strong>Methods:</strong> In this study, we use the HPLC method-based ISO 19343:2017 to compare the results from different samples of raw fish, canned fish, fish sauce and fishery derived products. This method allows the separation of histamine within the set of biogenic amines from fish and fishery products. The sample is extracted by mixing it with perchloric acid. A derivatization is carried out prior passing through a chromatographic column using dansyl chloride to do histamine detectable under UV light. Biogenic amines and components in the solution are separated by HPLC using a chromatographic column, with UV detection. Histamine concentration is calculated from the ratio between the areas of histamine peaks and internal standard, using a calibration curve.<br>In the validation process, veracity of the method is studied, examining parameters such as accuracy, recovery and correction. The work interval is defined between the lower and upper quantification limit. The specificity / selectivity of the method is studied and verified by performing exercises in different matrices with different compositions and checking that the quality criteria are met independently of the matrix composition. In this way, and if the parameters are within the limits established by the standard, the method would be validated [3].</p> Manuel Jiménez Peña Fernando Andrada Franco María Menta Ballesteros Martín Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-06 2020-04-06 9 In silico discovery of Acinetobacter baumannii genes involved in microaerobiosis resistance <p><strong>Motivation:</strong> the infectious ability of Acinetobacter baumannii combined with its antibiotic resistant profile turn this bacteria into a objective with global priority, being currently highlighted by the World Health Organization as one of the most relevant resistant bacteria. Thanks to the development of Next-Generation Sequencing Methods, we can apply bioinformatics tools to analyse data that give us information about the behaviour of the bacteria under different conditions, which gives us the opportunity to discover new pharmacological targets that allow us to fight against infections by A. baumannii.<br><strong>Methods:</strong> We use data from RNA-Seq technique, obtained from A. baumannii ATCC 17978 growth in two different conditions: normoxia (21% O2) and microaerobiosis (0.1-0.3% O2). The first one is the regular situation, while the second one is the condition that the bacteria suffer when an infection occurs, especially across an injury, during the inflammatory phase. The results of this transcriptomic experiment were subjected to a bioinformatic workflow, starting with the quality analysis and trimming process, followed by the alignment of the reads and their quantification, until the differential expression analysis, whose results were filtered according to fold change value (R2&gt;=1) and p-value (padj&lt;0.05). Additionally, we want to improve the current annotation from A. baumannii ATCC 17978 genome. For that purpose, three sources of candidates were combined: GenBank available information, Prokka predictions and AnaBlast predictions. With this annotation, we can associate possible paths and processes in which the differential expression genes could be implicated, by a functional enrichment analysis using GO terms and KEGG, and the packages from R programming languages, such as Bioconductor and Clusterprofiler.Results: Resultados obtenidos. No tienen porqué aparecer todos los apartados (se puede prescindir de alguno de ellos, o todos). No más de 2500 caracteres el total del resumen. Incluir aparte (en el apartado de debajo) 1-3 referencias bibliográficas.<br><strong>Conclusions:</strong> The differentially expressed genes are enriched in X and Y, and these pathways have later been reviewed and completed by manual annotation using specific proteins databases. This could give us key information about the behaviour of the bacteria when it is found in hypoxia during the infection, and we could even find some factors involved in its virulence.</p> Nuria Fernández Rivera Alejandro Rubio María Luisa Gil Marques Maria Eugenia Pachón-Ibáñez Jerónimo Pachón Younes Smani Antonio Pérez Pulido Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-06 2020-04-06 9 Roll of Mrf4 gen in homeostasis of adult skeletal muscle. <p><strong>Myogenesis is the process that controls skeletal muscle growth during embryonic and postnatal development, and it is orchestrated by a family of four transcription factors (TFs) known as MRFs (Myogenic Regulatory Factors)(1). Our group is working on the function of two of these genes: Mrf4 and Myf5. We and others have established a link between MRFs function and muscle atrophy/hypertrophy (2).</strong></p> <p><strong>&nbsp;</strong></p> <p><strong>Mrf4 is the only MRF highly expressed in all adult skeletal muscles, but a complete understanding of its function is lacking as previous KOs models affect Myf5 expression in cis (3). Recently, our group has generated two new KO alleles using CRISPR technology (Mrf4 L1/L1 and Mrf4 L2/L2). Preliminary data shows that both alleles develop mild muscle hypertrophy, without affecting Myf5 expression in cis. Experiments in vivo indicate that the two alleles have alterations in muscle metabolism.</strong></p> <p><strong>&nbsp;</strong></p> <p><strong>This project is focused on elucidating how this TF is involved in muscle growth, function, and homeostasis. Furthermore, as adult satellite cells emerge from embryonic founder cells in which Mrf4 expression was activated, we are also studying Satellite Cell biology and function in the absence of Mrf4.</strong></p> <p><strong>&nbsp;</strong></p> <p><strong>Surprisingly, we have identified important phenotypic differences between the two alleles; one hypothesis is that one or both may generate a small peptide that modifies the phenotype. </strong></p> <p><strong>&nbsp;</strong></p> <p><strong>Methods: We dissected the extensor digitorium longus muscle, isolated myofibers from young (3 months) and old (24 months), WT and Mrf4-/- mice, and divided these in three groups: 1- Fibers were fixed and permeabilised for immunostaining with Pax7 antibody to study the myofiber size and content in satellite cells/nuclei; 2- Fibers were plated for bioenergetic analysis using a XF24e Seahorse Analyzer that provides oxygen consumption rate as indicator of mitochondrial respiration as a proxy for metabolic function; and 3- Fibers were plated in proliferation medium to allow their associated Satellite Cells to abandon the niche, migrate and proliferate, and we studied parameters of Satellite Cell proliferation and differentiation.</strong></p> <p><strong>&nbsp;</strong></p> <p><strong>In order to study the potential effect of small peptides generated by one or both alleles, we generated overexpression plasmids containing the sequences of interest. C2C12 myogenic cells were co-transfected with a GFP-expression vector using the electroporator BTX Gemini and then selected to determine if there were significant differences in proliferation and differentiation</strong></p> Elena Gonzalez-Lozano Cristina Vicente-García Juan Diego Hernández-Camacho Jaime Carvajal Derechos de autor 2020 2020-03-19 2020-03-19 9 Study of PMT target specificity in Ustilago maydis <p>Ustilago maydis is a pathogenic fungi responsible for the corn smut fungus disease, which causes a significant loss in mayze production every year. The PMT is a family of well-conserved O-mannosylating proteins. In Ustilago maydis, the deletion of Pmt2 has been shown to be deleterious and the deletion of Pmt4 disrupts completely the infectious process; on the other hand, the deletion of Pmt1 doesn't manifest any phenotype. All PMT proteins have three domains, named PMT, MIR and 4TMC, and several transmembrane regions. We hipothesized that one of these domains of Pmt4 is responsable for the substrate specificity that confers the virulence phenotype in Ustilago maydis Pmt4 and, if this would be the case, we could develop an antifungal drug specific to this domain. To check this, we built three chimerical protein strains, chanching one domain in Pmt4 for the same domain of Pmt1 at a time, and measure the tumor formation in 3 independent experiments. We also built a full Pmt4 length strain as a positive control. The results indicate that either the protein requires all three Pmt4 domains to infect or the chimerical protein is not working properly.<br>Furthermore, we also wanted to check what region gives Pmt2 its specificity for growth. In Schizosaccharomyces pombe, a ortholog of Pmt2, named Ogm2, present the same essencial-for-growth phenotype than in U. maydis. Since U. maydis doesn't have any repressable promoter, we built a S. pombe strain with the shut-off promoter nmt81 in the endougenous Ogm2 gene and will complement the phenotype with mutagenized plasmids containing the U. maydis Pmt2 and measure its viability.<br>Finally, we also plan to build other three strains interchanching Pmt4 domains in the Pmt1 protein.</p> Álvaro Rodríguez Rivas Ramón Ramos Barrales María Dolores Pejenaute Ochoa Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-07 2020-04-07 9 Optimization of CRISPR-Cas systems in ectothermal organisms <p>CRISPR-Cas systems have been traditionally optimized for gene editing in different types of models, such as in mammalian cell lines. However, their implementation in animal models requires additional work. In addition, another impediment is the number of genomic targets of CRISPR-Cas systems since it is limited due to PAM (Protospacer adjacent motif) sequence restriction. PAM sequence is a short DNA sequence, characteristic of each CRISPR-Cas system, near to the target DNA region and required for each Cas nuclease to recognize the target and cut it. Therefore, the possibility of having different CRISPR-Cas systems with different PAM sequences allows us to have a greater number of targets in the genome, which would simplify and increase our possibilities of carrying out a gene editing anywhere in the genome and in a more precise manner.<br>In the laboratory we are interested in implementing new CRISPR-Cas systems in vivo such as CRISPR-Cas12b and a Type I CRISPR-Cas, using zebrafish embryos as a model in animals. First, we are optimizing CRISPR-Cas12b system that has been recently characterized in cell culture (1,2) and allows gene editing in genome locations rich in AT regions where other endonucleases already characterized such as Cas12a or Cas9 cannot target. Optimizing this system in zebrafish embryos will increase the number of genomic targets that can be used in vivo. Second, type I CRISPR-Cas system is formed by Cas3 and a ribonucleoprotein complex (RNP) called Cascade. This system has been able to trigger long-range deletions (3) but its activity in vivo has not been shown yet. Therefore in this project we are implementing type I CRISPR-Cas tool in vivo to facilitate the elimination of large regions in the genome with potential role during embryogenesis.<br>To evaluate the activity of both CRISPR-Cas systems, we are generating different expression vectors by cloning, to subsequently obtain the mRNA of the protein. On the other hand, we are designing specific guide RNAs (gRNAs) for these systems whose targets are genes involved in the pigmentation of zebrafish that facilitates the analysis of these CRISPR-Cas systems. We are currently co-injecting mRNAs and gRNAs of each system in the zebrafish embryos and evaluating the in vivo activity of each system. All together, we will optimize novel CRISPR-Cas systems that will be used for precise gene editing in vivo.</p> Carlos Sánchez Bolaños Miguel Ángel Moreno Mateos Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-07 2020-04-07 9 Bacterial Characterization of Sourdough from a Local Factory <p><strong>Motivation</strong>: Sourdough is the fermentation product of flour and water dough by yeasts and lactic acid bacteria (LAB). Depending on the origin of the microorganisms it can be classified in Type I (spontaneous and with backslopping), Type II (uses a starter culture without backslopping) and Type III (uses a starter culture and backslopping)1.</p> <p>Sourdough-based products have improved properties, which have been attributed to the LAB and their metabolism 2. This study is focused on the characterization of LAB in an industrial sourdough at two different moments.</p> <p><strong>Methods:</strong> To isolate LAB from sourdough, independent samples were taken from a homogenized portion. Appropriate dilutions were plated on mMRS agar and grown on aerobiosis at 30ºC. Cell concentration was calculated as CFU/g of sourdough.&nbsp;</p> <p>Morphologies of Catalase negative isolates were determined by optic microscopy. For molecular identification, DNA was extracted by a method described by Cold Spring Harbour Laboratory3. Fragments from rRNA 16S gene were amplified by PCR and sent for sequencing. Sequences were compared with databases using the BLASTn tool4.</p> <p>Non-pathogen S. aureus and L. innocua were used as indicator strains to detect antimicrobial capacity on BHI medium. Clear zones around colonies were rated as positive results.</p> <p><strong>Results:</strong> LAB were present in the two sourdough samples at 2.75*10^7 CFU/g and 4.3*10^7 CFU/g.</p> <p>The dominant morphologies were long, medium and short bacilli. They presented percentages of 41.5%, 18.9% and 39.6% in the first sample and 13.5%, 30.8% and 55.8% in the second, respectively. The dominant species were Lactobacillus crustorum (corresponding to long bacilli), Lb. rossiae (short bacilli) and Lb. plantarum (medium bacilli).</p> <p>Antagonistic activity was detected only against S. aureus just in 5% of candidates from the second sample.</p> <p><strong>Conclusions:</strong> LABs present in two sourdough sample´s from the same factory have been characterized. Cell concentrations were similar to that described in the bibliography. Dominant species identified were Lb. crustorum, Lb. rossiae and Lb. plantarum. However, their proportion was different in the two sourdough samples. Antagonistic activity against S. aureus was detected in the second sample</p> Moisés López López Belén Floriano Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-07 2020-04-07 9 Cellulose from Posidonia oceanica using clean technologies <p>Due to climate change, environmental conscience is becoming increasingly important today. Deforestation contributes 15-20% to greenhouse emissions, therefore, with the aim of minimizing the felling of trees, there are many investigations that focus on the search for alternatives to the use of wood such as the use of agricultural waste or alternative raw materials to conventional. Among the alternative raw materials to conventional ones to get cellulose, one of the most industrially produced polymers with 1.5 × 10 ^ 12 tons per year, there are tidal residues, composed of algae and marine plants of which there are few studies.[1] After all, cellulose extraction has been considered, since ancient times, terrestrial raw materials as the main route. After storms with large waves this biomass causes large accumulations that form uplands in the coasts with depositions of about 125 Kg of dry matter per kilometer of grassland [2] on the coasts in the form of sidewalks or uprights that provide organic matter and food support for many species of insects, birds, etc. However, its decomposition and rot can affect the adequacy of the beaches and pose problems for tourism due to its visual impact, bad smells, etc. that force the local administration to withdraw to landfills in the summer months. In the present study a chemical characterization of Posidonia oceanica waste collected on the beach of Almerimar (Almería) by the ECOWAL group is carried out. A standard protocol is followed that analyzes moisture content (TAPPI T 257 Standard), ash (TAPPI T 211 Standard), hot water solubility (TAPPI T 257 Standard [2]), soda solubility (TAPPI T 212 Standard [2] ), extractable with ethanol-benzene (TAPPI Standard T 204 [2]), lignin (TAPPI Standard T 222 [2]), holocellulose (Method of Wise et al. [3]) and α-cellulose (TAPPI Standard T203 0S- 61 [2]) . The results obtained from the chemical characterization confirm that Posidonia oceanicais a very good alternative to other conventional raw materials, due to its high content in cellulose and its low-level of ligninalo which makes cellulose extraction relatively simple, economical and with much conditions less energetic so that in future research the study of cellulose extraction from P. oceanica will be expanded, seeking to maximize energy efficiency and the reduction of chemical reagents</p> A Pipió Ternero A Moral Rama M.M Ballesteros Martin Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-15 2020-04-15 9 Study of the expression of ecfG1 and ecfG2, two extracytoplasmic function sigma factors (ECFs) in Sphingopyxis granuli estirpe TFA <p><strong>Motivation:</strong> In bacteria, the initiation of transcription requires a specific multi-domain subunits of RNA polymerase (RNAP) called sigma (σ) factors that binds to its core that play critical roles, including the recognition and opening of promoters for the RNA synthesis (Paget, 2015). One type of sigma factors are extracytoplasmic function sigma factors (ECF) which provide a means of regulating gene expression in response to a wide range of environmental changes (Feklistov et al., 2014). Sphingopyxis granuli TFA is a Gram-negative Alphaproteobacteria that is one of the few strains able to grow on the organic solvent tetralin as a sole carbon and energy source and able to grow respiring nitrate under anaerobic conditions (Gonzalez-Flores et al., 2016). In Sphingopyxis granuli TFA two ECF σ factors have been described, EcfG1 and EcfG2, that have a critcal biological role in the General Stress Response (GSR) in this bacterium (de Dios et al., 2020)<br><strong>Methods:</strong> With the aim of studying the transcriptional and postranscriptional regulation of each ecfG genes, a recombinant protein was built in which each EcfG have a FLAG-tag fused which allowed us to quantified the amount of each sigma factor by Western Blot studies in different growth conditions. This protein was constructed using a DNA-recombination method based on a double-strand break caused by SceI nuclease. Flanking regions of each ecfG genes were cloned in a multiple cloning site (MCS) of a non-replicative vector, this MCS is flanked by two SceI target sites. When this integrative vector is integrated into the chromosome of TFA, a broad host range vector including SceI gene downstream of an inducible promoter must be introduced and a double-strand break is caused in the chromosome. The final repair of this break results with a high frequency in the deletion of the target gene (ecfG). Thus we only get the gene with the FLAG-tag<br><strong>Results:</strong> In this work we analyzed the levels of expression of each sigma factors both at the transcriptional by RNA-seq and at translational level by quantifying the levels of each protein through Western Blot studies under different growth conditions</p> Rosario Elena Armijo Miranda Ruben De Dios Barranco Francisca Reyes-Ramírez Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-15 2020-04-15 9 In vivo testing of safety and immunogenicity of new vaccine candidates against PCV2 designed for a better performance than that showed by currently marketed vaccines <p>Porcine circovirus type 2 (PCV2) is a globally distributed virus causing considerable economic losses. It affects mainly piglets after the weaning, leading to the disease known as postweaning multisystemic wasting syndrome, whose consequences are death or a significant reduction of pigs fattening rate. PCV2 also affects pregnant sows, leading to abortions. There are four commercial vaccines against PCV2, two killed vaccines and two recombinant vaccines. All of them are based on PCV2 strains of genotype a, which was predominant in the field when the vaccines were marketed. Since live replicative virus could not be used as vaccines, these vaccines lead to prevention of symptoms but not to complete viral clearance. Vaccinated and infected pigs can still infect other pigs. Today the predominant genotypes are PCV2 b and d. Although the marketed vaccines showed cross-protection against heterologous genotypes, it is suboptimal and there are increasing cases of vaccine escape by new PCV2 strains. ADL Bionatur Solutions designed and produced three new generation recombinant vaccine candidates, BNT029, 030, and 031 with two goals: improve antigenic presentation to achieve better viral clearance and to raise specific immunity against the new genotypes. In this project, the vaccine candidates have been tested in vivo for safety and immunogenicity in comparison with the current commercial leader, Ingelvac CircoFLEX® from Boehringer Ingelheim. In mice, the new candidates raised cell-mediated immunity of higher intensity than that raised by the commercial gold standard, and with a more significant Th1-specific component biased to the PCV2b and PCV2d genotypes. In an experimental trial in piglets, immunization with BNT029, 030, and 031 did not lead to any adverse effect. The three new candidates led to seroconversion, with antibody titers significantly higher than those raised by the commercial gold standard. While the antisera raised in piglets by Ingelvac CircoFLEX predominantly recognized viral antigens derived from a PCV2a strain, the antisera raised by BNT029, 030, and 031 predominantly recognized viral antigens derived from PCV2b and PCV2d strains. Therefore, we have confirmed the initial hypotheses aimed for the new candidates. Currently, we are carrying out serum neutralization assays to confirm that the immune response raised by the new candidates in piglets can neutralize effectively the infection by the currently predominant viral strains of PCV2.</p> I Rodríguez-Barrios R Cid J.J Infante Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-15 2020-04-15 9 Expression and Purification of Cas13d <p><strong>The class 2 type VI CRISPR Cas13 systems, an RNA targeting CRISPR-Cas effector, has been originally involved in adaptive prokaryotic immunity by protecting bacteria against invading RNA phages, It is currently used as a tool to cut and degrade RNA in a precise manner in yeasts, mammalian and plant cell lines. In the Moreno-Mateos lab the CRISPR-RfxCas13d system has been recently shown as an efficient and specific system in zebrafish and other models targeting mRNA in animal embryos (Kushawah et al., Biorxiv 2020). Our lab is interested in understanding an early embryogenesis process called the maternal-to-zygotic transition (MZT). This process implies the activation of a silent embryonic genome by the maternal mRNA products deposited in the oocyte. We have used our CRISPR-Cas13d technology to knockdown mRNAs from the maternal contribution with a role in the zygotic genome activation (ZGA). For example, we injected mRNA coding Cas13d and gRNAs targeting nanog, a maternally-provided factor crucial for the ZGA, and we observed classical phenotypes when MZT is altered. However, we found that the penetrance of the targeting and the observed knockdown phenotypes can be optimized. To achieve this, we reasoned that the injection of the purified protein instead the mRNA coding Cas13d could have an earlier targeting of these and other maternal RNAs.</strong></p> <p><strong>In this work, we have generated a bacterial expression vector and successfully purified RfxCas13d endonuclease. The RfxCas13d protein was expressed in E. coli cells and purified by affinity and ion-exchange chromatography methods. In vivo tests in zebrafish embryos demonstrate that the purified protein is biologically active and the complex Cas13-gRNA triggers efficient mRNA knock-down showing higher targeting RNA depletion than injections of Cas13 mRNA. We are currently analysing the specificity of the Cas13d protein in vivo by RNAseq experiments and performing in vitro cleavage assays to complement the results obtained in vivo.</strong></p> <p><strong>All together, these results demonstrate that the use of purified protein for maternal RNA targeting is an efficient and convenient system to potentially uncover novel maternal RNA functions. This new optimized tool will open up new avenues that will illuminate our knowledge in early vertebrate embryogenesis.</strong></p> Dahiana E. Monges Luis Hernandez-Huertas Miguel Ángel Moreno-Mateos Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-03-19 2020-03-19 9 Strategies to improve the robustness of acentrosomal spindle formation in female meiosis <p>In meiosis, centrosomes are so important because they organize microtubules and nucleation of the spindle for a correct chromosomal segregation in eukaryotics cells. Human female oocytes lack centrosomes, so microtubules must self-assembly, which can cause mistakes in the process and diseases to the embryo. To study the molecular mechanisms supporting acentrosomal spindle, we are using the fission yeast Saccharomyces pombe as model scenario. In this organism, spindle pole bodies (SPBs), the functional equivalents of centrosomes, are sitting on the nuclear envelope (NE), which is dissasembled in each cell cycle by activating proteins like Sad1 and Bqt1, that mediate chromosome-NE contacts (Pineda-Santaella &amp; Fernández-Álvarez, 2019). Based on these findings, our aims are making the acentrosomal spindle more robust and minimizing chromosomal segregation errors. In order to get then, we want to analyze the effects of overexpression of Cls1p, a cytoplasmic linker associated protein (CLASP) that stabilizes specific groups of MTs in S. pombe and has two homologous proteins in humans, CLASP1 and CLASP2. They contribute to the formation and maintenance of the spindle midzone by promoting MT rescue events (Al-Bassam et al., 2010). On the other hand, there are another important proteins in this process, like Klp6, a kind of kinesin-8, whose homologous proteins in humans are Kif18A, Kif18B, and Kif19. An in vivo study suggets that Klp6 binds to the tubulin triggering the birth of new MTs and promoting nucleation and catastrophe at the growing MT tip (Erent et al., 2012). Deletion or knockdown of Klp6 leads to longer spindles and defects in its assembly and position in many cases (Gergely et al., 2017), but we suggest that a longer acentrosomal spindle could also be stronger and more stable. So, we also pretend to observe the impact of deletion of klp6 on the spindle behavior and chromosome movements. To perform that experiments, we have obtained two different mutants for klp6 and cls1 in a bqt1Δ sad1.2 background by crossing some strains with these characteristics and we are studying what happens in the cell nucleus by fluorescent microscopy, using a DeltaVision microscope. As a result, we expect that chromosomal segregation in mutants for cls1 and klp6 will be more efficient with respect to the mutant control, which has only bqt1Δ sad1.2, and, ultimately, improve the meiotic process in this context.</p> Nazaret Fernández Castillo Alberto Pineda-Santaella Ángela Sánchez Gómez Ana María Brokate Alfonso Fernández Álvarez Derechos de autor 2020 Biosaia: Revista de los másteres de Biotecnología Sanitaria y Biotecnología Ambiental, Industrial y Alimentaria 2020-04-05 2020-04-05 9